Inwardly rectifying chloride channels have been described

in several tissues (Chesnoy-Marchais, 1983; Thieman et al.1992; Chesnoy-Marchais & Fritsch, 1994; Komwatana etal. 1994; Arreola et al. 1996; Carew & Thorn, 1996; Fritsch

& Edelman, 1996; Kajita & Brown, 1997; Clark et al. 1998;

Park et al. 1998; Tarran et al. 2000; Fava et al. 2001;

Mohammad-Panah et al. 2001). These native channels share

many properties with the cloned ClC-2 channel, including

a requirement for strong hyperpolarization to open. ClC-2

cloned from heart and brain (Thieman et al. 1992) is a

widely expressed channel that induces the appearance of

inwardly rectifying Cl_ currents when expressed in Xenopusoocytes. ClC-2 is expressed in many tissues where ClC-2-like

currents are present, although the function of this protein

in these organs is unclear. The inwardly rectifying Cl_

current in Sertoli and Leydig cells requires the ubiquitously

expressed Clcn2 gene (Bösl et al. 2001). Moreover, targeted

disruption of Clcn2 produces severe postnatal degeneration

of photoreceptor cells in the retina and of primary

spermatocytes and spermatogonia in the testis, suggesting

that ClC-2 plays a critical role in regulating the ionic

environment of these cells (Bösl et al. 2001). Using a gene

ablation approach, we have recently shown that the

inwardly rectifying Cl_ current in mouse parotid acinar

cells also requires expression of the Clcn2 gene (K. Nehrke,

J. Arreola, H.-V. Nguyen, J. Pilato, L. Richardson, G.

Okunade, G. Shull, R. Baggs & J. E. Melvin, unpublished

observations). Heterologously expressed ClC-2 channels

(Jordt & Jentsch, 1997) and native inwardly rectifying

(Kajita & Brown, 1997; Ferroni et al. 2000; Tarran et al.2000) currents in some cells are sensitive to changes in pH.

Since cells in the parotid gland are exposed to considerable

changes in the pH environment when stimulated to secrete

(Melvin et al. 1988; Okada et al. 1991), the H+ sensitivity of

this channel may play an important physiological role.

Although no direct evidence exists, several studies suggest

that ClC-type Cl_ channels undergo conformational changes

during gating. For example, following the binding of Cl_

ions to the closed state of ClC-0, a rearrangement to a Cl_-

bound closed state was proposed to explain the voltage

dependence of opening (Chen & Miller, 1996). Also, it was

suggested that an increase in affinity of the H+ binding site

upon channel opening leads to further activation of ClC-0

by H+ (Hanke & Miller, 1983). The temperature dependence

of ClC-0 and ClC-1 also suggests conformational changes

of the protein structure during closed to open transitions

(Pusch et al. 1997; Bennetts et al. 2001). Finally, using the

projection structure of a ClC-type Cl_ channel from E. coli,

Conformation-dependent regulation of inward rectifierchloride channel gating by extracellular protonsJorge Arreola*†, Ted Begenisich† and James E. Melvin*†

*Center for Oral Biology in the Aab Institute of Biomedical Sciences and †Department of Pharmacology and Physiology,University of Rochester Schoolof Medicine and Dentistry, Rochester, NY 14642, USA

We have investigated the gating properties of the inward rectifier chloride channel (Clir) from

mouse parotid acinar cells by external protons (H+o) using the whole-cell patch-clamp technique.

Increasing the pHo from 7.4 to 8.0 decreased the magnitude of Clir current by shifting the open

probability to more negative membrane potentials with little modification of the activation

kinetics. The action of elevated pH was independent of the conformational state of the channel. The

effects of low pH on Clir channels were dependent upon the conformational state of the channel.

That is, application of pH 5.5 to closed channels essentially prevented channel opening. In contrast,

application of pH 5.5 to open channels actually increased the current. These results are consistent

with the existence of two independent protonatable sites: (1) a site with a pK near 7.3, the titration of

which shifts the voltage dependence of channel gating; and (2) a site with pK = 6.0. External H+

binds to this latter site (with a stoichiometry of two) only when the channels are closed and prevent

channel opening. Finally, block of channels by Zn2+ and Cd2+ was inhibited by low pH media. We

propose that mouse parotid Clir current has a bimodal dependence on the extracellular proton

concentration with maximum activity near pH 6.5: high pH decreases channel current by shifting

the open probability to more negative membrane potentials and low pH also decreases the current

but through a proton-dependent stabilization of the channel closed state.

(Resubmitted 4 January 2002; accepted after revision 6 March 2002)

Corresponding author J. Arreola: Center for Oral Biology in the Aab Institute of Biomedical Sciences, University of RochesterSchool of Medicine and Dentistry, 601 Elmwood Avenue, Box 611, Rochester, NY 14642, USA. Email: Jorge-Arreola@urmc.rochester.edu

Journal of Physiology (2002), 541.1, pp. 103–112 DOI: 10.1113/jphysiol.2002.016485

© The Physiological Society 2002 www.jphysiol.org

it was reasoned that transmembrane segment 4 lies near

the pore entrance in the closed state and possibly

undergoes a conformational change during gating to form

part of the pore (Mindell et al. 2001).

