molecular pharmacologymolpharm.aspetjournals.org/content/molpharm/early/2016/0... · this article...

MOL#104158

Channels and volume changes in the life and death of the cell

Herminia Pasantes-Morales

División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de

México, Mexico City, Mexico

This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158

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Running title: channels and cell volume

Prof. Herminia Pasantes-Morales

Instituto de Fisiología Celular

Av. Universidad 3000, Ciudad Universitaria, Mexico City, Mexico

Tel. 0155 5622 5608

0155 5622 5588

Fax. 0155 5622 5607

e-mail: [email protected]

Text pages:

Table number: 0

Figure number: 5

Abstract words: 196

Text words: 7513

Reference number: 146

Abbreviations: AMPA, α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate; DCPIB, 4-

[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid; DIDS,

4,4B-diisothiocyanostilbene-2,2B-disulfonic acid; NKCC, Na+/K+/2Cl co-transporter; NMDA, N-

Methyl-D-aspartic acid; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; OREBP, osmotic

Response Element-binding Protein; KCC, K+/Cl− co-transporter; Kv, voltage-gated K+ channels; KCa,

Ca2+

-activated K+ channels; KIR, inwardly rectifying K

+ channels; K2P, two-pore domain K

+ channels;

SITS, 4-acetamido-4'-isothiocyanatostilbene-2, 2'-disulfonic acid; SPAK, STE20/SPS1-related

proline/alanine-rich kinase kinases; SUR1, sulfonylurea receptor 1; TBOA, DL-threo-β-

benzyloxyaspartic-acid; TNF, tumor necrosis factor; TonEBP, tonicity-responsive enhancer binding

protein; TRPC, transient receptor potential channels; TRPM4, transient receptor potential

melastatin-4 channel; VSOAC, volume-sensitive organic osmolyte and anion channel; VRAC,

volume-regulated anion channel.


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Address correspondence to: Herminia Pasantes-Morales, División de Neurociencias, Instituto de

Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico.

[email protected]

ABSTRACT

Volume changes deviating from original cell volume represent a major challenge for homeostasis.

Cell volume may be altered either by variations in the external osmolarity or by disturbances in the

transmembrane ion gradients that generate an osmotic imbalance. Cells respond to anisotonicity-

induced volume changes by active regulatory mechanisms that modify the

intracellular/extracellular concentrations of K+, Cl

-, Na⁺ and organic osmolytes, in the necessary

direction to reestablish the osmotic equilibrium. Corrective osmolyte fluxes permeate across

channels that have a relevant role in cell volume regulation and participate as causal actors in

necrotic swelling and apoptotic volume decrease. This review provides an overview of the types

of channels involved in either corrective or pathological changes in cell volume. The review also

underlines the contribution of TRP channels, notably TRPV4, in volume regulation after swelling,

and describes the role of TRPs in volume changes linked to apoptosis and necrosis. Lastly we

discuss findings showing that multimers derived from LRRC8A (leucine-rich repeat containing 8A)

gene are structural components of the volume-regulated Cl- channel (VRAC)and underlines the

intriguing possibility that different heteromer combinations comprise channels with different

intrinsic properties as to permeate the heterogenous group of molecules acting as organic

osmolytes.


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Introduction

Cell volume is characteristic of each cellular lineage and is maintained with few variations through

the cell life. Conditions inducing changes in cell volume represent a major challenge for the cell

homeostasis, and this may even culminate in the cell death. In order to keep the cell volume

constant, water fluxes should be in equilibrium between the intracellular and extracellular

compartments. This occurs under conditions of isotonicity, but any fluctuations in the osmolarity

of the extracellular milieu or in the distribution of osmotically active solutes drives water fluxes in

the necessary direction to reach a new equilibrium, which disturbs cell volume. Except for cells in

the kidney and intestine, which normally face large variations in external osmolarity, the

extracellular milieu maintains a well-controlled osmolarity. This is modified only under

pathological conditions leading to hyponatremia or in other pathologies that alter the distribution

of osmolytes in the extra-intracellular compartments (Song and Yu, 2014; Pedersen et al., 2016).

The intracellular osmolarity also endures continuous variations due to transient and local osmotic

microgradients generated during physiological processes, such as uptake and release of

metabolites, cytoskeletal remodeling, synthesis and degradation of macromolecules, and

exocytosis and secretion (Pedersen et al., 2001; Strbák, 2011; Platonova et al., 2015), but even

these localized changes in cell volume should be regulated to prevent changes in the

concentration of molecules free in the cytosol, protein misfolding, or alterations in cell and

organelle architecture. Cell volume modifies during migration, proliferation and cell adhesion, and


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therefore, volume change has been proposed as a signaling event for these processes (Lang et al.,

2005; Stutzin and Hoffmann, 2006; Dubois and Rouzaire-Dubois, 2012).

When volume is perturbed due to changes in the extracellular osmolarity, cells respond by

activation of a plethora of signaling pathways and mechanisms intended to protect them from the

challenge of volume change; remodeling of the cytoskeleton, changes in adhesion molecules,

activation of stress and survival signals are among the adaptive mechanisms triggered by the

altered volume (Pasantes-Morales et al., 2006; Pedersen and Hoffmann, 2009). At the same time,

cells set in motion an active regulatory process and tend to partially or fully restore the original

volume (Cala, 1977; Olson et al., 1986; Hoffmann, 1992; Okada, 2004). This adaptive mechanism,

which has been highly preserved during evolution, essentially consists of the redistribution of

osmotically active solutes in the necessary direction to equilibrate water fluxes facing the new

osmotic condition (Lang, 2007) (see Hoffmann et al., 2009 for a comprehensive review on cell

volume regulation). Osmolytes that accomplish cell volume regulation are the ions present in high

concentrations in the intracellular or extracellular compartments, such as Na+, K

+ and Cl

-, and a

group of heterogeneous organic molecules, such as amino acids, polyamines and polyalcohols

(Burg and Ferraris, 2008; Verbalis and Gullans, 1991; Pasantes-Morales et al., 1998). Volume-

regulated channels and cotransporters translocate ion osmolytes, and cotransporters and pores,

although not yet fully identified, mobilize organic osmolytes; channels participate predominantly

in the volume regulatory process activated by cell swelling, while cotransporters play a more

important role in the cell response to shrinkage (Khale et al., 2015). Besides their role in volume

regulation, channels are involved in the pathophysiological cell volume changes generated in

isotonicity that lead to necrotic death, as well as in those associated with apoptosis (Okada et al.,

2006; Bortner and Cidlowski, 1998; 2007). This review focus on i) channels permeating ions and


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organic osmolytes during the regulatory volume decrease, ii) ion channels participating in the

volume regulatory increase, iii) channels implicated in normotonic cell volume changes leading to

apoptotic and necrotic cell death.

