· abstract properties and regulation of small-conductance anion channels in a rat rnicroglia cell...
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PROPERTIES AND REGULATION OF SMALL-CONDUCTANCE ANION CHANNELS IN A RAT MCROGLIA CELL LINE
Timothy Edward Mertens
A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology
University of Toronto
Q Copyright by Timothy Edward Mertens 1998
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Abstract
Properties and regulation of small-conductance anion channels in a rat rnicroglia cell line.
Master of Science, 1998.
Timothy Edward Mertens, Department of Physioiogy, University of Toronto.
Microglia, resident brain immune cells, are involved in brain pathologies; e.g., stroke. Using
appropriate bath and pipette solutions, we isolated a whole-cell anion current in a rat rnicroglia cell
line and created a biophysical and pharmacological fingerprint. We also investigated the regulation
of the underlying channels. The whole-cell current was outwardly rectified and displayed time- and
voltage-independent gating. The channels were volume sensitive, moduIated by intracellular ion
content, had a selectivity sequence similar to Eisenman I (I' > Br' = Cl') and were permeable to the
excitatory amino acid glutamate. The following traditional and non-traditional anion channel
blockers inhibited the current: NPPB, flufenamic acid, giibenclarnide, suramin and riluzole. Channel
activation depended on cellular ATP and was not supported by GTPyS alone. The channel properties
are consistent with a potential role in glutamate neurotoxicity during pathological conditions. The
biophysical and pharmacological properties of the anion channels are distinct from other known
chloride channels; e.g. CIC-2 and C1C-3.
Acknowledgements
I would like to thank Dr. Schlichter for her time, patience and her true passion
for science. Despite supervising several graduate students, she always found the
time to critique my work and provide invaluable feedback for which I am very
gratefil. I would also like to take this opportunity to thank my colleagues.
Francisco Cayabyab, Liane Chen, Martin Chang, Raj Khanna, Bob Kotecha and
Lipi Roy for their camaraderie (especially over lunch!) throughout the duration of
my Master's thesis. Furthermore, I would like to thank George Sakellaropoulos,
Frank Vidic and Brent Clark for their friendship and technical assistance without
which I would have been unable to complete my thesis. I would also like to thank
my family for their unconditional support and encouragement. Finally, I would like
to thank Rachel Rotenberg for her friendship, kindness and love (not to mention the
countless hours she spent helping and encouraging me). Thank you.
Table of Contents ............................................................................... Table of Contents iv
. . .................................................................... List of Tables and Boxes VII
... .................................................................................... List of Figures wii
Chapter 1 Introduction 1
........................................................................... What are microglia? 1
.......................................................... Where do microglia originate? -3
......................................................... What currents do they express? 4
.......................... Why are we interested in studying anion channels? 4
.................................... What is regulatory volume decrease (RVD)? 5
...... How might the channels contribute to glutamate neurotoxicity? 6
Chapter 2 Materials and Methods ................................................................................................... Cells 10
Preparation of rat primary microglia ............................ ... 10
.......................... Preparation of the rat microglia cell line 12
Preparation of human T lymphocytes .............................. 12 ............................................................................................ Solutions 13
................................................................................................ Drugs 1 6
Guanine nucleotide experiments ...................................... 16 Pharmacological compounds ........................................ 16
................................................................ EIectrophysiology ........ ...... 18
............................................................................. Pipettes 18
Junction potentials ................................................... 1 8
...................................................................... Acquisition 1 9
............................................................................... Digitized Images -20
............................................................................................ Perfusion 20
............................................................................................. Analysis -20
......................................................... Current-voltage data 20
Statistical Analysis ........................................................... 21
Chapter 3 Biophysics .......................................................................................... 22
Isolation o f the chloride current ....................................... 22
Time and voltage independence ......... .... ................... -23
Current-voltage reiationship ............................................ 23
Rat primary microglia and human T lymphocytes .......... 24
Selectivity sequence ......................................................... 24 . . Glutamate perrneablllty ..... ........ ................................ 29
NaF-induced currents ....................................................................... - 3 2
Swelling and volume sensitivity ........................................................ 34
Ionic strength .......................................... -36
Pharmacology .................................................................................... 41
NPPB ................................................................................ 42
Flufenarnic acid ................................................................ 42
................................................................. Glibenclamide -42
Riluzole ............................................................................ 44
External application of 1 O m M ATP ................................. 44
............................................... Human T lymphocytes 47
List of Tables and Boxes
Table 1
Table 2
Tabie 3
Table 4
Table 5
Table 6
Table 7
Table 8
Table 9
Bath solutions ............................................................................... 15
....................................................................... Pipette solutions 1 5
................... Pharmacologicai compounds (source and handling) 17
..................... Junction potentials vs 125 mM NaCl agar bridges -19
Anion selectivity ........................... ... ........................................ 29
Inhibition of Gclrev by various pharmacological compounds ....... 50
Bonferroni t test for painvise comparisons of the means of the nucleotide experiments .................................................. - 3 6
Pauling radii and ionic hydration energies of the halides ............ 60
.................................... Comparison of whole-cell anion currents 89
..................... Box 1 Derivation of the modified GHK-voltage equation 30
........................................................................ Box 2 The Donnan effect 40
vii
Figure 1A
Figure 1B
Figure 2A Figure 2B Figure 2C
Figure 3A & 3B
Figure 4A Figure 4B
Figure 5
Figure 6A Figure dB
Figure 7
List of Figures
Time- and voltage-independent gating of the rat microglia cell line whole-cell anion current ............................. 25
Current-voltage relationship .................................................... -25
................. Whole-cell anion current for the microglia cell line 26 ............ Whole-cell anion current for the rat primary microglia 26 ............ Whole-cell anion current for the human T lymphocyte 26
Representative traces showing the halide selectivity of the rat microglia cell line anion channel ............................... 28
Current-voltage relationship of the NaF-induced currents ...... -33 NaF-induced currents in response to voltage steps .................. -33
Hoffman modulation-contrast (HMC) images of a microglia cell prior to and following exposure to a hypo-osmotic shock ........................................... -34
Activation of Icl in hypo-osmotic solution ............................... 35 Plot of instantaneous slope conductance at the experimentally measured reversal potential ........................ 35
Comparison of the spontaneous activation of GclRv using iso-osmotic and hypo.osmotic. 50 mM NMDGCl pipette solutions .............................. - ....................... -37
viii
Figure 8A
Figure 8B
Figure 9
Figure 10A Figure 10B
Figure 11
Figure 12
Figure 13
Figure 14
Figure 15
Figure 16
Figure 17
Representative traces comparing the spontaneous activation of G- with high and low ionic-strength pipette solutions ..... -39 Comparison of Gch with high and low ionic-strength pipette solutions during the time of half-maximal activation of G- when using the low ionic-strength pipette solution ..................................... -39
...................................................... Inhibition of G- by NPPB 43
Inhibition of G- by glibenclamide ........................................ -45 Voltage-independent inhibition of h by 500 pM glibenclarnide ........................................................................ 4 5
Inhibition of Go by riluzole ..................................... ... ........... 46
Voltage-dependent inhibition of L. by suramin ................... ..... 48
An illustration of the lack of block of the human T ....................... lymphocyte anion current by extracellular ATP 49
..................................... Effect of intracellular GTPyS on Gel ,, 52
Effect of intracellular GDPPS on GaW .................................... 54
Comparison of peak Germ with and without various nucleotides added to the pipette solution .................................. 55
Effect of intracellular GTP and GDP on the ................... whole-cell anion current in human T lymphocytes 57
........................................ kIC3 C1C-3 related current
.................................... IL- 1 ..interle*- 1
........................................ IL-6 interleukin-6
......................................... Kir w d - r e potassium channel
........................................ Kv.. d - r e potassium channel
...................................... K(Ca) c c i e e d e t potassium channel
KATp.. ...................................... ATP-sensitive potassium channel
MDR l ................................... .muhirug resistance gene i
MEM ............................... ..-..-.Minimum essential medium
..................................... MHC . m a r histocompatibility complex
NMDA .................................. N - m e t
............................... NMDGCI N-methyl-D-glucamine chloride . . NOS.. ................................... c oxide synthase
P-gp ....................................... .P-glycoprotein . . PKC ...................................... p r o t e b a s e C
. . Po, ...................................... ..open pro babdity
RSe,, ....................................... sene resistance
RVD ..................................... ..regulatory volume decrease
RVI.. ...................................... . r e l a t o volume increase
TNF-a .................................... tumo necrosis factor alpha
................................... Vse,is.. v o g e drop due to series resistance
VSOR.. .................................. vole-sensit ive outwardly rectifying
Chapter 1
Introduction
What are microglia?
The central nervous system (CNS), once considered immune privileged
because of the protective nature of the blood brain barrier (BBB), is now
considered to have resident immune cells known as microglia (Gehrmann et al.,
1995). Microglia make up 5 - 15% of the total cellular composition of brain
tissue and are capable of existing in multiple forms depending on the
developmental status and pathological condition of the CNS (Davis et al., 1993).
They are generally classified as having four functional and morphological states:
amoeboid, ramified, active and reactive, all of which appear to have specific roles
(Thomas, 1992).
The amoeboid state has a flat morphology with pseudopodia and is
predominantly found in the prenatal to early postnatal period of development
(Murabe and Sano, l982a, 1983; Giulian and Baker, 1986). Amoeboid microglia
are phagocytic and may be involved in removing dead cells and cellular debris
which occur during routine development (Ashwell et al., 1989; Hume, Perry and
Gordon, 1983 ; Linden, Cavaicante and Bmadas, 1 986).
In the healthy adult C N S microglia become ramified and are characterized by
5-10 p long bodies with long thin processes. Ramified microglia or "resting
microgiia" exhibit few macrophage properties (Thomas, 1992). However, in their
resting state they have a high level of endocytic activity (pinocytosis) (Ward et
al., 1991) and motility, potentially enabling them to migrate within a local region
within the CNS (Thomas, 1990; Ward et al., 1991). Therefore, resting microglia
may help cleanse the extracellular fluid of excess chemical transmitters and
modulators in normal brain tissue (Ward et al., 1 99 1 ; Thomas, 1992).
In the event of neurological trauma and disease, (e-g., hypoxic and ischemic
insult) resting microglia become activated and undergo distinct morphological
changes and mitotic activity. Activation occurs in two stages (Gehrmann.
Matsumoto and Kreutzberg, 1995). In the first stage, microglia are characterized
by stouter cell processes and a swollen cell body (Dei Rio-Hortega, 1932) and
express macrophage cell surface markers; e.g., CR3 receptors and major
histocompatibility complex (MHC) class I (Graeber, Streit and Kreutzberg, 1988;
Streit, Graeber and Kreutzberg, l989), however they are not phagocytic.
In the second stage of activation, the "activated microglia" are transformed
into fully phagocytic cells termed "reactive microglia" and are characterized by an
oval morphology without cell processes (Thomas, 1992). The transition from
active to reactive is enhanced by neuronal degeneration (Streit and Kreutzberg,
1988).
Almost all neurological m a may non-specifically activate microgIia
(Thomas, 1992). In their up-regulated state they are capable of secreting cytokines
that modulate astroglial proliferation (IL-I), neovascularization in brain trauma
(IL-I), nerve growth factor production (IL-6) and demyelination (TNF-a)
(Gehrmann, Matsumoto and Kreutzberg, 1995; Thomas, 1 992). Reactive
microglia also release potentially cytotoxic substances such as enzyme inhibitors
(cystatin C), complement components, binding proteins (ferritin, fibronectin),
reactive oxygen intermediates (Oyr H~OZ) and reactive nitric oxide (NO. O N 0 0 3
(Banati et al.. 1993). Therefore microglia can have both beneficial roles; e.g.,
wound healing (Thomas. l992), as weil as deleterious consequences; e.g.,
involvement in neuronal loss and functional neuronal impairment after spinal cord
ischemia (Giulian and Robertson, 1990).
Where do microglia originate?
The origin of microglia is controversial. Some reports suggested that
rnicroglia originate from the neuroepithelium similar to neurons and other glia of
the CNS (Kitamura, Miyake and Fujita, 1984; Matsumoto and Fujiwara, 1987;
Sirninia et al., 1987). However, the general consensus now is that microglia
originate from blood monocytes which enter the brain and retina during
- embryonic development (Hume, Perry and Gordon, 1983; Perry, Hume and
Gordon, 1985).
What currents do they express?
Microglia fiom several different species, including rat, mouse, human and
cattle, express a wide array of ion currents. The two most predominant currents
are an inward rectifier (Kir) and an outward rectifier (Kv). Less frequently
reported have been proton currents (Visentin et a1 ., 1993, Kv 1.5-like currents
(Pyo et al., 1997), non-selective cation currents activated by purinergic receptors
(Kettenrnann, Banati and Walz, 1993; Walz et al., 1993; Langosch et al., 1994;
Norenberg et al., 1994), cazf-dependent K' currents (Kc,) (McLarnon, Sawyer
and Kim, 1995; McLarnon et al., 1997), ~ a + currents (Norenberg, Illes and
Gebicke-Haerter, 1 994; Korotzer and Cotman, 1 992), ~ a " currents (Colton et al.,
1994) and anion currents (Visentin et al., 1995; Schlichter et al.. 1996; McLarnon
et d., 1997). There has also been one report from a collaboration with our lab of
a rat microglia cell line (the same cell line which is the focus of this thesis)
expressing HERG-like K' currents (Zhou et al., 1998).
Why are we interested in studying anion channels?
The role of anion channels in microglia has not been extensively
characterized. Putative roles include involvement in proliferation (Schlichter et
al., 1 996) and generation of nitric oxide (Brown, Kozlowski and Perry, 1998). By
analogy with other immune cells, the microglial anion conductance may also
regdate membrane potential (Holevinsky, Jow and Nelson, 1994), intracellular
pH (Holevinsky, low and Nelson, 1 994), intracellular ca2+ concentrations
(Matthew, Neher and Pemer, 1989) and volume (Lewis, Ross and Cahalan,
r 993).
