1521-0111/90/3/358–370$25.00 http://dx.doi.org/10.1124/mol.116.104158 MOLECULAR PHARMACOLOGY Mol Pharmacol 90:358–370, September 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

MINIREVIEW—A LATIN AMERICAN PERSPECTIVE ON ION CHANNELS

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 Received March 3, 2016; accepted June 22, 2016 Downloaded from

ABSTRACT Volume changes deviating from original cell volume represent a the types of channels involved in either corrective or pathologic major challenge for cellular homeostasis. Cell volume may be changes in cell volume. The review also underlines the contri- altered either by variations in the external osmolarity or by bution of transient receptor potential (TRP) channels, notably

disturbances in the transmembrane ion gradients that generate TRPV4, in volume regulation after swelling and describes the molpharm.aspetjournals.org an osmotic imbalance. Cells respond to anisotonicity-induced role of other TRPs in volume changes linked to apoptosis and volume changes by active regulatory mechanisms that modify necrosis. Lastly we discuss findings showing that multimers the intracellular/extracellular concentrations of K1,Cl–,Na1, and derived from LRRC8A (leucine-rich repeat containing 8A) gene organic osmolytes in the direction necessary to reestablish are structural components of the volume-regulated Cl– channel the osmotic equilibrium. Corrective osmolyte fluxes permeate (VRAC), and we underline the intriguing possibility that different across channels that have a relevant role in cell volume regula- heteromer combinations comprise channels with different in- tion. Channels also participate as causal actors in necrotic trinsic properties that allow permeation of the heterogenous swelling and apoptotic volume decrease. This is an overview of group of molecules acting as organic osmolytes. at ASPET Journals on September 29, 2021 Introduction compartments (Song and Yu, 2014; Pedersen et al., 2016). The intracellular osmolarity also endures continuous variations Cell volume is characteristic of each cellular lineage and is owing to transient and local osmotic microgradients generated maintained with few variations through the cell life. Condi- during physiologic processes, such as uptake and release of tions inducing changes in cell volume represent a major metabolites, cytoskeletal remodeling, synthesis and degrada- challenge for cell homeostasis and may even culminate in cell tion of macromolecules, exocytosis and secretion (Pedersen death. To keep cell volume constant, water fluxes should be in et al., 2001; Strbák, 2011; Platonova et al., 2015). Even these equilibrium between the intracellular and extracellular com- localized changes in cell volume should be regulated to partments. This occurs under conditions of isotonicity, but any prevent changes in the concentration of molecules free in the fluctuations in the osmolarity of the extracellular milieu or in cytosol, protein misfolding, or alterations in cell and organelle the distribution of osmotically active solutes drives water architecture. Cell volume is modified during migration, fluxes in the necessary direction to reach a new equilibrium, proliferation, and cell adhesion; therefore, volume change which disturbs cell volume. Except for cells in the kidney has been proposed as a signaling event for these processes and intestine, which normally face large variations in exter- (Lang et al., 2005; Stutzin and Hoffmann, 2006; Dubois and nal osmolarity, the extracellular milieu maintains a well- Rouzaire-Dubois, 2012). controlled osmolarity. This is modified only under pathologic When volume is perturbed as a result of changes in the conditions leading to hyponatremia or in other pathologies extracellular osmolarity, cells respond by activation of a that alter the distribution of osmolytes in the extra-intracellular plethora of signaling pathways and mechanisms intended to protect them from the challenge of volume change; remodeling Work in the author’s laboratory is supported by Dirección General de of the cytoskeleton, changes in adhesion molecules, activation Asuntos del Personal Académico (DGAPA) at the Universidad Nacional Autónoma de México (UNAM) [Grant No. PAPIIT IN205916 ]. of stress and survival signals are among the adaptive mecha- dx.doi.org/10.1124/mol.116.104158. nisms triggered by the altered volume (Pasantes-Morales et al.,

ABBREVIATIONS: AMPA, a-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate; AQP4, aquaporin 4; AVD, apoptotic volume decrease; BK, big potassium; DCPIB, 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid; DIDS, 4,49-diisothiocyanostilbene-2,29- 21 1 1 2 disulfonic acid; IKCa, intermediate-conductance (channels); IVR, isovolumetric regulation; KCa,Ca -activated K (channels); KCC, K /Cl cotransporter; 1 1 1 1 1 KIR,inwardlyrectifyingK channels; K2P,two-pore–domain K channels; Kv, voltage-gated K (channels); NKCC, Na /K /2Cl cotransporter; NMDA, N-methyl-D-aspartic acid; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; RVD, regulatory volume decrease; RVI, regulatory volume increase; SITS, 4-acetamido-49-isothiocyanatostilbene-2, 29-disulfonic acid; TBOA, DL-threo-b-benzyloxyaspartic-acid; TRP, transient receptor potential; TRPM4, transient receptor potential melastatin-4 channel; VRAC, volume-regulated anion channel.

