Volume-sensitive Organic Osmolyte-anion Channel Research Articles

Biophysics and Physiology of the Volume-Regulated Anion Channel (VRAC)/Volume-Sensitive Outwardly Rectifying Anion Channel (VSOR).

The volume-regulated anion channel (VRAC), also known as the volume-sensitive outwardly rectifying (VSOR) anion channel or the volume-sensitive organic osmolyte/anion channel (VSOAC), is essential for cell volume regulation after swelling in most vertebrate cell types studied to date. In addition to its role in cell volume homeostasis, VRAC has been implicated in numerous other physiological and pathophysiological processes, including cancer, ischemic brain edema, cell motility, proliferation, angiogenesis, programmed cell death, and excitotoxic glutamate release. Although VRAC has been extensively biophysically, pharmacologically, and functionally characterized, its molecular identity was highly controversial until the recent identification of the leucine-rich repeats containing 8A (LRRC8A) protein as essential for the VRAC current in multiple cell types and a likely pore-forming subunit of VRAC. Members of this distantly pannexin-1-related protein family form heteromers, and in addition to LRRC8A, at least another LRRC8 family member is required for the formation of a functional VRAC. This review summarizes the biophysical and pharmacological properties of VRAC, highlights its main physiological functions and pathophysiological implications, and outlines the search for its molecular identity.

Letter to the editor: AVD1, AVDT, AVD2 in cisplatin-induced apoptosis: differences between WT EATC and MDR EATC resting on differing expression levels of type-1 porin/VDAC in cell membranes?

Combining data on mammalian VDAC with those of Poulsen and colleagues (5), a testable model of interpretation arises: cisplatin first opens type-1 porin at the cell membrane to initiate AVD1. After about 12 h, AVDT starts, showing similarity to regulatory volume increase. Finally, cisplatin reaches VDAC at OMM, thus stimulating caspase-dependent AVD2. Differences between WT EATCs and MDR EATCs under study might just rest on different expression levels of VDAC in their cell membranes; however, differences in the expression levels of plasmalemmal VDAC have been stated for normal or prostate cancer cells, respectively, taken from a single patient (3). Effects observed under the influence of the anion channel blocker NS3728 should also be seen under VDAC blockers. Finally, it might be worthwhile to refer to data on a putative opener of VDAC in cell membranes (10).

Subnanomolar Concentrations of Thrombin Enhance the Volume-Sensitive Efflux of Taurine from Human 1321N1 Astrocytoma Cells

The ability of subnanomolar concentrations of thrombin to protect both neurons and glia from ischemia and other metabolic insults has recently been reported. In this study, we demonstrate an additional neuroprotective property of thrombin; its ability to promote the release of the organic osmolyte, taurine, in response to hypoosmotic stress. Incubation of human 1321N1 astrocytoma cells with hypo-osmolar buffers (320-227 mOsM) resulted in a time-dependent release of taurine. Inclusion of thrombin (EC(50) = 60 pM) resulted in a marked increase in taurine efflux that, although evident under isotonic conditions (340 mOsM), was maximal at an osmolarity of 270 mOsM (3-4-fold stimulation). Thrombin-stimulated taurine efflux was dependent upon its protease activity and could be mimicked by addition of the peptide SFLLRN, a proteinase activated receptor-1 (PAR-1) subtype-specific ligand. Inclusion of anion channel blockers known to inhibit the volume-sensitive organic osmolyte anion channel attenuated thrombin-stimulated taurine release. Depletion of intracellular Ca(2+) with either 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or thapsigargin, or alternatively, inhibition of protein kinase C (PKC) with bisindolylmaleimide or chelerythrine resulted in a 30 to 50% inhibition of thrombin-stimulated taurine efflux. Under conditions in which intracellular Ca(2+) was depleted and PKC activity inhibited, thrombin-stimulated taurine efflux was reduced by >85%. The results indicate that activation of PAR-1 receptors by thrombin facilitates the ability of 1321N1 astrocytoma cells to release osmolytes in response to a reduction in osmolarity via a mechanism that is dependent on intracellular Ca(2+) and PKC activity.

