Mechanisms of Activation of LRRC8 Volume Regulated Anion Channels
Sara Bertellia,b Alessia Remigantea Paolo Zuccolinia Raffaella Barbieria
Loretta Ferreraa,c Cristiana Piccoa Paola Gavazzoa Michael Puscha
aIstituto di Biofisica, Consiglio Nazionale delle Ricerche, Genova, Italy, bScuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy, cU.O.C. Genetica Medica, Istituto G. Gaslini, Genova, Italy
Key Words
LRRC8 • Volume regulation • Phosphorylation • Ionic strength
Abstract
The structure of LRRC8 VRAC channels
The composition of the intracellular milieu is vastly different from that of the immediately adjacent extracellular environment in cells from all organisms. Highly specialized transmembrane proteins determine the tightly controlled transport of substrates across the plasma membrane. In this review we are considering transport mediated by so-called volume regulated anion channels (VRACs). A most intriguing feature of VRACs is their involvement in disparate processes including volume regulation, cell to cell signaling, metabolic sensing, apoptosis, membrane potential regulation, and chemotherapeutics uptake.
Anion currents activated by extracellular hypotonicity have been first described by Lewis and Cahalan in T lymphocytes [1] and by Hazama and Okada in intestinal epithelial cells [2]. Since then, similar “volume regulated anion channels” (VRACs) have been described and extensively characterized in practically all vertebrate cell types [3-6]. Several names have been assigned to such hypotonicity activated anion currents [3, 6]. Here, for simplicity, we will stick to the “VRAC” nomenclature, and refer to the reviews by Jentsch and Strange et al. regarding other nomenclatures [3, 6].
In 2014 two independent groups identified the “Leucine Rich Repeat Containing 8 A” (LRRC8A) gene as encoding an essential VRAC subunit [7, 8]. Voss et al. additionally showed that VRACs are heteromeric proteins that contain the LRRC8A subunit in an obligatory manner, and additionally one or more subunits encoded by the homologous genes LRRC8B-E [7]. Single-molecule photo bleaching studies and biochemical assays suggested that the stoichiometry is not fixed and that VRAC channels may be composed of more than two different subunits [9, 10]. LRRC8 proteins are about 800 amino acids long. The first ~400 amino acids are organized in four transmembrane domains and show homology to pannexin channel subunits [11], while the last ~400 amino acids are unique to LRRC8 proteins and contain 17 cytosolic leucine rich repeats (LRRs) [11]. Four different high-resolution cryo-EM structures of homomeric LRRC8A assemblies and lower resolution structures of LRRC8A-LRRC8C heteromers [12-15] fully confirmed the predictions of Abascal & Zardoya [11]. Three of these structures were obtained from detergent solubilized proteins [12-14]. The structure obtained by Deneka et al. [12] is illustrated in Fig. 1A. The protein can be divided in several domains including an extracellular domain (ED), which is not fully resolved, and in which several beta strands are stabilized by intra-subunit disulfide bonds. The ED forms the most narrow part of the pore. The transmembrane domain (TMD) is formed by four helices (TM1-TM4) from each subunit with TM1 and TM2 contributing to pore lining with mostly hydrophobic residues. The ED and TMD exhibit six-fold symmetry as illustrated in Fig. 1B and the protein fold is similar not only to that of pannexins but also to that of connexins, even though there is no sequence homology with connexins [11, 12]. The cytosolic subdomain (CSD) is located below the membrane and is formed by the intracellular loop connecting TMD2 and TMD3 and the stretch connecting TMD4 with the leucine rich repeat domain (LRRD). The CSD exhibits a complex fold and the six fold symmetry of the ED and TMD is broken at the level of the CSD resulting in a three-fold symmetry at the level of the LRRD (according to three out of four published structures - see below), which form a trimer of dimers (Fig. 1C). Several, possibly important parts of the protein are not resolved, including the first 14 residues at the N-terminus, a 23 amino acid stretch of the ED and a relatively long stretch in the intracellular loop connecting TMD2 and TMD3 in the CSD [12]. All three structures obtained using detergent solubilized protein are similar [12-14]. The structures reported by Kern et al. exhibit some differences to these and were obtained using protein incorporated into lipid nanodiscs [15]. Classification of the cryo-EM images predicted two different structures, a “constricted” structure and a “relaxed” structure, which might be related to gating movements [15]. Surprisingly, the “nanodisc structures” are considerably different from the “detergent structures” in two key aspects. First, in the nanodisc structures the TMD is more loosely packed, with several lipid molecules intercalating into the membrane pore wall. Second, the nanodisc structures are overall six-fold symmetric, i.e. do not show a trimer of dimers configuration of the LRRD [15]. Which of the two classes of structures is more relevant to native heteromeric LRRC8 structures remains to be determined.
It needs to be underlined that all four high resolution structures are obtained from non-physiological homomeric LRRC8A complexes. Even though homomeric LRRC8A channels exhibit residual function [12, 13, 15], the functional properties appear to be quite different from those of physiological VRACs [16]. For example currents obtained from LRRC8 KO cells transfected with only LRRC8A are poorly volume sensitive and also much less sensitive to low intracellular ionic strength than regular VRAC channels [16, 17]. Interestingly, Yamada & Strange found that homomeric functional volume-sensitive channels could be formed by LRRC8C, D or E subunits in which the intracellular loop connecting TMD2 and TMD3 was replaced with that from LRRC8A [16]. The necessary region to achieve functional homomeric channels could be narrowed down to the stretch from residues D182 to D206. Such a homomeric construct could be useful to obtain a structure of a functional homo hexameric channel.
More recently the structure of a homomeric LRRC8D assembly was resolved using cryo-EM of protein solubilized in detergent [18]. Even though the overall architecture is similar to that of the homomeric LRRC8A structures, the homo-hexameric LRRC8D structure exhibits 2-fold dimer of trimers symmetry, both in the membrane as well as the intracellular domains [18]. The outer pore entry is wider compared to the structures of homomeric LRRC8A, consistent with the higher permeability of LRRC8D containing heteromeric LRRC8A/LRRC8D complexes for organic osmolytes [9, 19, 20]. Yet, similar to the homomeric LRRC8A structures, the relevance of the homomeric LRRC8D structure remains to be determined.
Emergent physiological roles of LRRC8 VRAC channels
The fact that VRACs are activated by hypotonicity indicates that the channels are important for cellular volume regulation, and indeed pharmacological inhibition impairs the ability of cells to achieve a regulatory volume decrease (RVD) [3, 4, 21]. This result was fully confirmed in LRRC8A knock-out cells [7]. In addition to chloride, VRACs are permeable to small organic, mostly anionic or uncharged osmolytes like taurine [4, 6, 9, 10, 22]. This permeability likely plays an important role in volume regulatory processes like RVD or apoptotic volume decrease (AVD) [23]. In addition, release of excitatory amino acids or ATP through VRAC channels has been proposed to be important for signaling and toxicity in glia and other cell types [24-26].