358 Channels and Cell Volume 359

2006). At the same time, cells set in motion an active regulatory cells is consistently Ca21-dependent owing to the predomi- 1 process directed to partially or fully restore the original volume nant role of KCa channels in the volume-corrective K fluxes. 1 (Cala, 1977; Olson et al., 1986; Hoffmann, 1992; Okada, 2004). The high- and intermediate-conductance K channels (BKCa, This adaptive mechanism, which has been highly preserved IKCa) are the channels mainly implicated in RVD. High during evolution, essentially consists of the redistribution of conductance or big potassium (BK) channels are activated by osmotically active solutes in the necessary direction to equili- swelling in the intestine, kidney, and bronchial epithelia, brate water fluxes facing the new osmotic condition (Lang, chondrocytes, pituitary tumor cells, and osteoblast-like cells 2007; see Hoffmann et al., 2009 for a comprehensive review of (reviewed in Hoffmann et al., 2009). Activation of BK channels cell volume regulation). Osmolytes that accomplish cell volume occurs by Ca21 influx mediated by the TRPV4 subtype, regulation are the ions present in high concentrations in the establishing a link between BKCa channels and some tran- intracellular or extracellular compartments—such as Na1,K1 sient receptor potential (TRP) channels (discussed later) – and Cl —and a group of heterogeneous organic molecules, such (Jin et al., 2012). IKCa channels participate in RVD in as amino acids, polyamines, and polyalcohols (Verbalis and T lymphocytes, erythrocytes, hepatocytes, osteosarcoma cells, Gullans, 1991; Pasantes-Morales et al., 1998; Burg and intestine 407 cells, proximal tubule cells, and lens epithelia Ferraris, 2008). Volume-regulated channels and cotransporters (rev. in Hoffmann et al., 2009). In other cells—such as brain translocate ion osmolytes, and cotransporters and pores, as yet cells—following hypotonic swelling, depolarization generated fully identified, mobilize organic osmolytes; channels partici- by the Cl– efflux occurs across the volume-regulated Cl– channel, Downloaded from pate predominantly in the volume-regulatory process activated and in these cells RVD is essentially Ca21-independent by cell swelling, whereas cotransporters play a more important (Pasantes-Morales et al., 1994; Morán et al., 1997) and the role in the cell response to shrinkage (Kahle et al., 2015). K1 regulatory fluxes permeate across voltage-gated K1 chan- Besides their role in volume regulation, channels are involved nels. Subtypes of Kv involved in the different cell types include in the pathophysiologic cell volume changes generated in Kv1.3 in lymphocytes and murine spermatozoa, Kv1.4.2 and isotonicity that lead to necrotic death, as well as in those Kv1.4.3 in myocytes, and Kv1.5 in lymphocytes and spermatozoa. molpharm.aspetjournals.org associated with apoptosis (Bortner and Cidlowski, 1998, KCNQ1 (Kv7.1), in various subunit arrangements, partici- 2007; Okada et al., 2006). This review focuses on: 1) channels pates in RVD in human mammary epithelial cells, rat hepato- through which ions and organic osmolytes permeate during cytes, cells from the inner ear, and in mouse trachea epithelium regulatory volume decrease, 2) ion channels participating in (reviewed in Hoffmann et al., 2009). The inwardly rectifying K1 volume regulatory increase, and 3) channels implicated in channels Kir4.1 and Kir4.5, which sense small changes in normotonic cell volume changes leading to apoptotic and osmolarity (Grunnet et al., 2003; Soe et al., 2009), colocalize necrotic cell death. with aquaporin 4 (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 extracellu- at ASPET Journals on September 29, 2021 lar K1 homeostasis during the K1 spatial buffering through the The Role of Channels in Regulatory Volume 1 Decrease glial syncytium, and this transcellular K transport is accom- panied by transmembrane water fluxes, likely via a Kir4.1/AQP4 A decrease in external osmolarity modifies the water complex (Nagelhus et al., 1999; Dibaj et al., 2007). The concentration in the extracellular space, which activates two-pore–domain K1 channels participate in RVD in lympho- water inward-directed fluxes and increases the cell volume cytes, Ehrlich ascites cells, astrocytes, and kidney cells (Niemeyer with a magnitude proportional to the extent of the osmolarity et al., 2001; Kirkegaard et al., 2010; Andronic et al., 2013). change. As mentioned in the Introduction, most animal cells The variety of K1 channels in different cell types that are respond to this situation with an active process of volume involved in RVD and triggered by stimuli concurrent with cell recovery known as regulatory volume decrease (RVD) (Cala, swelling raises the question about the existence of a purely 1977), which is accomplished by extrusion of the major swelling-gated K1 channel. A recent study, in which AQP1 intracellular ions, K1 and Cl–, and organic osmolytes. Most and a number of K1 channel types were coexpressed in 1 – of the corrective fluxes occur via K and Cl channels and Xenopus oocytes (Tejada et al., 2014) identified the Kca 4.1 diffusion pathways for the organic osmolytes (Hoffmann and channel (Slick; Slo2.1) as the most sensitive to volume changes. Lambert, 1983; Sánchez-Olea et al., 1991; Okada and Maeno, Slick currents increased markedly in hypotonicity and de- 2001; Hoffmann et al., 2009) (Fig. 1). creased in hypertonicity, and these changes were not observed K1 Channels. K1 is a major intracellular osmolyte and in the absence of AQP1, indicating that channel activity follows contributes substantially to RVD. The osmosensitive K1 the changes in cell volume and not in osmolarity. Thus, Slick 1 1 fluxes permeate across a large variety of K channels, that may be a purely volume-sensitive K channel. Although Kca 4.1 are involved in multiple physiologic processes in the cell other channel was originally included in the Kca channel nomencla- than volume regulation, but are triggered via signals evoked ture, it is now known that it is activated by internal Na1 and by swelling. Four main classes of K1 channels are activated Cl– and not by Ca21. during RVD: voltage-gated K1 (Kv) channels, Ca21-activated The Volume-Regulated Cl– Channel. Volume-sensitive 1 1 – K (KCa) channels, inwardly rectifying K (KIR) channels, Cl fluxes play a key role in RVD; in contrast to the diversity of 1 1 – and two-pore–domain K channels (K2P) (Fig. 1). The kind of K channels implicated in RVD in various cell types, Cl fluxes channel involved differs in the various cell types and responds permeate across only one type of channel, the volume-regulated to one or several of the signals elicited by the volume change, anion channel (VRAC), also named volume-sensitive outwardly such as depolarization, membrane stretch, and elevation of rectifying anion channel. The channel is also referred to as 21 21 intracellular Ca [Ca ]i. In epithelial and other cell types, volume-sensitive organic osmolyte and anion channel to denote 21 swelling evokes a large increase in [Ca ]i, and RVD in these the possibility that it permeates organic osmolytes. VRAC will 360 Pasantes-Morales Downloaded from molpharm.aspetjournals.org