We have investigated the regulation of the inwardly

rectifying Cl_ current associated with the Clcn2 gene in

mouse parotid acinar cells. We found that pHo had a

bimodal effect on this current. Both low and high pHo

inhibited channel current, whilst near-neutral pHo

facilitated opening. The current inhibition by pHo 8.0 was

independent of the conformational state of the channel.

Conversely, the inhibition observed at pHo 5.5 occurred

only when the channel was in the closed state. These data

are consistent with the presence of two independent H+

binding sites, one of which is not available when the

channel is in the open state. Channel block by the divalent

cations Cd2+ and Zn2+ was H+ dependent, but the data do

not allow a determination of whether this effect involves

one of the two H+ binding sites that regulate channel gating,

or if inhibition is associated with a third protonatable site

present on these channels.

METHODS Single cell dissociationThe single acinar cells were dissociated from mouse parotid glandsfollowing a protocol approved by the University of Rochesteron Animal Resources, as previously described (Arreola et al.1996). Briefly, glands were dissected from exsanguinated mice(BlackSwiss w 129/SvJ hybrid mice) after CO2 anaesthesia. Glandswere minced in Ca2+-free minimum essential medium (MEM)(Gibco BRL, Gaithersburg, MD, USA) supplemented with 1 %bovine serum albumin (BSA) (Fraction V, Sigma Chemical Co., StLouis, MO, USA). The tissue was treated for 20 min (37 °C) with a0.02 % trypsin solution (MEM-Ca2+ free + 1 mM EDTA (ethylene-diaminetetraacetic acid) + 2 mM glutamine + 1 % BSA). Digestionwas stopped with 2 mg ml_1 of soybean trypsin inhibitor (Sigma)and the tissue further dispersed by two sequential treatments of60 min each with collagenase (100 units ml_1 of type CLSPA,Worthington Biochemical Corp., Freehold, NJ, USA) in MEM-Ca2+ free + 2 mM glutamine + 1 % BSA. The dispersed cells werecentrifuged and washed with basal medium Eagle (BME)(Gibco)/BSA-free. The final pellet was resuspended in BME/BSA-free + 2 mM glutamine, and cells were plated onto poly-L-lysine-coated glass coverslips for electrophysiological recordings.

Electrophysiological recordingsChloride currents were recorded at room temperature (20–22 °C)using the conventional whole-cell patch-clamp configuration(Hamill et al. 1981) and an Axopatch 200B amplifier (AxonInstruments). Patch pipettes were pulled to have a resistancebetween 2 and 4 MV when filled with the standard pipette(internal) solution containing (mM): 140 TEA-Cl, 10 or 20 EGTAand 20 Hepes; pH 7.2. Cells were bathed in the standard externalsolution containing (mM): 140 NMDG-Cl, 0.5 CaCl2, 50D-mannitol and 10 Hepes; pH 7.4 (or 8). Other pHo values wereadjusted by using 20 mM CAPS (pH 10), AMPSO (pH 9), MOPS(pH 6.75), or MES (pH 6.5, 6.0 and 5.5). The internal and externalsolutions were designed to have nearly 0 free [Ca2+] and to beslightly hypertonic, respectively, to avoid the activation of Ca2+-

dependent and volume-sensitive chloride channels present inmouse parotid acinar cells (J. Arreola, T. Begenisich, K. Nehrke,H.-V. Nguyen, K. Park, L. Richardson, B. Yang, F. Lamb,B. C. Shutte & J. E. Melvin, unpublished observations; Iwatsuki etal. 1985). To assay the effects of [Cl_]o on reversal potentials,glutamate was used for equimolar substitution of Cl_. Chloridechannels were activated by delivering square pulses from aholding potential of 0 mV to membrane potentials from _120 to+60 mV in 10 or 20 mV steps. Instantaneous current-voltagerelationships were constructed from data collected from tailcurrents recorded between +60 and –100 mV after opening thechannels by a 6 s prepulse potential to –100 mV. Currents werefiltered at 1 kHz using an 8 db/decade low-pass Bessel filter andsampled using pCLAMP software (Axon Instruments).

Inward rectifier chloride currents were nearly absent afterachieving the whole-cell configuration; instead, they appearedwith time and reached a maximum within 15–20 min. Thisphenomenon has been previously observed in T84 cells andastrocytes and it appears to be related to the presence ofintracellular ATP (Fritsch & Edelman, 1996; Fava et al. 2001).However, there is no definitive explanation for this effect and wedid not investigate it further. All the data presented in this paperwere sampled in the absence of intracellular ATP and after morethan 15 min of cell dialysis.

To determine whether the reduction in Clir current induced bychanges in [H+]o was state dependent (closed vs. open), dualpulses were given to –100 mV separated by a 5 s depolarization to+60 mV. The first hyperpolarization to –100 mV lasted 50 s andwas used to examine whether open channels were sensitive tochanges in [H+]o. The second 4 s long hyperpolarization was usedto assay the fraction of channels inhibited after being in the closedstate for 5 s at pHo of 8.0, 7.4 or 5.5. Means ± S.E.M. of currentvalues without leak correction are given.