Volume‐Sensitive Organic Osmolyte/Anion Channels in Cancer: Novel Approaches to Studying Channel Modulation Employing Proteomics Technologies

Key elements of tumor development include proliferation, migration, invasiveness, and angiogenesis. Activation of the volume-sensitive organic osmolyte/anion channel (VSOAC) has been suggested to play a role in all of these processes. VSOACs may therefore represent an important therapeutic target in the etiology of cancer. However, pharmacological inhibitors of VSOAC are nonselective and of low potency, highlighting the importance of identifying novel regulators of the channel. The use of electrophysiological methods coupled with techniques such as pull-down assays, yeast 2-hybrid, and functional protein arrays have already proved valuable in studying protein-protein interactions in a variety of systems. Some of these methods have been used to identify small molecules that modulate the function of other types of ion channels. Given that several proteins have already been identified as putative modulators of VSOACs, proteomics technologies may prove useful in elucidating the molecular identity of VSOACs and helpful in identifying novel modulators of channel function. In this paper, we review the involvement of VSOACs in tumor development processes and its regulation by pharmacological agents and cellular proteins. Proteomic approaches to study protein-protein interactions and how such approaches may be used to study VSOACs are also discussed. We speculate on how modulation of protein-protein interactions may result in the identification of a novel class of compounds for modulating VSOACs.

Regulation of Myo-inositol homeostasis in differentiated human NT2-N neurons.

We have investigated the possible role of second messengers on inositol homeostasis in NT2-N cells, human central nervous system neurons obtained by terminal differentiation of teratocarcinoma precursors. Uptake of inositol into NT2-N neurons was inhibited approximately 10% by protein kinase C (PKC) activation but was unaffected by either the presence of cyclic nucleotide analogs or changes in the intracellular concentration of Ca2+. Efflux of inositol from NT2-N neurons was enhanced in hypotonic buffer but virtually eliminated by inclusion of the Cl- channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, a result which indicates the involvement of a volume-sensitive organic osmolyte-anion channel. Volume-sensitive inositol efflux was stimulated approximately 30% following activation of PKC or elevation of the cytosolic Ca2+ concentration but was unaffected by protein kinase A activation. These results suggest that whereas inositol uptake into NT2-N neurons is relatively refractory to regulation, volume-sensitive inositol efflux may be significantly affected by intracellular signaling events.

Intracellular ATP depletion inhibits swelling-induced d-[ [formula omitted]]aspartate release from primary astrocyte cultures

Volume expansion-sensing outward rectifier (VSOR) anion channel, also referred to as volume-sensitive organic osmolyte-anion channel (VSOAC), appears to be responsible for cell swelling-induced amino acid release in a variety of cells. One prominent feature of the VSOR/VSOAC is that non-hydrolyzed intracellular ATP binding to the channel or an accessory protein is required for its activation. In this study, the effect of intracellular ATP depletion on the swelling-induced release of d-[ 3 H ]aspartate from rat primary astrocyte cultures due to exposure to either high K + or hypotonic media was studied. When the cells were pretreated for 10 min with a combination of the metabolic inhibitors 2-deoxyglucose and rotenone, 100 mM K + media- or hypotonic media-induced d-[ 3 H ]aspartate release was completely suppressed. Added separately, each inhibitor showed only partial or no inhibition of d-[ 3 H ]aspartate release, which correlated with its relative effectiveness in decreasing intracellular ATP levels. These data are consistent with the view that during high [K +] o or hypotonic media-induced swelling of primary astrocyte cultures an ATP-dependent swelling-activated VSOAC channel is responsible for d-[ 3 H ]aspartate release and close to normal ATP is required for full channel activation.

Volume-activated efflux pathway for taurine (TAU) in reptilian renal brush-border membranes: is it a volume-sensitive organic osmolyte-anion channel?

Volume-activated efflux pathway for taurine (TAU) in reptilian renal brush-border membranes: is it a volume-sensitive organic osmolyte-anion channel?