Fig. 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 at ASPET Journals on September 29, 2021 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 toward 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 diffuse pathways, possibly molecular variants of the VRAC. K+/Cl2 cotransporter also participates in the ion extrusion. (B) Time course of the changes in cell volume (gray solid line) and the efflux of 125I– as tracer for Cl– (u), 86Rb (tracer for K+)(n), and 3H-taurine (s). Data from cultured astrocytes (C) Swelling and cell volume recovery in mesenchymal cells exposed to a reduction in external osmolarity (unpublished). be the name used throughout this review. VRAC, widely The search for VRAC-specific blockers has been largely expressed in animal cells, is essentially inactive under isotonic unsuccessful, but the long list of nonspecific VRAC blockers conditions and opens slowly by cell swelling or by a decrease in (reviewed in Pedersen et al., 2016) include: 1) conventional anion ionic strength under isosmotic conditions (Emma et al., 1997; channel inhibitors, such as DCPIB, DIDS, SITS, NPPB, and the Voets et al., 1999; Pedersen et al., 2016). In biophysical terms, acidic di-aryl-urea NS3728; 2) transporter blockers, such as – – the Cl current (ICl swell) carried by VRAC exhibits mild fluoxetin, phloretin, and mefloquine; 3) the antiestrogens outward rectification and voltage inactivation at positive tamoxifen, clomiphene, and nafoxidine; 4) tyrosine kinase membrane potentials, and this differs among cell types (Nilius inhibitors, such as genistein, tyrphostin A23, and PD98059; 5) et al., 1994; Nilius and Droogmans, 2001; Okada, 2004; Akita purinergic receptor blockers suramin, reactive blue 2, and and Okada, 2014). This outward rectification defines VRAC and PPADS; and 6) carbenoxolone and riluzol. However, in addi- establishes clear differences from other volume-sensitive Cl– tion to inhibiting VRAC, these inhibitors block a variety of channels, such as channels with no rectification (maxi-anion other Cl– channels, transporters, or receptors to some extent channel), inward rectification (CLC2), steep outward rectifica- (Pedersen et al., 2016). The failure to find a selective VRAC tion (Cl–C3 and anoctamine), or no rectification (bestrophin). blocker has complicated efforts to establish the molecular The single-channel VRAC conductance is 50–80 pS at positive identity of this channel. and 10–20 pS at negative membrane potentials. VRAC exhibits The VRAC gating mechanisms are not fully understood, but a low field-strength anion permeability (I.Br.Cl.F). In this allosteric nonhydrolyzed-bound ATP and permissive cytosolic respect, VRAC differs from the ClC family members, which are Ca21 are necessary for channel activation. It has been pro- typically selective for chloride over iodide. VRAC permeates posed that G protein-mediated signal transduction pathways large molecules such as gluconate, glutamate, and benzoate, and membrane structures, such as caveolin, cholesterol, and and the pore size of this channel has been estimated to be about actin cytoskeleton influence the channel activation mecha- 11–17 Å (Nilius et al., 1997). nism. It has been suggested that the Ras-Raf-MEK-ERK Channels and Cell Volume 361 pathway and the Rho/Rho kinase are modulators of VRAC The possibility that LRRC8A constitutes the anion-conducting activity (Nilius et al., 1999). A volume-transduction pathway pore domain of VRAC is questioned (Akita and Okada, 2014) mediated by the epidermal growth factor receptor and NAD(P) on the basis of the lack of influence on currents across the H oxidase-derived H2O2 has been proposed as a signaling LCRR8 complexes of charged amino acid mutations in pre- pathway for VRAC activation (Varela et al., 2004). Epidermal dicted transmembrane domains. This suggests that the growth factor receptor is activated by hyposmolarity and is postulated VRAC structure may require a regulator molecule. an early signal that modulates osmolyte efflux pathways Decrease, and not an increase, in IClswell following over- (Pasantes-Morales et al., 2006). Additionally, reduction in expression of LCRR8A also is suggestive of a regulator ionic strength is a key element for VRAC activation; early molecule. Recently, Syeda et al. (2016) assembled models of studies by the Nilius group (Nilius et al., 1998; Sabirov different combinations of LCRR8A and LCRR8BD subunits in et al., 2000) demonstrated that reducing ionic strength which the pore-forming channels were incorporated into activates a Cl– current, with biophysical and pharmacolog- bilayers. Using this approach it was possible to demonstrate ical features identical to those of VRAC, even without cell that, as previously shown (Qiu et al., 2014; Voss et al., 2014), swelling. This mechanism of VRAC activation is of high the different combinations of LCRR8A with LCRR8B–D physiologic relevance. Under physiologic conditions, the subunits determine intrinsic channel properties, such as osmolality changes are small and gradual and, in most rectification, inactivation kinetics, and relative anion perme- cases, insufficient to generate significant changes in cell ability. The study also showed that reducing intracellular Downloaded from volume or in membrane distension, but the osmotic dis- ionic strength directly gates the channel, even in the absence equilibrium still should be corrected. Corrective Cl– fluxes of mechanical changes in the membrane or the cellular permeating across VRAC, activated by local changes in the components, such as cytoskeletal structures or elements of ionic environment, appear as the best option for an iso- signaling chains. The relevance of these findings is that volumetric regulation (see following). More direct evidence differently composed heteromers formed by combinations of on the role of ionic strength in VRAC gating is further LRRC8 subunits may result in a large variety of channels with molpharm.aspetjournals.org discussed in the context of the identification of LRRC8 different intrinsic properties and different regulatory mecha- proteins as components of the VRAC structure. nisms, which helps explain the differences in the inactivation In 2014, two independent groups (Qiu et al., 2014; Voss rate of VRAC in various cell types and the possibility of et al., 2014), using a similar fluorescence assay for a screen- permeating molecules as heterogeneous as the group of ing of human genome small-interfering RNA libraries, organic osmolytes. It also points to the possibility that one or identified a multispan transmembrane protein derived from some of the LCRR8 subunits forming VRAC contain the sensor a gene of unknown function named LRRC8A (leucine-rich for changes in ionic strength, the proposed channel gating repeat containing 8A). The LRRC8A subunit protein was (Cannon et al., 1998; Nilius et al., 1998; Sabirov et al., 2000). postulated to be an integral component of the VRAC struc- The relevance of these findings to define the molecular at ASPET Journals on September 29, 2021 ture. LRRC8A belongs to the LRRC8 family, which consists of identity of VRAC is extensively discussed in several excellent five members, LRRC8A–LRRC8E. All the LRRC8 proteins recent reviews (Pedersen et al., 2015, 2016; Stauber, 2015; have four putative transmembrane domains and contain Jentsch et al., 2016). up to 17 leucine-rich repeats. Studies by the groups men- Organic Osmolytes. Organic osmolytes, which contribute tioned earlier suggest that LRRC8 isoforms, similar to the to cell volume regulation in most animal cells (Kinne, 1993), pannexins to which they are related, organize in heteromeric are small molecules of heterogeneous structure that include complexes to form a functional channel with VRAC proper- amino acids and derivatives (taurine, glutamate, glycine, ties. It was also shown that the presence of LRRC8A in the GABA, and N-acetylaspartate), polyalcohols (myo-inositol multimeric complex is essential, but not sufficient alone and sorbitol), and amines (glycerophosphorylcholine, betaine, for VRAC activity, since at least one of the other LRRC8 creatine/P-creatine, and phosphoethanolamine). The concentra- isoforms should be present together with LRRC8A. Genetic tion of organic osmolytes varies in the different tissues; gluta- ablation of LRRC8B–LRRC8E did not separately affect mate, myo-inositol, creatine, taurine, and N-acetylaspartate are VRAC currents, but disruption of the five genes abolished present in the highest concentration in the brain (Verbalis VRAC activity. The functional channel can be restored by and Gullans, 1991), whereas glycerophosphorylcholine, betaine, expression of LRRC8A together with one of the other four myo-inositol, and sorbitol are the major organic osmolytes in LRRC8 subunits (Qiu et al., 2014; Voss et al., 2014). The renal cells (Beck et al., 1992). – largest reductions in ICl swell amplitudes are observed Organic osmolytes are important in the process of volume in LRRC8E2/2 and in LRRC8(C/E)2/2 cells and the lowest regulation; in contrast to ions, which generate adverse effects reductions in LRRC8B2/2, LRRC8D2/2 cells (Voss et al., that disturb the structure of macromolecules or affect neuro- 2014). Different combinations of LRRCA and other hetero- nal excitability, many organic osmolytes are compatible mers define intrinsic properties of the channel. Coexpression molecules. Thus, in the long term, organic osmolytes tend to of LRRC8A and LRRC8E induces faster inactivation at less replace ionic osmolytes (Verbalis and Gullans, 1991). In the positive potentials, whereas coexpression of LCRRC8A and nervous system, glutamate, g-aminobutyric acid, and glycine LRRC8C has the opposite effect; this coexpression markedly are an exception since they play a dual role as osmolytes and reduces the inactivation rate (Voss et al., 2014). Different neurotransmitters, and, therefore, changes in their concen- combinations of LCRR8 heteromers also strikingly modify tration at the extracellular space disturb nervous excitability VRAC permeability. As discussed in detail later, VRAC or lead to neuronal death by excitotoxicity (discussed later). permeability to taurine has a strict requirement of LCRR8A Taurine is particularly suitable for an osmolyte role since it is and LCRR8D, whereas LCRR8BCE subunits appear un- largely free in the cytosol, is not a protein amino acid, and necessary (Planells-Cases et al., 2015) (Fig. 2). participates in few reactions in the cell (Pasantes-Morales 362 Pasantes-Morales