AnalysisThe dependence of reversal potential shifts (DEr) with the externalchloride concentration ([Cl_]o) was fitted with the Nernst equation:

RT [Cl_]oDEr = Er([Cl_]o) _ Er (141) = —— log —— , (1)zF 141

where Er([Cl_]o) and Er (141) are the reversal potentials experimentallydetermined in the presence of X [Cl_]o and 141 mM chloride,respectively, and R, T, z and F have their usual thermodynamicmeanings. Whole-cell Cl_ conductance (gCl) was calculated as:

IClgCl = ———— , (2)

Vm _ Er

where ICl is the whole-cell current, and Vm is the membranevoltage. Whole-cell conductance–voltage relationships were fittedto a Boltzmann distribution function:

gmaxgCl = —————— , (3)

1 + e((Vm _ V0.5)/s)

where V0.5 is the membrane voltage necessary to obtain 50 % of themaximal conductance (gmax), and parameter s reflects the voltagesensitivity of the conductance. The extrapolated gmax was thenused to normalize the whole-cell conductance at each potential topool normalized conductance from different cells. Time constantsof chloride channel activation were estimated by fitting entireraw traces with a double exponential function according to theLevenberg-Marquardt method.

J. Arreola, T. Begenisich and J. E. Melvin104 J. Physiol. 541.1

RESULTS Inward rectifier chloride channels (Clir) in mouseparotid acinar cellsThe native inward rectifier chloride channels (Clir) have

not been previously described in mouse parotid acinar

cells. Therefore, initial studies characterized the inward

rectifier chloride currents in this cell type by analysing

voltage sensitivity and kinetics. Figure 1A displays inwardly

rectifying, whole-cell chloride currents recorded from a

representative acinar cell. Upon hyperpolarization, there

was an immediate increase in current, which was followed

by a further, slow activation with no significant inactivation

by the end of the test pulse (up to 50 s, e.g. Fig. 5B). The

current jump observed at negative voltages probably

represents a small fraction of open channels. No current

was detected at positive voltages up to +60 mV with

symmetrical [Cl_] solutions, demonstrating that under

our experimental conditions both Ca2+-dependent and

volume-sensitive outward-rectifying chloride channels

present in this cell type were insignificant, and the majority

of the current was due to the activation of Clir.

The strong rectification displayed by Clir is clearly seen in

the current–voltage (I–V) relationship shown in Fig. 1B. I–Vcurves were normalized to the current recorded at –120 mV.

The average current at –120 mV was –338 ± 74 pA, whereas

the current at +40 mV was +3 ± 0.5 pA (n = 12).

Bimodal pH dependence of the ClC-2 chloride channelJ. Physiol. 541.1 105

Figure 1. Inward rectifier chloride currentsA, whole-cell chloride currents recorded from +60 to –120 mV in 10 mV increments. A 5 s interval wasallowed between sweeps. B, normalized current–voltage relationship. Current recorded at –120 mV was usedto normalize each curve and then the normalized curves were averaged (n = 12). C, instantaneouscurrent–voltage relationships determined from cells bathed in solutions with pH of 7.4 (n = 6). A prepulse to–100 mV was used to open the channels. Tail currents were generated by repolarizing the membrane to theindicated voltages and its magnitudes were normalized to that obtained at +60 mV. The continuous line is alinear regression. D, reversal potential shifts obtained from tail current experiments are plotted as a functionof [Cl_]o. The filled squares are experimental data and the continuous line reflects expected values for achloride-selective channel. Experimental data were fitted by the Nernst equation with a slope of –45 mV.

The open channel current–voltage relationship was approx-

imately linear. Tail currents were sampled immediately

after a 3 s hyperpolarizing pulse to –100 mV. Instantaneous

current–voltage relationships were constructed by plotting

the initial magnitude of the tail currents against the

repolarizing voltage and were approximately linear

(Fig. 1C).

We measured the current reversal potential in solutions in

which Cl_ was replaced by the ‘impermeant’ anion

glutamate. The shifts of the reversal potentials induced

by the respective changes in extracellular chloride

concentrations are plotted in Fig. 1D. The continuous line

is the predicted slope for a chloride-selective channel

according to the Nernst equation (eqn (1)), while the

squares represent the experimental determinations. Reversal

potential shifts exhibited a slope of –45 mV/decade of [Cl_]

(dashed line), close to the predicted slope (_58 mV/decade),

and consistent with a channel that is predominantly

permeable to Cl_ ions.

The voltage dependence of channel activation was estimated

by calculating the normalized whole-cell conductance as

described in Methods (eqn (2)). Figure 2A shows the

resulting macroscopic normalized whole-cell conductance,

an index of the open probability, as a function of the

membrane potential. Clir started to open around –20 to

–30 mV, and reached half-maximal activation at –85 mV

with a slope factor of 18 mV as deduced from the

Boltzmann fit (eqn (3); continuous line). The results of

previous studies showed that the kinetics of Clir activation

by hyperpolarization have two time constants (Ferroni

et al. 1997; Cid et al. 2000). In agreement, a single

exponential function did not adequately fit our data. At

least two exponential components were needed (data not

shown). The resulting values of the fast (t1, •) and slow

(t2, 8) time constants are plotted in Fig. 2B. The fast and

slow time constants were around 100–200 and 1000 ms,

respectively, at the most negative voltages. The relative

contributions of the fast and slow components to the total

currents at –100 mV were 0.34 ± 0.03 and 0.66 ± 0.03

(n = 8), respectively. A modest voltage sensitivity was

observed at less negative potentials.