Mice in which Lrrc8a is knocked out are characterized by increased mortality before and after birth and have several severe other defects including growth retardation and defective lymphocyte function [27]. However, the severity of the phenotype allows little conclusions because indirect effects cannot be distinguished from specific ones. Since the discovery that LRRC8 proteins constitute VRAC, more specific knockout strategies have revealed novel functions of LRRC8 proteins. Several of these have been excellently reviewed by Chen et al. [28]. Among others, these include resistance to cancer drugs [20, 29-31], insulin secretion [32, 33], spermiogenesis [34, 35], astrocyte mediated signaling [36-38], and ATP release [39]. In astrocytes, an additional role has been proposed regarding the interaction of LRRC8 with the cell adhesion molecule GlialCAM and the membrane protein of unknown function MLC1, mutations of which lead to megalencephalic leukoencephalopathy with subcortical cysts (MLC) [40].
More recently discovered roles include myogenesis [41, 42], glucose metabolism [42], reduction of intracellular chloride concentration [43], release and uptake of cyclic di-nucleotides, which are important in the STING defense pathway [44, 45], and the permeability to glutathione proposed to modulate epithelial-to-mesenchymal transition [46]. Interestingly, the involvement of VRAC in migration and proliferation [3, 4] could not be confirmed in several cell lines [47]. This important aspect requires further investigation. Further physiological roles of VRAC, possibly related to specific LRRC8 subunits, are likely to emerge in the future.
Several functional roles of LRRC8 proteins appear to be related to VRAC’s ability to decrease cell volume by allowing chloride and osmolyte efflux. For example, in spermiogenesis, cells have to reduce their volume and LRRC8A defects lead to swollen plasma and deformations of late spermatids [34]. In contrast, other functions appear to be unrelated to changes in cell volume but closely linked to the ability of VRACs to translocate specific large anionic molecules, like cyclic di-nucleotides [44, 45] or excitatory amino acids [36, 37].
In any case, it is clear that cells have to control tightly the activity of VRAC, because excessive activity would lead to the loss of cellular small organic molecules and would induce cell shrinkage. Indeed, transfection of constitutively active VRAC constructs lead to shrunken cells that could not anymore be patch-clamped [16]. In addition, an unexpected role of VRAC for lysosomal ion homeostasis was recently proposed [48], and this finding significantly extends the possible roles of VRAC for cellular physiology.
Surprisingly, despite these important physiological and pathophysiological insights, and the necessity of cells to tightly control VRAC activity, the fundamental question of which are the molecular and cellular mechanisms that underlie the activation of VRAC are incompletely understood. One of the few certain aspects regarding these mechanisms is that VRAC activation does not involve exocytotic insertion of “novel” VRAC channels into the plasma membrane [49]. In the following sections we first review which structural parts of VRAC proteins have been implicated in channel activation, and finally we provide an overview on the proposed mechanisms of VRAC activation and modulation in various cell types.
Structural parts of LRRC8 proteins involved in channel activation
Classically, ion channel functionality is described in terms of “closed” and “open” states, i.e. the channel can be in a non-conducting, closed state or in a partially or fully open state [50]. This paradigm has been confirmed for VRAC channels both in native systems and in heterologous expression systems by single channel recordings [9, 51-55]. The transition between closed and open states, called channel activation or channel gating, necessarily implies conformational changes of the channel protein complex. The question of how VRAC activation occurs can be further divided into three overlapping sub-questions:
1) Which protein domains are critically important for channel activation?
2) Which are the relevant cellular stimuli for VRAC activation?
3) How are these cellular stimuli conveyed to the relevant protein segments?
We first summarize the current knowledge on protein domains implicated in channel gating.
An extracellular region involved in channel inactivation
A distinctive feature of VRACs composed of different subunits is the degree and kinetics of current inactivation at positive voltage, as illustrated in Fig. 2. LRRC8A/LRRC8E complexes exhibit profound and rapid inactivation at positive voltages [7, 9] (Fig. 2A), similar to what is seen for example in endogenous VRAC in glioblastoma cells [56] (Fig. 2D), and also in several others. Conversely, LRRC8A/LRRC8D channels exhibit similar but slower and less pronounced inactivation [7, 9] (Fig. 2B), which further decreases in LRRC8A/LRRC8C heteromers [7, 9] (Fig. 2C), similar to VRAC channels in lymphocytes [57, 58] or Panc-1, a cell line derived from pancreatic duct carcinoma (Fig. 2E).
The physiological relevance of the inactivation is unclear, however the comprehension of this phenomenon is important to elucidate the general gating mechanisms of VRAC. Exploiting the kinetic differences between LRRC8A/8E and LRRC8A/8C heteromers and employing a chimeric strategy, Ullrich et al. identified the C-terminal half of the first extracellular loop as a major determinant of VRAC inactivation [19]. In Fig. 2F the corresponding region is highlighted in red in one LRRC8A subunit of the homomeric structure determined by Kefauver et al. [13]. The residue R103, which defines the narrowest part of the pore, is highlighted in pink and the residue corresponding to D102 of LRRC8C, the most “extracellularly” exposed residue within the resolved part of the homomeric LRRC8A structure and a major determinant of inactivation [19], is highlighted in green (Fig. 2F). Thus, the functional results of Ullrich et al. [19] and the structural information suggest that inactivation involves a conformational change of the external pore constriction. We speculate that inactivation reflects a closure of the outer pore entrance. Whether positive voltages induce inactivation directly by acting on charged VRAC protein segments, or indirectly, for example by interaction of permeating substrates with the protein remains to be investigated.
While inactivation might be relevant to determine the degree of VRAC channel activity in specific physiological settings, it is unlikely that the inactivation process is mechanistically related to the general mechanism of VRAC activation by hypotonicity. However, as we still don’t know the basic VRAC activation mechanism, a possible involvement of the inactivation process should be kept in mind.
Activation involves a separation of the C-terminal LRRDs
Compared to connexins and pannexins, a unique structural feature of LRRC8 subunits is their extended C-terminal cytosolic LRRD. Thus, since channels formed by LRRC8 subunits are volume sensitive, while connexins and pannexins are not, it is likely that the LRRDs are involved in VRAC activation. Two lines of evidence suggest indeed that channel activation is accompanied by a widening, i.e. increased spatial separation of the LRRDs. First, attaching fluorescent proteins like GFP or mCherry to the C-terminus of LRRC8 subunits resulted in channels that showed a constitutive activity in the absence of hypotonic stimulation [9, 59]. Addition of such ~27 kD proteins to each of the six subunits is expected to induce significant sterically induced displacement of the C-termini. Fig. 3 highlights the relative size of a GFP molecule with respect to the LRRDs. Importantly, these tagged channels could be further stimulated by hypotonicity demonstrating that tagging did not fully open the channels and that the basic mechanism of channel activation remained intact. Interestingly, attaching a 3xHA epitope to the C-termini led to a much smaller activation [9] (see Fig. 3 for a comparison in size between a 3xHA epitope and GFP), thus suggesting that the size of the tag, and the degree of LRRD separation determine the degree of channel activation.
A second line of evidence that LRRD separation or reorientation accompanies channel activation was provided by FRET measurements [60]. König et al. attached FRET donor and acceptor fluorescent proteins to the C-termini in different LRRC8 subunits and measuring FRET in transfected cells in response to hypotonic stimulation they observed a robust decrease of the signal between the donor/acceptor pairs [60].