Fig. 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– (s)is fast and inactivates rapidly, whereas taurine efflux (u) is sustained (see also Fig. 1B). (C) Effects on – 3 ICl swell and on H-taurine efflux of LCRR8 hetero- mer knockout in HEK cells. (D) Suggested LCRR8 multimeric composition of the two pathways, on the basis of the effect of LCRR8 heteromer knockout on ICl– and taurine efflux. ICl– is abolished

swell swell Downloaded from in LCRR8A2/2 (black), markedly reduced in LCRR8B2/2 and LCRR8C2/2 (gray), and mildly de- creased in LCRR8D2/2 and LCRR8E2/2 (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; Qiu et al., 2014; Voss et al., 2014; Planells-Cases et al., 2015. molpharm.aspetjournals.org at ASPET Journals on September 29, 2021 et al., 1998). Compared with other osmolytes, taurine shows Stutzin et al., 1999) (Fig. 2); Studies showed that I– efflux is the largest efflux (9- to 22-fold) and the lowest osmolarity fast and rapidly inactivating, whereas taurine efflux is slower threshold (Tuz et al., 2001), which could reflect a higher and sustained (Fig. 2). As clearly underlined in Shennan efficiency of the taurine efflux pathway or more availability of (2008) and Stutzin et al. (1999), if taurine and Cl– hypotonic taurine pools for release compared with osmolytes involved in fluxes were occurring via an identical pathway, such large other cell functions. differences in the time course should not be observed. Though The decrease in intracellular concentration of organic similar to VRAC-mediated currents, isotonic taurine efflux is osmolytes as part of RVD is accomplished largely by solute evoked by a decrease in ionic strength (Cardin et al., 1999; extrusion rather than by molecular degradation. Efflux of Guizouarn and Motais, 1999). The recent discovery of the organic osmolytes occurs via energy-independent, bidirec- LRRC8 family of proteins forming volume-sensitive hetero- tional leak pathways with net solute movements driven by meric channels with different permeability offers a real the concentration gradient (Hoffmann and Lambert, 1983; possibility to establish the molecular identity of the organic Sánchez-Olea et al., 1991). It has been reported consistently osmolyte efflux pathway(s). Studies by Qiu et al. (2014), Voss that organic osmolyte fluxes are greatly reduced by VRAC et al. (2014), and Planells-Cases et al. (2015) show that in blockers, but it is still not known whether Cl– and organic LRRC8A2/2 HEK, HCT116, and HeLa cells, the osmosensi- osmolytes permeate across a common pathway, most likely an tive taurine efflux is essentially abolished, stressing the anion channel like VRAC. The pore size of VRAC is sufficiently importance of this LCRR8 family isoform for the taurine large to allow such osmolytes as taurine and glutamate to efflux pathway, which is similar to VRAC (Qiu et al., 2014; permeate, and indeed currents carried by glutamate and Voss et al., 2014). Interestingly, also in 2014, a study by anionic taurine across VRAC have been demonstrated Hyzinski-García et al. (2014) showed similar results in (Banderali and Roy, 1992). However, taurine is an electro- cultured astrocytes from the mouse brain cortex. The rele- neutral zwitterion and at physiologic pH, has no net charge; vance of this study is that astrocytes from primary cultures therefore, it is unable to permeate across an anion channel. are not cell lines but originate from unmodified tissue. In this The same is true for polyols and most organic osmolytes. A respect it is also noteworthy that LCRR8A is present in a number of differences between VRAC and the osmosensitive variety of mouse tissues (Qiu et al., 2014). The effects of taurine efflux pathway have been documented (Lambert and genetic ablation of the different LCRR8 family isoforms on Hoffmann, 1994) and discussed in detail in reviews by IClswell andontaurineeffluxshowninstudiesbyVossetal. Shennan (2008) and Hoffmann et al. (2009). A most noticeable (2014) and Planells-Cases et al. (2015) revealed that de- – 125 – difference is the time course of Cl (traced by I ) and taurine letion of LCRR8A abolished both IClswell and taurine, efflux in various cell types (Pasantes-Morales et al., 1994; deletion of LCRR8E and C reduced IClswell (taurine efflux Channels and Cell Volume 363 was not examined), and deletion of LCRR8D did not affect Muller cells, TRPV4 is functionally linked to AQP4 (Benfenati IClswell but markedly decreased the hypotonic taurine et al., 2011; Jo et al., 2015), and this association is a requirement efflux (Planells-Cases et al., 2015). Comparison of results in for RVD. TRPV4 associates with AQP5 in acinar and salivary LRRC8(B,D,E)2/2 and LRRC8(B,C,D)2/2 cells confirmed that gland cells (Liu et al., 2006; Hosoi, 2016) and with AQP2 in LRRC8D is dispensable for IClswell but indispensable for renal cells (Galizia et al., 2008). taurine fluxes. As predicted by Stutzin et al. (1999) and The activation of TRPV4 by cell swelling is still unclear Shennan (2008), these results indicate that the combination (Pedersen and Nilius, 2007), but the following possibilities of LCRR8 heteromers, particularly the presence or absence have been proposed: 1) direct membrane stretch with integ- of the LCRRL8D subunit, confers two different molecular rins as intermediate molecule (Matthews et al., 2010); 2) entities. In fact, the LCRRL8D subunit, or lack of a subunit, interaction with supramolecular complexes containing regu- confers two different channels, one for permeation of anions latory kinases and cytoskeletal proteins (Becker et al., 2009); and the other with high permeability to a neutral