Intracellular ionic strength regulates the volume sensitivity of a swelling-activated anion channel

Cell swelling activates an outwardly rectifying anion channel termed VSOAC (volume-sensitive organic osmolyte/anion channel). Regulation of VSOAC by intracellular electrolytes was characterized in Chinese hamster ovary cells by whole cell patch clamp. Elevation of intracellular CsCl concentration from 40 to 180 mM resulted in a concentration-dependent decrease in channel activation. Activation of VSOAC was insensitive to the salt gradient across the plasma membrane, the intracellular concentration of specific anions or cations, and the total intracellular concentration of cations, anions, or electrolytes. Comparison of cells dialyzed with either CsCl or Na2SO4 solutions demonstrated directly that VSOAC activation is modulated by intracellular ionic strength (microi). The relative cell volume at which VSOAC current activation was triggered, termed the channel volume set point, decreased with decreasing ionic strength. At microi = 0.04, VSOAC activation occurred spontaneously in shrunken cells. The rate of VSOAC activation was nearly 50-fold higher in cells with microi = 0.04 vs. those with microi = 0.18. We propose that microi modulates the volume sensor responsible for channel activation.

New Findings Concerning Vertebrate Porin

Eukaryotic porin can be considered to be a good candidate for forming the channel component of the protein complex which, depending on the approach used, may realize its expression either as the outwardly-rectifying depolarization-induced chloride channel or as the volume-sensitive organic osmolyte-anion channel. As a basis for this proposition, we point to a series of correspondences in properties between mammalian porin and the ORDIC channel complex. Specifically, mammalian porin is expressed in the plasmalemma of different cells and chloride channels can be blocked by anti-human porin antibodies in astrocytes and endothelial cells. There is an indication of colocalisation of human porin and the cystic fibrosis (CF) gene product, CFTR, in the apical region of epithelial cells. The primary structure of porin from a CF patient was found to be normal. Cytosol and amniotic fluid fractions influence the channel characteristics of mammalian porin. Channel-active mammalian porin binds ATP and the stilbene disulphonate grouping of the chloride channel inhibitor DIDS. Human porin in black membranes is a pathway for taurine, and biogenic polyamines reduce the voltage dependence of human porin. Assuming the relationship between human porin and the ORDIC channel/VSOAC complex, studies on plasmalemma-integrated human porin have a relevance for CF research. In addition, we refer to a case study on a child with encephalomyopathy in which porin could not be detected using monoclonal anti-human porin antibodies. Our studies were based on purified and sequenced human porin from different cells and from different cell compartments. In addition, we raised antibodies against mature human porin or synthetic parts of the molecule. This provided a firm foundation for our topochemical work with which we were able to establish the multi-topological expression of eukaryotic porin channels. The data are summarized and discussed.

Identification of the volume-sensitive organic osmolyte/anion channel in human glial cells.

Cellular use of small organic solutes known as organic osmolytes for regulation of the volume of the cell is a universal biological phenomenon. During swelling, cells open an anion channel which allows for efflux of these solutes. This channel is known as the volume-sensitive organic osmolyte/anion channel (VSOAC). Anion channels with properties identical to VSOAC were found in human brain cells obtained following pediatric neurosurgical procedures. The tissues examined included tumors as well as putatively normal gray and white matter astrocytes. The cells activated an anion conductance when swollen by hypotonic shock. Baseline currents in these cells were generally small and increased up to 30-fold within 10 min following the onset of swelling. The anion channel activated by swelling was outwardly rectified and inactivated by depolarization, both characteristic of the VSOAC. These observations indicate that human glial cells and tumor cells activate VSOAC following cellular swelling. This suggests that organic osmolyte efflux can be modulated during brain swelling by pharmaceuticals which modulate VSOAC.

Single channel properties of a volume sensitive anion channel: Lessons from noise analysis

Swelling activated anion channels have recently been recognized to play an important role in not only volume regulatory electrolyte movement, but also in organic osmolyte transport. A swelling-activated, outwardly rectifying anion channel termed VSOAC (volume-sensitive organic osmolyte/anion channel) is a major pathway for swelling-induced loss of organic osmolytes and organic anions from mammalian cells. VSOAC has been described in numerous cell types. Until recently, however, the unitary conductance and gating kinetics of VSOAC were uncertain. Stationary noise analysis and single channel measurements have produced estimates for the unitary conductance of swelling-activated, outwardly rectifying anion channels that vary by > 15-fold. This review describes our current understanding of the single channel properties of VSOAC.