The Role of Channels in Regulatory Volume Decrease

A decrease in external osmolarity modifies the water concentration in the extracellular space,

which activates water inward-directed fluxes and increases the cell volume with a magnitude

proportional to the extent of the osmolarity change. As mentioned above, most animal cells

respond to this situation with an active process of volume recovery, known as regulatory volume

decrease (Cala, 1977), which is accomplished by extrusion of the major intracellular ions, K+ and

Cl-, and organic osmolytes. Most of the corrective fluxes occur via K

+ and Cl

- channels and diffusion

pathways for the organic osmolytes (Okada and Maeno, 2001; Hoffmann and Lambert, 1983;

Sánchez-Olea et al., 1991; Hoffmann et al., 2009)(Fig. 1).

K+

channels. K+ is a major intracellular osmolyte and contributes substantially to RVD. The

osmosensitive K+ fluxes permeate across a large variety of K

+ channels, and are involved in

multiple physiological processes in the cell other than volume regulation, but are triggered via

signals evoked by swelling. Four main classes of K+ channels are activated during RVD: voltage-

gated K+ (Kv) channels, Ca

2+-activated K

+ (KCa) channels, inwardly rectifying K

+ (KIR) channels, and

two-pore domain K+ (K2P) channels (Fig. 1). The kind of channel involved differs in the various cell

types and responds to one or several of the signals elicited by the volume change, such as

depolarization, membrane stretch, and elevation of intracellular Ca2+

[Ca2+

]i. In epithelial and

other cell types, swelling evokes a large increase in [Ca2+

]I, and RVD in these cells is consistently

Ca2+

-dependent due to the predominant role of KCa channels in the volume-corrective K+ fluxes.


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The high- and intermediate conductance K+ channels (BKCa, IKCa) are the channels mainly

implicated in RVD. BK channels are activated by swelling in the intestine, kidney and bronchial

epithelia, chondrocytes, pituitary tumor cells, and osteoblast-like cells (rev. in Hoffmann et al.,

2009). The activation of BK channels occurs by Ca2+

influx mediated by the TRPV4 subtype,

establishing a link between BKCa channels and some transient receptor potential (TRP) channels

(discussed below) (Jin et al., 2012). Intermediate-conductance (IKCa) channels participate in RVD in

T lymphocytes, erythrocytes, hepatocytes, osteosarcoma cells, intestine 407 cells, proximal tubule

cells, and lens epithelia (Rev. in Hoffmann et al., 2009). In other cells, such as brain cells, following

hypotonic swelling, depolarization generated by the Cl- efflux across the volume-regulated Cl

-

channel occurs, and in these cells, RVD is essentially Ca2+

independent (Pasantes-Morales et al.,

1994; Moran et al., 1997) and the K+ regulatory fluxes permeate across voltage-gated K

+ channels.

Subtypes of Kv involved in the different cell types include Kv1.3 in lymphocytes and murine

spermatozoa, Kv1.4.2 and Kv1.4.3 in myocytes, and Kv1.5 in lymphocytes and spermatoza. KCNQ1

(Kv7.1), in various subunit arrangements, participates in RVD in human mammary epithelial cells,

rat hepatocytes, cells from the inner ear and in mouse trachea epithelium (Rev. in Hoffmann et al.,

2009). The inwardly rectifying K+ channels Kir4.1 and Kir4.5, which sense small changes in

osmolarity (Grunnet et al., 2003; Soe et al., 2009), co-localize with AQP4 in the glial endfeet of the

blood-brain barrier, suggesting that they play a role in swelling and/or volume control in these

cells. Kir channels regulate extracellular K+ homeostasis during the K

+ spatial buffering through the

glial syncytium, and this transcellular K+ transport is accompanied by transmembrane water fluxes,

likely via a Kir4.1/AQP4 complex (Nagelhus et al. 1999; Dibaj et al., 2007). The two-pore domain K⁺

channels (K2P) participate in RVD in lymphocytes, Ehrlich ascites cells, astrocytes, and kidney cells

(Andronic et al., 2013; Niemeyer et al., 2001; Kirkegaard et al., 2010).


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The variety of K⁺ channels involved in RVD in different cell types that are triggered by stimuli

concurrent with cell swelling raises the question about the existence of a purely swelling-gated K⁺

channel. A recent study, where AQP1 and a number of K⁺ channel types were co-expressed in

Xenopus oocytes (Tejada et al., 2014), identified the Kca 4.1 channel (Slick; Slo2.1) as the most

sensitive to volume changes. Slick currents increased markedly in hypotonicity and decreased in

hypertonicity, and these changes were not observed in the absence of AQP1, indicating that

channel activity follows the changes in cell volume and not in osmolarity. Thus, slick may be a

purely volume-sensitive K⁺ channel. Although Kca 4.1 was originally included in the Kca

nomenclature, it is now known that it is activated by internal Na⁺ and Cl- and not by Ca

2+.

The volume-regulated Cl-

channel. Volume-sensitive Cl-

fluxes play a key role in RVD; in

contrast to the diversity of K⁺ channels implicated in RVD in various cell types, Cl- fluxes permeate

across only one type of channel, the volume-regulated anion channel (VRAC), also named VSOR

(volume-sensitive outwardly rectifying anion channel). The channel is also referred to as VSOAC

(volume -sensitive organic osmolyte and anion channel) to denote that possibility that it

permeates organic osmolytes, but we will use VRAC throughout this review. VRAC, widely

expressed in animal cells, is essentially inactive under isotonic conditions and opens slowly by cell

swelling or by a decrease in ionic strength under isosmotic conditions (Emma et al., 1997; Voets et

al., 1999; Pedersen et al., 2016). In biophysical terms, the Cl- current (ICl

-swell) carried by VRAC

exhibits mild outwardly rectification and voltage inactivation at positive membrane potentials, and

this differs among cell types (Nilius et al., 1994; Nilius and Droogmans, 2001; Okada, 2004; Akita

and Okada, 2014). This outward rectification defines VRAC, and establishes clear differences from

other volume-sensitive Cl- channels, such as channels with no rectification (maxi-anion channel),

inward rectification (CLC2), steep outward rectification (Cl-C3 and anoctamine), or no rectification


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(bestrophin). The single-channel VRAC conductance is 50–80 pS at positive and 10–20 pS at

negative membrane potentials, and VRAC exhibits a low field strength anion permeability

(I>Br>Cl>F). In this respect, VRAC differs from the ClC family members, which are typically selective

for chloride over iodide. VRAC permeates large molecules such as gluconate, glutamate, and

benzoate, and the pore size of this channel has been estimated to be about 11-17 A (Nilius et al.,

1997).

The search for VRAC specific blockers has been largely unsuccessful, but the long list of nonspecific

VRAC blockers (rev. in Pedersen et al., 2016) include: 1) conventional anion channel inhibitors,

such as DCPIB, DIDS, SITS, NPPB, and the acidic di-aryl-urea NS3728; 2) transporter blockers, such

as fluoxetin, phloretin and mefloquine; 3) the anti-estrogens tamoxifen, clomiphene, and

nafoxidine; 4) tyrosine kinase inibitors as genistein, tyrphostin A23, and PD98059; 5) purinergic

receptors blockers suramin, reactive blue 2 and PPADS; and 6) carbenoxolone and riluzol.

