Channels and Volume Changes in the Life and Death of the Cell Molpharm.Aspetjournals.Org Herminia Pasantes-Morales

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Channels and volume changes in the life and death of the cell molpharm.aspetjournals.org Herminia Pasantes-Morales

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

México, Mexico City, Mexico

at ASPET Journals on October 1, 2021

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 Downloaded from 0155 5622 5588 Fax. 0155 5622 5607 e-mail: [email protected] molpharm.aspetjournals.org

Text pages: Table number: 0 Figure number: 5 Abstract words: 196 Text words: 7513 at ASPET Journals on October 1, 2021 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, 4,4-diisothiocyanostilbene-2,2-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, 2+ + + + Ca -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.

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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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.

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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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, molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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, Downloaded from 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 molpharm.aspetjournals.org 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; at ASPET Journals on October 1, 2021

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-

+ 2+ + + gated K (Kv) channels, Ca -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

2+ 2+ depolarization, membrane stretch, and elevation of intracellular Ca [Ca ]i. In epithelial and

2+ other cell types, swelling evokes a large increase in [Ca ]I, and RVD in these cells is consistently

2+ + Ca -dependent due to the predominant role of KCa channels in the volume-corrective K fluxes. Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

+ 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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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). Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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

- 2+ nomenclature, it is now known that it is activated by internal Na⁺ and Cl and not by Ca . molpharm.aspetjournals.org

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 at ASPET Journals on October 1, 2021 (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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

(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). Downloaded from 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 molpharm.aspetjournals.org 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. at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org

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). at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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) Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

(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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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

2+ Ca influx evoked by swelling and the ensuing activation of KCa channels involved in RVD Downloaded from (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; molpharm.aspetjournals.org

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 at ASPET Journals on October 1, 2021 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). Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org 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, at ASPET Journals on October 1, 2021 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. Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org 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); at ASPET Journals on October 1, 2021 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; Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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; Downloaded from 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 molpharm.aspetjournals.org 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, at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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. Downloaded from 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, molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 efflux from swollen astrocytes (Mongin, 2016). The consequent overactivation of AMPA and

NMDA ionotropic receptorscauses Na+ and Ca2+ 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+, Ca2+, ATP, or adenosine, among others. Hemichannels are expressed in the membrane of astrocytes, neurons, oligodendrocytes, and vascular endothelium. Unregulated hemichannels have been implicated in Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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, molpharm.aspetjournals.org 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, at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org

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, at ASPET Journals on October 1, 2021 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⁺ Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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. Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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 Downloaded from 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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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 Downloaded from moves following its concentration gradient and the cell swells. Immediately after, intracellular signals activate the channels and transporters mobilizing the main intracellular osmotically active molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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 125I- as tracer for Cl- (), 86Rb (tracer for K+)(Δ) and 3H-taurine (). 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- () is fast and inactivates rapidly, while taurine efflux ()

- 3 is sustained (see also Fig. 1B). C. Effects on ICl swell and on H-taurine efflux of LCRR8 heteromer knockout in HEK cells. D. Suggested LCRR8 multimeric composition of the two pathways, based on Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

- - 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.

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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 molpharm.aspetjournals.org 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 at ASPET Journals on October 1, 2021 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)

86 - and 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 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 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. MOL#104158

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- Downloaded from 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 molpharm.aspetjournals.org

NMDA and AMPA neuronal receptors generates Na+ and Ca2+ 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. at ASPET Journals on October 1, 2021 Time course of astrocyte swelling (gray solid line) and glutamate () (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.

Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2021 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2021 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2021 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2021 Molecular Pharmacology Fast Forward. Published on June 29, 2016 as DOI: 10.1124/mol.116.104158 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2021