Is low ionic strength the relevant physical-chemical parameter inducing VRAC activation?
Despite the nomenclature “volume-regulated anion channel”, it is clear that volume per se, not being an intensive physical parameter, cannot be directly involved in channel activation. Assuming that VRACs are directly activated by a hypotonic shock (and not via interaction with other proteins or phosphorylation, as discussed below), in principle other intensive parameters related to cell volume like membrane tension, concentration of a specific molecule or cytosolic ionic strength (among many other possibilities) have to be considered as relevant parameters [61]. Intuitively, the idea of a mechanical activation of VRAC through membrane stretch might appear as the most reliable hypothesis, given the nature of a channel activated by an increase in cell volume. However, several studies argued that cells have sufficient amounts of extra-plasma membrane in form of invaginations which may unfold upon hypotonic challenge. Based on these observations, a direct activation of the channel by stretch is unlikely [62, 63]. Importantly, VRAC can indeed be activated by low intracellular ionic strength in isotonic conditions [55, 64-67]. Even recombinant LRRC8 proteins incorporated in lipid droplet bilayers were shown to be activated by low ionic strength [68]. Since in the droplet bilayer system a purified protein had been employed, the latter result demonstrated that low ionic strength directly acts on the channel protein [68]. Since the LRRDs contain numerous positively and negatively charged residues, one hypothesis is that low ionic strength alters the electrostatic interaction of the LRRDs because the charges are less shielded by solvent ions, leading to channel opening.
However, as discussed in detail by Strange et al. [6], it is unlikely that low ionic strength is indeed the physiologically relevant stimulus. First, the reductions of ionic strength necessary to activate VRAC in patch-clamp recordings is much larger than what would be expected from physiological activation by hypotonicity. Second, in whole cell patch clamp recordings in normal ionic strength, even upon application of a hypotonic extracellular solution, the intracellular ion concentrations are practically “clamped” through the patch pipette, even close to the membrane [6] (Bertelli & Pusch, unpublished results). Thus, in these conditions, activation of VRAC occurs without reduction of intracellular ionic strength. Third, VRAC can be activated in a variety of conditions not associated with alteration of ionic strength or extracellular osmolarity. For example application of GTPγS though the patch pipette activates VRAC in chromaffin cells [69]. The potential physiological role of intracellular ionic strength needs to be determined.
Intracellular and extracellular loops are involved in VRAC activation
By extensive mutagenesis and chimeric constructs, Yamada & Strange [16] found that the extracellular loop connecting TMD1 and TMD2 and the intracellular loop (IL) connecting TMD2 and TMD3 are essential for VRAC function. Several chimeric constructs showed changes in inactivation properties, or exhibited constitutive activity. Most interestingly, homomeric functional channels could be obtained by a chimera of LRR8C containing the IL of LRRC8A [16]. The chimera gave rise to a certain degree of constitutive activation, which could be further stimulated by hypotonicity [16], similar to the addition of GFP tags at the C-terminus [9]. Fig. 4 highlights the IL in the structure of homomeric LRRC8A in orange. Yamada & Strange [16] could narrow down the sequence to an essential region of IL sufficient to generate functional homomeric chimeric LRRC8C channels to the stretch comprising residues 182-206 (numbering referring to LRRC8A). Surprisingly, this region is not resolved in any of the homomeric LRRC8A structures [12-15] nor in the structure of homomeric LRRC8D [18] (Fig. 4).
Activation of LRRC8A-LRRC8E heteromers by oxidation
VRAC activity is also dependent on reactive oxygen species (ROS) in several cellular systems [70-72]. In addition, hypotonicity has been reported to lead to increased ROS production [73]. On the other hand, VRAC activation impacts on the oxidation status, probably by the release of reduced glutathione [46]. An important question is whether LRRC8 proteins are directly or indirectly affected by oxidation. Gradogna et al. [58] found that LRRC8 channels are differentially affected by oxidation depending on the subunit composition. While LRRC8A/LRRC8C and LRRC8A/LRRC8D heteromers are inhibited by chloramine-T oxidation, LRRC8A/LRRC8E heteromers are dramatically activated by chloramine-T and tert-butyl hydroperoxide mediated oxidation [58]. In addition, using membrane permeable and impermeable cysteine modifying reagents the authors concluded that oxidation of intracellular cysteines is responsible for the effect. The relevant cysteines have yet to be identified.
Phosphorylation as an essential event for VRAC activation: an open question
Another event that has often been considered among the mechanisms inducing VRAC activation is phosphorylation. Few years after its discovery, in the early 90’s, several laboratories spent their efforts to identify subfamilies of kinases and putative upstream pathways involved in the modulation of the channel.
Many studies linked tyrosine phosphorylation and VRAC activation upon swelling, showing that various protein tyrosine kinase (PTKs) inhibitors (like e.g. tyrphostin B46, tyrphostin A25 and genistein), when added prior to the hypotonic stimulus, could prevent in a time- and concentration-dependent manner the activation of a current with a biophysical and pharmacological profile of VRAC [74-78]. However, it remained unclear which tyrosine kinase is specifically responsible for the hypotonicity-induced phosphorylation. Lang and collaborators found that the transfection of p56lck, a gene encoding a member of the Src family, involved in the maturation of developing T cells, could restore the osmotic activation of VRAC in lck-deficient lymphocytes [79].
In line with the hypothesis of a PTKs activator effect, it was found that the protein tyrosine phosphatase (PTP) inhibitors, Na3VO4 and dephostatin potentiated VRAC when applied after activation of the current by a mild hypotonic shock [76, 77]. Overall, these data suggested a reversible role of tyrosine phosphorylation as a critical step in the regulation of VRAC activity. However, despite the apparent consistency of these conclusions it has to be highlighted that other studies could not detect any effect of such PTK inhibitor compounds on VRAC [80-83]. To make the whole story even more contradictory, other studies reported an increase of VRAC in atrial and ventricular myocytes upon the application of PTK inhibitors [84, 85], and protein tyrosine phosphatase (PTP) inhibitors were shown to suppress VRAC in bovine chromaffin cells and mouse fibroblasts [86, 87].
Even though at first these findings suggested an inhibitory effect of PTK on VRAC, further experiments in myocytes unveiled an antagonistic mechanism of two distinct PTK families, with Src and EGFR kinases showing inhibitory or activating effects, respectively [85]. It was concluded that an interplay of PTKs and their corresponding signaling cascades differentially modulates the gating of the channel in ventricular myocytes. Table 1 summarizes kinase effects and the respective cellular background investigated.
Several hypotheses to justify the difficulty in determining a unique effect of the phosphorylation state of tyrosine residues on VRAC activity have been proposed so far. First, it was suggested that regulatory processes may be connected to tissue- or species-specific mechanisms [62]. Also, broad-spectrum PTK inhibitors, as the most commonly used genistein, can mask antagonistic effects of specific PTKs subfamilies, given that in the very same cell line divergent effect were observed [85]. It has to be stressed that all the above studies were performed before the discovery of the heteromeric nature of VRAC channels and thus, one possible explanation of divergent results may partially rely on different cell-type specific channel composition and stoichiometry.