However, in addition to inhibiting VRAC, these inhibitors block a variety of other Cl-

channels,

transporters, or receptors to some extent (Pedersen et al. 2016). The failure to find a selective

VRAC blocker has complicated efforts to identify the molecular basis and proteins comprising for

this channel.

The VRAC gating mechanisms are not fully understood, but allosteric non-hydrolyzed-bound ATP

and permissive cytosolic Ca2+

are necessary for channel activation. It has been proposed that G-

protein-mediated signal transduction pathways and membrane structures, such as caveolin,

cholesterol, and actin cytoskeleton influence the channel activation mechanism, and it has been

suggested that the Ras-Raf-MEK-ERK pathway and the Rho/Rho kinase are modulators of VRAC

activity (Nilius et al., 1999). A volume-transduction pathway mediated by the epidermal growth

factor receptor and NAD(P)H oxidase-derived H2O2 has been proposed as a signaling pathway for


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VRAC activation (Varela et al., 2004). Epidermal growth factor receptor is activated by

hyposmolarity and is an early signal that modulates osmolyte efflux pathways (Pasantes-Morales

et al., 2006). Additionally, reduction in ionic strength is a key element for VRAC activation; early

studies by the Nilius group (Nilius et al., 1998; Sabirov et al., 2000) demonstrated that reducing

ionic strength activates a Cl- current with biophysical and pharmacological features identical to

those of VRAC, even without cell swelling. This mechanism of VRAC activation is of high

physiological relevance. Under physiological conditions, the osmolality changes are small and

gradual, and in most cases, insufficient to generate significant changes in cell volume or in

membrane distension, but the osmotic disequilibrium still should be corrected. Corrective Cl-

fluxes permeating across VRAC, activated by local changes in the ionic environment, appear as the

best option for an isovolumetric regulation (see below). More direct evidence on the role of ionic

strength in VRAC gating is further discussed in the context of the identification of LRRC8 proteins

as components of the VRAC structure.

In 2014, two independent groups (Voss et al., 2014; Qiu et al., 2014), using a similar fluorescence

assay for a screening of human genome siRNA libraries, identified a multispan transmembrane

protein derived from a gene of unknown function named LRRC8A (leucine-rich repeat containing

8A). The LRRC8A subunit protein was postulated to be an integral component of the VRAC

structure. LRRC8A belongs to the LRRC8 family, which consists of five members, LRRC8A-LRRC8E.

All the LRRC8 proteins have four putative transmembrane domains and contain up to 17 leucine-

rich repeats. Studies by the above mentioned groups suggest that LRRC8 isoforms, similar to the

pannexins to which they are related, organize in heteromeric complexes to form a functional

channel with VRAC properties. It was also shown that the presence of LRRC8A in the multimeric

complex is essential, but not sufficient alone for VRAC activity, since at least one of the other


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LRRC8 isoforms should be present together with LRRC8A. Genetic ablation of LRRC8B-LRRC8E did

not separately affect VRAC currents, but disruption of the five genes abolished VRAC activity. The

functional channel can be restored by expression of LRRC8A together with one of the other four

LRRC8 subunits (Voss et al., 2014; Qiu et al., 2014). The largest reductions in ICl(swell) amplitudes are

observed in LRRC8E−/−

and in LRRC8(C/E)−/−

cells and the lowest reductions in LRRC8B−/−

, LRRC8D−/−

cells (Voss et al., 2014). Different combinations of LRRCA and other heteromers define intrinsic

properties of the channel. Co-expression of LRRC8A and LRRC8E induces faster inactivation and

less positive potentials, while co-expression of LCRRC8A and LRRC8C has the opposite effect; this

co-expression markedly reduces the inactivation rate (Voss et al., 2014). Different combinations of

LCRR8 heteromers also strikingly modify VRAC permeability. As later discussed in detail, VRAC

permeability to taurine has a strict requirement of LCRR8A and LCRR8D, while LCRR8BCE subunits

appear unnecessary (Planells-Cases et al., 2015) (Fig. 2).

The possibility that LRRC8A constitutes the anion conducting pore domain of VRAC is questioned

(Akita and Okada, 2014) based on the lack of influence on currents across the LCRR8 complexes of

charged amino acids mutations in predicted transmembrane domains. This suggests that the

postulated VRAC structure may require a regulator molecule; a decrease, and not an increase, in

ICl(swell) following overexpression of LCRR8A also is suggestive of a regulator molecule. Recently,

Syeda et al. (2016) assembled models of different combinations of LCRR8A and LCRR8BD subunits

where the pore-forming channels were incorporated into bilayers. Using this approach, it was

possible to demonstrate that, as previously shown (Voss et al., 2014; Qiu et al., 2014), the

different combinations of LCRR8A with LCRR8B-D subunits determines intrinsic channel properties

such as rectification, inactivation kinetics, and relative anion permeability. The study also showed

that reducing intracellular ionic strength directly gates the channel, even in the absence of


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mechanical changes in the membrane or the cellular components, such as cytoskeleton structures

or elements of signaling chains. The relevance of these findings is that differently composed

heteromers formed by combinations of LRRC8 subunits may result in a large variety of channels

with different intrinsic properties and different regulatory mechanisms, which helps explain the

differences in the inactivation rate of VRAC in various cell types and the possibility to permeate

molecules as heterogeneous as the group of organic osmolytes. It also points to the possibility that

one or many of the LCRR8 subunits forming VRAC contain the sensor for changes in ionic strength,

the proposed channel gating (Nilius et al., 1998; Sabirov et al., 2000; Cannon et al., 1998). The

relevance of the molecular identity of VRAC is extensively discussed in several excellent recent

reviews (Pedersen et al., 2015, 2016; Stauber, 2015; Jentsch et al., 2016).

Organic osmolytes. Organic osmolytes, which contribute to cell volume regulation in most

animal cells (Kinne, 1993), are small molecules of heterogeneous structure including amino acids

and derivatives (taurine, glutamate, glycine, GABA, and N-acetylaspartate), polyalcohols (myo-

inositol and sorbitol), and amines (glycerophosphorylcholine, betaine, creatine/P-creatine, and

phosphoethanolamine). The concentration of organic osmolytes varies in the different tissues;

glutamate, myo-inositol, creatine, taurine, and N-acetylaspartate are present in the highest

concentration in the brain (Verbalis and Gullans, 1991), whereas glycerophosphoryl choline,

betaine, myo-inositol and sorbitol are the major organic osmolytes in renal cells (Beck et al., 1992).

Organic osmolytes are important in the process of volume regulation; in contrast to ions, which

generate adverse effects that disturb the structure of macromolecules or affect neuronal

excitability, many organic osmolytes are compatible molecules. Thus, in the long term, organic

osmolytes tend to replace ionic osmolytes (Verbalis and Gullans, 1991). In the nervous system,

glutamate, GABA, and glycine are an exception since they play a dual role as osmolytes and


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neurotransmitters, and therefore, changes in their concentration at the extracellular space disturb

nervous excitability or lead to neuronal death by excitotoxicity (discussed below). Taurine is

particularly suitable for an osmolyte role since it is largely free in the cytosol, is not a protein

amino acid, and participates in few reactions in the cell (Pasantes-Morales et al., 1998). Compared

to other osmolytes, taurine shows the largest efflux (9- to 22-fold) and the lowest osmolarity

threshold (Tuz et al., 2001), which could reflect a higher efficiency of the taurine efflux pathway or

more availability of taurine pools for release compared to osmolytes involved in other cell

functions.