Effects of elevated external pH on Clir

As described in the Introduction, cloned ClC-2 and some

native inwardly rectifying Cl_ channels are modulated by

external pH. We found that the inwardly rectifying currents

in mouse parotid acinar cells were reduced by elevations in

external pH with little or no change in gating kinetics.

Figure 3A shows whole-cell chloride currents recorded from

a cell exposed to a bath solution of pH 7.4 (left-hand traces)

and then superfused with a pH 8.0 solution (right-hand

traces). The instantaneous current–voltage relationship

(not shown) remained linear as for pH 7.4 (Fig. 1C). The

reduction in current at pH 8.0 appeared to be a result of a

shift of the conductance–voltage relationship along the

voltage axis as illustrated in Fig. 3B. Figure 3B shows that

increasing the pHo to 8 produced a negative shift of the

channel activation of approximately 28 mV, with no

apparent change in the voltage sensitivity.

J. Arreola, T. Begenisich and J. E. Melvin106 J. Physiol. 541.1

Figure 2. Macroscopic open probability and kinetics of ClirA, the open probability as a function of Vm was estimated by calculating the macroscopic conductance usingeqn (2) and normalized to the estimated gmax (see Methods). V0.5 and s parameters were determined by fittingthe average normalized conductance curve with the Boltzmann function (eqn (3); n = 12). B, kinetics of Clir.Traces at each membrane potential were fitted with a two-exponential function to estimate both fast(•, n = 9) and slow (8, n = 9) time constants.

Effects of reduced external pH on Clir

In contrast to the activation shift of high pH solutions, the

actions of low pH on the inwardly rectifying Cl_ channels

were rather more complex. As illustrated in Fig. 4A,

modest reduction in pH, e.g. to 6.5, increased current with

little kinetic change; pH 6.0 solutions significantly slowed

channel gating with little change in magnitude, and very

little current was seen in pH 5.5 solutions. We measured

the two time constants for channel activation (e.g. Fig. 2B)

and found no significant change in solutions of pH

between 6.5 and 8.0. In contrast, decreasing the pH to 6.0

slowed fast and slow time constants from 0.21 ± 0.01 to

2.48 ± 1.07 s (n = 6) and from 1.49 ± 0.15 to 7.74 s

(n = 8), respectively. At low pHo the kinetics became so

slow that even with pulses of 25 s it was not always possible

to measure the current amplitude in steady state.

We measured the amplitude of the current at the end of a

pulse to –100 mV in solutions of varying pH. Figure 4Bdemonstrates that the current amplitude (normalized to

the value at pH 7.4) had a bimodal dependence on the

external proton concentration with maximum activity

near pH 6.5. Reductions in the current amplitude by low

pH solutions were much more sensitive to proton

concentrations than were changes by high pH solutions.

Actions of pH on Clir: a simple modelIn order to begin a quantitative analysis of these data, we

consider a simple, generic model in which the channel

contains two independent H+ binding sites (S1 and S2):

k1

S1 + n1H ™ S1Hn1

k_1

k2

and S2 + n2H ™ S2Hn2,

k_2

where n1 and n2 represent, in these simple schemes, the

number of H+ that interact with the two sites, and k1, k_1, k2

and k_2 are the protonation rate constants. We associate

the actions of high pH with deprotonation of site S1 and the

actions of low pH with protonation of S2. Thus the

bimodal relationship of channel current with pH would be

consistent with protonation of site S1 and no protonation

of S2. Therefore, the current at any pH would be proportional

to:

(1/(1 + (K1/[H])n1)) w (1/(1 + ([H]/K2)n2)), (4)

where [H] is the external proton concentration and K1

(k_1/k1) and K2 (k_2/k2) are the dissociation constants of

protonation.

The continuous line in Fig. 4B is the best fit of eqn (4) to

the data with values of n1 and n2 of 0.61 and 2.2, respectively,

K1 = 5.4 w 10_8M (pK1 = 7.3) and K2 = 9.6 w 10_7

M (pK2 =

6.0). The simplest interpretation of this analysis is that

channels are ‘activated’ by the binding of a single H+ to a

site with an apparent pK of 7.3 and ‘inhibited’ by the

binding of 2 H+ to a second site with an apparent pK of 6.0.

The requirement for 2 H+ binding to the second site

accounts for the increased steepness of the pH sensitivity at

low pH values.