A recent important contribution came from the group of Stauber, who used a FRET sensor to monitor VRAC activation. They proposed protein kinase D (PKD) activity as a possible candidate that is critically involved in hypotonicity-induced activation of VRAC [60]. This kinase converts transient diacylglycerol (DAG) signals into physiological effects downstream of PKC, suggesting that serine/threonine phosphorylation may be the final event for VRAC regulation.
There is a large body of literature on a primary role of PKC in VRAC activation. Here, we can only highlight a selected number of contributions. As a general remark it has to be reminded that the use of different drugs to interfere with PKC signaling may lead to different observations as shown by Mongin and colleagues [88, 89]. Application of PKC blockers was able to prevent channel activation under hypotonic conditions, which could be rescued by the PKC activator phorbol 12-myristate,13-acetate (PMA) [90-93]. In particular, two independent studies provided a direct indication of a swelling-induced translocation of PKC-α in the membrane/cytoskeleton fraction and its subsequent phosphorylation in the cell membrane [91, 93]. Furthermore, experiments with a cell line stably expressing kinase dominant negative PKC variants suggested an essential role of PKCα isoforms in swelling-dependent VRAC activation [93].
The molecular identification and the recent determination of VRAC structures may provide a solid tool to revisit previous finding, also taking advantage of the availability of cell lines depleted of endogenous LRRC8 genes. This may help in addressing unsolved questions, on top of which is whether phosphorylation constitutes an obligatory part of swelling induced VRAC activation. Indeed, phosphorylation is one of the first reactions to swelling [22, 76, 94]. However, it remains to be established whether the target of phosphorylation is VRAC itself or another regulatory protein [95]. Structure function studies could be key to uncover residues that may be the final target where single, or different signaling pathways may convey.
Few years before the molecular identity of VRAC was known, in a pioneering work, Abascal & Zardoya not only correctly predicted LRRC8 proteins to form hexameric ion channels, but highlighted also the importance of the intracellular loop connecting the TM pore to the LRR domain as a potential target of post-translational modification [11]. In particular, given the abundance of serine, threonine and tyrosine amino acids in this region and taking advantage of high-throughput proteomic studies, they speculated that such residues in the intracellular loop may undergo phosphorylation.
Recently, as already mentioned above, the group of Strange identified a sequence of the first intracellular loop of the LRRC8A subunit (8A IL), as essential for channel functioning [16]. Their data show that the homomeric chimera of 8C was still functional after the intracellular loop was replaced with residues from 8A IL. Most interestingly, a short minimal stretch of 8A IL was sufficient to give rise to functional homomeric chimeric LRRC8D(8A IL) and LRRC8E(8A IL) as well. From a PONDR analysis (Predictor of Natural Disordered Regions), Yamada & Strange uncovered that this region of the IL, which is not resolved in any of the structures described so far, significantly diverges among LRRC8A, LRRC8C, LRRC8D, and LRRC8E proteins [16]. The authors concluded that such dips in the PONDR score of intrinsically disordered protein regions may predict the existence of molecular recognition elements or other features [6]. In any case, even assuming that such post-translational modifications may occur, it should be assessed whether they are necessary for channel gating, or if they may be required for folding, membrane targeting, protein-protein interaction and/or other physiological roles.
Role of GTPγS in VRAC modulation
The role of GTPγS was deeply investigated by several groups. Application of the non-hydrolysable GTP analogue guanosine 5'-O-(3-thiotriphosphate) into the cell pipette was shown to induce activation of VRAC even in the absence of swelling in several cells type [77, 83, 96]. On the contrary, application of GDPβS, a non-hydrolysable analogue of GDP that competes with GTP for the nucleotide binding sites on G proteins, caused a time-dependent inhibition of VRAC, which was more pronounced when the current was activated by mild hypotonicity. Moreover, some studies reported that treatment of cells with recombinant Clostridium Botulinum or Clostridium Difficile, which catalyzes the UDP-glycosylation of the Rho subfamily of monomeric G proteins, resulted in a strong reduction of osmo-sensitive Cl- current efflux [97, 98]. These data suggest a role of GTP binding proteins, and specifically of the Rho pathway in the signal transduction leading to VRAC activation. Given that activation of the Ras-related GTPase p21rho was accompanied by a transient reorganization of the F-actin cytoskeleton, Tilly and coworkers [97] proposed an involvement of a Rho signaling cascade and the subsequent rearrangement of actin filaments in the activation of VRAC.
Nevertheless, transfection of calf pulmonary artery endothelial cells with constitutively active isoforms of Gα (a Rho activating heterotrimeric G protein subunit), Rho, or Rho kinase alone failed to induce VRAC activation [98]. Furthermore, GTPγS alone has no effect when applied in Xenopus oocytes in isotonic conditions [99], and, the application of GDPβS could not prevent activation of anion currents in retinal pigment epithelial cells [100]. Altogether, these results make it unlikely that GTPγS signaling has an essential role in VRAC activation and weighs in favor of a permissive or cell subtype specific role rather than an obligatory activation path upon hypotonic stimulation.
Role of intracellular calcium in VRAC activation
The role of intracellular calcium, [Ca2+]int, in the activation of VRAC remains rather enigmatic. [Ca2+]int impacts the function of many enzymes, and therefore it is difficult to distinguish direct from indirect effects on VRAC [91, 92, 101]. In several systems, exposure of cells to hypotonic conditions leads to a rise of [Ca2+]int [62]. However, unlike Ca2+-activated Cl− channels (e.g. bestrophin and TMEM16A), whose activation require a rise in [Ca2+]int as a fundamental for activation, canonical volume-regulated Cl− channels directly activate in response to swelling, and no direct evidence of a compulsory role of intracellular Ca2+ has been reported so far [24, 102, 103].
It is generally assumed that a non-zero baseline concentration of [Ca2+]int is necessary for VRAC activation [104]. Nevertheless, at least in some systems, VRAC can be activated even under conditions of heavy buffering of [Ca2+]int [90, 105], suggesting that [Ca2+]int is not a primary parameter determining VRAC activity.
In our unpublished whole cell experiments in HEK293 LRRC8 KO cells transfected with LRRC8A and LRRC8E, using a pipette solution with 1 µM free Ca2+ resulted in a slight but only transient activation of VRAC currents in isotonic conditions. In agreement with the above cited results [90, 105]. In another set of experiments, using an intracellular solution without Ca2+ and heavy buffering did not prevent VRAC activation upon hypotonic perfusion (data not shown).
Dependence of VRAC on intracellular ATP
It has been reported in several cell types that activation of VRAC requires a minimal presence of intracellular ATP [90, 106, 107]. As reviewed in detail by Okada’s group, ATP acts in a non-hydrolytic manner and presumably binds directly to VRAC channels [62, 108]. However, the relevant ATP binding site has yet to be identified and it cannot be excluded that ATP is acting indirectly on other essential proteins.