The decrease in intracellular concentration of organic osmolytes as part of RVD is accomplished

largely by solute extrusion rather than by molecular degradation. Efflux of organic osmolytes

occurs via energy-independent, bidirectional leak pathways with net solute movements driven by

the concentration gradient (Hoffmann and Lambert, 1983; Sánchez-Olea et al., 1991). It has

consistently been reported that organic osmolyte fluxes are greatly reduced by VRAC blockers, but

it is still not known whether Cl- and organic osmolytes permeate across a common pathway, most

likely an anion channel like VRAC. The pore size of VRAC is sufficiently large as to permeate

osmolytes such as taurine and glutamate, and indeed currents carried by glutamate and anionic

taurine across VRAC have been demonstrated (Banderali and Roy, 1992). However, taurine is an

electroneutral zwitterion and at physiological pH, has no net charge, and is therefore unable to

permeate across an anion channel. The same is true for polyols and most organic osmolytes. A

number of differences between VRAC and the osmosensitive taurine efflux pathway have been

documented (Lambert and Hoffmann, 1994) and discussed in detail in reviews by Shennan (2008)

and Hoffmann et al. (2009). A most noticeable difference is the time course of Cl- (traced by

125I-)

and taurine efflux in various cell types (Stutzin et al., 1999; Pasantes-Morales et al., 1994; 1997)


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(Fig.2); Studies showed that I- efflux is fast and rapidly inactivating, whereas taurine efflux is

slower and sustained (Fig. 2). As clearly underlined in Stutzin et al. (1999) and Shennan (2008), if

taurine and Cl- hypotonic fluxes were occurring via an identical pathway, such large differences in

the time course should not be observed. Though similar to VRAC-mediated currents, isotonic

taurine efflux is evoked by a decrease in ionic strength (Guizouarn and Motais, 1999: Cardin et al.,

1999). The recent discovery of the LRRC8 family of proteins forming volume-sensitive heteromeric

channels with different permeability offers a real possibility to establish the molecular identity of

the organic osmolyte efflux pathway(s). Studies by Voss et al. (2014), Qiu et al., (2014), and

Planells-Cases et al. (2015) show that in LRRC8A-/-

HEK, HCT116, and HeLa cells, the osmosensitive

taurine efflux is essentially abolished, stressing the importance of this LCRR8 family isoform for the

taurine efflux pathway, which issimilar to VRAC (Voss et al., 2014; Qiu et al., 2014) Interestingly,

also in 2014, a study by Hysinski-García et al. (2014) showed similar results in cultured astrocyte

from the mouse brain cortex. Astrocytes from primary cultures are not cell lines, but originate

from unmodified tissue, and therefore in this respect is noteworthy that LCRR8A is present in a

variety of mouse tissues (Qiu et al., 2014). The effects of genetic ablation of the different LCRR8

family isoforms on IClswell and on taurine efflux shown in studies by Voss et al. (2014) and Planells-

Cases et al. (2015) revealed that deletion of LCRR8A abolished both IClswell and taurine, deletion of

LCRR8E and C reduced IClswell (taurine efflux was not examined), and deletion of LCRR8D did not

affect IClswell, but markedly decreased the hypotonic taurine efflux (Planells-Cases et al., 2015).

Comparison of results in LRRC8(B,D,E)−/−

and LRRC8(B,C,D)−/−

cells confirmed that LRRC8D is

dispensable for IClswell, but indispensable for taurine fluxes. As predicted by Stutzin et al. (1999)

and Shennan (2008), these results indicate that the combination of LCRR8 heteromers, particularly

the presence or absence of the HCRRL8D subunit, confers two different molecular entities. In fact,

the HCRRL8D subunit, or lack of a subunit, confers two different channels, one for permeation of


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anions and the other with high permeability to a neutral molecule, such as taurine. While both

channels have LCRR8A as an integral element and are activated by swelling/ion strength reduction,

these channels exhibit different intrinsic properties, such as inactivation and permeability,

suggesting that the pore structure must be significantly different in these two molecular entities; a

larger pore and differences in the amino acid residues between different combinations of LCRR8

subunits are necessary to permit the flow passage of molecules like taurine and other neutral

organic osmolytes. The predictable existence of a spectrum of channels constructed by multiple

combinations of LCRR8 subunits is relevant to understanding the puzzling existence of

permeability pathways for organic osmolytes that are not anions and have dissimilar molecular

structures, but are still gated by reduced ionic strength and inhibited by VRAC blockers.

The variety of LCRR8-based channels specific to permeate different osmolyte groups, including Cl-,

represent a useful tool to estimate the contribution of these different osmolyte pathways to cell

volume regulation. Presently, it has been shown that in LCRR8A-/-

HEK293, HeLa, and HCT116 cells,

RVD is attenuated, and in the cancer cell line BHY, RVD is abolished (Voss et al., 2014; Qiu et al.,

2014; Siriniant et al., 2016). These likely reflect differences in the contribution to the regulatory

process of other LCRR8A-independent mechanisms, such as the cotransporter KCC. Parallel

analysis of the effect of LCRR8A genetic ablation, together with blockers of KCCs as in HeLa cells in

the study by Siriniant et al. (2016), would help to clarify this question.

TRP channels. Transient Receptor Potential (TRP) channels are widely expressed in

vertebrate tissues and are cellular sensors for a large variety of stimuli. Most of the channels are

non-selective cation channels, with the exception of a few members that are Ca2+

selective. TRPs

of the mammalian superfamily are grouped into several subfamilies by sequence similarity (rev. in

Pedersen et al., 2005; Owsianik et al., 2006; Nilius and Owsianik, 2011; Nilius and Szallasi, 2014). A


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member of the vanilloid subfamily, the TRPV4 channel, is an osmosensitive channel that

contributes to RVD in numerous cell types (Vriens et al., 2004; Plant, 2014), including

chondrocytes (Lewis et al., 2011.), corneal epithelial cells (Pan et al., 2008), keratinocyte cell lines

(Becker et al., 2005), salivary gland cells (Aure et al., 2010), and airway epithelial cells (Arniges et

al., 2004). In these cell types, RVD is Ca2+

-dependent and TRPV4 is proposed as the pathway for

Ca2+

influx evoked by swelling and the ensuing activation of KCa channels involved in RVD

(Fernández-Fernández et al., 2002, 2008; Jin et al., 2012). In astrocytes and retinal Muller cells,

TRPV4 is functionally linked to AQP4 (Benfenati et al., 2011; Jo et al., 2015), and this association is

a requirement for RVD. TRPV4 associates with AQP5 in acinar and salivary gland cells (Hosoi, 2016;

Liu et al., 2006) and with AQP2 in renal cells (Galizia et al., 2008).