By studying the regulation and properties of the volume-sensitive anion
channels, we hope to gain insight into some of these potential functions. For
example, the anion conductance may be involved in the process of regulatory
volume decrease (RVD) and by a similar process may inadvertently exacerbate
ischemia-induced neurotoxicity.
What is regulatory volume decrease (RVD)?
RVD is the process by which swollen cells decrease their volume while under
a constant hypo-osmotic stress (Haussinger, 1996). At rest. water is in
thermodynamic equilibrium across the cell membrane (Strange. Emma and
Jackson, 1996). When there is an increase in the internal or externa! osrnolality,
water moves in or out of the celI passively in the direction of the osmotic gradient.
Due to the compliance of the cell membrane, the cell shrinks or swells (Strange,
Emma and Jackson, 1996). To control the composition of the internal milieu of
the cell, the cell must be able to counter these volume changes. By increasing the
uptake o f ions or osmolytes (e-g., taurine, inositol, and sorbitol) the cell increases
its volume, (RVT). Conversely, by releasing ions or osmolytes, cells decrease
their volume (EWD) (Haussinger, 1996).
In lymphocytes, it is proposed that RVD is mediated by the activation of
volume-sensitive anion channels (Lewis, Ross and Cahaian, 1 993). In this model,
cell-swelling activates the volume-sensitive anion channels, which causes
membrane depolarization. This in turn activates voltage-dependent Kf channels
(Deutsch, Krause and Lee, 1986), resulting in an efflux of K' and C1-. The net
efflux of KCI creates an osmotic gradient favourable for water loss that shrinks
the cell thus restoring the cells' volume. A similar role can be envisioned for the
volume-sensitive anion channels (Schlichter et al., 1996) in microglia.
How might the channels contribute to glutamate neurotoxicity?
It is generally accepted that neuronal damage during hypoxia and ischemia is
the result of increased ca2' influx (Garthwaite and Garthwaite, 1986; Choi. 1987)
which activates enzymes deleterious to cell survival; e-g., lipases. proteases. and
endonucleases (Meldrum and Garthwaite. 1 990). One means by which neuronal
~ a " influx increases is through agonist-operated calcium channels (AOCCs);
e.g., N-methyl-D-aspartate (NMDA) glutamate-receptor channels (Siesjo and
Bengtsson, 1989; Choi and Rothman, 1990; Lipton and Rosenberg, 1994;
Szatkowski and Attwell, 1994). For example, when there is a severe reduction in
blood flow to the CNS, neuronal ATP levels decline (Raichle, 1983) due to the
attenuation of oxidative phosphorylation (Sweeny et al., 1995). Initially this
decline can be compensated for by anaerobic metabolism (Sweeny et al., 1995)
however, if the ischemic insult persists then ca2+-activated K' channels or ATP-
inhibited K+ channels open and there is a large K+ efflux fiom neurons (Walz,
Klimaszewski and Patterson, 1993). The increase in extracellular K' depolarizes
adjacent cells; e.g., neurons and astrocytes (Walz, Klimaszewski and Patterson,
1993) an event termed anoxic depolarization (Szatkowski and Attwell, 1994).
Shortly thereafter, extracellular glutamate concentrations increase due to
excessive release, abnormal leakage and/or impaired uptake by surrounding cells
(Benveniste et al., 1984). The combination of neuronal membrane depolarization
(necessary to alleviate M ~ ~ + blockade of the NMDA-receptor channel) and the
increase in extracellular glutamate activates NMDA-receptor channels that allow
ca2+ influx (Siesjo and Bengtsson, 1989). In this way, glutamate enhances
neuronai death (Szatkowski and Attwell, 1994).
One potential source for the increase in extracellular glutamate is efflux
through volume-sensitive anion channels (Kimelberg et ai., 1990; Bausch and
Roy, 1996). Consequently, it has been demonstrated that anion-channel blockers
can inhibit the release of excitatory amino acids fiom swollen astrocytes
(Kimelberg et al., 1990). Analogously, efflux of glutamate from volume-sensitive
anion channels in microglia (Schlichter et al.. 1996) may also contribute to
glutamate neurotoxicity. One difficulty with this hypothesis, however, is that
many volume-sensitive anion channels require intracellular ATP for their
activation and continued functioning (Strange, Emma and Jackson, 1996; Okada,
1997). Therefore, in conditions where cellular ATP levels are compromised the
rnicroglia anion channels may be inactive. Hence, they would not provide a
pathway for glutamate efflux.
Therefore, one objective of this thesis was to characterize the properties and
regulation of the anion channels to determine if the channel properties are
consistent with a role in glutamate neurotoxicity ; e. g., are the small-conductance
anion channels volume sensitive and are they permeable to glutamate?
Also, very little is known about the molecular identity of volume-sensitive
anion channels. It is unclear whether the whole-cell C1- current seen in a variety
of cell types (see Okada, 1997) represents a single channel type or whether there
is more than one channel type (Clapham, 1998). To make comparisons between
the anion channels in microglia and potential molecular candidates, it is important
to characterize the anion conductance as fully as possible; e-g., biophysical,
pharmacological and regulatory properties. By doing so. a fingerprint for the
channel can be established and compared to other channels.
Finally, based on the following observations we hypothesized that G proteins
may modulate the anion channels in microglia. First, it has been demonstrated
that G proteins can modulate anion channels involved in RVD (Schwiebert et al.,
1990; Schwiebert et al., 1992). Second, G proteins may regulate some small-
conductance anion channels (Matthews. Neher and Penner, 1 989). Third, our
laboratory has found that the anion current in human T lymphocytes is modulated
by guanine nucleotides; i.e., GTPyS increased the peak CI- conductance and
decreased the rate of conductance rundown (Schlichter et al., unpublished).
Finally, both microglia and lymphocytes have G-protein coupled receptors; e.g.,
CXCR4 chernokine receptors (Hesselgesser et ai., 1998; Jiang et al., 1998) thus
providing a biochemical pathway whereby G proteins are activated.
Therefore the thesis objectives were:
(I) to characterize the biophysics of the anion conductance
(2) to characterize the role of volume and intracellular ionic composition in
regulating the anion channels
(3) to determine the pharmacology of the anion channels and
(4) to determine if the anion channels are regulated by guanine and adenine
nucleotides.
Throughout the thesis. comparisons with the anion channels in primary
microglia have been made where possible. In addition: comparisons with anion
channels in another immune cell (human T lymphocytes), which we have also
extensively characterized, have been made.
Chapter 2
Materials and Methods
Cells
Preparation of rat primary microglia
Primary microglia were isolated from litters of 2-3 day old Wistar rat pups.
The pups were decapitated and the heads were transferred to a beaker of 1 : 1 95%
alcohol/bleach for sterilization. The heads were then placed in a petri dish of
Minimum Essential Medium (MEW (Gibco Laboratories, Grand IsIand, NY)
where the brains were isolated. The newly isolated brains were separated into two
cerebral hemispheres and the meninges, hippocampus, cerebellum and olfactory
bulb were removed. The remaining tissue, the neopallium, was transferred to a
flask containing 40 ml of MEM containing 0.25% trypsin and 25 &ml DNAse I
(all from Sigma, St. Louis, MO). The tissue was stirred for 30 min at room
temperature and gently triturated after which the cell suspension was centrifuged
for 10 rnin at 1000 rpm to remove any cell debris. The MEM/trypsin solution was
I0
aspirated and the remaining pellet was re-suspended in 5 mI of culture medium.
Two 75 cm2 flasks (containing 30 ml of medium) were seeded with the
dissociated cells. Cells were incubated at 37OC with 5% COz. Seven days after
the initial plating, the cells were fed with 10 rnl of culture medium (MEM + 5%
fetal bovine serum (FBS) + 5% horse serum + 0.05 pg/ml gentamicin). After 12
days, the flasks were shaken at 180 rpm for 30 min to dislodge adherent microglia
£iom the astrocyte bed. The floating microglia celIs were re-plated and allowed to
adhere for 1.5-2 h before shaking for a fiuther 5 min to remove any remaining
astrocytes. Cell cultures were >95% microgiia, as determined by staining with the
microglia-binding lectin from GrzBnia simplic~oliu (GSA EB4) that binds to an
a-D-galactose residue (Streit and Kreutzberg, 1988; Streit, Graeber and
Kreutzberg, 1989). The cells also stained positively with the OX-42 antibody
(specific for the CR3 complement receptor) and ED1 antibody (specific for a
cytoplasmic antigen) (Graeber, Streit and Kreutzberg, 1988). Astrocytes were
selectively stained with an antibody directed against glial fibrillary acidic protein
(GFAP) (Bignarni et al., 1972). All antibodies were from Sigma. In preparation
for electrophysiological studies, microglia cells were plated onto 15 rnm diameter
cover slips (Bellco Glass, Vineland, NJ) 1-2 days prior to the start of an
experiment.
The isolation and preparation of the rat primary microglia was performed by
the cell-culture technician, Brent Clark. Brent Clark and a MSc. student, Bob
Kotecha, performed the immunocytochemistry. Immediately before an
experiment, a microglia-covered coverslip was placed into a 150 p1, small
volume, perfusion chamber (see Perfusion) on an inverted microscope.
11
Preparation of the rat microglia cell line
Initially, to enhance cell proliferation, weekly feedings of the primary rat
- cultures were supplemented with the supernatant £iom a mouse fibroblast cell line
(LM 10-5) (gift from Dr. S. Fedoroff, University of Saskatchewan, Canada)
known to contain high concentrations of colony stimulating factor (CSF-I).
CSF-1 is a stimulus of microglia proliferation (Fedoroff et at., 1993). Having
stimulated microglia proliferation in this manner, a bloom of cells was isolated.
These cells were highly proliferative and did not require fiather addition of
growth factors and as such were established as a microglia cell line. The cell line
stained positively with the microglia markers OX42 (Graeber, Streit and
Kreutzberg, 1988) and GSA I-&. (Streit and Kreutzberg, 198 8; Streit, Graeber
and Kreutzberg, 1989). Preparation of the microglia cell line for
electrophysiologicai studies was the same as that described for the primary
microglia. The imrnunocytochemical characterization and preparation of the
microglia cell line was performed by the cell culture technician, Brent Clark.
Preparation of human T lymphocytes
Peripheral whole blood, 10-1 5 ml, was drawn from healthy
into sterile vacutainers. The whole blood was diluted with RPMI
student donors
1 : 1 and pipetted
into two 50 ml centrifuge tubes containing Ficoll Hypaque (Sigma). The whole-
blood Ficoll Hypaque mixture was centrifuged for 30 min at 1500 rpm to isoiate
the lymphocytes from other contaminating cells; e.g., erythrocytes and platelets.
After centrihgation the lymphocytes were aspirated fiom the b u m coat and
placed in 15 ml centrifuge tubes and diluted in a 1: 1 ratio with RPMI 1640
medium (Gibco). Following further centrifugation (8 min at 1000 RPM), the
supernatant was aspirated and discarded. The remaining pellet containing the
lymphocytes was resuspended in complete culture medium (RPMI + 10% FBS +
0.05 gg/rnl gentamkin). To remove B cells and fiuther enhance the purity of the
T cell population the lymphocyte-RPMI mixture was incubated on a nylon wool
column for 30 m i . at 37OC with 5% C02. Non-adherent T cells were eluted with
complete culture medium while B cells adhered to the nylon wool. The eluted T
lymphocytes were washed and suspended in AIM-V lymphocyte medium and
used for same-day experiments or incubated overnight at 37OC with 5% C02.
Using this isolation method our lab has previously shown that the cells are z 98%
T cells as determined by fluorescence-activated cell sorter (FACS) analysis with
the anti-CD3 Ab OKT3 (Ortho Pharmaceuticals, Raritan, NJ).
Solutions
The chemical constituents of the salt solutions are outlined in Tables 1 and 2.
Bath and pipette solutions were titrated to pH 7.4 and pH 7.2, respectively, using
a hydroxide comprised of the major cation of the solution (except for
N-methyl-D-glucamine chloride (NMDGCI) solutions, which were titrated using
NMDG). Pipette solutions were filtered using a sterile syringe filter (0.22-micron
pore size) (Sarstedt, St. Leonard, QC). Bath solutions were filtered using a
0.22-micron cellulose-acetate membrane disposable bottle-top filter. Solutions
were stored at 2-8°C under sterile conditions for future use. The osmolalities of
the solutions were measured using a freezing-point depression osmometer (model
3M0, Advanced Instruments Inc., Norwood, MA).
Hypossmotic solutions were made by diluting the bath solution with a
"dilution solution" containing 1 mM MgC12, 1 rnM CaC12 and 10 mM HEPES, pH
7.4 (see Table 1). The osmolaiity of the hypo-osmotic NMDGCI bath solution
typically ranged korn 220 to 255 mOsm/kgH20. It was observed that a smaller
hypo-osmotic shock; i.e., 255 mOsrn/kgHzO, produced less variable results.
Dilution of the iso-osmotic, 140 rnM NMDGCl bath solution to 255
mOsm/kgH20, effectively reduced the NMDGCl concentration to -120 mM. The
alteration in NMDGCl concentration was taken into consideration for all
calculations performed.
In experiments requiring adenosine triphosphate (ATP) in the pipette solution,
ATP was either weighed out and added to 5 ml of the pipette solution prior to the
beginning of the experiment or frozen 10 mM ATP aliquots were thawed and
diluted to 2 mM ATP for use that day. Solutions containing nucleotides were
kept on ice for the duration of the experiment.