molecule 3) interaction with the arachidonic acid metabolite 5,6-EET such as taurine. Although both channels have LCRR8A (Watanabe et al., 2003); and 4) phosphorylation by WNKs, as an integral element and are activated by swelling/ion particularly WNK4 (Fu et al., 2006). Results of TRPV4 genetic strength reduction, these channels exhibit different intrin- manipulation suggest that its role is not restricted to cellular sic properties, such as inactivation and permeability, sug- processes of volume regulation, but that it is also implicated gesting that the pore structure must be significantly in systemic osmosensing; TRPV4 genetic ablation alters Downloaded from different in these two molecular entities; a larger pore and functions, such as drinking behavior, serum osmolarity, differences in the amino acid residues between different and antidiuretic hormone plasma levels (Liedtke and combinations of LCRR8 subunits are necessary to permit Friedman, 2003). Osmosensing neurons and cells in kid- the passage of molecules like taurine and other neutral ney epithelium express TRPV4 and may represent its site of organic osmolytes. The predictable existence of a spectrum influence on systemic osmoregulation (Ciura and Bourque, of channels constructed by multiple combinations of LCRR8 2006; Cohen, 2007). molpharm.aspetjournals.org subunits is relevant to understanding the puzzling exis- Other members of the transient receptor potential channels tence of permeability pathways for organic osmolytes that family, such as TRPV1, TRPV2, TRPC1, TRPC5 and TRPC6, are not anions and have dissimilar molecular structure but TRPM3, TRPM7 are possibly involved in RVD; these channels are still gated by reduced ionic strength and inhibited by are osmotically activated channels, but detailed studies on VRAC blockers. their role in volume regulation are largely missing (Plant, The variety of LCRR8-based channels specific to perme- 2014). ate different osmolyte groups, including Cl–,representa useful tool to estimate the contribution of these different osmolyte pathways to cell-volume regulation. Presently, it at ASPET Journals on September 29, 2021 2 2 Isovolumetric Regulation has been shown that in LCRR8A / HEK293, HeLa, and HCT116 cells, RVD is attenuated, and in the cancer cell line In most animal species, extracellular osmolarity is tightly BHY, RVD is abolished (Qiu et al., 2014; Voss et al., 2014; regulated and abrupt, and large changes in osmolarity rarely Sirianant et al., 2016). These likely reflect differences in the occur. In contrast, small and gradual changes in osmolarity contribution to the regulatory process of other LCRR8A- associate with a number of metabolic reactions and cell independent mechanisms, such as the K1/Cl2 cotrans- functions. Exposing cells to small and gradual osmolarity porter KCC. Parallel analysis of the effect of LCRR8A reductions have shown that cells are able to maintain a genetic ablation, together with blockers of KCCs as in HeLa constant volume over a wide range of tonicities via an active cells in the study by Sirianant et al. (2016), would help to mechanism of continuous volume adjustment. This adaptive clarify this question. response, known as isovolumetric regulation (IVR), is accom- Transient Receptor Potential Channels. Transient re- plished by the efflux of intracellular osmolytes (Lohr and ceptor potential (TRP) channels are widely expressed in Grantham 1986, Mountian and Van Driessche, 1997; Souza vertebrate tissues and are cellular sensors for a large variety et al., 2000). The osmolytes involved in IVR are the same as in of stimuli. Most of the channels are nonselective cation RVD, i.e., Cl–,K1 and organic molecules. Fig. 3 illustrates as channels, with the exception of a few members that are an example the Cl– currents in glioma cells evoked by gradual Ca21-selective. TRPs of the mammalian superfamily are or sudden reductions in osmolarity (Fig. 3B). This Cl– current grouped into several subfamilies by sequence similarity activates early, at osmolarity reductions of about 3% and is (reviewed in Pedersen et al., 2005; Owsianik et al., 2006; blocked by niflumic acid and NPPB (Fig. 3B), which suggests Nilius and Owsianik, 2011; Nilius and Szallasi, 2014). A that it’s similar to the current across VRAC. This point should member of the vanilloid subfamily, the TRPV4 channel is an be clarified in light of new findings regarding the LCRR8 osmosensitive channel that contributes to RVD in numerous family members as integral elements of VRAC. K1 conduc- cell types (Vriens et al., 2004; Plant, 2014), including tance is also activated in these cells during IVR with chondrocytes (Lewis et al., 2011.), corneal epithelial cells (Pan different osmolarity thresholds in the presence or absence et al., 2008), keratinocyte cell lines (Becker et al., 2005), of Ca21 (Ordaz et al., 2004). In a variety of cell types, gradual salivary gland cells (Aure et al., 2010), and airway epithe- changes in osmolarity activate taurine efflux with different lial cells (Arniges et al., 2004). In these cell types, RVD is efflux threshold (Pasantes-Morales et al., 2000; Souza et al., Ca21-dependent and TRPV4 is proposed as the pathway 2000; Ordaz et al., 2004). The lowest efflux is observed in for Ca21 influx evoked by swelling and the ensuing activa- cerebellar granule neurons where taurine efflux increases tion of KCa channels involved in RVD (Fernández-Fernández significantly at osmolarity reductions of only 2 mOsm et al., 2002, 2008; Jin et al., 2012). In astrocytes and retinal (Fig. 3A) (Tuz et al., 2001). This compensatory mechanism 364 Pasantes-Morales