Swelling-activated organic osmolyte efflux: A new role for anion channels

Efflux of intracellular electrolytes and small organic solutes termed organic osmolytes is a universal response to cell swelling. While volume-activated ion efflux pathways have been studied extensively, relatively little was known until recently about the mechanisms and regulation of organic osmolyte release. An accumulating body of evidence, however, now indicates that structurally dissimilar organic osmolytes are lost from cells via a swelling-activated anion channel. We have termed this channel VSOAC for volume-sensitive organic osmolyte-anion channel. The purpose of this review is to summarize our current understanding of VSOAC and the process of volume regulatory organic osmolyte efflux.

Single-channel properties of a volume-sensitive anion conductance. Current activation occurs by abrupt switching of closed channels to an open state.

Swelling-induced loss of organic osmolytes from cells is mediated by an outwardly rectified, volume-sensitive anion channel termed VSOAC (Volume-Sensitive Organic osmolyte/Anion Channel). Similar swelling-activated anion channels have been described in numerous cell types. The unitary conductance and gating kinetics of VSOAC have been uncertain, however. Stationary noise analysis and single-channel measurements have produced estimates for the unitary conductance of swelling-activated, outwardly rectified anion channels that vary by > 15-fold. We used a combination of stationary and nonstationary noise analyses and single-channel measurements to estimate the unitary properties of VSOAC. Current noise was analyzed initially by assuming that graded changes in macroscopic current were due to graded changes in channel open probability. Stationary noise analysis predicts that the unitary conductance of VSOAC is approximately 1 pS at 0 mV. In sharp contrast, nonstationary noise analysis demonstrates that VSOAC is a 40-50 pS channel at +120 mV (approximately 15 pS at 0 mV). Measurement of single-channel events in whole-cell currents and outside-out membrane patches confirmed the nonstationary noise analysis results. The discrepancy between stationary and nonstationary noise analyses and single-channel measurements indicates that swelling-induced current activation is not mediated by a graded increase in channel open probability as assumed initially. Instead, activation of VSOAC appears to involve an abrupt switching of single channels from an OFF state, where channel open probability is zero, to an ON state, where open probability is near unity.

The volume-sensitive organic osmolyte-anion channel VSOAC is regulated by nonhydrolytic ATP binding.

Efflux of intracellular organic osmolytes to the external medium is a ubiquitous response to cell swelling. Accumulating evidence indicates that volume regulatory loss of structurally unrelated organic osmolytes from cells is mediated by a relatively nonselective volume-sensitive anion channel. In C6 cells, we have termed this channel VSOAC for volume-sensitive organic osmolyte-anion channel. Swelling-induced activation of VSOAC required the presence of ATP or nonhydrolyzable ATP analogues [adenosine 5'-O-(3-thiotriphosphate), adenylylmethyl-enediphosphonate (AMP-PCP), or 5'-adenylylimidodiphosphate] in the patch pipette. Sustained activation of VSOAC also required ATP. Channel rundown was observed when cellular ATP levels were lowered by intracellular dialysis with the patch pipette solution. Rundown was prevented by the ATP analogue AMP-PCP. Passive swelling-induced myo-[3H]inositol and [3H]taurine efflux was blocked by metabolic inhibitors that decreased cellular ATP levels. Titration of cellular ATP levels with azide demonstrated that the apparent dissociation constant (Kd) for ATP of both myo-inositol and taurine efflux was approximately 1.7 mM. The high Kd for ATP indicates that cellular metabolic state plays an important role in modulating organic osmolyte loss. Regulation of VSOAC activity by ATP prevents depletion of metabolically expensive organic osmolytes when cellular energy production is reduced. In addition, ATP-dependent regulation provides essential feedback to minimize the loss of energy-producing carbon sources such as pyruvate, short-chain fatty acids, ketone bodies, and amino acids, which readily permeate this channel.