Table 1 Bath Solutions
IHEPESI [sucrose] [glucose]
125 m M NaCl 140 m M NMDGCI Dilution Soiution
osmolaIity (mOsm) 296 295 26 pH 7.4 7.4 7.4 titrated with NaOH NMDG NMDG
All concentrations are in mM
Table 2 Pipette Solutions
[NMDGCI] W s p l [KC11 [MgC121 [CaC121 [HEPES] [EGTA] [sucrose]
fiee [ ~ g " ] 0.08 0.08 eee [ca2'] 16 x 10" 16 x lod PH 7.2 7.2 ionic strength (mM) I46 76 osmoiality (mOsm) 262 295
All concentrations are in mM Free magnesium and caIcium concentrations were calculated using the CaBuffer program written by Jochen KIeinschmidt (NYU Medical Center, NY) When necessary, bath and pipette osmolalities were increased by adding sucrose
Drugs
Guanine nucleotide experiments -
Guanosine 5'-G(2-thiodiphosphate) (GDPPS) and guanosine
5'-0-(3-thiotriphosphate) (GTPyS) were purchased from Sigma in 5 mg
quantities. 20 m M stock solutions of the guanine nucleotides were made up in
distilled water and aliquoted in 10 p1 quantities for future use. The aliquots were
stored in -20°C refrigeration for up to two weeks before being discarded. During
experiments, solutions containing GTPyS or GDPPS were kept on ice. The h a 1
working concentration, 200 pM, of the GTPyS and GDPPS was obtained by
diluting the aliquot 100 times with the appropriate pipette solution.
Phanacological compounds
Stock solutions of the following pharmacological blockers were dissolved in
dimethylsulfoxide (DMSO): 5-nitro-2-(3-phenylprop ylamino) benzo ic acid
(WPB, 500 mM), flufenamic acid (50 mM),
5-chloro-n-[2- [ 4 [ [ [ ( c y c l o h e x y l a m i n o ) c ~ y l ] - 2 -
rnethoxybenzamide (glibenclamide, 200 mM) and 2-amino-6-(trifluoromethoxy)-
benzothiazole (riluzole, 150 mM). The purinergic receptor blocker, suramin, was
dissolved in distilled water (25 mM). All chemicals were aliquoted into 50 p1
volumes and stored at -20°C for future use (see Table 3).
Table 3 Pharmacological Compounds
Chemical Source Handling
Stock [ ] (mM) Solvent
Glibenclarnide RBI, Natick, MA 200 DMSO
NPPB RBI 500 DMSO
Riluzole R B I I50 DMSO
Suramin Sigma, St. Louis, MO 25 dHzO
Flufenarnic acid Sigma 50 DMSO
Electrophysiology
Pipettes
Thin-walled borosilicate capillary glass was purchased from World Precision
Instruments (Sarasota, FL). The pipettes were pulled to 2-7 MR resistances
using a Narishige pipette puller (Narishige Scientific Instnunent Lab, Setagaya-
Ku, Tokyo). The pipette resistances varied depending on the solutions being
used. Due to NMDGCl's large size and low diffusion coefficient the pipette
resistances tended to be higher, 8-17 MR, when recording in the low ionic-
strength 50 mM NMDGCl solutions. The glass pipettes were pulled prior to the
start of an experiment.
Junction potentials
The junction potential between the Ag/AgCl ground electrode and the bath
solution was minimized by using agar bridges made with bath solution. Liquid-
liquid junction potentials behveen the pipette and the bath solution were measured
using an Axopatch 200A integrating patch-clamp amplifier (Axon Instruments,
Foster City, CA). The amplifier was set to the current-clamp mode and a pipette
containing 3 M KC1 was immersed in the bath solution and the voltage was set to
zero. The bath was then completely exchanged with pipette solution and the
resulting potential was read off the voltage meter on the amplifier (see Table 4).
Junction potentials were subtracted from digital recordings post acquisition,
during analysis.
Table 4 Junction Potentials (VJ) vs 125 mM NaCl Agar Bridges
Solution Vr (mV)
pipette1
80 mM Kasp
50 mM NMDGCl
~ a t h '
120 mM NaI
120 mM NaBr
120 mM NaCI
120 mM NaGlutamate
hypo-osmotic NMDGC l3
140 mM NMDGCl
I see Table 2 2 see Table 1 3 ~ h e junction potential for the hypossrnotic solution varied depending on the degree of dilution of the iso-osmotic, 140 rnM NMDGCI, bath solution (see Materials and Methods).
Acquisition
All electrophysiological recordings were made in the whole-cell configuration
using either the Axopatch 1B or the Axopatch 200A integrating patch-clamp
amplifier at room temperature, 20-25°C. Digitized recordings were acquired
using the Clarnpex acquisition utility of the pCLAMP software package (version
6.04, Axon Instruments) and follow the electrophysiological convention of
upward deflections representing outward currents. Pipette and whole-cell
capacitance were compensated during the experiments using the amplifier
circuitry before digitization. Series resistance (R,,,,) was measured for each
recording but was not compensated prior to acquisition. Recordings were filtered
at 5 kHz using the patch-clamp amplifier's four-pole Bessel filter.
19
To enhance seal formation, recordings were begun in 125 NaCl saline solution
(see Table 1). In the cell attached configuration, when a gigaohm seal had
- formed, the 125 NaCl bath solution was completely exchanged with 140 mM
NMDGCl bath solution.
Digitized Images
Images of the rnicroglia cell line were captured using a video camera (Model
WVBL202, KM Video and Security, Mississauga, ONT) attached to the inverted
microscope and recorded on videotape. Analog images were digitized using the
Video Blaster software and acquisition board (Creative Labs, Inc., Milpitas, CA).
Perfusion
A 150 pi, small volume, perfusion chamber (Model RC-25, Warner
Instrument Corp., Hamden, CT) was routinely used for electrophysiological
experiments. Bath solutions were rapidly exchanged using a gravity perfusion
system with a home-made six-port manifold. The perfusion flow rate was
maintained between 1-2 mumin and was periodically checked using dye-
exchange rates.
Analysis
Current-voltage data
In the whole-cell configuration of the patch-clamp technique, R s c n s (access
resistance + pipette resistance) acts as a voltage divider and introduces an error
20
into the measurement of the membrane potential. To decrease the magnitude of
this error, two strategies were employed. First, thin-walled glass was used which
decreased the pipette resistance and consequently reduced &,,. Second, the
instantaneous slope conductance at the experimentally measured reversal potential
was plotted vs time. Based on Ohm's law the voitage drop (Vseaa) due to
declines as current declines, V,,,, = I x &,~,. At the reversal potential the net
current is zero, therefore V,&, is zero.
Digitized EV data were acquired using Clampex 6.04 (Axon instruments).
The data were imported into the Clampfit utility of the pCLAMP software
package and were fit with monoexponentials. For W e r analysis the
monoexponential fits were exported into Microcal Origin (Microcal Software,
Northampton, MA) where the instantaneous slope conductance was calculated by
taking the derivative of the monoexponential fits at the reversal potential. The
instantaneous slope conductance was normalized to cell membrane capacitance.
To easily visualize changes in the conductance throughout the course of an
experiment, the instantaneous slope conductance was plotted against time using
Microcal Origin.
Stafistical Analysis
GraphPad InStat (GraphPad Software, V2.04, O Dr. E.E. Daniel, McMaster
University) was used for statistical calculations. For comparison of two means,
unpaired t tests were used. Data are given as mean values _t SD unIess otherwise
stated.
Chapter 3
Results
Biophysics
isolation of the chloride current
To study the biophysics of the Cf current (Icl) and to eliminate other
potentially contaminating cation currents during patch-clamp recordings (e.g.,
HERG-like K' currents) ~+-f?ee bath and pipette solutions were used. The bath
and pipette solutions contained 140 m M NMDGCl (Table 1) and 50 mM
NMDGCl (Table 2) respectively. By substituting K' with the bulky, non-
permeating cation, NMDG', the only major permeating ion was Cl-. Based on the
experimentally measured reversal potential (EEv = -22 mV), which is close to the
theoretical electrochemical equilibrium for C1- predicted by the Nemst equation
hemst = -25 mV), and inhibition of the whole-cell current with C1--channel
blockers (see Pharmacology), we concluded that the whole-cell current was
predominately carried by Cl-. Furthermore, exchanging 140 mM NMDGCl with a
hypo-osmotic NaCl solution did not shift the reversal potential once junction
potentials had been compensated for (see Selectivity sequence). Therefore, the
current is not a cationic current carried by ~ a + ions. Further support for the
selectivity is that the only other potentially permeating ions, M ~ ~ + and ca2+, had
positive reversal potentials, +3 2 mV and + 13 9 mV respectively.
The observed discrepancy between the predicted and observed EEv was likely
due to non-selective leak between the pipette tip and cell-membrane interface
shunting the reversal potential towards 0 mV.
Time and voltage independence
Icl showed neither time-dependent activation nor inactivation over 2-sec long
voltage steps. At all of the potentials tested, including potentials as high as 140
mV (data not shown) there was no voltage-dependent inactivation (Figure 1A).
The lack of voltage-dependent inactivation is an important biophysical
characteristic when comparing Icl to other volume-sensitive Cl- currents in the
literature (see Discussion).
Current-voltage relationship
Icl displayed outward rectification of the current-voltage (I-V) relationship in
response to a 400 msec-long voltage ramp between +80 mV and -100 mV. This
ramp protocol was used routinely and shall be referred to as the standard ramp
protocol. The current crossed the x-axis at -21 mV (ENcmst = -25 mV). In Figure
1B the whole-cell I-V relationship was recorded immediately after the set of
voltage steps in Figure 1A. Superimposed on the I-V relationship, obtained with
the ramp protocol, are the corresponding currents recorded from the voltage steps.
The high degree of similarity between the two protocols justified the use of the
ramp protocol as a true measure of conductance and indicated that the previous
history of membrane potentials did not affect the instantaneous conductance; i-e.,
there was no current hysteresis.
When we used an 80 KAsp/40 KC1 pipette solution (see Table 2), the current
exhibited greater rectification (1.67 + 0.18 times more current at 60 mV above
E, than 60 mV below ERv (n=12) vs 1.38 + 0.23 times using the 50 mM
NMDGCI pipette solution ( ~ 5 ) ) .
Rat primary microglia and human T lymphocytes
The whole-cell anion current in rat primary rnicroglia and human T
lymphocytes also displayed outward rectification of the I-V relationship and had
time- and voltage-independent gating (Figures 2B and 2C). In Figure 2B, the rat
primary microglia anion current appears more rectified as compared to the anion
current in the rat microglia cell line (Figure 2A). The greater apparent
rectification is likely due to the use of the 80 KAsp/40 KC1 pipette solution. In
Figures 2A and 2C the 50 mM NMDGCl pipette solution was used (see above).
Selectivity sequence
The channel is permeable to anions other than C1-. The relative permeability
of the channel to the halides, I' and Br', was tested. Due to the activation of an
unexpected current, the relative permeability of F- could not be determined. For a
description of this novel current refer to the end of this section.
Voltage (mV)
Figure 1 (A) The current displayed time- and voltage-independent gating over 2- sec long voltage steps ranging fiom -80 mV to +80 mV fiom a holding potential of -10 mV (see inset). (B) In response to the standard ramp protocol (see text) the I-V relationship was outwardly rectified and reversed (E, = 21 mV) near the Nernst potential for CI- (EN- = -25 mV). Superimposed on the I-V relationship are the corresponding currents fiom the voltage steps. Solutions were 140 m M NMDGCL saline bath and SO mM NMDGCl pipette.
(A) Microglia cell line
1
(B) Primary microglia
1
m 250 rns
(C) Human T lymphocyte
Figure 2 The whole-cell anion currents for the microglia cell line (A), primary microglia (B), and the human T lymphocyte (C) all displayed time- and voltage- independent gating. 140 mM NMDGCl saline bath solution was used for all recordings. For the microglia cell line and the human T lymphocyte the currents were recorded using a 50 m M NMDGCI pipette solution. The current in the primary microglia was recorded using an 80 KAsp pipette solution.
To determine the relative permeability of the test anions, a hypo-osmotic
(-260 mOsm/kgH20) 80 KAsp/40 KC1 pipette solution (Table 2) was used to
prevent the spontaneous activation of Icl (see Ionic strength). After establishing
a whole-cell recording in 140 rnM NMDGCI solution, cells were swollen with a
hypo-osmotic 120 mM NaCl bath solution (-250 mOsmkgH20). At the peak of
the volume-induced C1- current the bath was exchanged with a hypo-osmctic
solution containing an equimolar concentration of the Na' salt of the test anion;
e.g., 120 mM NaI or I20 mM NaBr. The permeability ratio (Px/PcIj of the
channel was determined from changes in the reversal potential using the modified
Goldman-Hodgkin and Katz (GHK) voltage equation (see Box 1-Equation 6).
Chemical activities of Cl-, Asp-, Br-, I', and C r were determined by multiplying
their respective concentrations by activity coefficients (y) calculated from the
Debye-Huckel equation:
Equation 7
where 3 and C, are the valences and concentrations (M) of the ions in solution and
z, is the valence of the ion whose activity is to be determined. Chemical activities
were used instead of concentrations to compensate for the nonidealities of ionic
solutions; e.g., the intermolecular attractive forces between ions and their
counterions that effectively reduce their potential energy (Hille, 1994).
Table 5 lists the reversal potentials and relative permeabilities of the test
anions. Figures 3A and 3B show representative whole-cell I-V traces from two
different cells for the NaCl-NaI and NaCl-NaBr solution exchanges. The I-V
Point of intersection
/'
NaCl /
Figure 3 Representative current traces showing the halide selectivity of the anion channel. The halide selectivity, as judged by shifts in the reversal potential, was I.> Bi 2 C1'. (A) At the peak of a hypo-osmotic 120 mM NaCl bath solution was exchanged with a hypoosmotic 120 m M Nal solution. At depolarized potentials the anion current intersected icl (for an explanation see text). (B) Hypo-osmotic 120 m M NaCl was exchanged with a hypoosmotic 120 mM NaBr solution. The reversal potentials and relative permeabilities of the halides are listed in Table 5.
traces have been corrected for junction potentials between the agar bridge and the
test anion solution (see Table 4). At hyperpolarized potentials, when the majority
of the current was carried by intracellular Cf irrespective of the extracellular
solution, kl and the test anion current (Imion) converged as expected. However, in
Figure 3A Iaion intersected with Icl at --+SO mV. This crossover was consistent
with a subtle inhibition of the anion channel at depolarized potentials by I-.
Several other anion channels; e-g., CFTR and hCIC-1, are inhibited at depolarized
potentials by I- (Fahlke, Durr and George, 1997; Tabcharani, Linsdell and
Hanrahan, 1 997).