State-dependent effects of external pHWe investigated a possible state-dependent action of

external pH by applying solutions of different pH at

voltages at which the channels were either mostly closed or

mostly open. We used a double pulse protocol shown in

Fig. 5A. The initial –100 mV pulse lasted 50 s, sufficient

time to examine the effects of raising or lowering the

external pH on channel activity in the open conformation

state. We included a second hyperpolarizing pulse

preceded by a 5 s depolarization to +60 mV to evaluate the

inhibition of the channel after spending 5 s in the closed

state. Figure 5B shows a control trace where the extra-

cellular pH was held constant at 7.4; the channels

Bimodal pH dependence of the ClC-2 chloride channelJ. Physiol. 541.1 107

Figure 3. Shift in Clir activation induced by changes in pHo

A, cells were bathed in solutions with the pH adjusted to theindicated values. Control currents (pHo = 7.4) are shown in theleft-hand panel and test currents are shown in the right-hand panel(pHo = 8.0). Membrane potential was changed from –120to +40 mV in 20 mV steps. B, voltage dependency of channelactivation at pH 7.4 (0, n = 5) and 8.0 (8, n = 5). The analysis ofthese curves obtained from paired experiments was as described inFig. 2B. The resulting average curves were fitted with theBoltzmann function to obtain the parameters V0.5 and s displayedin the inset.

remained open during the entire 50 s hyperpolarizing

pulse to –100 mV, closed when depolarized to +60 mV,

and then re-opened normally during the second test

pulse to –100 mV. In the following sweep (Fig. 5C),

approximately 10 s after the channels were in the open

state at –100 mV (the final current is independent of the

previous channel state), the pHo was changed from 7.4 to

5.5. Surprisingly, the channel current amplitude was not

decreased by this low pH (see pH 5.5 trace in Fig. 4 for

comparison). In fact, we consistently observed a relatively

fast increase in the current upon decreasing the bath pH,

followed by a much slower reduction. After closing the

channels in the pH 5.5 solution at +60 mV, reapplication

of the –100 mV pulse did not result in the reactivation of

current, as if low pH ‘locked’ the channels in the closed

conformation. Current recorded during the second

hyperpolarization pulse was reduced by 85.6 ± 3.3 %

(n = 6) compared with the current during the initial

hyperpolarization at pH 7.4. Current inhibition at pHo 5.5

was readily reversed by returning the pHo to 7.4 (Fig. 5D).

We used the same protocol illustrated in Fig. 5A to test for

a possible state dependence of the actions of elevated pH

on Clir channels. Applying pH 8.0 at –100 mV (when the

channels are open) produced the expected reduction in

current magnitude (Fig. 6B; 47.6 ± 3.2 %, n = 3). In the

continued presence of pH 8.0, reapplication of the

–100 mV activating pulse after closing the channels at

+60 mV reactivated the current to the same level. The

reduction of current at pH 8.0 reversed after returning the

cell to pH 7.4 (Fig. 6C).

Thus it appears that the strong inhibitory effects of low pH

were state dependent and occurred only if the channels

J. Arreola, T. Begenisich and J. E. Melvin108 J. Physiol. 541.1

Figure 4. Bimodal regulation of Clir by pHo at –100 mVA, recordings obtained from the same cell. External solutions ofindicated pH were continuously perfused throughout theexperiment. B, normalized current versus [H+]o relationship. Datalike those shown in A obtained at –100 mV were normalized to theabsolute current value (measured at the end of the pulse) obtainedat pH 7.4. The values plotted at pH 6.0 and 5.5 were obtained using20 or 50 s pulses. Number of observations are indicated inparenthesis. Fitting of data with eqn (4) is shown as continuousline. Parameters obtained from the fit are given in the text.

Figure 5. State-dependent inhibition of Clir by pHo 5.5A, pulse protocol used to assess the effects of external H+ on openchannels (first 50 s hyperpolarization) and after closing (second 4 shyperpolarization). Two consecutive hyperpolarizations to–100 mV separated by a depolarization to +60 mV were used asstimuli. The first and longest hyperpolarization allowed theexchange of the bath solution while the channel was open. Thesecond and shortest hyperpolarization evaluated the fraction ofchannels inhibited after a 5 s pulse to +60 mV that closes thechannels. Closed to open confirmation transitions are indicatedbelow the pulse protocol. Bath volume was about 0.2 ml, flow ratewas about 4 ml_1, resulting in a typical time for solution exchangeof 10–15 s. B, C and D depict traces obtained from the same cell.B shows a control record obtained at pH 7.4 with the protocolshown in A. C shows the effects of changing the bath pH from 7.4to 5.5. D shows the reversibility of the inhibition by changing bathpH from 5.5 to 7.4.

were simultaneously closed and exposed to the low pH

solution. In contrast, high pH solutions decreased channel

current independently of the conformational state.

pH-dependent actions of divalent cationsDivalent cations block heterologouosly expressed ClC-1 in

a pH-dependent manner (Rychokov et al. 1997; Kürz et al.1999). We tested for this effect on mouse Clir channels. The

application of Zn2+ (Fig. 7A) and Cd2+ (Fig. 7B) reversibly

inhibited the inward chloride currents from mouse

parotid acinar cells at pH 7.4. At –100 mV, 72.1 ± 3.5

and 87.4 ± 1.9 % of the control current was inhibited by

50 mM Zn2+ (n = 5) and 500 mM Cd2+ (n = 4), respectively,

comparable to results previously documented in other

preparations (Chesnoy-Marchais & Fritsch, 1994; Fritsch

& Edelman, 1996; Ferroni et al. 1997; Clark et al. 1998).

