Volume-activated Anion Channels Research Articles

Cell Volume-Activated and Volume-Correlated Anion Channels in Mammalian Cells: Their Biophysical, Molecular, and Pharmacological Properties.

There are a number of mammalian anion channel types associated with cell volume changes. These channel types are classified into two groups: volume-activated anion channels (VAACs) and volume-correlated anion channels (VCACs). VAACs can be directly activated by cell swelling and include the volume-sensitive outwardly rectifying anion channel (VSOR), which is also called the volume-regulated anion channel; the maxi-anion channel (MAC or Maxi-Cl); and the voltage-gated anion channel, chloride channel (ClC)-2. VCACs can be facultatively implicated in, although not directly activated by, cell volume changes and include the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, the Ca2+-activated Cl- channel (CaCC), and the acid-sensitive (or acid-stimulated) outwardly rectifying anion channel. This article describes the phenotypical properties and activation mechanisms of both groups of anion channels, including accumulating pieces of information on the basis of recent molecular understanding. To that end, this review also highlights the molecular identities of both anion channel groups; in addition to the molecular identities of ClC-2 and CFTR, those of CaCC, VSOR, and Maxi-Cl were recently identified by applying genome-wide approaches. In the last section of this review, the most up-to-date information on the pharmacological properties of both anion channel groups, especially their half-maximal inhibitory concentrations (IC50 values) and voltage-dependent blocking, is summarized particularly from the standpoint of pharmacological distinctions among them. Future physiologic and pharmacological studies are definitely warranted for therapeutic targeting of dysfunction of VAACs and VCACs.

Microglial Contact Prevents Excess Depolarization and Rescues Neurons from Excitotoxicity.

Microglia survey and directly contact neurons in both healthy and damaged brain, but the mechanisms and functional consequences of these contacts are not yet fully elucidated. Combining two-photon imaging and patch clamping, we have developed an acute experimental model for studying the role of microglia in CNS excitotoxicity induced by neuronal hyperactivity. Our model allows us to simultaneously examine the effects of repetitive supramaximal stimulation on axonal morphology, neuronal membrane potential, and microglial migration, using cortical brain slices from Iba-1 eGFP mice. We demonstrate that microglia exert an acute and highly localized neuroprotective action under conditions of neuronal hyperactivity. Evoking repetitive action potentials in individual layer 2/3 pyramidal neurons elicited swelling of axons, but not dendrites, which was accompanied by a large, sustained depolarization of soma membrane potential. Microglial processes migrated to these swollen axons in a mechanism involving both ATP and glutamate release via volume-activated anion channels. This migration was followed by intensive microglial wrapping of affected axons and, in some cases, the removal of axonal debris that induced a rapid soma membrane repolarization back to resting potentials. When the microglial migration was pharmacologically blocked, the activity-induced depolarization continued until cell death ensued, demonstrating that the microglia–axon contact served to prevent pathological depolarization of the soma and maintain neuronal viability. This is a novel aspect of microglia surveillance: detecting, wrapping, and rescuing neuronal soma from damage due to excessive activity.

Letter to the editor: “The origin of free glutamate in milk: a role for anionic amino acid transporters”

Letter to the editor: “The origin of free glutamate in milk: a role for anionic amino acid transporters”

The research progress of volume-sensitive Cl- channel in tumor

Cell swelling activates or upregulates a number of anion channels. Of the volume-activated or-regulated anion channels, the volume-sensitive outwardly rectifying anion channel (VSOR) is most prominently activated and ubiquitously expressed. Volume regulated chloride channels play significant roles in a variety of physiological processes including volume regulation, proliferation, differentiation, migration,apoptosis and resistance of tumor cells. Molecular identification and activation mechanisms of VSOR are poorly understood. Many signaling molecules have been shown to regulate VSOR activity in research, playing permissive or modulatory roles. Therefore, VSOR may be a new anticancer target by identifying its signaling molecules and activation mechanisms. Key words: Chloride channels; Neoplasms; Research

Nonsynaptic communication through ATP release from volume-activated anion channels in axons.

The release of neuronal messengers outside synapses has broad biological implications, particularly with regard to communication between axons and glia. We identify a mechanism for nonsynaptic, nonvesicular release of adenosine triphosphate (ATP) from axons through volume-activated anion channels (VAACs) activated by microscopic axon swelling during action potential firing. We used a combination of single-photon imaging of ATP release, together with imaging for intrinsic optical signals, intracellular calcium ions (Ca(2+)), time-lapse video, and confocal microscopy, to investigate action potential-induced nonsynaptic release of this neurotransmitter. ATP release from cultured embryonic dorsal root ganglion axons persisted when bafilomycin or botulinum toxin was used to block vesicular release, whereas pharmacological inhibition of VAACs or prevention of action potential-induced axon swelling inhibited ATP release and disrupted activity-dependent signaling between axons and astrocytes. This nonvesicular, nonsynaptic communication could mediate various activity-dependent interactions between axons and nervous system cells in normal conditions, development, and disease.