The activation of TRPV4 by cell swelling is still unclear (Pedersen and Nilius, 2007), but the

following possibilities have been proposed: i) direct membrane stretch with integrins as

intermediate molecule (Matthews et al., 2010); ii) interaction with supramolecular complexes

containing regulatory kinases and cytoskeletal proteins (Becker et al., 2009); iii) interaction with

the arachidonic acid metabolite 5,6-EET (Watanave et al., 2003); and iv) phosphorylation by WNKs,

particularly WNK4 (Fu et al., 2006). Results of TRPV4 genetic manipulation suggest that its role is

not restricted to cellular processes of volume regulation, but that it is also implicated in systemic

osmosensing; TRPV4 genetic ablation alters functions, such as drinking behavior, serum

osmolarity, and antidiuretic hormone plasma levels (Liedtke and Friedman, 2003). Although the

mechanism is unclear, osmosensing neurons and cells in kidney epithelium express TRPV4 and

may represent its site of influence on systemic osmoregulation (Ciura and Bourke, 2006; Cohen,

2007).


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Other members of the TRPC family, such as TRPV1, TRPV2, TRPC1, TRPC5 and TRPC6, TRPM3,

TRPM7 are possibly involved in RVD; these channels are osmotically activated channels, but

detailed studies on their role in volume regulation are largely missing (Plant, 2014).

Isovolumetric Regulation

In most animal species, extracellular osmolarity is tightly regulated and abrupt, and large changes

in osmolarity rarely occur. In contrast, small and gradual changes in osmolarity associate with a

number of metabolic reactions and cell functions. Exposing cells to small and gradual osmolarity

reductions have shown that cells are able to maintain a constant volume over a wide range of

tonicities via an active mechanism of continuous volume adjustment. This adaptive response,

known as isovolumetric regulation (IVR), is accomplished by the efflux of intracellular osmolytes

(Lohr and Grantham 1986, Mountian and Van Driessche, 1997; Souza et al., 2000). The osmolytes

involved in IVR are the same as in RVD, i.e., Cl-, K

+ and organic molecules, and as shown in Figure 3,

the Cl- currents in glioma cells are evoked by gradual or sudden reductions in osmolarity. The Cl

-

current evoked by gradual osmotic reductions activates early, at osmolarity reductions of about

3%, and is blocked by niflumic acid and NPPB (Figure 3), which suggests that it’s similar to the

current across VRAC. This point should be clarified in light of new findings regarding the LCRR8

family members as integral elements of VRAC. K+ conductance also activates in these cells during

IVR with different osmolarity thresholds in the presence or absence of Ca2+

(Ordaz et al., 2004). In

a variety of cell types, gradual changes in osmolarity activate taurine efflux with different efflux

threshold (Souza et al., 2000; Pasantes-Morales et al., 2000, Ordaz et al., 2004), and the lowest

efflux is observed in cerebellar granule neurons where taurine efflux increases significantly at

osmolarity reductions of only 2 mOsm (Figure 3)(Tuz et al., 2001). This compensatory mechanism

is then likely to be preferred by neurons facing physiological changes in cell volume.


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Channel-Transporter Interactions in RVI

Increase in external osmolarity activates intracellular water exit driven by osmotic disequilibrium,

and accordingly, this leads to a decrease in cell volume. If this is not corrected, cells undergo

apoptotic death. In healthy cells, hypertonicity activates a regulatory mechanism that aims to

restore normal cell volume even if the external anisotonic condition persists. This adaptive

mechanism, known as regulatory volume increase (Burg et al., 2007), occurs once a set of

mechanisms that increase the concentration of intracellular osmolytes are set in motion. This

tends to re-establish the osmotic equilibrium between extracellular and intracellular

compartments. Electroneutral cotransporters and ion exchangers both participate in this process

to increase the cytosolic levels of Na⁺ and Cl- (Figure 4), while NCC and NKCC family cotransporters

(Arroyo et al., 2013) and Na⁺/H exchangers are the main contributors to RVI (Cala et al., 1980;

Alexander and Grinstein 2006). Organic osmolytes are also significantly involved in RVI (Figure 4);

cell shrinkage increases the expression of Na⁺-dependent cotransporters known to operate for

various molecules acting as organic osmolytes (Sánchez-Olea et al., 1992; Burg and Ferraris, 2008).

In contrast to RVD where the corrective fluxes of osmolytes permeate essentially across ion

channels and to diffusive pathways for organic osmolytes, in RVI, channels play only a minor role

and the regulatory process is essentially accomplished by cotransporters and exchangers. It has

been proposed that unidentified hypertonicity-activated cation channels participate in RVI

(Wehner et al. 2006). A study in HeLa cells (Wehner et al. 2006) showing RVI reduction by SKF-

96365, a blocker of TRP channels, raised the question of whether the hypertonicity-induced cation

channels are actually TRP channels, and recent evidence pointed to the TRPM2 channel. A recent

report, also in HeLa cells (Numata et al., 2012), showed that nucleotides adenosine diphosphate

ribose (ADPr) and cyclic ADPr, reported as typical TRPM2 activators (Pedersen et al., 2005;


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Pedersen and Nilius, 2007), generate cation currents similar to those elicited by hypertonicity,

previously known as hypertonicity-activated cation channels. In the same line, siRNA silencing of

TRPM2 expression or the precursor enzyme of the TRPM2 nucleotide activators abolish the

hypertonicity-elicited cation current and mildly reduce RVI. Cloning of TRPM2 identified the ΔC-

splice variant as the molecular entity corresponding to the nucleotide-activated current (Numata

et al., 2012), which suggests that TRPM2 may represent the molecular identity of the

hypertonicity-activated cation channels. Another member of the TRP channel family, TRPV2,

seems to participate in RVI via an interesting connection with the electroneutral cotransporter

NKCC, which as previously mentioned, is a main effector in RVI. The suggested mechanistic chain

of events relating TRPV2 channels and RVI involves a TRPV2-dependent accelerated depolarization

followed by Ca2+

release from intracellular sources and the subsequent phosphorylation of

STE20/SPS1-related proline/alanine-rich kinase kinases (SPAK), which is essential for activation of

NKCC. This proposed pathway is supported by studies in skeletal muscle showing that expression

of TRPV2 negative dominant reduces the RVI efficiency at the time that depolarization is impaired

and the Ca2+

response is diminished (Zanou et al., 2015). Interestingly, the upregulation of all

transporters involved in RVI, ions and organic osmolyte transporters, occurs by the concerted

action of transcription factor TonEBP/OREBP. As part of the regulatory response to cell volume

decrease, TonEBP/OREBP increases the expression of osmoprotective genes, including Na⁺-

dependent transporters, responsible for the accumulation of organic osmolytes (Ferraris and Burg,

2006).