Glutamate permeability
The anion channel is permeable to glutamate. The permeability of glutamate
was determined as described above for the halide selectivity sequence using the
modified GHK-voltage equation and glutamate activity*. The relative
permeability of glutamate (Pglum,JPcl) is 0.12 & 0.02, n=7 (Table 5).
*Depending on the pH of the solution it is sometimes necessary to adjust the effective concentration of glutamate, prior to calculating activity, to allow for the incomplete dissociation of weak acids; i-e., glutamic acid. However, at pH 7.4 a correction was not necessary.
Table 5 Anion Selectivity
Anion E, (mV) k SD P x f f a + SD Number of Cells
The permeability of I' is significantly greater than Bi (p < 0.05).
Box 1. Derivation of the modified GHK-voltage equation
Scenario 1
In the first scenario, the only permeable anions are aspartate* and chloride.
*For a rigorous derivation of the equation it is assumed that aspartate permeates the channel.
( W i m 1 = intracellular chloride activity
(cl?mt = extracellular chloride activity
(Asp-)intl = intracelldar aspartate activity
(Asp?,, I = extracellular aspartate activity
Therefore, the reversal potential can be predicted by the Goldman-Hodgkin and
Katz (GHK) voltage equation:
(Equation 1)
Where R is the gas constant, T is absolute temperature and F is Faraday's
:onstant.
f (Asp-),,, 1 = 0 then
(Equation 2)
In the second scenario the test anion, X-, with a chemical activity denoted by (X3,
is perfused into the recording chamber. Therefore, the reversal potential predicted
by the GHK-voltage equation is:
(Equation 3)
(Equation 4)
(Equation 5)
f (CI-)int 2 = (Cl-)inI and 2 = 1 then equation 5 cu be re-arranged
o express Ps/Pcl as a function o~AE, , , .
(Equation 6)
:quation 6 was used to calculate the relative permeabilities of the test anions.
NaF-induced currents
Superfbsion of 120 mM NaF into the recording chamber activated an
unexpected current. The current built with time and displayed outward
rectification at depolarized potentials and inward rectification at hyperpolarized
potentials (Figure 4A). E,, was 2 + 1 1 mV (n=4). The current showed time- and
voltage-independent gating (Figure 4B). Activation of the outwardly rectifying
component (I,,,) preceded the development of the inward component (Iin) by - 40-60 sec. After the initial build-up, &,,, exhibited some run-down over an 8-min
acquisition whereas Iin continued to increase (4/4 cells).
The temporal dissociation of development suggests that Iin and I,., represent
two distinct currents through different channels. In this regard, pulsing the
membrane potential to increasingly hyperpolarized potentials increased the
amplitude of the noise for Iin. Conversely, depolarizing the membrane potential
did not alter the noise level for I,.,. Therefore the underlying Iin channels may
have a greater single-channel conductance or a decreased open clian~el
probability (P,,,,) and hence exhibit greater variability compared to I,,,.
Alternatively, Iin may experience a fast flickery block at hyperpolarized
potentials. No attempt was made to filly characterize these currents.
50 100 150 I I J
Voltage (mV)
Figure 4 Currents induced by extracellular 120 m M NaF. (A) The I-V relationship is inwardly rectified at hyperpolarized potentials and outwardly rectified at depolarized potentials. Over the course of the acquisition (standard voltage ramp pulsed every 20 sec) the outward current (I,,,) decreased with time whereas the inward current (Iin) continued to increase (denoted by the arrows). E, was 2 & I I mV (n=4). (B) The current displayed time- and voltage-independent gating. At hyperpolarized potentials the current was considerably noisier than at depolarized potentials.
Swelling and volume sensitivity
During whole-cell patch-clamp recordings, the microglia cells swelled when
they were exposed to a hypo-osmotic shock (-220-250 mOsmkgH20) and there
was visible expansion of the cell membrane and cytoplasm (Figure 5). Under
these conditions, the whole-cell current increased with time until it reached a
quasi-stationary plateau. If the cells were then exposed to an iso-osmotic (-300
mOsm/kgHzO) or a hyperosmotic bath solution (-340 mOsm/kgH20) the current
declined to its pre-swelling level (Figure 6A and 6B). Figure 6A shows the
increase of the swelling-induced CI- current in response to the standard voltage
ramp protocol applied every 20 sec. Figure 6B shows the instantaneous slope
conductance measured at the reversal potential (Gcrrev) (see Materials and
methods) as a b c t i o n of time for the same cell.
Figure 5 Hofkan modulation-contrast (HMC) images of a microglia cell prior to (left) and 7 min after exposing the cell to a hypo-osmotic shock (right).
-
0 20 40 6Q 80
Time (min)
Figure 6 (A) Activztion of Icl after exposing the cell to a hypo-osmotic shock (-220-255 mOsrn/kgHzO) using an 80 KAsp/40 KC1 pipette solution. The current activated with time and reached a quasi-stationary plateau (left). When the cell was exposed to an iso-osmotic or hyper-osmotic bath solution the current declined to its pre- swelling level (right). (B) Instantaneous slope conductance plotted at the experimentally measured reversal potential by fitting the traces in (A) with monoexponentials and taking the derivative at &,,.
Ionic strength
To study the effects of intracellular ionic strength (Ti) on the swelling-
activated whole-cell anion conductance, we used a low ionic-strength and a high
ionic-strength pipette solution. The low ionic-strength pipette solution, 50 mM
NMDGCI, had an ionic strength of 76 mM (Table 2). The high ionic-strength
pipette solution, 80 KAsp/40 KCl, had an ionic strength of 146 mM (Table 2).
Ionic strength was calcuiated using the following equation:
1 ionic strength = 7X ci (zi ) 2 Equation 8
where zi and Ci are the valences and concentrations (M) of any ions in the
solution. With the low ionic-strength pipette solution Gel,, activated
spontaneously upon establishing a whole-cell recording in nominally iso-osmotic
solutions. To compensate for potential osmof c imbalances between the pipette
and bath due to large intracellular osmolytes (Donnan effects - see Box 2), the
osmolality of the pipette solution was decreased from -300 mOsmflcgH20 to
-260 rnOsm/kgH20 (vs -300 mOsrn/kgHzO bath solution) thus decreasing the
likelihood of cell swelling. Despite the alteration in osmolality the conductance
still developed spontaneously. Figure 7 shows the spontaneous activation of
Gclrev in nominally iso-osmotic solution versus the spontaneous activation of
Gclmv with the hypo-osmotic pipette solution. When using the hypo-osmotic
pipette solution, Gclm reached half-maximal activation with the same time
constant (tin) (149 + 25 sec vs 160 + 26 sec, n=5) and reached the same peak
L. 1 I I
0 I
100 I
200 I
300 400 500 600
Time (s)
Figure 7 Comparison of the spontaneous activation of Go,, with an iso-osmotic (n=5) vs a hypo-osmotic. 50 m M NMDGCI. pipette solution (n=j). Pipette osmolality was adjusted by removing sucrose. Open circles - iso-osmotic pipette solution (-300 mOsm/kgHzO). Filled circles - hypo-osmotic pipene solution (-260 mOsm/kgH20). The bath solution (-300 mOsm/kgHzO) was 140 mM NMDGCI in both cases.
conductance (0.68 + 0.14 nS/pF vs 0.67 f 0.16 nS/pF, n=5) as Gel, when the
nominally iso-osmotic pipette solution was used.
In contrast, when Ti was increased fiom 76 mM to 146 mM spontaneous
activation of the current was significantly reduced. Figure 8A shows a
representative trace comparing Gel, with low and high ionic-strength pipette
solutions. There was a significantly lower spontaneous activation over t ~ n
(p<0.001) when I'i was increased to 146 mM (0.06 f 0.08 nS/pF, n=12) fiom 76
rnM (0.35 -t 0.07 nSIpF, n=5) (see Figure 8B). Although the higher ionic-
strength pipette solution inhibited the spontaneous conductance the swefling-
induced conductance was unaffected (1 l / 1 1 cells).
On two occasions (211 1 cells) the conductance activated in the absence of an
applied osmotic shock despite using the high ionic-strength pipette solution. On
both occasions the cells had swelled spontaneously with visibIe expansion of the
cell membrane. These cells were included in the results.
Since the C1- conductance was less likely to develop spontaneously when the
higher ionic-strength pipette solution was used. this protocol was adopted for the
pharmacological and regulation studies. The ability to prevent the conductance
from activating was advantageous for several reasons. First. it allowed us to
determine the amount of spontaneous conductance upon establishing a whole-cell
recording. Second, it provided time for nucleotides or other substances to either
diffuse in (e.g., guanine nucleotides) or diffuse out of the cell (e.g.. endogenous
ATP when conducting the adenine nucleotide regulation studies) prior to the cell
being swollen.
Time (s)
Time (s)
Figure 8 (A) The spontaneous development of Gckcv (solid curve) was inhibited when r, was increased to 146 m M (dashed curve). However, increasing T i did not inhibit the swelling induced conductance (1 I l l 1 cells). t In represents the time for half-maximal activation of Go, with low 1; (76 mM) (B) Comparison of Gclre, with the low (filled circles) and high (open circles) ionic-strength pipette solutions during t ! !~ ( tlrz =I60 k 26 sec, n=S). * p < 0.00 1.
Box 2. The Donnan Effect
The term "nominally iso-osmotic" acknowledges that due to
impermeant proteins within the cell the intracellular solution may
contain more osmotically active substances than the extraceiIular
solution. To illustrate, imagine a cell with a semi-permeable
membrane and with K', Cl- and impermeant proteins on the
intracellular side, X, but only K' and Cl- on the extracellular side,
Y. The serni-permeable membrane allows the fiee movement of
K+ and Cl', but is impermeable to the proteins. To maintain
electroneutrality, the concentrations of the anions and cations on
each side are initially equal. If the ions are then allowed to freely
diffuse. CI' moves down its concentration gradient from side Y to
side X. To maintain neutrality, Kt also moves from side Y to side
X. At equilibrium. therefore. [Cl-s] + [K's] + [impermeant
proteins] > [ClmY] + [K'yj. Therefore, even if the pipette and bath
solutions were initially iso-osmotic, the presence of intracellular
impermeant proteins may alter the osmotic balance making the
inside of the cell hyperosmotic relative to the bath.
Pharmacology
We investigated the pharmacology of the anion channels using the traditional
C1' channel blockers, NPPB, flufenamic acid and glibenclamide. We also tested
extracellular ATP and the experimental inhibitor, riluzole, for their ability to
block the anion channeis.
After establishing a whole-cell recording using the standard 80 KAsp/40 KC1
pipette solution, we swelled the cell (-220-255 mOsm/kgH20 hypo-osmotic
shock). When Gckv was stable, we introduced the same hypo-osmotic medium
containing the inhibitor into the recording chamber. We perfused the bath with 2-
3 rnl to ensure complete exchange (I 50 11 recording chamber - see Materials and
methods).
All data represent the percent inhibition of Gel, (f SD, # of cells) unless
otherwise stated. Percent inhibition was calculated using the following equation:
where Gel,, is the conductance remaining after inhibition and Gam, is the
maximum conductance prior to inhibition. Ga,, was determined by fitting Gclrty
vs time with a sigrnoidd curve using the Boltzmann distribution. Gel,, was then
determined from the fit parameters. The Boltzmann distribution was chosen as it
consistently yielded low chi-squared values.
We did not construct Ml dose-response curves because at concentrations
where there was less than 50 % inhibition, the inhibition was variable and it was
difficult to distinguish inhibition from current rundown. At higher concentrations
the inhibition was sufficiently fast that current rundown was not a problem.
The results of the pharmacology experiments are summarized in Table 6.
NPPB
NPPB potently and reversibly inhibited Gel,,, ( S O % inhibition with 1 25, 250
and 500 CIM) Figure 9 demonstrates the rapid, reversible inhibition of GclRv.
When we tested a lower concentration (50 pM) the results were variable (n=5),
with one cell showing no apparent inhibition. Ir? 215 cells the inhibition occurred
over a much longer time period and current rundown could not be ruled out. In
the remaining cells Gclrev was slightly inhibited (data not shown). For all of the
concentrations tested there was no apparent voltage dependence.
Flufenamic acid
Flufenamic acid (1 00 and 200 pM) reversibiy inhibited Gclrev by 53.1 k 13.4
% (n=3) and 78.4 f 9.3 % (n=3) respectively. Similar to NPPB. a lower
concentration of flufenarnic acid (50 pM) produced variable results (data not
shown).
Glibenclamide
We examined the effect of two concentrations of glibenclamide, 80 and 500 pM.
80 p M glibenclamide was chosen because this concentration is well above the Ki
values for CFTR (Ki = 17-26 pM) (Sheppard and Welsh, 1992).
Time (min)
Figure 9 A representative experiment illustrating the rapid, reversible inhibition of Gae" by 125 p M NPPB. The standard 80 KAsp140 KC1 pipette solution was used. Data are summarized in Table 6.
Gclrev was slightly inhibited by 80 p M glibenclamide (1 6.3 k 1 1.5 %, n=4). In
a separate set of experiments, 500 p M glibenclamide inhibited 79.2 + 5.9 % ( ~ 3 )
of Gclrcv (see Figure 10A). In both cases, the inhibition was completely
reversible within minutes of washing out the inhibitor. To determine if 500 @I
inhibited the Cl- current in a voltage-dependent manner we plotted the current at
+50 mV and -100 mV for each cell. The inhibition of Icl at +50 mV was not
significantly different from inhibition at -100 mV (a=0.05, n=3). Figure 10B
shows a representative experiment demonstrating the voltage-independent
inhibition of Icl by 500 pM glibenclarnide.
Rilutole
Application of 300 pM riluzole, a glutamate release inhibitor (Malgouris et
al., 1989; Pratt et al., 1992), reversibly inhibited -90% of the conductance
(Figure 11).
External application of 10 mM ATP
Within 2-3 min of perfusing ATP into the recording chamber, an inwardly
rectifying current developed that was followed by the development of a large
outwardly rectifying current. Due to the activation of these currents it was not
determined conclusively whether Gc[, was inhibited by extracellular ATP (data
not shown).