Taking advantage of our finding that pHo 5.5 does not

inhibit the current through open channels, we next

evaluated inhibition by Zn2+ and Cd2+ at pH 7.4 and 5.5.

We used long hyperpolarizing pulses and exposed the cells

to Zn2+ or Cd2+ after the channels reached the open state.

Figure 7C and D shows whole-cell current traces recorded

from two cells bathed in a solution of pH 7.4, and exposed

to 500 mM Zn2+ and 500 mM Cd2+, respectively. A relatively

rapid inhibition of current (comparable to that in Fig. 7Aand B; 61 ± 4.8 % with Zn2+ and 84 ± 1.8 % with Cd2+) was

observed after applying the divalent cations. Figure 7E and

F shows that these same concentrations of divalent cations

had no significant effect on the current when applied in a

pH 5.5 solution. That is, the simultaneous application of

low pH and divalent cations affected the current no

differently than application of only low pH (see Fig. 5C).

DISCUSSION Targeted disruption of the Clcn2 gene has recently verified

that the inwardly rectifying Cl_ current in mouse parotid

acinar cells is associated with ClC-2 expression (K. Nehrke,

J. Arreola, H.-V. Nguyen, J. Pilato, L. Richardson, G.

Okunade, G. Shull, R. Baggs & J. E. Melvin, unpublished

observations). Thus the hyperpolarization-activated Cl_

current in this cell type is almost certainly mediated by the

ClC-2 Cl_ channel protein, providing an opportunity to

characterize this channel in its native environment. Both

native and cloned inwardly rectifying chloride channels

are extremely sensitive to [H+]o (Jordt & Jentsch, 1997;

Kajita & Brown, 1997; Ferroni et al. 2000; Tarran et al.2000). We found that mouse parotid Clir channels were

inhibited by both high and low extracellular pH solutions,

demonstrating a novel bimodal mechanism of regulation

by external pH. The simplest interpretation of our data

suggests the existence of two separate mechanisms for

regulating the pH dependency of ClC-2 channel gating. In

contrast, Kajita & Brown (1997) found that the pH

dependence of ClC-2-like currents from rat choroid

plexus cells was monotonic, with inhibition of activity

observed at low extracellular pH. They suggested that a

single proton acting at an extracellular site blocked the rat

choroid plexus channel. The reason for this difference is

not clear, but may be due to different gene products, as

suggested by the relatively rapid activation kinetics and

cAMP dependence of the chloride channels in the choroid

plexus.

The inhibition of current magnitude by high pHo can be

explained by a shift in the voltage dependency of channel

gating. This effect is consistent with changes in membrane

surface potential resulting from protonation of a site with

pK near 7.3. This type of mechanism has been proposed

for the cloned rat ClC-2 channel (Jordt & Jentsch, 1997)

and the ClC-2-like currents in cultured rat cortical

astrocytes (Ferroni et al. 2000). Alternatively, OH_ could

conceivably block the channels by tight binding, as has

been suggested for Ca2+-dependent Cl_ channels (Qu &

Hartzell, 2000). A voltage-dependent k_1/k1 ratio in scheme 1

resulting from either mechanism will be sufficient to

explain the negative shift in channel activation.

Bimodal pH dependence of the ClC-2 chloride channelJ. Physiol. 541.1 109

Figure 6. State-independent inhibition by pHo 8.0 at–100 mVThe protocol was as described in Fig. 5A. A, B and C show rawtraces obtained from the same cell. A, control trace obtained fromcell bathed in a solution with pH 7.4. B, current recorded whilechanging the bath solution pH from 7.4 to 8.0. C, current recordedwhile changing the bath solution pH from 8.0 to 7.4.

The actions of low pH on the mouse Clir channels were

more complex than a shift of channel activation. We found

that a pH level of 6.5 increased current amplitude

(compared with pH 7.4) and levels below 6.5 decreased the

current and substantially slowed channel kinetics. The

inhibition of current by low pH required that the channels

be in a closed conformation. A detailed mechanistic

analysis of these effects is beyond the scope of this

investigation. However, we suggest that two protons may

bind to a site with a pK near 6.0 and stabilize a closed

conformation of the channel. That is, when the channel is

closed and the H+ binding site is protonated, hyper-

polarization may be unable to move a mobile part of the

channel because of the additional positive charge, thus

resulting in immobilization of the gating machinery. This

mechanism is consistent with the slowing produced by low

pH solutions as well as the steep dependence of current

magnitude on pH.