Concluding remarks
Surprisingly, even after decades of study, and almost six years after the availability of molecular structures of VRAC, the essential molecular mechanism underlying the activation by hypotonicity and other triggers remains largely unknown. Putting it the other way round – the investigation of these mechanisms remain an important and interesting aspect of fundamental research with a significant relevance in several pathological situations involving VRAC channel activity.
S.B. and M.P. prepared the first draft of the manuscript, all authors contributed to finalizing the manuscript and to the preparation of figures.
This work was supported by a grant from the Fondazione AIRC per la Ricerca sul Cancro (grant # IG 21558) and the Italian Research Ministry (PRIN 20174TB8KW) to M. Pusch. S. Bertelli is supported by the PhD program of SISSA.
The authors have no ethical conflicts to disclose.
References
|
1 Cahalan MD, Lewis RS: Role of potassium and chloride channels in volume regulation by T lymphocytes. Soc Gen Physiol Ser 1988;43:281-301. |
|
|
|
|
|
2 Hazama A, Okada Y: Ca2+ sensitivity of
volume-regulatory K+ and Cl- channels in cultured human epithelial cells. J
Physiol 1988;402:687-702. |
|
|
|
|
|
3 Jentsch TJ: VRACs and other ion channels and
transporters in the regulation of cell volume and beyond. Nat Rev Mol Cell
Biol 2016;17:293-307. |
|
|
|
|
|
4 Pedersen SF, Okada Y, Nilius B: Biophysics and
physiology of the volume-regulated anion channel (VRAC)/volume-sensitive
outwardly rectifying anion channel (VSOR). Pflugers Arch 2016;468:371-383. |
|
|
|
|
|
5 Okada Y, Numata T, Sato-Numata K, Sabirov RZ,
Liu H, Mori SI, et al.: Roles of volume-regulatory anion channels, VSOR and
Maxi-Cl, in apoptosis, cisplatin resistance, necrosis, ischemic cell death,
stroke and myocardial infarction. Curr Top Membr 2019;83:205-283. |
|
|
|
|
|
6 Strange K, Yamada T, Denton JS: A 30-year
journey from volume-regulated anion currents to molecular structure of the
LRRC8 channel. J Gen Physiol 2019;151:100-117. |
|
|
|
|
|
7 Voss FK, Ullrich F, Munch J, Lazarow K, Lutter
D, Mah N, et al.: Identification of LRRC8 heteromers as an essential
component of the volume-regulated anion channel VRAC. Science
2014;344:634-638. |
|
|
|
|
|
8 Qiu Z, Dubin AE, Mathur J, Tu B, Reddy K,
Miraglia LJ, et al.: SWELL1, a plasma membrane protein, is an essential
component of volume-regulated anion channel. Cell 2014;157:447-458. |
|
|
|
|
|
9 Gaitán-Peñas H, Gradogna A, Laparra-Cuervo L,
Solsona C, Fernández-Dueñas V, Barrallo-Gimeno A, et al.: Investigation of
LRRC8-mediated volume-regulated anion currents in Xenopus oocytes. Biophys J
2016;111:1429-1443. |
|
|
|
|
|
10 Lutter D, Ullrich F, Lueck JC, Kempa S,
Jentsch TJ: Selective transport of neurotransmitters and modulators by
distinct volume-regulated LRRC8 anion channels. J Cell Sci
2017;130:1122-1133. |
|
|
|
|
|
11 Abascal F, Zardoya R: LRRC8 proteins share a
common ancestor with pannexins, and may form hexameric channels involved in
cell-cell communication. Bioessays 2012;34:551-560. |
|
|
|
|
|
12 Deneka D, Sawicka M, Lam AKM, Paulino C,
Dutzler R: Structure of a volume-regulated anion channel of the LRRC8 family.
Nature 2018;558:254-259. |
|
|
|
|
|
13 Kefauver JM, Saotome K, Dubin AE, Pallesen J,
Cottrell CA, Cahalan SM, et al.: Structure of the human volume regulated
anion channel. eLife 2018;7:e38461. |
|
|
|
|
|
14 Kasuya G, Nakane T, Yokoyama T, Jia Y, Inoue
M, Watanabe K, et al.: Cryo-EM structures of the human volume-regulated anion
channel LRRC8. Nat Struct Mol Biol 2018;25:797-804. |
|
|
|
|
|
15 Kern DM, Oh S, Hite RK, Brohawn SG: Cryo-EM
structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in
lipid nanodiscs. eLife 2019;8:e42636. |
|
|
|
|
|
16 Yamada T, Strange K: Intracellular and
extracellular loops of LRRC8 are essential for volume-regulated anion channel
function. J Gen Physiol 2018;150:1003-1015. |
|
|
|
|
|
17 Yamada T, Figueroa EE, Denton JS, Strange K:
LRRC8A homohexameric channels poorly recapitulate VRAC regulation and
pharmacology. Am J Physiol Cell Physiol 2020; DOI:10.1152/ajpcell.00454.2020. |
|
|
|
|
|
18 Nakamura R, Numata T, Kasuya G, Yokoyama T,
Nishizawa T, Kusakizako T, et al.: Cryo-EM structure of the volume-regulated
anion channel LRRC8D isoform identifies features important for substrate
permeation. Commun Biol 2020;3:240. |
|
|
|
|
|
19 Ullrich F, Reincke SM, Voss FK, Stauber T,
Jentsch TJ: Inactivation and anion selectivity of volume-regulated anion
channels (VRACs) depend on C-terminal residues of the first extracellular
loop. J Biol Chem 2016;291:17040-17048. |
|
|
|
|
|
20 Planells-Cases R, Lutter D, Guyader C,
Gerhards NM, Ullrich F, Elger DA, et al.: Subunit composition of VRAC
channels determines substrate specificity and cellular resistance to Pt-based
anti-cancer drugs. EMBO J 2015;34:2993-3008. |
|
|
|
|
|
21 Okada Y, Sato K, Numata T: Pathophysiology
and puzzles of the volume-sensitive outwardly rectifying anion channel. J
Physiol 2009;587:2141-2149. |
|
|
|
|
|
22 Nilius B, Eggermont J, Voets T, Buyse G,
Manolopoulos V, Droogmans G: Properties of volume-regulated anion channels in
mammalian cells. Prog Biophys Mol Biol 1997;68:69-119. |
|
|
|
|
|
23 Maeno E, Ishizaki Y, Kanaseki T, Hazama A,
Okada Y: Normotonic cell shrinkage because of disordered volume regulation is
an early prerequisite to apoptosis. Proc Natl Acad Sci U S A
2000;97:9487-9492. |
|
|
|
|
|
24 Akita T, Okada Y: Characteristics and roles of
the volume-sensitive outwardly rectifying (Vsor) anion channel in the central
nervous system. Neuroscience 2014;275:211-231. |
|
|
|
|
|
25 Mongin AA: Volume-regulated anion channel--a