Fig. 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 on the cell are gradual and small, the cell volume remains un- changed, as result of an active process of osmolyte extrusion known as isovolumetric regulation (IVR). Efflux of taurine, glutamate, K+, and Cl–, all contrib- ute to the cell volume adjustment. Efflux thresholds in these cells were –2% and –19%, for [3H]taurine and 3

D-[ H]aspartate (as marker for glutamate), respec- Downloaded from tively, and –25% and –29%, respectively, for Cl– (125I) and + 86 – K ( 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 2

Cl channel blockers niflumic acid (NA) and NPPB molpharm.aspetjournals.org on the Cl2 current elicited by gradual reductions in osmolarity. GOR, Gradual osmolarity reduction; Vt/N0, represents volume changes with time. Details in Tuz et al., 2001; Ordaz et al., 2004. at ASPET Journals on September 29, 2021 is then likely to be preferred by neurons facing physiologic and diffuse pathways for organic osmolytes, in RVI, channels changes in cell volume. play only a minor role, and the regulatory process is essen- tially accomplished by cotransporters and exchangers. It has been proposed that unidentified hypertonicity-activated cat- Channel-Transporter Interactions in Regulatory ion channels participate in RVI (Wehner et al., 2006). A study Volume Increase in HeLa cells (Wehner et al., 2006) showing RVI reduction by Increase in external osmolarity activates intracellular SKF-96365, a blocker of TRP channels, raised the question of water exit driven by the osmotic disequilibrium, and, accord- whether the hypertonicity-induced cation channels are actu- ingly, this leads to a decrease in cell volume. If this is not ally TRP channels, and recent evidence pointed to TRPM2 as corrected, cells undergo apoptotic death. In healthy cells, the channel involved. A recent report, also in HeLa cells hypertonicity activates a regulatory mechanism that aims to (Numata et al., 2012), showed that nucleotides adenosine restore normal cell volume even if the external anisotonic diphosphate ribose (ADPr) and cyclic ADPr, reported as condition persists. This adaptive mechanism, known as typical TRPM2 activators (Pedersen et al., 2005; Pedersen regulatory volume increase (RVI) (Burg et al., 2007), occurs and Nilius, 2007), generate cation currents similar to those when mechanisms that increase the concentration of intracel- elicited by hypertonicity, previously known as hypertonicity- lular osmolytes are set in motion. This tends to re-establish activated cation channels. In the same line, small-interfering the osmotic equilibrium between extracellular and intra- RNA silencing of TRPM2 expression or the precursor enzyme cellular compartments. Electroneutral cotransporters and of the TRPM2 nucleotide activators abolish the hypertonicity- ion exchangers both participate in this process to in- elicited cation current and mildly reduce RVI. Cloning of crease the cytosolic levels of Na1 and Cl– (Fig. 4), and NCC and TRPM2 identified the DC–splice variant as the molecular Na1/K1/2Cl cotransporter (NKCC) family cotransporters (Arroyo entity corresponding to the nucleotide-activated current et al., 2013) and Na1/H exchangers are the main contributors (Numata et al., 2012), which suggests that TRPM2 may to RVI (Cala, 1980; Alexander and Grinstein 2006). Organic represent the molecular identity of the hypertonicity- osmolytes are also significantly involved in RVI (Fig. 4); cell activated cation channels. Another member of the TRP shrinkage increases the expression of Na1-dependent cotrans- channel family, TRPV2, seems to participate in RVI via an porters known to operate for various molecules acting as interesting connection with the electroneutral cotransporter organic osmolytes (Sánchez-Olea et al., 1992; Burg and NKCC, which as previously mentioned is a main effector in Ferraris, 2008). In contrast to RVD, in which the corrective RVI. The suggested mechanistic chain of events relating fluxes of osmolytes permeate essentially across ion channels TRPV2 channels and RVI involves a TRPV2-dependent Channels and Cell Volume 365

Fig. 4. Channels and transporters involved in RVI. (A) Schematic representation of channels and trans- porters involved in the volume adjustment after hypertonicity-induced cell shrinkage. (B) Cell vol- ume reduction and RVI in a renal cell line mIMCD3 exposed to media made hypertonic by addition of NaCl. (Inset table) Increase in intracellular osmolyte amino acid levels in astrocytes cultured in hyper- tonic medium. Details in Ruiz-Martínez et al., 2011; Sanchez-Olea et al., 1992. Downloaded from molpharm.aspetjournals.org accelerated depolarization followed by Ca21 release from Necrotic Volume Increase. Necrosis associates with the intracellular sources and the subsequent phosphoryla- extensive cell loss that accompanies pathologic conditions, tion of STE20/SPS1-related proline/alanine-rich kinases such as ischemia and ischemia/reperfusion, cardiovascular (SPAKs), which is essential for activation of NKCC. This diseases, and several neurodegenerative conditions. Cytotoxic proposed pathway is supported by studies in skeletal swelling in ischemia results from the arrest of oxygen- muscle showing that expression of TRPV2 negative domi- dependent ATP synthesis, which reduces the activity of the nant reduces the RVI efficiency at the time that depolar- Na1/K1 ATPase, impedes the transmembrane Na1/K1 ex- 1 ization is impaired and the Ca2 response is diminished changes, and results in dissipation of ion gradients. In the

(Zanou et al., 2015). Interestingly, the upregulation of all brain, swelling during ischemia is the first step in a cascade of at ASPET Journals on September 29, 2021 transporters involved in RVI, ions and organic osmolyte ion gradient disturbances between the intravascular and transporters, occurs by the concerted action of transcrip- extracellular compartments that culminates in blood brain tion factor tonicity-responsive enhancer binding protein/ barrier injury and vasogenic edema (Annunziato et al., 2013, osmotic response element–binding protein. As part of the Kahle et al., 2015). Besides ischemic swelling, brain cells also regulatory response to cell volume decrease, tonicity- swell during trauma, seizures, and spreading depression. responsive enhancer binding protein/ osmotic response Swelling is essentially owing to Na1,K1 and Cl– overload, element–binding protein increases the expression of osmo- which drives inwardly-directed water fluxes, and both chan- 1 protective genes, including Na -dependent transporters, nels and cotransporters participate in this process (Fig. 5). responsible for the accumulation of organic osmolytes Characteristic of brain ischemia is the marked increase in 1 1 (Ferraris and Burg, 2006). extracellular K (K o), which is often elevated from a 1 physiologic level of around 3 mM up to 60 mM. This high K o — Channels Involved in Volume Changes in level reverses the KCC direction, and together with the ischemia-activated expression of NKCC—results in large Necrotic and Apoptotic Death inward ion and water fluxes (Kahle et al., 2015). Na1 influx Changes in cell volume are characteristic features of cell also permeates across a nonspecific cation channel, which, on death and represent one of the main differences between the basis of recent evidence, is identified as transient receptor necrosis and apoptosis. In necrotic death, cell swelling and potential melastatin-4 channel (TRPM4). The TRPM4 pore is depletion of intracellular ATP are distinctive traits. During selective for monovalent cations with similar permeability for 1 1 the necrotic process, water accumulates in the cytosol and K and Na and is impermeable to divalent cations. Evidence 1 organelles, and the membrane surface architecture is de- in support of TRPM4 as a major mechanism for Na influx in formed by prominent, stationary blebs that eventually lead to ischemia includes the following observations: 1) Two main 1 membrane rupture and cell death (Jurkowitz-Alexander et al., regulators of TRPM4 (i.e., ATP and intracellular Ca2 ) are 1992; Barros et al., 2001). In contrast, in apoptosis, reduction deeply disturbed by conditions promoting necrosis. This de- 1 in cell volume is a central event and a hallmark in this regulation favors the opening of TRPM4, Na overload and program of cell death (Bortner and Cidlowski, 1998; Yu and NVI. 2) Redox imbalance by excessive generation of free Choi, 2000). Coined phrases for these cell volume changes radicals, a condition related to pathologies leading to necrosis, include “apoptotic volume decrease” (Hughes et al., 1997; induces a sustained activity of TRPM4 (Simon et al., 2010). 3) Maeno et al., 2000) and “necrotic volume increase,” also known Pharmacological or genetic manipulation of TRPM4 has a as oncotic swelling or cytotoxic swelling. A variety of channels marked influence on the swelling phase of the necrotic process. are involved in these changes in cell volume. 4) activation of TRPM4 channel by ATP depletion in COS-7 366 Pasantes-Morales