We found that divalent cations Zn2+ and Cd2+ decreased

Clir channel currents when they are applied at pH 7.4,

consistent with previous observations (Chesnoy-Marchais

& Fritsch, 1994; Fritsch & Edelman, 1996; Ferroni et al.1997; Clark et al. 1998). Moreover, low pHo drastically

reduced the ability of these divalent cations to inhibit Clir

channel currents. This is quite similar to the phenomenon

observed for the ClC-1 chloride channel, where important

cysteine residues have been identified (Kürz et al. 1999),

J. Arreola, T. Begenisich and J. E. Melvin110 J. Physiol. 541.1

Figure 7. Low pHo interferes with Clir inhibition by Zn2+ and Cd2+

A and B, inhibition of Clir current by 0.05 mM Zn2+ and by 0.5 mM Cd2+, respectively, at –100 mV. Divalentcations were applied in a solution of pH 7.4 while the cell was held at 0 mV. C and D, inhibition of Clir currentby 0.5 mM Zn2+ (n = 5) or 0.5 mM Cd2+ (n = 3), respectively, at pH 7.4 and –100 mV. E and F, lack ofinhibition by 0.5 mM Zn2+ (n = 5) or 0.5 mM Cd2+ (n = 3), respectively, at pH 5.5 and –100 mV. The bathsolution pH in A, B, C and D was 7.4 throughout the experiments. Rectangles in E and F indicate the exchangeof the standard pH 7.4 bath solution containing no blocker with a pH 5.5 bath solution containing 0.5 mM ofZn2+ or Cd2+. The arrows in C, D, E and F indicate when the blockers were present.

suggesting that a comparable mechanism may be responsible

for this behaviour in ClC-2.

In summary, the inward rectifier ClC-2 Cl_ channels in

mouse parotid acinar cells were regulated by [H+]o in a

bimodal fashion. This regulation may have a physiological

consequence. Salivary gland acinar cells are exposed to

dramatic changes in their pH environment in response to

secretion-inducing agonists (Melvin et al. 1988; Okada etal. 1991). When acinar cells are stimulated, bicarbonate

and H+ efflux occurs across the apical and basolateral

membranes, respectively (Melvin et al. 1988). If ClC-2 is

located in the basolateral membrane of this cell, then low

pHo in the range needed for this form of inhibition might

occur when Na+–H+ exchange is upregulated during

stimulated secretion (Melvin et al. 1988; Evans et al. 1999).

Na+–H+ exchanger activity is enhanced several fold by

muscarinic stimulation. The extent of the resulting

extracellular acidification is unknown, but is probably

quite large near the plasma membrane. The secretion of

bicarbonate across the apical membrane of acinar cells is

likely to produce an alkaline pH near the external apical

surface, and if the ClC-2 channel is targeted to this

membrane, the resulting alkaline pH would produce

negative feedback on channel gating.

REFERENCESARREOLA, J., PARK, K., MELVIN, J. E. & BEGENISICH, T. (1996). Three

distinct chloride channels control anion movements in rat parotid

acinar cells. Journal of Physiology 490, 351–362.

BENNETS, B., ROBERTS, M. L., BRETAG, A. H. & RYCHKOV, G. Y. (2001).

Temperature dependence of human muscle ClC-1 chloride

channel. Journal of Physiology 535, 83–93.

BÖSL, M. R., STEIN, V., HUBNER, C., ZDEBIK, A. A., JORDT, S. E.,

MUKHOPADHYAY, A. K., DAVIDOFF, M. S., HOLSTEIN, A. F. &

JENTSCH, T. J. (2001). Male germ cells and photoreceptors, both

dependent on close cell-cell interactions, degenerate upon ClC-2

Cl_ channel disruption. EMBO Journal 20, 1289–1299.

CAREW, M. A. & THORN, P. (1996). Identification of ClC-2-like

chloride currents in pig pancreatic acinar cells. Pflügers Archiv 433,

84–90.

CHEN, T.-Y. & MILLER, C. (1996). Non-equilibrium gating and

voltage dependence of the ClC-0 Cl_ channel. Journal of GeneralPhysiology 108, 237–250.

CHESNOY-MARCHAIS, D. (1983). Characterization of a chloride

conductance activated by hyperpolarization in Aplysia neurones.

Journal of Physiology 342, 277–308.

CHESNOY-MARCHAIS, D. & FRITSCH, J. (1994). Activation by

hyperpolarization and atypical osmosensitivity of a Cl_ current in

rat osteoblastic cells. Journal of Membrane Biology 140, 173–188.

CID, L. P., NIEMEYER, M. I., RAMIREZ, A. & SEPULVEDA, F. V. (2000).

Splice variants of a ClC-2 chloride channel with differing

functional characteristics. American Journal of Physiology 279,

C1198–1210.

CLARK, S., JORDT, S. E., JENTSCH, T. J. & MATHIE, A. (1998).

Characterization of the hyperpolarization-activated chloride

current in dissociated rat sympathetic neurons. Journal ofPhysiology 506, 665–678.

EVANS, R. L., BELL, S. M., SCHULTHEIS, P. J., SHULL, G. E. &

MELVIN, J. E. (1999). Targeted disruption of the Nhe1 gene

prevents muscarinic-induced upregulation of Na+/H+ exchange in

mouse parotid acinar cells. Journal of Biological Chemistry 274,

29025–29030.

FAVA, M., FERRONI, S. & NOBILE, M. (2001). Osmosensitivity of an

inwardly rectifying chloride current revealed by whole-cell and

perforated-patch recordings in cultured rat cortical astrocytes.

FEBS Letters 492, 78–83.

FERRONI, S., MARCHINI, C., NOBILE, M. & RAPISARDA, C. (1997).