Inability of volatile anesthetics to inhibit oxygen–glucose deprivation-induced glutamate release via glutamate transporters and anion channels in rat corticostriatal slices

Ischemia-induced extracellular glutamate accumulation and the subsequent excitotoxicity contribute significantly to ischemic brain injury. Volatile anesthetics have been shown to reduce ischemic brain injury. Here, we showed that oxygen–glucose deprivation (OGD, to simulate ischemia in vitro) increased extracellular glutamate accumulation in the corticostriatal slices of adult rats. This increased accumulation was reduced by dihydrokinate, a glutamate transporter type 2 inhibitor, and 4,4′-dinitrostilbene-2,2′-disulfonic acid, a blocker for volume-activated anion channels. The volatile anesthetics isoflurane, sevoflurane and desflurane at clinically relevant concentrations did not affect the OGD-induced extracellular glutamate accumulation from brain slices of adult rats. Isoflurane also did not change the OGD-induced extracellular glutamate accumulation from brain slices of newborn/young rats. These results suggest that the OGD-induced glutamate accumulation involves reversed transport of glutamate via glutamate transporters and volume-activated anion channels. Volatile anesthetics may not inhibit this extracellular glutamate accumulation.

Volume-activated chloride channels in mice Leydig cells

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume-activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of approximately 75 mOsm. These currents display the typical biophysical signature of volume-activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(- )> Cl(- )> F(-)). Staurosporine (200 nM) did not block the activation of I(Cl,swell). The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 microM), SITS (200 microM), ATP (500 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 100 miccroM), and Suramin (10 microM) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(do) and fractional distance of the binding site (delta) of 334 microM and 47 %, 880 microM and 35 %, 2,100 microM and 49%, 188 microM and 27%, and 66.5 microM and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that I(Cl,swell) in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

H-ras transformation sensitizes volume-activated anion channels and increases migratory activity of NIH3T3 fibroblasts

The expression of the H-ras oncogene increases the migratory activity of many cell types and thereby contributes to the metastatic behavior of tumor cells. Other studies point to an involvement of volume-activated anion channels (VRAC) in (tumor) cell migration. In this paper, we tested whether VRACs are required for the stimulation of cell migration upon expression of the H-ras oncogene. We compared VRAC activation and migration of wild-type and H-ras-transformed NIH3T3 fibroblasts by means of patch-clamp techniques and time-lapse video microscopy. Both cell types achieve the same degree of VRAC activation upon maximal stimulation, induced by reducing extracellular osmolarity from 300 to 190 mOsm/l. However, upon physiologically relevant reductions in extracellular osmolarity (275 mOsm/l), the level of VRAC activation is almost three times higher in H-ras-transformed compared to wild-type fibroblasts. This increase in VRAC sensitivity is accompanied by increased migratory activity of H-ras fibroblasts. Moreover, the high-affinity VRAC blocker NS3728 inhibits migration of H-ras fibroblasts dose-dependently by up to about 60%, whereas migration of wild-type fibroblasts is reduced by only about 35%. Consistent with higher VRAC activity in H-ras than in wild-type fibroblasts, more VRAC blocker is needed to achieve a comparable degree of inhibition of migration. We suggest that H-ras modulates the volume set point of VRAC and thus facilitates transient changes of cell volume required for faster cell migration.

Astrocytic swelling in cerebral ischemia as a possible cause of injury and target for therapy

In this viewpoint article, I summarize data showing that the astrocytic swelling that occurs early after the acute CNS pathologies ischemia and traumatic brain injury is damaging. We have proposed that one reason may be the release of excitatory amino acids (EAA) via volume-activated anion channels (VRACs) that are activated by such swelling. This release could be a target for therapy, which could involve blocking the astrocytic swelling or the release mechanisms. The transport mechanisms likely causing the early astrocytic swelling are therefore summarized. In terms of targeting the release mechanisms, we have found a potent inhibitor of VRACs, tamoxifen, to be strongly neuroprotective in focal ischemia with a therapeutic window of 3 h after initiation of the ischemia. The question, however, of whether neuroprotection by tamoxifen can be solely attributed to VRAC inhibition in astrocytes has yet to be resolved.