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 K+/Cl2 cotransporter reversal. Ischemia-induced ex- pression 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 further in- creases the extracellular glutamate levels. Overfunc- tion of the NMDA and AMPA neuronal receptors Downloaded from generates Na+ and Ca2+ overload, together with Cl– influx driven by VRAC and 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 3 glutamate (s) (traced by H-D-aspartate) efflux from cultured cortical astrocytes. (C) Glutamate efflux is molpharm.aspetjournals.org abolished by simultaneous treatment with the VRAC blockers DIDS and phloretin (Phl) and transporter blocker (TBOA). ISOS, isosmotic medium. Details in Perez-Dominguez et al. (2014). at ASPET Journals on September 29, 2021 cells leads to depolarization and progressive bleb formation consistent with the idea that the TRPM4 channel plays a role (Chen and Simard, 2001; Chen et al., 2003), but this is not in cell swelling and necrotic death. Other channels, including observed in cells lacking the channel (Simard et al., 2012). 5) members of the TRP family (TRPV4, TRPM2, and TRPM7) Induced upregulation of TRPM4 protein in endothelial cells and the acid-activated cation channels, also increase in from human umbilical vein causes Na1 overload, cell volume expression and functional activity after an ischemic episode, increase, and necrotic death, effects which are prevented by and their blockade reduces the size of the infarct. However, pharmacologic inhibition of the channel (Gerzanich et al., their role in inducing ischemic injury is more related to Ca21 2009; Becerra et al., 2011). It should be noted that ADP and overload and protease activation than to cytotoxic swelling. AMP are reported to block TRPM4b currents (Nilius et al., In brain cells, VRAC participates in cytotoxic swelling and 2004); however, during the first minutes of ischemia, ADP ulterior necrotic neuronal death, particularly in the penumbra levels decrease in parallel to the increase in ATP and AMP where intracellular ATP that is necessary for VRAC activation levels (Guarnieri et al., 1993; Phillis et al.,1995). Regardless, still remains. Neuronal death results from the increase in the concentration of AMP is so low (3% and 5% of ATP levels in extracellular glutamate driven by the reversal of Na1-energy- heart and brain, respectively) (Guarnieri et al., 1993; Phillis dependent transporters and the swelling-activated, VRAC- et al.,1995) that the increase may not affect TRPM4 activa- mediated glutamate efflux from swollen astrocytes (Mongin, tion. TRPM4 is not constitutively present in the central 2016). The consequent overactivation of AMPA and NMDA nervous system but is transcriptionally upregulated in neu- ionotropic receptors causes Na1 and Ca21 overload at the rons, astrocytes, oligodendrocytes, and microvascular endo- same time that the ischemic depolarizing condition activates thelial cells after the onset of ischemia (Gerzanich et al., 2009). VRAC in neurons, moving Cl– into the cell driven by its Necrotic death of endothelial cells caused by TRPM4 activa- electrochemical potential. All this culminates in NVI and a tion is also responsible for the capillary fragmentation, known necrotic cascade. The effect of VRAC blockers on reducing both as progressive hemorrhagic necrosis, observed in traumatic swelling and neuronal death (Inoue et al., 2007; Mongin, 2016) injury of the spinal cord. The capillary damage is prevented in suggests that VRAC has a role in this deleterious effect. TRPM42/2 or by antisense oligodeoxynucleotides directed Necrosis is a delayed pattern of excitotoxicity, but NMDA against TRPM4 (Gerzanich et al., 2009). TRPM4 channel also receptor overfunction also activates the apoptotic machinery contributes to cytotoxic swelling, cell death, blood-brain (Linnik et al., 1993). barrier breakdown, and vasogenic edema in ischemia and Hemichannels, the constituent elements of the Gap junc- trauma (Simard et al., 2006; Zweckberger et al., 2014; tions, allow transmembrane movements of large groups of Martinez-Valverde et al., 2015). Together, these findings are heterogeneous molecules of different sizes, such as water, Channels and Cell Volume 367