Characterization of an inwardly rectifying chloride conductance

expressed by cultured rat cortical astrocytes. Glia 21, 217–227.

FERRONI, S., NOBILE, M., CAPRINI, M. & RAPISARDA, C. (2000). pH

modulation of an inward rectifier chloride current in cultured rat

cortical astrocytes. Neuroscience 100, 431–438.

FRITSCH, J. & EDELMAN, A. (1996). Modulation of the

hyperpolarization-activated Cl_ current in human intestinal T84

epithelial cells by phosphorylation. Journal of Physiology 490,

115–128.

HAMILL, O. P., MARTY, A., NEHER, E., SAKMAN, B. & SIGWORTH, F.

(1981). Improved patch-clamp techniques for high-resolution

current recording from cells and cell-free membrane patches.

Pflügers Archiv 391, 85–100.

HANKE, W. & MILLER, C. (1983). Single chloride channels from

Torpedo electroplax. Activation by protons. Journal of GeneralPhysiology 82, 25–45.

IWATSUKI, N., MARUYAMA, Y., MATSUMOTO, O. & NISHIYAMA, A.

(1985). Activation of Ca2+-dependent Cl_ and K+ conductances in

rat and mouse parotid acinar cells. Japanese Journal of Physiology35, 933–944.

JORDT, S. E. & JENTSCH, T. J. (1997). Molecular dissection of gating in

the ClC-2 chloride channel. EMBO Journal 16, 1582–1592.

KAJITA, H. & BROWN, P. D. (1997). Inhibition of the inward-

rectifying Cl_ channel in rat choroids plexus by a decrease in

extracellular pH. Journal of Physiology 498, 703–707.

KOMWATANA, P., DINUDOM, A., YOUNG, J. A. & COOK, D. I. (1994).

Characterization of the Cl_ conductance in the granular duct cells

of mouse mandibular glands. Pflügers Archiv 428, 641–647.

KÜRZ, L. L., KLINK, H., JAKOB, I., KUCHENBECKER, M., BENZ, S.,

LEHMANN-HORN, F. & RUDEL, R. (1999). Identification of three

cysteines as targets for the Zn2+ blockade of the human skeletal

muscle chloride channel. Journal of Biological Chemistry 274,

11687–11692.

MELVIN, J. E., MORAN, A. & TURNER, R. J. (1988). The role of HCO3_

and Na+/H+ exchange in the response of rat parotid acinar cells to

muscarinic stimulation. Journal of Biological Chemistry 263,

19564–19569.

MINDELL, J. A., MADUKE, M., MILLER, C. & GRIGORIEFF, N. (2001).

Projection structure of a ClC-type chloride channel at 6.5 Å

resolution. Nature 409, 219–224.

MOHAMMAD-PANAH, R., GYOMOREY, K., ROMMENS, J.,

CHOUDHURY, M., LI, C., WANG, Y. & BEAR, C. E. (2001). ClC-2

contributes to native chloride secretion by a human intestinal cell

line, Caco-2. Journal of Biological Chemistry 276, 8306–8313.

OKADA, M., SAITO, Y., SAWADA, E. & NISHIYAMA, A. (1991).

Microfluorimetric imaging study of the mechanism of activation

of the Na+/H+ antiport by muscarinic agonist in rat mandibular

acinar cells. Pflügers Archiv 419, 338–348.

PARK, K., ARREOLA, J., BEGENISICH, T. & MELVIN, J. E. (1998).

Functional properties of a voltage-activated chloride channel

expressed in a mammalian cell line. Journal of Membrane Biology163, 87–95.

Bimodal pH dependence of the ClC-2 chloride channelJ. Physiol. 541.1 111

PUSCH, M., LUDEWIG, U. & JENTSCH, T. J. (1997). Temperature

dependence of fast and slow gating relaxations of ClC-0 chloride

channels. Journal of General Physiology 109, 105–116.

QU, Z. & HARTZELL, H. C. (2000). Anion permeation in Ca2+-

activated Cl_ channels. Journal of General Physiology 116, 825–844.

RYCHKOV, G. Y., ASTILL, D. ST J., BENNETTS, B., HUGHES, B. P.,

BRETAG, A. H. & ROBERTS, M. L. (1997). pH-dependent

interactions of Cd2+ and a carboxylate blocker with the rat ClC-1

chloride channel and its R304E mutant in the Sf-9 insect cell line.

Journal of Physiology 501, 335–362.

TARRAN, R., ARGENT, B. E. & GRAY, M. A. (2000). Regulation of a

hyperpolarization-activated chloride current in murine

respiratory ciliated cells. Journal of Physiology 524, 353–364.

THIEMAN, A., GRUNDER, S., PUSCH, M. & JENTSCH, T. J. (1992).

A chloride channel widely expressed in epithelial and non-

epithelial cells. Nature 356, 57–60.

AcknowledgementsWe thank Dr Patricia Perez-Cornejo for critical reading of thismanuscript and Jodi Pilato for excellent technical assistance. Thiswork was supported in part by NIH grants DE09692 and DE13539(J. E. M.).

J. Arreola, T. Begenisich and J. E. Melvin112 J. Physiol. 541.1