Recently, Walz et ai., (1993) reported that extracellular ATP activates a
desensitizing cation conductance and a potassium conductance in cultured
0 Hypossrnotic solution (120 mM NMDGCI)
Hypwsmotic solution + 500 pfvl glibendarnide
0.0 - -=,-- I I I 1 I I 1
0 5 10 15 20 25 30
Time (min)
n Hypo-osmotic solution (120 mM NMDGCI)
Hypwsrnotic solution r 500 fl glibendarnide
Time (min)
Figure 10 (A) Inhibition of Gcl, by 500 pM glibenclamide. (£3) An illustration of the voltage independence of inhibition of I. For the same cell as in (A), the current was plotted at +SO mV and at -IOOmV, using the standard ramp protocol (see Table 6).
1 ( Hypo-osmotic solution (1 20 rnM NMDGCI)
I Hypo-osmotic solution + 300 pM n'luzole
Time bin)
Figure 11 Inhibition of Go, by 300 pM riluzole. For this particular cell. there was a small amount of spontaneous current upon establishing the whole-cell recording. The standard 80 KAsp/40 KC1 pipette solution was used.
microgiial cells fiom mouse brain. Therefore, the authors proposed the
involvement of P2 purinergic receptors. Primary rat microglia (Kettenma~,
- Banati and Walz, 1993; Norenberg et al., 1994) and two mouse microglia cell
lines (Ferrari et al., 1997) have also been reported to have purinergic receptors.
To prevent the probable activation of these receptors on the rnicroglia cell
line, we added a purinergic receptor antagonist, suramin, to the iso-osmotic bath
solution as well as to the hypo-osmotic solution containing the 10 m M ATP.
When the hypo-osmotic medium was introduced into the recording chamber Gel,,
did not activate. Therefore, we hypothesized that s u r d inhibited the swelling-
induced current. We tested this hypothesis using the same protocol outlined for
the other pharmacological blockers and discovered that 30 pM suramin inhibits
Ia in a voltage dependent manner (Figure 12).
Human T lymphocytes
Since we did not conclusively determine whether the microglia cell line anion
conductance was inhibited by extracellular ATP, we were interested in whether
the similar whole-cell anion current in human T lymphocytes would be inhibited.
The human T lymphocyte current was not inhibited by 5 (n=l), 10 (n=4) or 20
mM extracellular ATP (n=4) (Figure 13); nor was there activation of another
current; i.e., via activation of purinergic receptors.
1 OOC
0 Hypo-osmotic solution (120 rnM NMDGCI)
Time (min)
Figure 12 Voltage-dependent inhibition of Icl by the purinergic receptor antagonist, surarnin. At positive potentials (+50 mV) 30 pM suramin greatly inhibited the current. However, at negative potentials (-1 00 mV) there was much less inhibition (see Table 6).
-- 10 mM ATP added
Time (sec)
Figure 13 Bolus addition of 10 m M ATP to the bath solution did not inhibit the normal activation or hasten the rundown of the anion current in human T lymphocytes (see Discussion - Comparison...). To illustrate the lack of voltage-dependent block, normalized current was plotted at +80 mV and -80 mV. The pipette solution contained 50 mM NMDGCI and the bath contained 140 mM NMDGCI.
Table 6 Inhibition of Gel, by various pharmacological compounds
Drug Concentration % Inhibition SD Number of cells
Flufenamic acid
Glibenclamide
Riluzole
Suramin
variable
86.9
83 -3
92.8
variable
53.1
78.4
16.3
79.2
78.5
67.1
90.6
70.5
18.0
The voltage dependence of inhibition of glibenclamide and suramin was determined by plotting Icl at +50 mV and -100 mV. All other data represent the inhibition of Gcrrcv.
Chapter 4
Nucleotide Regulation
G-protein regulation
We investigated G-protein regulation of the microglia anion conductance by
adding 200 pM GTPyS to the low ionic-strength, 50 m M NMDGCl pipette
solution. Since we used the low ionic-strength pipette solution GclEv activated
spontaneously. Despite the spontaneous activation, Gel, was significantly
increased by GTPyS (1.05 k 0.29 nS/pF, n=8) as compared to controls (0.66 f
0.16 nS/pF, n=5; p4I.05) (Figure 14).
To determine if 200 pM GDPPS inhibits G c , , we used the high ionic-
strength, 80 KAsp/40 KCl, pipene solution. This was done to allow sufficient
time for GDPPS to accumulate in the cell prior to activation. After 3-5 min the
cells were exposed to a hypo-osmotic 120 rnM NMDGCl bath solution (-220-255
mOsm/kgH20). Loading cells with 200 pM GDPPS did not significantly inhibit
Figure 14 The spontaneous activation of Gctxv is significantly increased when 200 p M GTPyS is added to the pipette solution, as compared to controls (p<0.05). The solutions were 50 rnM NMDGCl pipetre and 140 mM NMDGCl saline bath. In both cases the pipette solutions contained 2 rnM ATP. * p<0.05.
Gclrcv (0.83 f 0.33 nS/pF, n=7) as compared to control (0.77 f 0.38 nS/pF, n=6)
(Figure 15).
Adenine nucleotide regulation
The role of adenine nucleotides in regulating the anion channels was
investigated by excluding ATP fiom the pipette solution. After establishing a
whole-cell recording, we waited 5-10 min before swelling the cell to allow
endogenous ATP to diffuse out of the cell. In the absence of cellular ATP, GaW
did not activate in response to a hypo-osmotic shock (-220 mOsm/kgH20)
(Figure 16). However, by excluding ATP &om the pipette solution we
concomitantly increased the free intracellular magnesium concentration fiom 0.08
m M to 0.6 mM. To ensure that this increase was not responsible for the inhibition
of Gclrcv, we lowered the magnesium concentration to 0.08 rnM in subsequent
experiments. As illustrated in Figure 16 there was no significant difference in the
amount of swelling-induced conductance between the high and low magnesium
solutions.
Finally, to determine if other nucleotides could support the activation of GclRv
in the absence of cellular ATP, we loaded cells, via the patch pipette. with 200
pM GTPyS. GTPyS alone did not support the activation of the swelling-induced
C1--conductance (Figure 16).
For multiple comparisons of the means of the nucieotide experiments we
conducted a one-way analysis of variance (ANOVA). Planned (a priori) painvise
Figure 15 The swelling-induced Gck, was not inhibited by 200 p M GDPPS (a=0.05). To allow sufficient time for GDPPS to accumulate in the cell prior to activation of Gch, the 80 KAsp/40 KC1 pipette solution was used. After 3-5 rnin the cells were exposed to a hypoosmotic shock (-220-255 mOsmlkgHrO) and the peak conductances were compared. Cells were swollen with a hypo-osmotic, -120 m M NMDGCI bath solution. The pipette solution contained 2 m M ATP.
1 1 2 rnM ATP (0.08 mM Mg2+)
No ATP - (0.6 rnM Mg2+)
$ 1 No ATP - (0.08 mM Mg2+)
NO ATP - 200 p M GTPyS
Figure 16 Comparison of peak G~~~~ with and without nucleotides added to the pipette solution. When 2 mM ATP is excluded from the pipette solution Gcke, does not activate despite lowering the free intacellular M~'' concentration or adding 200 pM GTPyS to the pipette solution. The asterisk (*) denotes statistical significance at the p~0.05 level compared to the 2 m M ATP-loaded cells (summarized in Table 7).
comparisons between the conditions were then made using a Bonferroni t test.
The results of the t test are summarized in TabIe 7.
Table 7 Bonferroni t test for pairwise comparisons o f the means of the nucleotide ex~eriments
Comparison Difference of means t p < 0.05
2 rnM ATP vs No ATP-200 p M GTPyS 1.270 1 1.860 Yes
2 mM ATP vs No ATP (0.08 rnM ~ ~ ' 3 1.2 10 10.107 Yes
2 rnM ATP vs No ATP (0.6 m M Mg23 1 270 15.138 Yes
No ATP (0.6 mM ~ g ' ' ) vs No ATP-200 p M GTPyS
No ATP (0.6 rnM Mg23 vs No ATP (0.08 m M ~ ~ ' 3
No ATP (0.08 mM ~~~3 vs No ATP-200 pM GTP/S
G-protein regulation - human T lymphocytes
For comparison, we further investigated G-protein regulation of what appears
to be the same anion current in human T lymphocytes. To do this we used the
hydrolyzable guanine nucleotides GTP and GDP. When 100 p M GTP was
included in the pipette solution the peak CI' current amplitude at +40 mV was
greatly enhanced (187 k 41 PA, n=5) as compared to control (70 + 8 PA, n=4;
p<0.001). However, loading cells with GDP did not significantly inhibit the
current (a=0.05; n=8) (Figure 17).
n 100 pM GTP
(1 100 pM GDP
Figure 17 GTP increases the peak Cf current in human T lymphocytes (p<0.001; n=5), whereas GDP has no effect (a=0.05; n=8). The 50 m M NMDGCI pipette solution contained either 100 pM GTP, GDP or no nucleotide. In all cases the bath solution was 140 mM NMDGCI. Current was plotted at +40 mV. * p<0.00 1.
Chapter 5
Discussion
Using appropriate bath and pipette solutions we isolated a volume-sensitive
whole-cell anion current in a microglia cell line and constructed a biophysical and
pharmacological fingerprint. We also examined the role of volume, ionic
composition and intracellular nucleotides in regulating the underlying channels.
Based on the properties and regulation we are able to speculate about the
molecular identity of the channels and provide some insight into a possible
physiological role.
Biophysics
The first objective of our study was to characterize some of the biophysics of
the whole-cell anion current. This was done in order to be able to make
comparisons between the underlying channels in the microglia cell line and anion
channels in the literature whose molecular identity is known. To accomplish this,
we looked at several properties that are often used to distinguish between anion
channels; e-g., time- and voltage-dependence, rectification and selectivity
sequence. We also looked at glutamate permeability to determine if the anion
channels may provide a pathway for glutamate efflux during hypoxia and
ischemia-
Comparison of the anion channels, with anion channels in the literature, will
be made at the end of the discussion when all of the regulatory, pharmacological
and biophysical data are consolidated.
I-V relationship
The rectification of the I-V relationship was enhanced when the 80 KAsp/40
KC1 pipette solution was used compared to the 50 mM NMDGCl pipette solution.
Since both pipette solutions had similar C1' concentrations it is unlikely that the
change in rectification was due to an alteration in Goldman rectification.
Alternatively, the increased rectification may indicate that intracellular aspartate
partially blocks the C1' conductance. Block of the small-conductance anion
channels in human T lymphocytes by bulky anion species (e-g., aspartate) has
been proposed (Schumacher et al., 1995).
Halide selectivity sequence
The selectivity of the anion channels is most similar to the Eisenman I
sequence; i.e., I- > Br- > CI- > P. Selectivity depends on two factors: (1) the
electrostatic energy of attraction of the anion to a positively charged site in the
channel and (2) the hydration energy of the anion. The Eisenman I sequence
suggests the anion permeabilities are dominated by dehydration energies (Table
8). From Coulomb's law, the energy (per mole) of interaction (Usik) between the
anion and the cationic binding site is inversely proportional to the sum of the radii
of the cationic binding site (r,) and the anion (ra) (Equation 9)(Hille, 1984).
Equation 9
4 and 4 are the valences of the anion and the cationic binding site
respectively, E is the dielectric constant, co is the polarizability of free space, N is
Avogadro's number and e is the elementary charge. Given that r, is constant for a
given anion (Table 8), Usit, decreases as rc increases. Therefore the Eisenman I
sequence implies that rc is large and consequently the electrostatic energy of
attraction between the anion and cationic binding site is small.
Table 8 PauIing radii and ionic hydration energies of the halides
Atom Radius (A) mhvdmdoa (kcaVmot)
Glutamate permeability
A family of ~a+-dependent excitatory amino acid transporters (EAAT) (1-5)
has recently been cloned. The EAATs are h i g h - e t y L-glutamate transporters
arid likely play a role in maintaining subtoxic levels of glutamate in the brain
(Kanai, Smith and Hediger, 1993). Two of the transporters (EMT4 and EAATS)
also behave predominantly as ~a*-dependent (Arriza et al., 1997) glutamate-gated
C1' channels (Fairman et al., 1995; Aniza et al., 1997)-
The observed glutamate permeability, therefore, could potentially be
explained by one of three possibilities. One, the pehsion of NaGlu into the
recording chamber activated an EAAT. Two, the observed permeability was due
to a combination of an EAAT and the volume-sensitive anion channels, or three,
the observed permeability was due entirely to the volume-sensitive anion
channels.
Based on the ce1Iula.r distributions of the EAATs it is unlikely that they
contributed to the glutamate permeability. For example, in the brain
EAAC I/EAAT3 is primarily neuron-specific (although some expression has been
found in the rat C6 glioma cell line, Palos et al., 1996) (Rothstein et al., 1994).
The expression of EAAT4 is found mainly in post-synaptic Purkinje cell spines
(Dehnes et al., 1998). EAATS expression appears to be retina-specific with very
little expression in the brain (Arriza et al., 1997). Recently, GLT I EAAT2 and
GLASTIEAATl mRNA expression has been found in microglia, however the
expression is not prominent (Kondo et al., 1995). These observations support the
proposal that mouse and rat microglia do not effectively take up glutamate
(Patrizio and Levi, 1994).
Furthermore, expression of EAAT (1-5) in Xenopus oocytes produces currents
which have linear or inwardly rectified I-V relationships (Fairman et al., 1995;
Wadiche, Amara and Kavanaugh, 1995; Arriza et al., 1997). The current we
observed in the presence of glutamate displayed strong outward rectification (data
not shown). This observation further supports the proposal that the glutamate
permeability was not due to an EAAT. In addition, if the glutamate permeability
represented a combination of an EAAT and the volume-sensitive anion channels,
then upon activation of the EAAT the volume-sensitive inward current should
have dramatically increased; Le., due to the addition of the Linear or inwardly
rectified EAAT current. Conversely, EAAT inactivation upon washing out NaGlu
should decrease the inward current. However, t b i s was not observed. When
NaGlu was pehsed into the bath, the inward current remained constant or
decreased slightly. When NaGlu was washed out, the current often increased
slightly. These observations suggest that a functional EAAT is not present in the
rnicroglia cell line and the observed glutarnate permeability was due to the
volume-sensitive anion channels.