frenemy within the brain. Pflugers Arch 2016;468:421-441. |
|
|
|
|
|
26 Burow P, Klapperstuck M, Markwardt F:
Activation of ATP secretion via volume-regulated anion channels by
sphingosine-1-phosphate in RAW macrophages. Pflugers Arch 2014:467:1215-1226. |
|
|
|
|
|
27 Kumar L, Chou J, Yee CS, Borzutzky A,
Vollmann EH, von Andrian UH, Park SY, Hollander G, Manis JP, Poliani PL, Geha
RS: Leucine-rich repeat containing 8A (LRRC8A) is essential for T lymphocyte
development and function. J Exp Med 2014;211:929-942. |
|
|
|
|
|
28 Chen LY, König B, Liu TB, Pervaiz S, Razzaque
YS, Stauber T: More than just a pressure relief valve: physiological roles of
volume-regulated LRRC8 anion channels. Biol Chem 2019;400:1481-1496. |
|
|
|
|
|
29 Gradogna A, Gaitan-Penas H, Boccaccio A,
Estevez R, Pusch M: Cisplatin activates volume sensitive LRRC8 channel
mediated currents in Xenopus oocytes. Channels (Austin) 2017;11:254-260. |
|
|
|
|
|
30 Ise T, Shimizu T, Lee EL, Inoue H, Kohno K,
Okada Y: Roles of volume-sensitive Cl- channel in cisplatin-induced apoptosis
in human epidermoid cancer cells. J Membr Biol 2005;205:139-145. |
|
|
|
|
|
31 Lee CC, Freinkman E, Sabatini DM, Ploegh HL:
The protein synthesis inhibitor blasticidin s enters mammalian cells via
leucine-rich repeat-containing protein 8D. J Biol Chem 2014;289:17124-17131. |
|
|
|
|
|
32 Kang C, Xie L, Gunasekar SK, Mishra A, Zhang
Y, Pai S, et al.: SWELL1 is a glucose sensor regulating beta-cell
excitability and systemic glycaemia. Nat Commun 2018;9:367. |
|
|
|
|
|
33 Stuhlmann T, Planells-Cases R, Jentsch TJ:
LRRC8/VRAC anion channels enhance beta-cell glucose sensing and insulin
secretion. Nat Commun 2018;9:1974. |
|
|
|
|
|
34 Lück JC, Puchkov D, Ullrich F, Jentsch TJ:
LRRC8/VRAC anion channels are required for late stages of spermatid
development in mice. J Biol Chem 2018;293:11796-11808. |
|
|
|
|
|
35 Bao J, Perez CJ, Kim J, Zhang H, Murphy CJ,
Hamidi T, et al.: Deficient LRRC8A-dependent volume-regulated anion channel
activity is associated with male infertility in mice. JCI Insight
2018;3:e99767. |
|
|
|
|
|
36 Hyzinski-García MC, Rudkouskaya A, Mongin AA:
LRRC8A protein is indispensable for swelling-activated and ATP-induced
release of excitatory amino acids in rat astrocytes. J Physiol
2014;592:4855-4862. |
|
|
|
|
|
37 Schober AL, Wilson CS, Mongin AA: Molecular
composition and heterogeneity of the LRRC8-containing swelling-activated
osmolyte channels in primary rat astrocytes. J Physiol 2017;595:6939-6951. |
|
|
|
|
|
38 Yang J, Vitery MDC, Chen J, Osei-Owusu J, Chu
J, Qiu Z: Glutamate-releasing SWELL1 channel in astrocytes modulates synaptic
transmission and promotes brain damage in stroke. Neuron 2019;102:813-827
e816. |
|
|
|
|
|
39 Fujii Y, Maekawa S, Morita M: Astrocyte
calcium waves propagate proximally by gap junction and distally by
extracellular diffusion of ATP released from volume-regulated anion channels.
Sci Rep 2017;7:13115. |
|
|
|
|
|
40 Elorza-Vidal X, Sirisi S, Gaitan-Penas H,
Perez-Rius C, Alonso-Gardon M, Armand-Ugon M, et al.: GlialCAM/MLC1 modulates
LRRC8/VRAC currents in an indirect manner: Implications for megalencephalic
leukoencephalopathy. Neurobiol Dis 2018;119:88-99. |
|
|
|
|
|
41 Chen L, Becker TM, Koch U, Stauber T: The
LRRC8/VRAC anion channel facilitates myogenic differentiation of murine
myoblasts by promoting membrane hyperpolarization. J Biol Chem
2019;294:14279-14288. |
|
|
|
|
|
42 Kumar A, Xie L, Ta CM, Hinton AO, Gunasekar
SK, Minerath RA, et al.: SWELL1 regulates skeletal muscle cell size, intracellular
signaling, adiposity and glucose metabolism. eLife 2020;9:e58941. |
|
|
|
|
|
43 Chen L, König B, Stauber T: LRRC8 channel
activation and reduction in cytosolic chloride concentration during early
differentiation of C2C12 myoblasts. Biochem Biophys Res Commun
2020;532:482-488. |
|
|
|
|
|
44 Zhou C, Chen X, Planells-Cases R, Chu J, Wang
L, Cao L, et al.: Transfer of cGAMP into bystander cells via LRRC8
volume-regulated anion channels augments STING-mediated interferon responses
and anti-viral immunity. Immunity 2020;52:767-781.e6. |
|
|
|
|
|
45 Lahey LJ, Mardjuki RE, Wen X, Hess GT,
Ritchie C, Carozza JA, et al.: LRRC8A:C/E heteromeric channels are ubiquitous
transporters of cGAMP. Mol Cell 2020;80:578-598.e5. |
|
|
|
|
|
46 Friard J, Corinus A, Cougnon M, Tauc M,
Pisani DF, Duranton C, et al.: LRRC8/VRAC channels exhibit a noncanonical
permeability to glutathione, which modulates epithelial-to-mesenchymal
transition (EMT). Cell Death Dis 2019;10:925. |
|
|
|
|
|
47 Liu T, Stauber T: The volume-regulated anion
channel LRRC8/VRAC is dispensable for cell proliferation and migration. Int J
Mol Sci 2019;20:2663. |
|
|
|
|
|
48 Li P, Hu M, Wang C, Feng X, Zhao Z, Yang Y,
et al.: LRRC8 family proteins within lysosomes regulate cellular
osmoregulation and enhance cell survival to multiple physiological stresses.
Proc Natl Acad Sci U S A 2020;117:29155-29165. |
|
|
|
|
|
49 Ross PE, Garber SS, Cahalan MD: Membrane
chloride conductance and capacitance in Jurkat T lymphocytes during osmotic
swelling. Biophys J 1994;66:169-178. |
|
|
|
|
|
50 Hille B: Ion channels of excitable membranes, ed 3. Sunderland, Massachusetts, Sinauer, 2001. |
|
|
|
|
|
51 Jackson PS, Strange K: Single-channel
properties of a volume-sensitive anion conductance. Current activation occurs
by abrupt switching of closed channels to an open state. J Gen Physiol
1995;105:643-660. |
|
|
|
|
|
52 Jackson PS, Strange K: Characterization of
the voltage-dependent properties of a volume-sensitive anion conductance. J
Gen Physiol 1995;105:661-676. |
|
|
|
|
|
53 Jackson PS, Strange K: Single channel
properties of a volume sensitive anion channel: lessons from noise analysis.