Channels Involved in Volume Changes in Necrotic and Apoptotic Death

Changes in cell volume are characteristic features of cell death and represent one of the main

differences between necrosis and apoptosis. In necrotic death, cell swelling and depletion of


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intracellular ATP are distinctive traits. During the necrotic process, water accumulates into the

cytosol and organelles, and the membrane surface architecture is deformed by prominent,

stationary blebs that eventually lead to membrane rupture and cell death (Jurkowitz-Alexander et

al., 1992; Barros et al., 2001). In apoptosis, reduction in cell volume is a central event and a

hallmark in this program of cell death (Yu and Choi, 2000; Bortner and Cidlowsi, 1998). Coined

phrases for these cell volume changes include Apoptotic volume decrease (Hughes et al., 1997;

Maeno et al., 2000) and necrotic volume increase , also known as oncotic swelling or cytotoxic

swelling. A variety of channels are involved in these changes in cell volume.

Necrotic volume increase. Necrosis associates with the extensive cell loss that

accompanies pathological conditions, such as ischemia and ischemia/reperfusion, cardiovascular

diseases, and several neurodegenerative conditions. Cytotoxic swelling in ischemia results from

the arrest of oxygen-dependent ATP synthesis, which reduces the activity of the Na+/K

+ ATPase,

impedes the transmembrane Na+/K

+ exchanges, and results in dissipation of ion gradients. In brain,

swelling during ischemia is the first step in a cascade of ion gradient disturbances between the

intravascular and extracellular compartments that culminates in blood brain barrier injury and

vasogenic edema (Annunziato et al., 2013, Kahle et al., 2015). Besides ischemic swelling, brain cells

also swell during trauma, seizures, and spreading depression. Swelling is essentially due to Na⁺, K⁺

and Cl- overload, which drives inwardly-directed water fluxes, and both channels and

cotransporters participate in this process (Figure 5). Characteristic of brain ischemia is the marked

increase in extracellular K⁺ (K+

o), which is often elevated from a physiological level of around 3 mM

up to 60 mM. This high K+

o level reverses the KCC direction, and together with the ischemia-

activated expression of NKCC, results in large inwards ion and water fluxes (Khale et al., 2015). Na⁺

influx also permeates across a non-specific cation channel, which based on recent evidence, is


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identified as TRPM4. The TRPM4 pore is selective for monovalent cations with similar permeability

for K⁺ and Na⁺, and is impermeable to divalent cations. Evidence in support of TRPM4 as a major

mechanism for Na+ influx in ischemia includes the following observations: i) two main regulators of

TRPM4, i.e., ATP and intracellular Ca2+

are deeply disturbed by conditions promoting necrosis. This

deregulation favors the opening of TRPM4, Na⁺ overload and NVI; ii) redox imbalance by excessive

generation of free radicals, a condition related to pathologies leading to necrosis, induces a

sustained activity of TRPM4 (Simon et al., 2010); iii) pharmacological or genetic manipulation of

TRPM4 has a marked influence on the swelling phase of the necrotic process; iv) activation of

TRPM4 channel by ATP depletion in COS-7 cells leads to depolarization and progressive bleb

formation (Chen and Simard, 2001; Chen et al., 2003), but this is not observed in cells lacking the

channel (Simard et al., 2012); and v) induced upregulation of TRPM4 protein in endothelial cells

from human umbilical vein causes Na⁺ overload, cell volume increase, and necrotic death, effects

which are prevented by pharmacological inhibition of the channel (Gerzanich et al., 2009; Becerra

et al., 2011). It should be noted that ADP and AMP are reported to block TRPM4b currents (Nilius

et al., 2004), however, during the first minutes of ischemia, ADP levels decrease in parallel to ATP

and AMP levels increase ( Guarnieri et al., 1993; Phillis et al.,1995). Regardless, the concentration

of AMP is so low (3% and 5% of ATP levels in heart and brain, respectively) (Muscari et al., 1993;

Phillis et al.,1995) that the increase may not affect TRPM4 activation. TRPM4 is not constitutively

present in the central nervous system, but is transcriptionally upregulated in neurons, astrocytes,

oligodendrocytes, and microvascular endothelial cells after the onset of ischemia (Gerzanich et al.,

2009). Necrotic death of endothelial cells caused by TRPM4 activation is also responsible for the

capillary fragmentation, known as progressive hemorrhagic necrosis, observed in traumatic injury

of the spinal cord. The capillary damage is prevented in TRPM4−/−

or by antisense

oligodeoxynucleotides directed against TRPM4 (Gerzanich et al., 2009). TRPM4 channel also


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contributes to cytotoxic swelling, cell death, blood-brain barrier breakdown, and vasogenic edema

in ischemia and trauma (Simard et al., 2014; Zbeckberger et al., 2014; Martinez-Valverde et al.,

2015). Together, these findings are consistent with the idea that the TRPM4 channel plays a role in

cell swelling and necrotic death. Other channels, including members of the TRP family (TRPV4,

TRPM2, and TRPM7) and the acidic-activated cation channels, also increase in expression and

functional activity after an ischemic episode, and their blockade reduces the size of the infarct.

However, their role in inducing ischemic injury is more related to Ca2+

overload and protease

activation than to cytotoxic swelling.

In brain cells, VRAC participates in cytotoxic swelling and ulterior necrotic neuronal death,

particularly in the penumbra where intracellular ATP that is necessary for VRAC activation still

remains. Neuronal death results from the increase in extracellular glutamate driven by the reversal

of Na+-energy-dependent transporters and the swelling-activated, VRAC-mediated glutamate

efflux from swollen astrocytes (Mongin, 2016). The consequent overactivation of AMPA and

NMDA ionotropic receptorscauses Na+ and Ca

2+ overload at the same time that the ischemic

depolarizing condition activates VRAC in neurons, moving Cl-

into the cell driven by its

electrochemical potential. All this culminates in NVI and a necrotic cascade. The effect of VRAC

blockers on reducing both swelling and neuronal death (Inoue et al., 2007; Mongin, 2016) suggests

that VRAC has a in this deleterious effect. Necrosis is a delayed pattern of excitotoxicity, but

NMDA receptor overfunction also activates the apoptotic machinery (Linnik et al., 1993).

Hemichannels, the constituent elements of the Gap junctions, allow transmembrane movements

of large groups of heterogeneous molecules of different sizes, such as water, Na⁺, K+, Ca

2+, ATP, or

adenosine, among others. Hemichannels are expressed in the membrane of astrocytes, neurons,

oligodendrocytes, and vascular endothelium. Unregulated hemichannels have been implicated in


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hypoxia, ischemia, and brain trauma where they generate disruption of intracellular ionic

homeostasis and cytotoxic swelling. Consistent with this action, downregulation of hemichannels

reduce cell swelling in astrocytes and reduce the ischemic injury (Davidson et al., 2015).