Hypotonic shock mediation by p38 MAPK, JNK, PKC, FAK, OSR1 and SPAK in osmosensing chloride secreting cells of killifish opercular epithelium

Hypotonic shock rapidly inhibits Cl(-) secretion by chloride cells, an effect that is osmotic and not produced by NaCl-depleted isosmotic solutions, yet the mechanism for the inhibition and its recovery are not known. We exposed isolated opercular epithelia, mounted in Ussing chambers, to hypotonic shock in the presence of a variety of chemicals: a general protein kinase C (PKC) inhibitor chelerythrine, Gö6976 that selectively blocks PKC alpha and beta subtypes, H-89 that blocks PKA, SB203580 that blocks p38 mitogen-activated protein kinase (MAPK), as well as serine/threonine protein phosphatase (PP1 and 2A) inhibitor okadaic acid, and finally tamoxifen, a blocker of volume-activated anion channels (VSOAC). Chelerythrine has no effect on hypotonic inhibition but blocked the recovery, indicating PKC involvement in stimulation. Gö6976 had little effect, suggesting that PKC alpha and PKC beta subtypes are not involved. H-89 did not block hypotonic inhibition but decreased the recovery, indicating PKA may be involved in the recovery and overshoot (after restoration of isotonic conditions). SB203580 significantly enhanced the decrease in current by hypotonic shock, suggesting an inhibitory role of p38 MAPK in the hypotonic inhibition. Okadaic acid increased the steady state current, slowed the hypotonic inhibition but made the decrease in current larger; also the recovery and overshoot were completely blocked. Hypotonic stress rapidly and transiently increased phosphorylated p38 MAPK (pp38) MAPK (measured by western analysis) by eightfold at 5 min, then more slowly again to sevenfold at 60 min. Hypertonic shock slowly increased p38 by sevenfold at 60 min. Phosphorylated JNK kinase was increased by 40-50% by both hypotonic and hypertonic shock and was still elevated at 30 min in hypertonic medium. By immunoblot analysis it was found that the stress protein kinase (SPAK) and oxidation stress response kinase 1 (OSR1) were present in salt and freshwater acclimated fish with higher expression in freshwater. By immunocytochemistry, SPAK, OSR1 and phosphorylated focal adhesion kinase (pFAK) were colocalized with NKCC at the basolateral membrane. The protein tyrosine kinase inhibitor genistein (100 micromol l(-1)) inhibited Cl(-) secretion that was high, increased Cl(-) secretion that was low and reduced immunocytochemical staining for phosphorylated FAK. We present a model for rapid control of CFTR and NKCC in chloride cells that includes: (1) activation of NKCC and CFTR via cAMP/PKA, (2) activation of NKCC by PKC, myosin light chain kinase (MLCK), p38, OSR1 and SPAK, (3) deactivation of NKCC by hypotonic cell swelling, Ca(2+) and an as yet unidentified protein phosphatase and (4) involvement of protein tyrosine kinase (PTK) acting on FAK to set levels of NKCC activity.

Pharmacological characterization of volume-sensitive, taurine permeable anion channels in rat supraoptic glial cells.

To characterize the volume-sensitive, osmolyte permeable anion channels responsible for the osmodependent release of taurine from supraoptic nucleus (SON) astrocytes, we investigated the pharmacological properties of the [(3)H]-taurine efflux from acutely isolated SON. Taurine release induced by hypotonic stimulus (250 mosmol l(-1)) was not antagonized by the taurine transporter blocker guanidinoethyl sulphonate, confirming the lack of implication of the transporter. The osmodependent release of taurine was blocked by a variety of Cl(-) channel inhibitors with the order of potency: NPPB>niflumic acid>DPC>DIDS>ATP. On the other hand, release of taurine was only weakly affected by other compounds (dideoxyforskolin, 4-bromophenacyl bromide, mibefradil) known to block volume-activated anion channels in other cell preparations, and was completely insensitive to tamoxifen, a broad inhibitor of these channels. Although the molecular identity of volume-sensitive anion channels is not firmly established, a few genes have been postulated as potential candidates to encode such channels. We checked the expression in the SON of three of them, ClC(3), phospholemman and VDAC(1), and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC(2). In conclusion, the osmodependent taurine permeable channels of SON astrocytes display a particular pharmacological profile, suggesting the expression of a particular type or subtype of volume-sensitive anion channel, which is likely to be formed by yet unidentified proteins.

Expression of volume-activated anion channels in exocrine acinar cells.

Expression of volume-activated anion channels in exocrine acinar cells.