In future, the potential contribution of an EAAT could be elucidated by
excluding ~ a ' from the bath solution.
NaF-induced currents
When we attempted to determine
activated one or more novel currents.
the permeablility of F' we unexpectedly
Although we did not characterize these
currents in detail some o f the mechanisms by which F- may have activated them
will be discussed.
NaF and/or aluminum fluoride complexes modulate a number of intracellular
s igding systems in intact cells (Kagaya et al., 1996; Goldman, Granot and Zor,
1995; Hauschildt, Hirt and Bessler, 1988). In rat C6 glioma cells, for example,
NaF transiently increased intracellular ca2+ and increased cGMP, possibly
through a ca2+- and nitric oxide synthase-dependent pathway (NOS) (Kagaya et
al., 1 996). In rnacrophages, NaF activates phospholipase C (Goldman, Grano t
and Zor, 1995) and membrane-associated protein b e C (Hauschlidt, Hirt and
Bessler, 1988). Other regulatory effects attributed to NaF in intact cells include
decreases in intracellular ATP, activation of mitogen-activated protein kinase and
activation of phospholipase A2. (Goidman, Granot and Zor, 1 995).
It is not surprising, therefore, that NaF has been linked to increases in ion
fluxes and alterations in membrane potential (Gofa and Davidson, 1996; current
shldy). In G292 osteoblastic cells, in the cell-attached configuration, adding 10
rnM NaF to the bath solution increased the amplitude and Po,, of a 73 pS
K'-selective ion channel (Gofa and Davidson, 1996). The effects depended on
ex~acellular ca2+ and were blocked by a combination of ca2'-channel blocking
agents. Therefore, it was proposed that NaF may directly modulate ca2' channels
or via the activation of second messengers may stimulate the release of ca2' from
internal stores. The influx in ca2* and/or depolarization may directly activate the
K' channel (Go fa and Davidson, 1996).
In rat hippocampal CAI pyramidal cells, extracellular NaF (10 mMJ
decreased the rate of rundown of high-voltage-activated (HVA) ca2+ currents
(Breakwell et al., 2995). The authors hypothesized that this effect may have been
mediated through a NaF-activated G protein. In these cells the NaF effect was
significantly reduced by the inclusion of 10 mM EGTA in the pipette solution
(Breakwell et al., 1995). As our pipette solution routinely included 10 mM
EGTA it is unlikely that the cunents were activated by transient increases in
intracellular ca2+. However, in future experiments, the removal of ca2+ from the
bath and pipette solutions would provide further clarification. Alternatively, the
calcium channel blocker nifedipine could be included in the bath solution to block
voltage-gated ~a~+-channels that have been reported in microglia (Colton et al.,
1994).
Many of the effects of NaF have been attributed to the activation of G
proteins. This is particularly true if there is aluminum contamination of the
recording solutions which can come from a number of sources including: the cell
culture media, borosilicate glass pipettes etched by fluoride solutions or impure
preparation of commercial NaF (Duszyk et al., 1995). Aluminum in the presence
of fluoride combines to form multifluorinated complexes; e.g., AIF, (Chabre,
1990). These complexes likely bind to the P-phosphate of GaGDP and mimic the
y-phosphate of bound GTP. In this regard, the GaGDP-AIF, complex is
analogous to active GaGTP (Bigay et al., 1985; Bigay et al., 1987) and can
activate G proteins. In the absence of aluminum contamination, P, in rnillimolar
concentrations in the presence of magnesium, can directly activate G proteins
(Antonny et al., 1993).
In future, to determine if aluminum contamination is responsible for the
activation of the new rrncharacterized currents an aluminum chelator; e.g.,
deferoxamhe mesylate, could be included in the recording solutions. To M e r
evaluate the role of G proteins, cells could be pre-incubated with pertussis toxin
prior to NaF exposure or loaded with GDPPS.
Based on our nucleotide regulation studies, it is unlikely that the activation of
these currents was directly caused by G protein activation. When we loaded cells
with GTPyS via the patch pipette no unusual currents activated.
What currents were activated by NaF?
We initially suspected the currents were through non-selective cation
channels. However, in a preliminary experiment, 100 p M gadolinium. a blocker
of non-selective cation channels, did not block the currents (data not shown).
In future, ion substitution experiments or the inclusion of selective channel
blockers in the recording solutions; e.g., K'-channel or Cl - -cha~el blockers,
would provide insight into the selectivity of the underlying channels.
Swelling and volume sensitivity
The second objective of the thesis was to characterize the swelling and
volume sensitivity of the whole-cell anion conductance. After the first response to
swelling and subsequent decline of Gel, in hyperosmotic or iso-osmotic solution,
a second exposure to a hypo-osmotic shock did not always fully restore Gclrm.
The inability to evoke the same peak conductance during multiple hypo-osmotic
exposures indicates one of three possibilities: (1) a decrease in the number of
available channels, (2) a decrease in single channel conductance or (3) a decrease
in Pow. We know fiom nucleotide regulation studies that Gclrev requires cellular
ATP (see Chapter 4). Thus, when considering the length of our experiments, 50-
80 min, the most likely explanation is a decrease in the number of available anion
channels, or Po, due to ATP hydrolysis. This type of hysteresis has been
observed inconsistently in another volume-sensitive anion channel and in
accordance with our findings, generally occurs for experiments longer than 45
min (Sorota, 1995). Furthermore, in rat C6 glioma cells the activity of volume-
sensitive anion channels decreases as cellular ATP levels decline (Jackson,
Momson and Strange, 1994) M e r supporting our hypothesis.
Dependence on ionic strength
The activity of the anion channels was inhibited by the 80 KAsp/40 KC1
pipette solution. Similar modulation of anion channeis by intracellular ionic
composition/ionic strength has been observed in a wide variety of cells including:
trout red cells (Motais, Guizouam and Garcia-Romeu, 199 1); skate hepatocytes
(Jackson et ai., 1996); rat C6 gliorna cells (Emma, McManus and Strange, 1997)
and bovine endothelid cells (Nilius et al., 1998).
In trout red cells taurine efflux via volume-sensitive anion channels (Nilius et
d., 1998) was decreased by increasing intracellular ionic strength (Motais,
Guizouarn and Garcia-Romeu, 1991). Later, it was proposed that intracellular CT
concentration (and possibly ionic strength) modulated anion channels (Jackson et
- al., (1996). Nilius et d. (1998) M e r clarified the role of ion content by
reducing intracellular ionic strength while keeping C1' concentration constant.
Under these conditions the current developed spontaneously. They also reported
that reductions of K' or Asp- did not cause spontaneous activation when ionic
strength was maintained. Therefore, it was concluded that ionic strength was the
key modulating factor and not specific effects of Kf or C1- ions.
Since we used pipette solutions with different ionic compositions; i.e.
potassium vs NMDG, we do not know if the spontaneous activation of the current
was due to a decrease in ionic strength or to an alteration of ionic composition.
Based on the observations of Nilius et al. (2998) it is reasonable to assume that
the reduced ionic strength activated the current. Furthermore, our low ionic-
strength pipette solution, in which there was substantial spontaneous current, had
a higher C1- concentmtion than our high ionic-strength pipette soiution which
evoked less spontaneous activity. Our results, therefore, suggest that ionic
strength arid not C1- concentration is the key modulating factor and supports the
findings of Motais, Guizouarn and Garcia-Romeu (I99 1) and Nilius et al., (1998).
Spontaneous activation of the chloride current
In 2/11 cells the current developed spontaneously even when the high ionic-
strength pipette solution was used. Despite also using an osmotic gradient that
should favour cell shrinkage, the cells had obviously swollen. Spontaneous cell
swelling has been observed in other studies examining volume-sensitive channels.
In the human colonic cell line T84, less than 2 min after establishing a whole-cell
recording the cells began to swell and transmembrane currents increased. The
current reached a steady state level over approximately 30 rnin. However, the
authors noted that the time to reach steady state was variable between cells
(Worrell et al., 1989). In canine atrial cells, C1- currents also deveIoped
spontaneously in nominally iso-osmotic solutions. The time course of
development was highly variable between cells, ranging from 5 to 25 min (Sorota,
1992). In these cells the current developed faster when the series resistance was
lower, similar to our observations in the present study. In both previous studies
(Sorota, 1992; Worrell et al., 1989) high ionic-strength pipette solutions were
used. In T84 cells the spontaneous current activation was not suppressed until the
the osmotic pressure difference (An, bath-pipette) was greater than 50
rnOsm/kgH20 (Worrell et al., 1989). In order to return the cell width to initial
values and decrease the C1' conductance in canine atrial cells, 50 - 75 mM
rnannitoI had to be added to the bath (Sorota, 1992).
Worrell et al. (1989) proposed that the cells swelled spontaneously owing to
slowly difising intracellular osmolytes. It is well known that after establishing a
whole-cell recording small solutes in the patch pipette equilibrate with the cell
interior. However, one hypothesis is that a cytoplasmic diffusion barrier restricts
the movement of iarger macromolecules from the cell into the pipette (Worrell et
al., 1989). If pipeae ions equilibrate with the cell interior and there is not a
corresponding efflux of cellular macromolecules, the cytoplasm would become
hyperosmotic with respect to the bath (Worrell et al., L989). As a result, the cells
would swell and the volume-sensitive C1- conductance would develop. As
discussed by Worrell et al. (1989) there is evidence for a cytoplasmic meshwork
that restricts intracellular solute movement in a size dependent manner (Luby-
Phelps, Taylor and Lanni, 1986). Luby-Phelps, Taylor and Lanni (1986) propose
that the cytoplasmic viscosity may decrease the rate of diffusion of monomeric
globular proteins by approximately 4 to 6 fold. Larger oligomeric proteins,
muitienyme complexes and long-chain polymers might not di&e at all (Luby-
Phelps, Taylor and Lanni, 1986) into the pipette. Therefore, even though we
decreased the osmolality of the pipette solution by 40 mOsmkgH20, the
adjustment may not have been sufficient to compensate for the slowly diffusing
intracellular osmolytes. Within the time span of a recording, prior to applying an
osmotic shock (5-10 min), only 2/11 cells visibly swelled; however, the time
course of cell swelling has been reported to be so variable that if we had waited
longer (30-40 min) more of the cells may have swollen spontaneously.
Pharmacology
We tested a wide array of compounds for several reasons. Most anion channel
blockers are not perfectly specific and have secondary effects involving
intracellular signaling pathways; e.g., NPPB in rat portal vein cells inhibits
nifedipine-sensitive ca2'-currents and stimulates a glibenclamide-sensitive
K'-current (Kirkup, Edwards and Weston, 1996). Therefore, in long term
functional assays, to demonstrate an anion channel-dependent fimction, several
blockers must be used to ensure that the observed effects are not mediated by
modulation of second messengers. Also, a panel of effective anion-channel
blockers can be exploited in other patch-clamp studies to eliminate anion currents
and isolate other currents of interest; e.g., voltage-gated or HERG-like K'-
currents.
Inhibition by NPPB
The inhibition of swelling-activated C1- currents by the carboxylate analog
NPPB is well documented (for a review see Okada, 1997). In rat primary
microglia NPPB inhibited the anion conductance with an ICso of 30 ph4
(Schlichter et al., 1996) which is similar to the majority of volume-sensitive anion
channels (Okada, 1997). In the microglia cell line, NPPB was less potent; i.e.,
with 50 p M NPPB the inhibition was variable with one cell showing no apparent
inhibition (see Pharmacology).
In human T lymphocytes, the anion conductance was irreversibly inhibited by
100 pM NPPB (> 95% inhibition) (Schumacher et al., 1995).
Inhibition by flufenamic acid (FFA)
The anion conductances in rat primary microglia, the rat microglia cell line
and human T lymphocytes are all blocked by FFA with similar potencies. For
example, in primary microglia, 50% of the conductance was inhibited by 80 pM
FFA. Similarly in the rat microglia cell line = 50 % of Gel,, was inhibited by
100 @I FFA. In human T lymphocytes complete channel block was obtained
with 50- 100 p M FFA.
lnhibiion by glibenclamide
The sulfonylurea compound glibenclamide is a blocker of ATP-sensitive
potassium channels (KAp). It is hypothesized that it binds to a sulfonylurea
receptor of KArp channels and inhibits their functioning (Sturgess et al., 1985).
Intrigued by functional similarities between the cystic fibrosis transmembrane
conductance regulator (CFTR) and KArp (for similarities see Sheppard and Welsh,
1992), Sheppard and Welsh (1 992) attempted to block CFTR using glibenclamide.
It was subsequently reported that in whole-cell recordings glibenclarnide was a
high affinity, irreversible blocker of CFTR. Prior to this date no high-affinity
CFTR blockers had been identified (Sheppard and Welsh, 1992).
We tested two concentrations of glibenclamide, 80 and 500 pM, to determine
their ability to block the rnicroglia cell line anion channels. In contrast to the high
affinity (ICSo = 20 pM), irreversible block of CFTR, 80 pM glibenclamide only
slightly and reversibly inhibited the microglia cell line anion conductance.
Recently, it has been suggested that in whole-cell recordings glibenclamide may
permeate the cell membrane and inhibit CFTR from the inside (Sheppard and
Robinson, 1997). However, based on the rapid reversible inhibition of the
rnicroglia anion conductance, the inhibition was most likely from the extracellular
side.
We tested the higher concentration of glibenclamide (500 pM), because it has
recently been demonstrated that a swelling-activated CI- current in guinea pig
atrial myocytes was inhibited by glibenclarnide (Yamazaki and Hurne, 1 997). The
inhibition displayed voltage dependence (I& at +50 mV = 193 pM; ICso at -100
mV = 470 CrM) and was reversible (Yamazaki and Hume, 1997). In contrast, the
block of the microglia cell line anion conductance was voltage independent and
was more potent (see Pharmacology). Our results, however, confim the
observations of Yamazaki and Hume (1997), as well as those of other researchers
(Meyer and Korbmacher, 1996), that glibenclarnide, apart from inhibiting ATP-
sensitive K' channels and CFTR, is also capable of inhibiting volume-sensitive
anion channels.