Kidney Int 1996;49:1695-1699. |
|
|
|
|
|
54 Shuba YM, Prevarskaya N, Lemonnier L, Van
Coppenolle F, Kostyuk PG, Mauroy B, et al.: Volume-regulated chloride
conductance in the LNCaP human prostate cancer cell line. Am J Physiol Cell
Physiol 2000;279:C1144-1154. |
|
|
|
|
|
55 Sabirov RZ, Prenen J, Tomita T, Droogmans G,
Nilius B: Reduction of ionic strength activates single volume-regulated anion
channels (VRAC) in endothelial cells. Pflugers Arch 2000;439:315-320. |
|
|
|
|
|
56 Caramia M, Sforna L, Franciolini F,
Catacuzzeno L: The volume-regulated anion channel in glioblastoma. Cancers
(Basel)2019;11:307. |
|
|
|
|
|
57 Lewis RS, Ross PE, Cahalan MD: Chloride
channels activated by osmotic stress in T lymphocytes. J Gen Physiol
1993;101:801-826. |
|
|
|
|
|
58 Gradogna A, Gavazzo P, Boccaccio A, Pusch M:
Subunit-dependent oxidative stress sensitivity of LRRC8 volume-regulated
anion channels. J Physiol 2017;595:6719-6733. |
|
|
|
|
|
59 Gaitán-Peñas H, Pusch M, Estevez R:
Expression of LRRC8/VRAC currents in Xenopus oocytes: Advantages and caveats.
Int J Mol Sci 2018;19:719. |
|
|
|
|
|
60 König B, Hao Y, Schwartz S, Plested AJ,
Stauber T: A FRET sensor of C-terminal movement reveals VRAC activation by
plasma membrane DAG signaling rather than ionic strength. eLife
2019;8:e45421. |
|
|
|
|
|
61 Pusch M, Jentsch TJ: Molecular physiology of
voltage-gated chloride channels. Physiol Rev 1994;74:813-827. |
|
|
|
|
|
62 Okada Y: Volume expansion-sensing
outward-rectifier Cl- channel: fresh start to the molecular identity and
volume sensor. Am J Physiol 1997;273:C755-789. |
|
|
|
|
|
63 Groulx N, Boudreault F, Orlov SN, Grygorczyk
R: Membrane reserves and hypotonic cell swelling. J Membr Biol
2006;214:43-56. |
|
|
|
|
|
64 Cannon CL, Basavappa S, Strange K:
Intracellular ionic strength regulates the volume sensitivity of a
swelling-activated anion channel. Am J Physiol 1998;275:C416-422. |
|
|
|
|
|
65 Nilius B, Prenen J, Voets T, Eggermont J,
Droogmans G: Activation of volume-regulated chloride currents by reduction of
intracellular ionic strength in bovine endothelial cells. J Physiol
1998;506:353-361. |
|
|
|
|
|
66 Voets T, Droogmans G, Raskin G, Eggermont J,
Nilius B: Reduced intracellular ionic strength as the initial trigger for
activation of endothelial volume-regulated anion channels. Proc Natl Acad Sci
U S A 1999;96:5298-5303. |
|
|
|
|
|
67 Best L, Brown PD: Studies of the mechanism of
activation of the volume-regulated anion channel in rat pancreatic
beta-cells. J Membr Biol 2009;230:83-91. |
|
|
|
|
|
68 Syeda R, Qiu Z, Dubin AE, Murthy SE, Florendo
MN, Mason DE, et al.: LRRC8 Proteins form volume-regulated anion channels
that sense ionic strength. Cell 2016;164:499-511. |
|
|
|
|
|
69 Doroshenko P: Second messengers mediating
activation of chloride current by intracellular GTP gamma S in bovine
chromaffin cells. J Physiol 1991;436:725-738. |
|
|
|
|
|
70 Browe DM, Baumgarten CM: Angiotensin II (AT1)
receptors and NADPH oxidase regulate Cl- current elicited by beta1 integrin
stretch in rabbit ventricular myocytes. J Gen Physiol 2004;124:273-287. |
|
|
|
|
|
71 Shimizu T, Numata T, Okada Y: A role of
reactive oxygen species in apoptotic activation of volume-sensitive Cl(-)
channel. Proc Natl Acad Sci U S A 2004;101:6770-6773. |
|
|
|
|
|
72 Varela D, Simon F, Riveros A, Jorgensen F,
Stutzin A: NAD(P)H oxidase-derived H(2)O(2) signals chloride channel
activation in cell volume regulation and cell proliferation. J Biol Chem
2004;279:13301-13304. |
|
|
|
|
|
73 Crutzen R, Shlyonsky V, Louchami K, Virreira
M, Hupkens E, Boom A, et al.: Does NAD(P)H oxidase-derived H2O2 participate
in hypotonicity-induced insulin release by activating VRAC in beta-cells?
Pflugers Arch 2012;463:377-390. |
|
|
|
|
|
74 Bryan-Sisneros A, Sabanov V, Thoroed SM,
Doroshenko P: Dual role of ATP in supporting volume-regulated chloride
channels in mouse fibroblasts. Biochim Biophys Acta 2000;1468:63-72. |
|
|
|
|
|
75 Sorota S: Tyrosine protein kinase inhibitors
prevent activation of cardiac swelling-induced chloride current. Pflugers
Arch 1995;431:178-185. |
|
|
|
|
|
76 Tilly BC, van den Berghe N, Tertoolen LG,
Edixhoven MJ, de Jonge HR: Protein tyrosine phosphorylation is involved in
osmoregulation of ionic conductances. J Biol Chem 1993;268:19919-19922. |
|
|
|
|
|
77 Voets T, Manolopoulos V, Eggermont J, Ellory
C, Droogmans G, Nilius B: Regulation of a swelling-activated chloride current
in bovine endothelium by protein tyrosine phosphorylation and G proteins. J
Physiol 1998;506:341-352. |
|
|
|
|
|
78 Crepel V, Panenka W, Kelly ME, MacVicar BA:
Mitogen-activated protein and tyrosine kinases in the activation of astrocyte
volume-activated chloride current. J Neurosci 1998;18:1196-1206. |
|
|
|
|
|
79 Lepple-Wienhues A, Szabo I, Laun T, Kaba NK,
Gulbins E, et al.: The tyrosine kinase p56lck mediates activation of
swelling-induced chloride channels in lymphocytes. J Cell Biol
1998;141:281-286. |
|
|
|
|
|
80 Gosling M, Smith JW, Poyner DR:
Characterization of a volume-sensitive chloride current in rat
osteoblast-like (ROS 17/2.8) cells. J Physiol 1995;485:671-682. |
|
|
|
|
|
81 Szücs G, Heinke S, De Greef C, Raeymaekers L,
Eggermont J, Droogmans G, et al.: The volume-activated chloride current in
endothelial cells from bovine pulmonary artery is not modulated by
phosphorylation. Pflugers Arch 1996;431:540-548. |
|
|
|
|
|
82 Strange K, Morrison R, Shrode L, Putnam R:
Mechanism and regulation of swelling-activated inositol efflux in brain glial
cells. Am J Physiol 1993;265:C244-256. |
|
|
|
|
|
83 Estevez AY, Bond T, Strange K: Regulation of
I(Cl,swell) in neuroblastoma cells by G protein signaling pathways. Am J
Physiol Cell Physiol 2001;281:C89-98. |
|
|
|
|
|
84 Du XL, Gao Z, Lau CP, Chiu SW, Tse HF,
Baumgarten CM, et al.: Differential effects of tyrosine kinase inhibitors on
volume-sensitive chloride current in human atrial myocytes: evidence for dual
regulation by Src and EGFR kinases. J Gen Physiol 2004;123:427-439. |
|
|
|
|
|
85 Ren Z, Baumgarten CM: Antagonistic regulation
of swelling-activated Cl- current in rabbit ventricle by Src and EGFR protein
tyrosine kinases. Am J Physiol Heart Circ Physiol 2005;288:H2628-2636. |
|
|
|
|
|
86 Doroshenko P: Pervanadate inhibits
volume-sensitive chloride current in bovine chromaffin cells. Pflugers Arch
1998;435:303-309. |
|
|
|
|
|
87 Thoroed SM, Bryan-Sisneros A, Doroshenko P:
Protein phosphotyrosine phosphatase inhibitors suppress regulatory volume
decrease and the volume-sensitive Cl- conductance in mouse fibroblasts.