Mechanisms of amino acid release from the isolated anoxic/reperfused rat heart

Loss of amino acids into the coronary artery perfusate, which is exacerbated during anoxic stress, may have important implications for the ability of hearts subjected to ischemia or anoxia to recover function during reoxygenation. This work investigates the mechanisms underlying the amino acid efflux. Rat Langendorff heart preparations were used to study amino acid loss into coronary artery perfusates during anoxia or anoxia/reoxygenation sequences. Coronary flow rates, heart rates and intra-aortic pressures were recorded. Changes in myocardial amino acid concentrations were equated with amino acid levels in collected anoxic perfusate. With the exception of taurine, the differences in amino acid levels between normoxic and anoxic hearts were smaller than the amounts lost into the coronary perfusates, indicating ongoing replenishment of most amino acids during the anoxic episode. Fifteen-minute periods of exposure to low oxygen levels ( P0 2 18–20 mmHg) resulted in large percentage increases in perfusate amino acid levels which returned slowly towards control levels upon reoxygenation. Anion channel blockers, anthracene-9-carboxylic acid, furosemide, and 4-acetamido-4-isothiocyanostilbene-2,2′-disulfonic acid (SITS), depressed anoxia-elicited increases in amino acid release. Phospholipase inhibition with quinacrine, 4-bromophenacyl bromide and 7,7-dimethyl-eicosadenoic acid (DEDA) depressed the anoxia-evoked release of amino acids. Combined applications of SITS and DEDA exhibited additive effects, virtually abolishing anoxia-evoked release of all the amino acids. The protein kinase C inhibitor, chelerythrine chloride, and the protein tyrosine kinase inhibitors, genistein and lavendustin A, inhibited anoxia-evoked amino acid release. Polyunsaturated fatty acids, arachidonic and linoleic, reduced anoxia-evoked amino acid release whereas monosaturated (oleic) and saturated (stearic) acids were ineffective. The glutamate transport inhibitor, dihydrokainate, depressed anoxia-evoked glutamate and aspartate release. These results suggest that at least three possible mechanisms for the anoxia-evoked amino acid efflux including (a) diffusional release through volume activated anion channels, (b) leakage across myocyte plasma membranes as a consequence of phospholipase activation and (c) reversal of Na + dependent high-affinity transporters.

Volume-sensitive taurine efflux from mammary tissue is not obliged to utilize volume-activated anion channels.

Cell-swelling, induced by a hyposmotic shock, activates the release of taurine from lactating rat mammary tissue explants. The degree of stimulation of taurine efflux was dependent upon the extent of cell-swelling. Volume-sensitive taurine release was attenuated by the anion transport inhibitors NPPB, DIOA, DIDS, niflumate, flufenamate, mefenamate and diiodosalicylate but not by salicylate. Cell-swelling, following a hyposmotic challenge, did not increase the unidirectional efflux of radiolabelled I- or D-asparate from mammary tissue explants. The results suggest that although mammary tissue expresses a volume-sensitive amino acid transport system which is inhibited by anion transport blockers the pathway has no identity with volume-activated anion channels.

Volume-activated anion channels in HTC rat hepatoma cells are permeable to taurine: [formula omitted], A Sostman, G Shimokura, JG Fitz, Div of Gastroenterology, Duke University Medical Center, Durham, NC 27710

Volume-activated anion channels in HTC rat hepatoma cells are permeable to taurine: [formula omitted], A Sostman, G Shimokura, JG Fitz, Div of Gastroenterology, Duke University Medical Center, Durham, NC 27710

Inhibition of volume-activated I- and taurine efflux from HeLa cells by P-glycoprotein blockers correlates with calmodulin inhibition.

P-glycoprotein, an active transporter that pumps a diverse range of hydrophobic compounds out of cells, has recently been proposed to function as, or regulate, a volume-activated, anion-selective channel (Valverde, M.A., Diaz, M., Sepulveda, F. V., Gill, D. R., Hyde, S. C., and Higgins, C. F. (1992) Nature 355, 830-833). In this study a number of compounds known to inhibit P-glycoprotein-mediated drug pumping were tested for their effect on the osmotically activated release from HeLa cells of I-, a known substrate of volume-activated anion channels, and taurine, a sulfonic amino acid that serves as an important organic osmolyte in many cell-types. Tamoxifen, 4-iodotamoxifen, and pyrrolidino-4-iodotamoxifen (idoxifene) were potent blockers of osmotically activated I- and taurine efflux. Other known P-glycoprotein inhibitors (verapamil, cyclosporin A, pimozide, trifluoperazine, ICI 164, and ICI 182) were less effective. For all compounds tested the effect on taurine release was the same as that on I- release, consistent with the hypothesis that swelling-activated taurine release is via anion-selective channels. There was no positive correlation between the effect of the inhibitors on osmotically activated solute release and their effect on P-glycoprotein-mediated drug transport. In contrast, there was a strong positive correlation between the IC50 values for the effect of the inhibitors on volume-activated solute release and those for their effect on calmodulin. These data raise doubts as to whether the effect of P-glycoprotein inhibitors on volume-activated channels is a consequence of their interaction with P-glycoprotein and indicate a possible role for calmodulin, or a cell component having at least some physical similarities, in controlling channel activity.