Apoptotic volume decrease. Apoptosis, or programmed cell death, is a physiological

mechanism that commits cells to individual death fate (Kerr, 1972). Apoptosis occurs normally

during development and aging to maintain optimal cell populations and eliminate excessive or

potentially harmful cells as an homeostatic mechanism. Apoptosis is characterized by changes in

the cell structure, notably cell nuclear condensation and DNA fragmentation, membrane blebbing,

and the formation of apoptotic bodies. Programmed cell death is activated by either intrinsic or

extrinsic stimuli. Intrinsic stimuli are molecules or situations of stress that result in mitochondrial

dysfunction, like UV radiation, nitric oxide, staurosporine, thapsigargin, and dexamethasone,

while extrinsic stimuli are ligands of death receptors such as Fas or other members of the tumor

necrosis factor receptor superfamily including CD95. As mentioned above, cell volume decrease is

a characteristic trait of apoptotic death, and depending on the cell type, the decrease in cell

volume may be in the range of 40-80%. The term apoptotic volume decrease was coined for this

unique condition (Hughes et al., 1997; Maeno et al., 2000). In terms of time-course, AVD occurs in

two phases, an initial phase which starts 0.5-2 h after exposure to the inductor and a late phase

detected about 3 h later. The two phases of AVD are accomplished by the same mechanism that

includes outwards fluxes of K⁺ and Cl- moving across specific channels and a decrease in osmolyte

intracellular concentration and water outflow resulting in AVD (Kondratskyi et al., 2014). It has yet

to be determined whether this decrease in cell volume is a passive factor or an active signal in the

induction of apoptosis, but evidence does exist that points to an explicit role of volume decrease


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in the apoptotic process; blockade of the main osmolyte pathways that reduce AVD impairs the

progression of apoptosis (Grishin et al., 2005; Yu et al., 2000).

The increase in K⁺ currents that were detected as an early event in the cell death program (Yu et

al., 1997) first suggested that K⁺ channels contribute to AVD. Regardless of the cell type or the

inductor stimulus, the role of K⁺ channels as the K⁺ efflux pathway leading to AVD and loss of

intracellular potassium is recognized as a distinctive feature in the apoptotic program, (rev in Burg

et al., 2006; Bortner and Cidlowski, 2007; Orlov et al., 2013). A diversity of K⁺ channels mediate the

K⁺ efflux during AVD in different cell types; voltage-gated, Ca2+

-activated of intermediate and

large-conductance K⁺ channels, inward rectifier, and two-pore domain K⁺ channels participate in

AVD. Different subtypes of Kv channels contribute to AVD including Kv 1.2 and Kv 2.1 in neurons,

Kv 1.3 in T-lymphocytes, and Kv1.5 in COS-7 and in vascular smooth muscle. Kv channels of not

defined subtypes account for AVD and K⁺ loss in hippocampal neurons, sympathetic neurons,

cerebellar granule neurons, corneal epithelial cells, and cardiomyocytes (Rev. in Remillard and

Yuan, 2004; Bortner and Cidlowski, 2007; Lang et al., 2007). The intermediate-conductance IKCa

channels mediate the apoptotic K⁺ current in lymphocytes, thymocytes, and glioblastoma cells,

and the large-conductance BK channels play this role in artery smooth muscle cells, glioma, and

superficial colonocytes, while inward rectifier IRK channels are involved in apoptosis in liver cells,

HeLa cells, and some neuronal cell lines (Rev. in Bortner and Cidlowski, 2007). Members of two-

pore-domain potassium channels TASK1 and TASK3 participate in apoptosis in cerebellar granule

neurons and in exocrine pancreatic cells (Patel and Landunski, 2004). Hydrogen peroxide-

mediated apoptosis is associated with activation of TREK channels and of HERG K⁺ channels. The

significance of intracellular K⁺ decrease in the apoptotic process is more complicated than simply

an involvement as an osmolyte and a main contributor to AVD. The decrease in intracellular K⁺


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levels has an effect on apoptotic mechanisms, independent of AVD, and is more related to the

release of cytochrome C (Yu, 2003; Remillard and Yuan, 2004). Various K⁺ channel subtypes

localized at the mitochondrial membrane regulate mitochondrial volume and contribute to the

organelle homeostasis.

Together with K+, Cl

- efflux is an essential component of AVD, during which K⁺ efflux hyperpolarizes

the cell and is followed by a Cl- outwards movement directed by its electrochemical gradient, thus

maintaining the ionic balance and electroneutrality of the cell. Activation of anion currents facing

intrinsic and extrinsic apoptotic stimuli is observed in a variety of cells (Okada et al., 2006), and

these anion currents are similar to those carried by VRAC regarding outward rectification, ATP

dependence, and pharmacological profile. However, VRAC is activated by a swelling and ionic

strength reduction, whereas volume decrease and ionic strength increase characterizes apoptosis.

Therefore, if VRAC participates in AVD, the channel volume set point must shift to a lower level or

the channel is gated by other mechanisms. Overproduction of reactive oxygen species, or a change

in cytosolic ATP, both concurrent with AVD, may be signals to either reduce the set point or to

modify an hypothetic modulator (Shimizu et al., 2008). While this critical point is not as yet

clarified, there is evidence showing that AVD induction is reduced by VRAC blockers in various cell

types. Although it should be considered that those blockers are not specific for VRAC or for Cl-

channels. The recent findings identifying LCCR8A as a structural element of VRAC provides

additional tools to define the VRAC contribution to AVD. The study by Planells-Cases et al. (2015)

in KBM7 and HAP1 cells showed that a channel formed by the combination of LCRR8A and LCRR8D

subunits participates in AVD and apoptosis. This channel composition is similar to that proposed to

permeate taurine flow, and dissimilar to the typical VRAC. In contrast, in HeLa cells, Sirianant et al.

(2016) reported no effect of LCRR8A knockdown in staurosporin-induced cell shrinkage.


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The significant role of organic osmolytes in cell volume regulation either in RVD and in RVI is well

documented, as above discussed. In contrast, evidence regarding organic osmolytes’ contribution

to volume decrease during apoptosis is rather scarce (Wehner et al., 2003). Although, the

importance of organic osmolytes was emphasized in the quantitative analysis made by Model

(2014), which demonstrated that the loss of organic molecules is necessary to account for the

osmotically obligated water exit in AVD. While taurine cell loss is documented in apoptosis in

Jurkat lymphocytes (Lang et al., 1998) and in cerebellar granule neurons (Moran et al., 2000), in

VRAC, taurine efflux is evoked by cell swelling or ionic strength reduction, two conditions absent in

apoptosis. A number of studies report antiapoptotic effects of taurine (Lambert et al., 2015), but

this seems unrelated to AVD. Recent findings that demonstrate resistance to cancer

chemotherapy by platinum-based drug show attenuated taurine efflux and reduced expression of

LCRR8D, suggesting a role for the LRRC8D containing VRAC entity in drug extrusion (Planells-Cases

et al., 2015; Voets et al., 2015). As for apoptosis, the normotonic activation of VRAC under these

conditions remains to be defined.