Inhibition by riluzole
Riluzole is a glutamate release inhibitor that protects CAI hippocampal cells
from ischemia-induced degeneration (Malgouris et al., 1989; Pratt et al., 1992)
and has anticonvdsant activity (Mizoule et al., 1985). We were interested in
whether riluzole would block the microglia cell line anion channels, since they are
significantly permeable to glutamate. It is proposed that volume-sensitive anion
channels are partially responsible for increases in extracellular glutamate
concentrations following ischemia (see Introduction). This idea was W e r
supported when Bausch and Roy (1996) demonstrated that riluzole blocked
volume-sensitive anion channels in a human glial cell line (U-138MG). The
results of our study are consistent with the hypothesis that one mechanism by
which riiuzole prevents ischemia-induced neuronal degeneration may be through
inhibition of volume-sensitive anion channels (Bausch and Roy, 1996). It has
also been proposed that riluzole inhibits voltage-dependent sodium channels
(Benoit and Escande, 1991), blocks ca2+ mobilization in brain neurons in
response to NMDA or glutamate (Hubert et al., 1994) and blocks glutamate
- release via stimulation of a G-protein dependent pathway (DobIe, Hubert and
Blanchard, 1992). Furthermore, riluzole may directly inhibit excitatory amino
acid receptors (Debono et al., 1993). The results of the present study suggest that
riluzole may also be neuroprotective by potentially inhibiting glutamate release
through volume-sensitive anion channels in microglia.
Block by extracellular AT?
In a study of human platelets, Manning and Williams (1 989) first reported that
ATP blocked anion-selective channels. Since then, a number of authors have
reported similar findings (Alton et al., 1991; Paulmichl et al., 1992; Venglarik et
al., 1993; Jackson and Strange, 1995; Tsumura et al., 1996). The inhibition of
volume-sensitive anion channels by ATP is proposed to be an extracellular, open-
channel block of the pore. Therefore susceptible channels are thought to have an
outer vestibule larger than the effective size of ATP but with a pore size that
prevents ATP permeation (Okada, 1 997).
The Icl, protein, once considered an anion channel, has a consensus
nucleotide-binding domain on what might be the extracellular region of the
protein (Paulmichl et al., 1992). This has led to the speculation that inhibition of
anion currents by extracellular nucleotides might result fiom Icl, forming part of
the channel or acting as a channel regulator.
Physiologically, there may be several important effects of nucleotides that
leak fiom neurons and glia into the extracellular space during cellular damage
(White and Hoehn, 1991). For instance, the activation of the novel currents by
extracellular ATP in the present study suggested that the microglia cell line had
purinergic receptors. This is consistent with a number of studies that
demonstrated purinergic receptors in primary microglia under both proliferating
and non-proliferating conditions (Kette~mann, Banati and Walz, 1993; Walz et
al., 1993; Norenberg et al., 1994) as well as in mouse rnicroglia cell lines (Ferrari
et al., 1997). In primary microglia, stimulation of P2y- and adenosine PI-
purinoceptors activated a desensitizing, non-selective cation conductance and a
more prolonged, outward K' conductance (Walz et al., 1993; Norenberg et al.,
1994).
The activation of an inward current followed by the development of an
outward current is similar to our findings. Rather than characterizing these
currents we attempted to eliminate them with the purinergic receptor antagonist,
suramin (Kennedy, 1990); however, surarnin blocked the anion current in a
voltage-dependent manner. This result supports the recent findings by Galietta et
al. (1997) that purinergic receptor antagonists block volume-sensitive anion
channels. Unfortunately, because of the presence of the purinergic receptors and
inhibition of the anion channels by surarnin, we did not conclusively determine
whether extracellular ATP inhibited the anion channels. Based on the similarities
between the rat rnicroglia and human T lymphocyte anion conductances which
suggest similar underlying channels (see Comparison.. .), we hypothesize that the
rnicroglia anion channels are not inhibited by extracellular ATP.
Nucleotide regulation
The final thesis objective was to evaluate the role of intracellular nucleotides
in regulating the anion channels. Since GTPyS increased the spontaneous CI-
conductance the channels may be regulated by G proteins. However, failure of
GDPPS to inhibit the swelling-induced conductance suggests that a G protein is
not required for channel activation. Based on these seemingly contradictory
results we did not fiuther pursue G-protein regulation. However, as the
conductance was supported by non-hydrolyzable ATP anaiogs (AMP-PNP,
Schlichter et al., unpublished) we hypothesized that the guanine nucleotides may
directly interact with the channels via nucteotide binding domains. It has already
been observed in Intestine 407 cells that the swelling-induced current is supported
by non-hydrolyzable ATP analogs as well as by ADP and GTP (Oiki, Kubo and
Okada, 1994). To determine if guanine nucleotides also support the activation of
the microglia anion conductance we added 200 pM GTPyS to the pipette solution.
GTPyS, however, did not suppoa channel activation. Therefore, the most likely
explanation for the seemingly contradictory results is that a G protein modulates
the anion channels but is neither sufficient nor essential for channel activation.
Comparison between the anion conductance in the rat microglia cell line, human T-lymphocytes and rat primary microglia
In human T lymphocytes the anion conductance developed spontaneously
upon establishing the whole-cell configuration using a low ionic-strength pipette
solution. The current activation did not require hypo-osmotic shock or pressure-
induced swelling. The microglia cell line anion conductance also activated
spontaneously using similar bath and pipette solutions. However, in contrast to
the exquisite volume sensitivity of the microglia anion conductance (Figure 6),
the human lymphocyte anion conductance, after the initial buildup, ran down over
several minutes and could not be reactivated by cell swelling despite adding 4
mM ATP to the pipette solution (Schumacher et al., 1995). It was proposed
therefore that current buildup and rundown resulted from a biochemical event
hggered by establishing the whole-cell recording (Schumacher et al., 1 995). The
present study suggests that spontaneous current activation in the human T
lymphocyte was a result of using a low ionic-strength pipette solution. If a high
ionic-strength pipette solution had been used, there may have been less
spontaneous current activation and the current may have exhibited greater volume
sensitivity. In contrast to the results of Schumacher et al. (1995), several
researchers who used high ionic-strength pipette solutions reported strong volume
sensitivity (Lewis, Ross and Cahalan, 1993). Another potential difference
between the rat microglia and human T lymphocyte anion conductance was that
after inhibiting the lymphocyte anion conductance with NPPB the block was not
reversible within 5 min of washing out the inhibitor (Schumacher et d., 1995).
However, since the current runs down with a time constant of 280 sec
(Schumacher et al., 1995), the washout of NPPB may have been sufficiently slow
that its reversibility was obscured by current rundown. Despite these potential
- differences, the microglia cell line and the human T lymphocyte anion
conductances share a number of similarities that suggest the whole-cell currents
have similar underlying channels. For example, both anion currents are
outwardly rectified, display time- and voltage-independent gating, have similar
selectivity sequences, have small singlechannel conductances and similar
pharmacological profiles (see Table 9). Furthermore, in both cell types the
spontaneous anion conductance was increased by GTPyS.
The anion conductance in rat primary microglia is also outwardly rectified,
displays time- and voltage-independent gating (Schiichter et al., 1996; present
study), likely has a small single-channel conductance, and a similar
pharmacological profile (Table 9) (Schlichter et al., 1996). Therefore all three
cell types may have similar underlying channels.
How do t h e anion channels in rat microglia and human T lymphocytes compare to volume-sensitive anion channels in t h e literature?
Recently, Okada (1 997) did a very thorough comparison of volume-sensitive
anion currents in several different cell types. In general, the volume-sensitive
outwardly rectiwing (VSOR) anion currents share a number of similarities with
the currents under investigation. For example, the VSOR anion currents are
characterized by outward rectification, have an Eisenman I selectivity sequence
and are inhibited by NPPB. Analogous to the microglia cell line currents, the
VSOR anion currents also require intracellular ATP and are sensitive to volume
changes.
In contrast to the channels under investivgation, VSOR anion currents are
blocked by extracellular ATP, inactivate at depolarized potentids and the
underlying channels have a Iarger single-channel conductance; i.e., 20-80 pS.
What is the molecular basis for the underlying channels?
Two likely candidates for volume-sensitive anion channels are ClC-2 and
CIC-3 (Strange, 1998) both of which belong to the expanding CIC gene family of
C1' channels. Since the initial discovery of CIC-0, nine other mammalian
members have been identified (Jentsch and Gunther, 1997). However, only C1C-2
and C1C-3 have exhibited volume sensitivity thus far. Furthermore, C1C-2 and
CIC-3 are abundantly expressed in rat brain as well as several other tissues
(Kawasaki et al., 1994; Thiemann et d., 1992).
Expression of CIC-2 in Xenopus oocytes leads to a C1--selective current. with
a C1- 2 Br- > I' selectivity sequence (Thiemann et al.. 1992). C1C-2 is nearly
completely closed at physiofogicd voltages (Thieman et al.. 1992), but can be
activated by strong hyperpolarization (< -90 mV) (Thiemann et al., 1992) or
hypo-osmotic shock (Grunder et al., 1992). If the channel is activated by
hyperpolarization the steady-state current activates over several seconds and has a
near linear I-V relationship (Thiemann et al., 1992; Grunder et al., 1992).
Alternatively, swelling-induced activation of ClC-2 leads to a steady state-current
which activates much faster and is inwardly rectified (Grunder et al., 1992). In
contrast, the current under investigation is outwardly rectified, has an I- > C1-
selectivity sequence and is constitutively active at all voltages tested. Therefore,
there are distinct biophysical differences bemeen CIC-2 and the anion currents in
rat microglia and human T lymphocytes. Recently, it has been demonstrated that
rabbit CIC-2 expression in Xenopus oocytes is associated with enhanced volume
regulation; i.e., in response to a hypo-osmotic shock, oocyte swelling was
minimized in cells in which the ClC-2 related current was large (Furukawa et al.,
1998). Based on the biophysical differences, however, it is unlikely that CIC-2
homomultimers represent the underlying channels in rat microglia and human T
lymphocytes.
Expression of the second candidate, CIC-3, in NIW3T3 fibroblasts produces a
volume-sensitive outwardly rectifying current with an 1- > C1- > Asp- selectivity
sequence that is inhibited by DIDS (100 pM), tarnoxifen (10 pM) and
extracellular ATP (10 mM) (Duan et al., 1997). Therefore, the characteristics of
ICIC-3 are similar to the VSOR anion currents (see Table 9). However, ICtC-3
diverges from Icl in rat microglia and human T lymphocytes in several respects.
For example, the microglia and human T lymphocyte currents are only weakly
blocked by DIDS (500 pM) (Schlichter et al., unpublished; Schurnacher et al.,
1995) and in the human T lymphocyte Icl was not blocked by extracellular ATP.
Furthermore, CIC-3 has a much larger single-channel conductance (-40 pS) and
ICICJ inactivates at potentials above +80 mV (Duan et al., 1997). Therefore, the
underlying channels in microglia and human T lymphocytes are most likely not
homomultimers of C1C-3. One intriguing possibility, however, is that CIC family
members form heteromultimers with novel properties. This has been
demonstrated by Lorem, Pusch and Jentsch (1996) where they showed that
coexpression of CIC-1 and CIC-2 in Xenopus oocytes led to the formation of
novel heteromultimeric channels. Therefore, the anion channels in rat microglia
and human T lymphocytes may be comprised of ClC heterornultimers.
In contrast to C1C-3 and VSOR anion currents, E1 in rat microglia and human
T lymphocytes, as well as a number of other cell types - e-g., human neutrophils,
(Stoddard, Steinbach and Simchowitz, 1993), skate hepatocytes (Jackson et al.,
1996) and the mouse muscle cell line, C2C12 (Voets et al., 1997) - does not
inactivate at depolarized potentials. It is unclear whether the voltage-
independence in these cells represent a distinct channel type or whether these
channels are differentially regulated. I t has been suggested that the threshold of
inactivation may be higher for anion currents in these cells (Okada, 1997).
However, the results of the present study do not support this hypothesis. At
potentials as high as 140 mV the current in microglia cell line showed no signs of
voltage-dependent inactivation (data not shown).
As discussed by Okada (1 997), it has also been demonstrated that alterations
in intracellular and extracellular free bAg2+ concentrations can alter inactivation
kinetics of swelling-activated anion channels. For example, in Intestine 407 cells,
increasing free intracellular M ~ " accelerated the rate of inactivation and after 111
inactivation there was less steady-state current. Furthermore, inactivation was
slower when the extracellular C1' concentration was increased. Based on these
observations it was proposed that intracellular M~~~ is an open-channel blocker of
the volume-sensitive anion channels in Intestine 407 cells (Oiki, Kubo and Okada,
1995). In contrast, Anderson, Jirsch and Fedida (1995) proposed that
extracellular M ~ " and other divalent cations (e-g., ca2> caused a voltage-
dependent block of the volume-sensitive anion channels in human small-cell lung
cancer (H69A.R) cells and cervical carcinoma (HeLa S5) cells. In these cells
replacing external monovalent and divalent cations, including M$+, with NMDG
removed almost all current relaxation at depolarized potentials. Since our standard
bath solution (140 m M NMDGCI - Table 1) contained 1 mM ~ g ' + and 1 m M
ca2+ and there was no evidence of current relaxation in any of the cell types
investigated (i.e., the rat microglia and human T lymphocytes) it is unlikely that
the anion channels under investigation are inhibited by extracellular M~".
In contrast to the results of Oiki and co-workers (1995), Anderson, Jirsch and
Fedida (1995) found that current relaxation was unaffected by changes in pipette
M ~ ~ ' concentration.