Na1,K1,Ca21, ATP, or adenosine, among others. Hemichannels Remillard and Yuan, 2004; Bortner and Cidlowski, 2007; are expressed in the membrane of astrocytes, neurons, Lang, 2007). The intermediate-conductance IKCa channels oligodendrocytes, and vascular endothelium. Unregulated mediate the apoptotic K1 current in lymphocytes, thymocytes, hemichannels have been implicated in hypoxia, ischemia, and glioblastoma cells, and the large-conductance BK chan- and brain trauma, where they generate disruption of in- nels play this role in artery smooth muscle cells, glioma, and tracellular ionic homeostasis and cytotoxic swelling. Con- superficial colonocytes, whereas inward rectifier IRK chan- sistent with this action, downregulation of hemichannels nels are involved in apoptosis in liver cells, HeLa cells, and reduce cell swelling in astrocytes and reduce the ischemic some neuronal cell lines (reviewed in Bortner and Cidlowski, injury (Davidson et al., 2015). 2007). Members of two-pore–domain potassium channels Apoptotic Volume Decrease. Apoptosis, or programmed TASK1 and TASK3 participate in apoptosis in cerebellar cell death, is a physiologic mechanism that commits cells to granule neurons and in exocrine pancreatic cells (Patel and individual death fate (Kerr et al., 1972). Apoptosis occurs Lazdunski, 2004). Hydrogen peroxide–mediated apoptosis is normally during development and aging as a homeostatic associated with activation of TWIK-related K1 (TREK) chan- mechanism to maintain optimal cell populations and elimi- nels and of hERG K1 channels. The significance of intracel- nate excessive or potentially harmful cells. Apoptosis is lular K1 decrease in the apoptotic process is more complicated characterized by changes in the cell structure, notably cell than simply an involvement as an osmolyte and a main nuclear condensation and DNA fragmentation, membrane contributor to AVD. The decrease in intracellular K1 levels Downloaded from blebbing, and the formation of apoptotic bodies. Programmed has per se an effect on apoptotic mechanisms, independent of cell death is activated by either intrinsic or extrinsic stimuli. AVD, and is more related to the release of cytochrome C (Yu, Intrinsic stimuli are molecules or situations of stress that 2003; Remillard and Yuan, 2004). Various K1 channel sub- result in mitochondrial dysfunction, like UV radiation, nitric types localized at the mitochondrial membrane regulate mito- oxide, staurosporine, thapsigargin, and dexamethasone, chondrial volume and contribute to the organelle homeostasis. whereas extrinsic stimuli are ligands of death receptors such Together with K1,Cl– efflux is an essential component of molpharm.aspetjournals.org as Fas or other members of the tumor necrosis factor recep- AVD, during which K1 efflux hyperpolarizes the cell and is tor superfamily, including CD95. As mentioned earlier, cell followed by a Cl– outward movement directed by its electro- volume decrease is a characteristic trait of apoptotic death, chemical gradient, which maintains the ionic balance and and depending on the cell type, the decrease in cell volume electroneutrality of the cell. Activation of anion currents may be in the range of 40–80%. The term “apoptotic volume facing intrinsic and extrinsic apoptotic stimuli is observed in decrease” was coined for this unique condition (Hughes et al., a variety of cells (Okada et al., 2006), and these anion currents 1997; Maeno et al., 2000). In terms of time-course, apoptotic are similar to those carried by VRAC regarding outward volume decrease (AVD) occurs in two phases, an initial phase rectification, ATP dependence, and pharmacological profile. that starts 0.5–2 hours after exposure to the inductor and a However, VRAC is activated by a swelling and ionic strength at ASPET Journals on September 29, 2021 late phase detected about 3 hours later. The two phases of reduction, whereas volume decrease and ionic strength in- AVD are accomplished by the same mechanism that includes crease characterizes apoptosis. Therefore, if VRAC partici- outward fluxes of K1 and Cl– moving across specific channels pates in AVD, the channel volume set point must shift to a and a decrease in osmolyte intracellular concentration and lower level or the channel is gated by other mechanisms. water outflow resulting in AVD (Kondratskyi et al., 2015). It Overproduction of reactive oxygen species, or a change in has yet to be determined whether this decrease in cell cytosolic ATP, both concurrent with AVD, may be signals to volume is a passive factor or an active signal in the induction either reduce the set point or to modify a hypothetic modulator of apoptosis, but evidence does exist that points to an (Shimizu et al., 2008). Although this critical point is not as yet explicit role for volume decrease in the apoptotic process; clarified, there is evidence showing that AVD induction is blockade of the main osmolyte pathways that reduce AVD reduced by VRAC blockers in various cell types. Although it impairs the progression of apoptosis (Yu and Choi, 2000; should be considered that those blockers are not specific for Grishin et al., 2005). VRAC or for Cl– channels. The recent findings identifying The increase in K1 currents that were detected as an early LCCR8A as a structural element of VRAC provides additional event in the cell death program (Yu et al., 1997) first suggested tools to define the VRAC contribution to AVD. The study by that K1 channels contribute to AVD. Regardless of the cell Planells-Cases et al. (2015) in KBM7 and HAP1 cells showed type or the inductor stimulus, the role of K1 channels as the that a channel formed by the combination of LCRR8A and K1 efflux pathway leading to AVD and loss of intracellular LCRR8D subunits participates in AVD and apoptosis. This potassium is recognized as a distinctive feature in the channel composition is similar to that of the channel proposed apoptotic program, (reviewed in Burg et al., 2006; Bortner to allow taurine to permeate, and dissimilar to the typical and Cidlowski, 2007; Orlov et al., 2013). A diversity of K1 VRAC. In contrast, in HeLa cells, Sirianant et al. (2016) channels mediate the K1 efflux during AVD in different exhibited no effect of LCRR8A knockdown in staurosporin- cell types; voltage-gated, Ca21-activated of intermediate induced cell shrinkage. and large-conductance K1 channels, inward rectifier, and The significant role of organic osmolytes in cell volume two-pore–domain K1 channels participate in AVD. Different regulation either in RVD and in RVI is well documented, as subtypes of Kv channels contribute to AVD, including Kv 1.2 discussed earlier. In contrast, evidence regarding organic and Kv 2.1 in neurons, Kv 1.3 in T-lymphocytes, and Kv1.5 in osmolyte contribution to volume decrease during apoptosis is COS-7 and in vascular smooth muscle. Kv channels of un- rather scarce (Wehner et al., 2003). However, the importance defined subtypes account for AVD and K1 loss in hippocampal of organic osmolytes was emphasized in the quantitative neurons, sympathetic neurons, cerebellar granule neurons, analysis made by Model (2014), which demonstrated that corneal epithelial cells, and cardiomyocytes (reviewed in the loss of organic molecules is necessary to account for the 368 Pasantes-Morales osmotically obligated water exit in AVD. Taurine cell loss is Authorship Contributions documented in apoptosis in Jurkat lymphocytes (Lang et al., Wrote or contributed to the writing of the manuscript: Pasantes- 1998) and in cerebellar granule neurons (Morán et al., 2000). Morales. Similar to VRAC, taurine efflux is evoked by cell swelling or ionic strength reduction, two conditions absent in apoptosis. References A number of studies report antiapoptotic effects of taurine Akita T and Okada Y (2014) Characteristics and roles of the volume-sensitive out- (Lambert et al., 2015), but this seems unrelated to AVD. wardly rectifying (VSOR) anion channel in the central nervous system. Neurosci- ence 275:211–231. Recent findings that demonstrate resistance to cancer chemo- Alexander RT and Grinstein S (2006) Na1/H1 exchangers and the regulation of therapy by a platinum-based drug, attenuated taurine efflux and volume. Acta Physiol (Oxf) 187:159–167. Andronic J, Bobak N, Bittner S, Ehling P, Kleinschnitz C, Herrmann AM, Zimmermann reduced expression of LCRR8D, suggest a role for the LRRC8D- H, Sauer M, Wiendl H, Budde T, et al. (2013) Identification of two-pore domain containing VRAC entity in drug extrusion (Planells-Cases et al., potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes. Biochim Biophys Acta 1828:699–707. 2015; Voets et al., 2015). As for apoptosis, the normotonic ac- Annunziato L, Boscia F, and Pignataro G (2013) Ionic transporter activity in astro- tivation of VRAC under these conditions remains to be defined. cytes, microglia, and oligodendrocytes during brain ischemia. J Cereb Blood Flow Metab 33:969–982. Comparing RVD and AVD mechanisms demonstrates that Arniges M, Vázquez E, Fernández-Fernández JM, and Valverde MA (2004) Swelling- 1 essentially the same type of K channels participate in both activated Ca21 entry via TRPV4 channel is defective in cystic fibrosis airway epithelia. J Biol Chem 279:54062–54068. processes in the same cell type. For instance, voltage- Arroyo JP, Kahle KT, and Gamba G (2013) The SLC12 family of electroneutral Downloaded from dependent Kv channels are those involved in both RVD and cation-coupled chloride cotransporters. Mol Aspects Med 34:288–298. 21 1 Aure MH, Røed A, and Galtung HK (2010) Intracellular Ca21 responses and cell AVD in neurons, whereas Ca -activated K channels are volume regulation upon cholinergic and purinergic stimulation in an immortalized implicated in epithelial cells. This selectivity is explained in salivary cell line. Eur J Oral Sci 118:237–244. Banderali U and Roy G (1992) Anion channels for amino acids in MDCK cells. Am J the thermodynamic analysis of ion fluxes described in Orlov Physiol 263:C1200–C1207. et al. (2013). The analysis in Orlov’s review refers only to AVD, Barros LF, Stutzin A, Calixto A, Catalán M, Castro J, Hetz C, and Hermosilla T but appears valid also for K1 and Cl– flux kinetics in RVD. (2001) Nonselective cation channels as effectors of free radical-induced rat liver cell necrosis. 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