Pflugers Arch 1999;438:133-140. |
|
|
|
|
|
88 Haskew-Layton RE, Mongin AA, Kimelberg HK:
Hydrogen peroxide potentiates volume-sensitive excitatory amino acid release
via a mechanism involving Ca2+/calmodulin-dependent protein kinase II. J Biol
Chem 2005;280:3548-3554. |
|
|
|
|
|
89 Mongin AA, Kimelberg HK: ATP regulates anion
channel-mediated organic osmolyte release from cultured rat astrocytes via
multiple Ca2+-sensitive mechanisms. Am J Physiol Cell Physiol
2005;288:C204-213. |
|
|
|
|
|
90 Meyer K, Korbmacher C: Cell swelling
activates ATP-dependent voltage-gated chloride channels in M-1 mouse cortical
collecting duct cells. J Gen Physiol 1996;108:177-193. |
|
|
|
|
|
91 Chou CY, Shen MR, Hsu KS, Huang HY, Lin HC:
Involvement of PKC-alpha in regulatory volume decrease responses and
activation of volume-sensitive chloride channels in human cervical cancer
HT-3 cells. J Physiol 1998;512:435-448. |
|
|
|
|
|
92 Gong W, Xu H, Shimizu T, Morishima S, Tanabe
S, Tachibe T, et al.: ClC-3-independent, PKC-dependent activity of
volume-sensitive Cl channel in mouse ventricular cardiomyocytes. Cell Physiol
Biochem 2004;14:213-224. |
|
|
|
|
|
93 Hermoso M, Olivero P, Torres R, Riveros A,
Quest AF, Stutzin A: Cell volume regulation in response to hypotonicity is
impaired in HeLa cells expressing a protein kinase Calpha mutant lacking
kinase activity. J Biol Chem 2004;279:17681-17689. |
|
|
|
|
|
94 Sadoshima J, Qiu Z, Morgan JP, Izumo S:
Tyrosine kinase activation is an immediate and essential step in hypotonic
cell swelling-induced ERK activation and c-fos gene expression in cardiac
myocytes. EMBO J 1996;15:5535-5546. |
|
|
|
|
|
95 Nilius B, Droogmans G: Amazing chloride
channels: an overview. Acta Physiol Scand 2003;177:119-147. |
|
|
|
|
|
96 Doroshenko P, Neher E: Volume-sensitive
chloride conductance in bovine chromaffin cell membrane. J Physiol
1992;449:197-218. |
|
|
|
|
|
97 Tilly BC, Edixhoven MJ, Tertoolen LG, Morii
N, Saitoh Y, Narumiya S, et al.: Activation of the osmo-sensitive chloride
conductance involves P21rho and is accompanied by a transient reorganization
of the F-actin cytoskeleton. Mol Biol Cell 1996;7:1419-1427. |
|
|
|
|
|
98 Carton I, Trouet D, Hermans D, Barth H,
Aktories K, Droogmans G, et al.: RhoA exerts a permissive effect on
volume-regulated anion channels in vascular endothelial cells. Am J Physiol
Cell Physiol 2002;283:C115-C125. |
|
|
|
|
|
99 Ackerman MJ, Wickman KD, Clapham DE:
Hypotonicity activates a native chloride current in Xenopus oocytes. J Gen
Physiol 1994;103:153-179. |
|
|
|
|
|
100 Botchkin LM, Matthews G: Chloride current
activated by swelling in retinal pigment epithelium cells. Am J Physiol
1993;265:C1037-C1045. |
|
|
|
|
|
101 Verdon B, Winpenny JP, Whitfield KJ, Argent
BE, Gray MA: Volume-activated chloride currents in pancreatic duct cells. J
Membr Biol 1995;147:173-183. |
|
|
|
|
|
102 Pedersen SF, Prenen J, Droogmans G, Hoffmann
EK, Nilius B: Separate swelling- and Ca2+-activated anion currents in Ehrlich
ascites tumor cells. J Membr Biol 1998;163:97-110. |
|
|
|
|
|
103 Hoffmann EK, Sorensen BH, Sauter DP, Lambert
IH: Role of volume-regulated and calcium-activated anion channels in cell
volume homeostasis, cancer and drug resistance. Channels (Austin)
2015;9:380-396. |
|
|
|
|
|
104 Szücs G, Heinke S, Droogmans G, Nilius B:
Activation of the volume-sensitive chloride current in vascular endothelial
cells requires a permissive intracellular Ca2+ concentration. Pflugers Arch
1996;431:467-469. |
|
|
|
|
|
105 Hoffmann EK, Dunham PB: Membrane mechanisms
and intracellular signalling in cell volume regulation. International review
of cytology 1995;161:173-262. |
|
|
|
|
|
106 Jackson PS, Morrison R, Strange K: The
volume-sensitive organic osmolyte-anion channel VSOAC is regulated by
nonhydrolytic ATP binding. Am J Physiol 1994;267:C1203-C1209. |
|
|
|
|
|
107 Oiki S, Kubo M, Okada Y: Mg2+ and ATP-dependence of volume-sensitive Cl- channels in human epithelial cells. Jpn J Physiol 1994;44:S77-79. |
|
|
|
|
|
108 Okada Y, Okada T, Sato-Numata K, Islam MR,
Ando-Akatsuka Y, Numata T, et al.: Cell volume-activated and
volume-correlated anion channels in mammalian cells: their biophysical,
molecular, and pharmacological properties. Pharmacol Rev 2019;71:49-88. |
|
|
|
|
|
109 Nilius B, Prenen J, Walsh MP, Carton I,
Bollen M, Droogmans G, et al.: Myosin light chain phosphorylation-dependent
modulation of volume-regulated anion channels in macrovascular endothelium.
FEBS Lett 2000;466:346-350. |
|