Since we used pipette solutions with low free M ~ ~ ' concentrations (i-e., 0.08
rnM) the results of the present study cannot exclude the possibility that the
microglia cell line anion channels are blocked by intracellular free M ~ " which
would confer voltage dependence. In the set of experiments where we did not
clamp the M~~~ concentration after excluding ATP from the pipette solution, there
was insufficient current to determine whether there was voltage-dependent
inactivation. In this regard, however, the human T lymphocyte anion current did
not display voltage-dependent inactivation despite using pipette solutions with an
intracellular free M ~ ~ + concentration of 0.68 mM (Schlichter et al., unpublished).
Alternatively, the lack of voltage-dependence in rat microglia and human T
lymphocytes may indicate that the anion channels under investigation do not
associate with an anion channel regulator that confers voltage dependence. The
two most likeiy candidates for anion-channel regulators are pIctn and P-
gl ycoprotein (P-gp).
pkln is an abundant, cytoplasmic protein (Krapivinsky et al., 1994) that was
isolated by expression cloning in Xenopus oocytes using mRNA from Madin
Darby canine kidney (MDCK) cells (Paulmichl et al., 1992). It was initially
thought to be an anion channel because its expression in Xenopus oocytes
activated a C1--selective current (Icln) with strong outward rectification that slowly
inactivated at depolarized potentials (Table 9). Furthermore pIcln had two
putative nucleotide binding sites near the proposed channel mouth and was
inhibited by extracellular nucleotides (Paulmichl et al., 1992). Based on the
observation that pIcrn was mainly cytoplasmic and highly soluble, Krapivinsky et
al. (1994) proposed that pIcln was an anion-channel regulator and not a C1-
channel. This hypothesis is W e r supported by the recent findings that
expression of picln in planar lipid bilayers does not reveal anion-channel activity
(Li et al., in press - cited in Strange, 1998). Furthermore, expression of CIC-6
(which is structurally unrelated to pIcln) in Xenopus oocytes activates the same
current as the expression of picln (Buyse et al., 1997). This suggests that the
currents initially described by Paulmichl et al. (1992) may have been endogenous
to Xenopus oocytes and artifactually activated by pIcl, expression (Strange, 1998).
Recently, it has been proposed that pIcln may associate with binding proteins that
are involved in cell-morphology regulation (Krapivinsky et al., 1 99 8). Therefore,
Krapivinsky et ai. (1998) suggest that pIcln may not be directly associated with
- anion channels but rather may be part of a pathway that is indirectly connected to
anion channels. An indirect role for pIcln modulation of anion channels is also
supported by Strange (1998).
However, the possibility remains that PIcr, directly or indirectly, may alter the
inactivation kinetics of volume-sensitive anion channels. Levitan et al. (1997)
tested this hypothesis by isolating Icl, cDNA from T84 or myeloma cells and
expressing it in mammary adenocarcinoma (TSA) cells. pkl. expression did not
alter the inactivation kinetics of the whole-cell anion current (Levitan et al.,
1997).
Another candidate for an anion-channel regulator is P-gp. P-gp is a
transporter that confers multidrug resistance on cells by pumping out cytotoxic,
hydrophobic drugs (Gottesman and Pastan, 1993). P-gp belongs to the ATP-
binding cassette (ABC) superfamily of transporters and is structurally similar to
another ABC family member, CFTR (Higgins, 1992). Once thought to be an
anion channel (Valverde et al., 1992), it is now considered an anion-channel
regulator (Okada, 1997; Nilius et al., 1996), possibly through direct protein-
protein interactions with the anion channels, the cytoskeleton or the anion
channels' volume sensors (Okada, 1997). Functionally, the association of an
anion channel with P-gp has been proposed to confer PKC dependence on channel
activity; i.e, swelling-induced channel activation was prevented by PKC
activators in NW3T3 or HeLa cells transfected with the P-gp gene, MDRl
(Hardy et al., 1995). P-gp expression has also been associated with increases in
volume sensitivity (Luclcie et al., 1994; Miwa, Ueda and Okada, 1997). For
example, Miwa and co-workers (1997) found that human epidermoid KB cells
transfected with MDRl (KB-G2) had larger current densities for a given relative
surface area than untransfected controls (KB-3-1). To the best of our knowledge,
however, it has not been reported that P-gp expression alters inactivation kinetics.
In summary, rat primary microglia, the rat microglia cell line and human T
lymphocytes have what appears to be the same anion current, suggesting that the
underlying channels are similar in these three cell types. In contrast to the VSOR
anion channels and the molecular candidate ClC-3, the channels have a much
smaller single-channel conductance, do not inactivate at depolarized potentials
and are probably not blocked by extracellular ATP. It remains to be determined if
these differences indicate a different channel type or whether these channels are
differentially regulated or lack an accessory subunit. The recent findings of
Levitan et al. (1997) suggest that pIa, may not confer voltage-dependence on
volume-sensitive anion channels.
A possible role in glutamate release?
The properties of the small-conductance anion channels in the rat microglia
cell Iine are consistent with a role in glutamate neurotoxicity; i.e., the anion
channels are permeable to glutamate, they are voIurne sensitive and they are
blocked by the glutamate release inhibitor riluzole. Their potential role in
glutamate neurotoxicity is further supported by the following observations. One,
microglia have a close association with neurons and synapses Ovfurabe and Sano,
l982b; Palacios, 1990). Two, microglia are capable of efficiently synthesizing
and releasing glutamate (Patrizio and Levi, 1 994). Three, ischemia-evoked release
of aspartate and glutamate in the rat cerebral cortex was dramatically reduced by
anion-channel inhibitors, including NPPB (Phillis, Song, and O'Regan, 1 998)
which inhibits the anion conductance in the rat microglia cell line @resent study)
and in rat primary microglia (Schlichter et al, 1996). Finally, in transient
forebrain ischemia, microglia are the first cells to be activated (Morioka, Kalehua
and Streit, 1991; Gehrmann et al., 1992). During activation and proliferation,
microglia undergo distinct morphological changes that have been characterized by
swelling of the cytoplasm and nucleus (Kitamura, Tsuchihashi and Fujita, 1978).
These morphological changes may be sufficient to activate the volume-sensitive
anion channels (Schlichter et al., 1996). In this regard, blocking the microglia
anion channels inhibits cell proliferation (Schlichter et al., 1996).
One difficulty with the hypothesis that microglia contribute to glutamate
neurotoxicity is that the microglia anion conductance requires intracellular ATP to
activate. Therefore, in conditions where cellular ATP levels are compromised
(e.g., ischemia and hypoxia) the channels may not open. In consideration of the
ATP dependence of volume-sensitive anion channels, Bausch and Roy (1996)
propose that gliai cells may swell prior to significant ATP decline in response to
the rapid increase in extracellular K* concentration during hypoxia and ischemia.
Alternatively, if a decline in cellular ATP inhibited the volume-sensitive anion
channels, during reperfusion when cellular ATP levels are restored, their
subsequent activation could potentially exacerbate neuronal damage.
Future directions
To determine conclusively that tk re is no voltagedependent inactivation in
rat primary microglia and the rnicroglia cell line, a series of pipette solutions
should be used with varying concentrations of free M ~ ~ * . This would help
determine whether the anion channels are blocked by intracellular M ~ " as has
been proposed for Intestine 407 cells (Oiki, Kubo and Okada, 1995).
The glutamate permeability of the channel was determined from shifts in the
reversal potential when NaGlu was perfused into the recording chamber. This
demonstrated that glutamate can permeate the channel &om the extracellular side.
To demonstrate that glutamate can permeate the channel from the intracellular
side, NMDGGlu pipette solutions should be used.
In the whole-cell patch-clamp configuration there was visible expansion of the
cell membrane and cytoplasm when the cells were exposed to a hypo-osmotic
shock (Figure 5). Under these conditions the current activated. However. the
question arises: ' Would the channeZs activate using a more physiological
experimentul paradigm?' To address this question, cell-attached recordings could
be used so that the cell maintains its ability to undergo a RVD and is not subjected
to the stress of establishing a whole-cell recording; i.e., the intracellular milieu of
the cell remains undisturbed. Alternatively, functional assays to assess the rate of
RVD (as a measure of channel activity) could be performed in the presence and
absence of anion-channel blockers. For example, we hypothesize that after a
hypo-osmotic shock, in the presence of 125 p M NPPB, RVD would be prolonged.
Cell volume could be determined in single-cell assays using fluorescent dyes such
as Calcein-acetoxymethyl ester. The advantage of using a single-cell assay is
that individual cell differences can be observed and correlated to biological
- parameters such as morphology. In contrast, light-scattering methods report
average changes of cell volume and provide no means of distinguishing individual
differences between cell responses (Crowe et d., 1995).
To determine if the anion channels are involved in ischemia-induced release
of glutamate, it is necessary to show that in conditions associated with ischemia
the anion channels activate. For example, does an increase in extracellular K'
concentration lead to cell swelling and anion-channel activation? This question
could be addressed in whole-cell recordings using bath solutions with high K'
concentrations. In high K+ solutions we hypothesize that the cells would swell in
the absence of an applied hypo-osmotic shock, subsequently activating the
volume-sensitive anion channels. To address the question of whether the anion
channels activate during hypoxia; i-e., in situations where the metabolic status of
the cell is compromised, anion-channel activity could be assessed using hypoxic,
aglycemic bath solutions. This would provide usehl idormation not only about
the activation of the volume-sensitive anion channels but also about their ability
to remain active.
In hc t ional assays, microglia cultures could be exposed to a hypo-osmotic
shock in the presence and absence of Cl--channel blockers. We hypothesize that
anion channel activation will increase the extracellular concentration of
glutamate. Extracellular glutamate concentrations could be measured using high-
pressure liquid chromatography W L C ) (Kimelberg et al., 1990). Ultimately,
microglia neurotoxicity under ischemic and hypoxic conditions should be
assessed using co-cultures of neurons and microglia in the presence and absence
of anion-channel blockers.
Table 9 Comparison of whole-cell anion currents
Rat primary microglia Rat microglia cell line Human T lymphocyte VSOR
Rectification
Single-channel conductance
Voltage dependence
Selectivity sequence
outward re~tification~"~'~
likely small'"
outward rectif~cation'~" outward rectification' outward rectification"
< 2 ps"
no voltage dependcr~ce'~,'~ no voltage dependence1'-" voltage-dependent inactivation' no voltage dependencc17
not determined
Volume sensitivity
Cytosolic ATP
less volume sensitive1' volunie sensitive" volume sensitive'
required" required' does not prevent spontaneous tun down'*
P 95 % inhibition with I00 @I" no apparent valtagc depcndend2 irreversible block"
;. 90% inhibition with 125 pM1" IC,,, a 30 p ~ ' " voltage-independent inhibition"'
NPPB a 80 % inhibition with 125 p ~ 1 7
no apparent voltage dependence" reversible"
ICM 25 pM voltage-independent inhibition'
DIDS
Flufenamic acid
weak voltage-dependent block with 500 pM1'
weak voltagedependent block with 500 p ~ l l
weak voltage-dependent block with 500 pMi1
ICW rs 20-150 pM a( positive potentink'
ICw .r 80 pM1" voltage-independent inhibition'" reversibility not tested"
ICx,s 100ph4 no apparent voltage de ndenceI7 contpletely revcrsiblc I P "
100% inhibition with 50-100 pM1' no apparent voltage dependence1'
inhibition of VSOH in mousc CCD cells (-58% inhibition wilh 100 CIM)' and mousc muscle cell lines (>75% inhibition with 500 PM)"
Glibenclarnide 16% inhibition with 80 CIMI'
s 80 % inhibition with 500 pMI7 voltage indcpendenl" inhibition cornplclely reversible"
inhibition of Imcll in guinea pig atrial myocytes 1Cw = 193 and 470 @I at t 50 mV and -100 mV respective~y'~ vollsge-dependent inhibition of Imdi in mouse CCD cells (23% inhibition with 100 pM)'
not tested
Riluzole
Extracellular ATP
Suramin
not tested
not tcsted
no1 Icslcd
= 90 ", inhibition with 300 pMi7 inhibition completely reversible
not tested
no black (20 m ~ ) "
not trsted
inhibition of l a d l in human glial cells I C M ~ 140 pM1
inconclusive" blocked by concentrations as low as 30 CIM'
voltage-dependent inhibitionI7 -70 and 18% inhibition with 30 pM aIt5O and - t 00 mV respeclivcly
Table 9 continued plcln CIC-3 CIC-2
Rectification
Single-channel conductance
Voltage dependence
Selectivity sequence
Cytosolic ATP
Volume sensitivity
DIDS
\O 0
Flufenamic acid
Glibenclamide
Riluzole
Extracellular ATP
Suramin
outward rect~fication~
no1 known
inactivates . v60 mVq
I + > cp4
nor dctermined'
not sensitive to volume"
not dctermined
not detcn\ined
riot dcternlined
blockcd by 1 - I 0 niM A'I'P'
not dctermincd
outward rectification'
-40 p ~ '
inactivnlcs -80 mVX
I' > CI' > ASP''
not determined
sensitive to volume'
not determined
95% and 37% block with I00 p M at t80 and -80 mV respcctivel J
not determined
not determined
not determined
voltagc-dependent block (10 m ~ ) '
not dclernlincd
linear"/inward rectification'
3-5 psb
activates over several seconds"
C1' 2 Br' > I"'
not determined'
ti, a 0.98 mM (rabbit CIC-2 expressed in Xenopus oocytes)'
almost no inhibition with 1 r n ~ "
not determined
not determined
not detemiined
not deternlined'
no! dcteniined
1 Bausch and Roy, 1996 6 Jentsch et al., 1995 10 Schlichter el al., 1996 14 Vmlsetal,, 1996 2 Duan et al., I997 7 Mcycr and Karbmachcr, 1996 I I Schlichtcr et al., unpublished 15 Vocls el al., I997 3 Furukawa et al., 1998 8 Oltada, 1997 12 Schumacher et al., 1995 16 Yamazaki and l lumc, 1997 4 Galietta er a]., 1997 9 Paillmiclil ct al , 1992 13 Thiemann e! al., I992 17 current study 5 Grundcr et al., 1992
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