Comparing RVD and AVD mechanisms demonstrates that essentially the same type of K⁺ channels

participate in both processes in the same cell type. For instance, voltage-dependent Kv channels

are those involved in both RVD and AVD in neurons while Ca2+

-activated K⁺ channels are

implicated in epithelial cells. This selectivity is explained in the thermodynamic analysis of ion

fluxes described in Orlov et al (2013). The analysis in Orlov`s review refers only to AVD, but

appears valid also for K+ and Cl

- fluxes kinetics in RVD. Interestingly, activation of channels in AVD

occurs under isotonic conditions, suggesting marked differences in the intracellular signals

responsible for the efflux of osmolytes during the two processes. Also, the sustained cell shrinkage

during AVD does not result in activation of RVI (Maeno et al., 2006) in cisplatin‐induced cell

shrinkage and subsequent apoptotic cell death


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Concluding Remarks

The key role of ion channels in together with cotransporters in the active processes of cell volume

regulation is now well-established. The intricate and complex signaling pathways directed to

activate/inactivate these channels represents a vast field for present and future research. The firm

establishment of the molecular identity of the volume-regulated anion channel and the pore(s) for

permeation of organic osmolytes are also intriguing aspects in the physiology of volume-related

channels. Identification of the mechanisms responsible for cytotoxic swelling may ideally

contribute to the formulation of drugs with the potential to prevent the chain of events disturbing

the ionic homeostasis that ultimately result in brain edema. Although there is extensive literature

reporting drugs and maneuvers directed to reduce swelling and injury due to ischemic episodes,

all have failed in clinical trials. There is, however, also strong evidence of their relevance in the ion

gradient disturbances generating cytotoxic and vasogenic brain edema. The ischemic phenomenon

is complex and involves a plethora of factors and signals, complicated further by those resulting

from reperfusion. A comprehensive understanding of the events concurrent with

ischemia/superfusion, including those related to the channels involved, is part of the integral

approach necessary to formulate effective therapeutic targets for this pathology

Work in the author’s laboratory is supported by Dirección General de Asuntos del Personal

Académico (DGAPA) at the Universidad Nacional Autónoma de México (UNAM) [grant number

PAPIIT IN205916 ]. I deeply appreciate the invaluable help of Dr. Gerardo Ramos-Mandujano in

the preparation of the manuscript and figures

Author contribution: This is a single author review.


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Figure Legends

Figure 1. Schematic illustration of the mechanisms mediating osmolyte fluxes during RVD. A.

Under isotonic conditions, water fluxes are in equilibrium and the cell volume corresponds

basically to that defined by the cell lineage. Upon a reduction in extracellular osmolarity, water

moves following its concentration gradient and the cell swells. Immediately after, intracellular

signals activate the channels and transporters mobilizing the main intracellular osmotically active

solutes: the ions K+, Cl

- and a number of organic molecules, mainly amines, amino acids and

polyalcohols. The massive efflux of osmolytes, drives osmotically obligated water and the volume

of the cell progressively decreases towards the original volume. K+ fluxes permeate through a

variety of channels, activated by events concurrent with cell swelling and are different in the

different cell types. Cl- fluxes are likely carried by one type of channel, identified as VRAC. Organic

osmolytes permeate through diffusive pathways, possibly molecular variants of the VRAC. KCC also

participates in the ion extrusion. B. Time course of the changes in cell volume (gray solid line) and

the efflux of 125

I- as tracer for Cl

- (B),

86Rb (tracer for K

+)(Δ) and

3H-taurine (B). Data from cultured

astrocytes C. Swelling and cell volume recovery in mesenchimal cells exposed to a reduction in

external osmolarity (unpublished).

Figure 2. Differences between the volume-sensitive efflux of Cl- and taurine. A.B. In HeLa cells (A)

and NIH3T3 cells (B), Cl- efflux traced by I

- (B) is fast and inactivates rapidly, while taurine efflux (B)

is sustained (see also Fig. 1B). C. Effects on ICl-swell and on

3H-taurine efflux of LCRR8 heteromer

knockout in HEK cells. D. Suggested LCRR8 multimeric composition of the two pathways, based on


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the effect of LCRR8 heteromer knockout on ICl-swell and taurine efflux. ICl

-swell is abolished in

LCRR8A-/-

(black), markedly reduced in LCRR8B-/-

and LCRR8C-/-

(gray) and mildly decreased in

LCRR8D-/-

and LCRR8E-/-

(white). The taurine efflux pathway requires LCRR8A and LCRR8D but

none of the other subunits of the complex. Details in Stutzin et al., 1999; Morán et al., 1997, Voss

et al., 2014, Qiu et al., 2014; Planells-Cases et al., 2015.

Figure 3. Cl- currents (C6 glioma cells) and taurine efflux (cerebellar granule neurons) evoked by

small and gradual osmolarity reductions . A. In cerebellar granule neurons, sudden 30% reduction

or increase in external osmolarity induces cell swelling or shrinkage, respectively, followed by RVD

or RVI. When osmolarity reductions imposed to the cell are gradual and small, the cell volume

remains unchanged, by the set in motion of an active process of osmolyte extrusion, known as

isovolumetric regulation (IVR). Efflux of taurine, glutamate, K+ and Cl

-, all contribute to the cell

volume adjustment. Efflux thresholds in these cells were -2% and -19%, for [3H]taurine and D-

[3H]aspartate (as marker for glutamate), respectively, and -25% and -29%, respectively, for Cl- (

125I)

and K+ (86

Rb). B. ICl-swell activated by small and gradual changes in osmolarity in glioma C6 cells.

Upper panel: ICl-swell at the osmolarity indicated in the conductance plot (middle panel). Lower

panel left, I-V curves in cells in isotonic medium and at 33% osmolarity reduction. Lower panel

right: effect of the Cl− channel blockers niflumic acid (NA) and NPPB on the Cl

− current elicited by

gradual reductions in osmolarity. Details in Ordaz et al., 2004; Tuz et al. 2001.

Figure 4. Channels and transporters involved in RVI. A. Schematic representation of channels and

transporters involved in the volume adjustment after hypertonicity-induced cell shrinkage. B. Cell

volume reduction and RVI in a renal cell line, mIMCD3, exposed to media made hypertonic by


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addition of NaCl. C. Increase in intracellular osmolyte amino acid levels in astrocytes cultured in

hypertonic medium. Details in Ruiz-Martinez et al., 2011, Sanchez-Olea et al., 1992.

Fig. 5. Role of ion channels and transporters on necrotic volume increase and excitotoxic

(necrotic) neuronal death at the penumbra area during brain ischemia. A. The chain of events

leading to NVI and cell death. The oxygen reduction leads to: energy failure, ATPase dysfunction,

dissipation of K+ and Na

+ transmembrane gradients, rise in K

+o levels and KCC reversal. Ischemia-

induced expression of TRPM4 and NKCC leads to massive Na+ overload. All this results in astrocyte

swelling and VRAC-mediated glutamate efflux. Reversal of the Na+- dependent glutamate

transporter increases further the extracellular glutamate levels. Overfunction of the neuronal

NMDA and AMPA neuronal receptors generates Na+ and Ca

2+ overload, together with Cl

- influx

through VRAC driven induced by the ischemic depolarizing condition. All this culminates in NVI

and the necrotic cascade. Noticeable, excitotoxicity also activates the apoptotic machinery. B.

Time course of astrocyte swelling (gray solid line) and glutamate (B) (traced by 3H-D- aspartate)

efflux from cultured cortical astrocytes. Glutamate efflux is abolished by simultaneous treatment

with the VRAC blockers DIDS and phloretin and transporter blockers (TBOA). Details in Perez-

Dominguez et al., 2014.


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