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At least 19 recordsLinked to original sources
PubMed2005

Characterization of a proton-activated, outwardly rectifying anion channel.

Anion channels are present in every mammalian cell and serve many different functions, including cell volume regulation, ion transport across epithelia, regulation of membrane potential and vesicular acidification. Here we characterize a proton-activated, outwardly rectifying current endogenously expressed in HEK293 cells. Binding of three to four protons activated the anion permeable channels at external pH below 5.5 (50% activation at pH 5.1). The proton-activated current is strongly outwardly rectifying, due to an outwardly rectifying single channel conductance and an additional voltage dependent facilitation at depolarized membrane potentials. The anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) rapidly and potently inhibited the channel (IC50: 2.9 microm). Flufenamic acid blocked this channel only slowly, while mibefradil and amiloride at high concentrations had no effect. As determined from reversal potential measurements under bi-ionic conditions, the relative permeability sequence of this channel was SCN-> I-> NO3-> Br-> Cl-. None of the previously characterized anion channel matches the properties of the proton-activated, outwardly rectifying channel. Specifically, the proton-activated and the volume-regulated anion channels are two distinct and separable populations of ion channels, each having its own set of biophysical and pharmacological properties. We also demonstrate endogenous proton-activated currents in primary cultured hippocampal astrocytes. The proton-activated current in astrocytes is also carried by anions, strongly outwardly rectifying, voltage dependent and inhibited by DIDS. Proton-activated, outwardly rectifying anion channels therefore may be a broadly expressed part of the anionic channel repertoire of mammalian cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Molecular physiology of anion channels.

Anion channels have diverse functions, ranging from regulation of cell volume to transepithelial transport and control of excitability. Three well established structural classes of plasma membrane chloride channels now exist: the ligand-gated chloride channels, the cAMP-stimulated cystic fibrosis transmembrane conductance regulator channel, and the voltage-gated (or swelling-activated) members of the CLC chloride channel family. Genetic defects leading to inherited disease are known for each of these classes. A combination of mutagenesis and biophysical analysis has been used to correlate their structure with function. Recently, the role of several molecules has been questioned; rather than being chloride channels themselves, they may be activators of endogenous channels in the cells used for expression.

Animals↗
PubMed1995

Anion-Channel Blockers Inhibit S-Type Anion Channels and Abscisic Acid Responses in Guard Cells.

The effects of anion-channel blockers on light-mediated stomatal opening, on the potassium dependence of stomatal opening, on stomatal responses to abscisic acid (ABA), and on current through slow anion channels in the plasma membrane of guard cells were investigated. The anion-channel blockers anthracene-9-carboxylic acid (9-AC) and niflumic acid blocked current through slow anion channels of Vicia faba L. guard cells. Both 9-AC and niflumic acid reversed ABA inhibition of stomatal opening in V. faba L. and Commelina communis L. The anion-channel blocker probenecid also abolished ABA inhibition of stomatal opening in both species. Additional tests of 9-AC effects on stomatal aperture in Commelina revealed that application of this anion-channel blocker allowed wide stomatal opening under low (1 mM) KCI conditions and increased the rate of stomatal opening under both low and high (100 mM) KCI conditions. These results indicate that anion channels can function as a negative regulator of stomatal opening, presumably by allowing anion efflux and depolarization, which prohibits ion up-take in guard cells. Furthermore, 9-AC prevented ABA induction of stomatal closure. A model in which ABA activation of anion channels contributes a rate-limiting mechanism during ABA-induced stomatal closure and inhibition of stomatal opening is discussed.

Journal Article↗
PubMed1993

Identification of High-Affinity Slow Anion Channel Blockers and Evidence for Stomatal Regulation by Slow Anion Channels in Guard Cells.

Slow anion channels in the plasma membrane of guard cells have been suggested to constitute an important control mechanism for long-term ion efflux, which produces stomatal closing. Identification of pharmacological blockers of these slow anion channels is instrumental for understanding plant anion channel function and structure. Patch clamp studies were performed on guard cell protoplasts to identify specific extracellular inhibitors of slow anion channels. Extracellular application of the anion channel blockers NPPB and IAA-94 produced a strong inhibition of slow anion channels in the physiological voltage range with half inhibition constants (K1/2) of 7 and 10 [mu]M, respectively. Single slow anion channels that had a high open probability at depolarized potentials were identified. Anion channels had a main conductance state of 33 [plus or minus] 8 pS and were inhibited by IAA-94. DIDS, which has been shown to be a potent blocker of rapid anion channels in guard cells (K1/2 = 0.2 [mu]M), blocked less than 20% of peak slow anion currents at extracellular or cytosolic concentrations of 100 [mu]M. The pharmacological properties of slow anion channels described here differ from those recently described for rapid anion channels in guard cells, fortifying the finding that two highly distinct types or modes of voltage- and second messenger-dependent anion channel currents coexist in the guard cell plasma membrane. Bioassays using anion channel blockers provide evidence that slow anion channel currents play a substantial role in the regulation of stomatal closing. Interestingly, slow anion channels may also function as a negative regulator during stomatal opening under the experimental conditions applied here. The identification of specific blockers of slow anion channels reported here permits detailed studies of cell biological functions, modulation, and structural components of slow anion channels in guard cells and other higher plant cells.

Journal Article↗
PubMed1994

Characterization of a voltage-dependent anionic channel in fused synaptosomes isolated from rat hippocampi.

The inside out configuration of the patch-clamp technique was used to study single-channel anionic currents from purified hippocampal synaptosomes fused into liposomes to form giant proteoliposomes. At least six different anionic channels with unitary conductances of 22-150 pS were found. The most frequently observed was the 32-pS conductance channel. This was voltage-dependent; the open probability increased from 0.20 at -40 mV to 0.46 at 40 mV. This channel may be involved in the repolarization of nerve terminal membranes after an action potential, thus, limiting the duration of the spike and the transmitter release.

Animals↗
PubMed2003

Amino acid-permeable anion channels in early mouse embryos and their possible effects on cleavage.

Effects of several Cl(-) channel blockers on ionic currents in mouse embryos were studied using whole-cell patch-clamp and microelectrode methods. Microelectrode measurements showed that the resting membrane potential of early embryonic cells (1-cell stage) was -23 mV and that reduction of extracellular Cl(-) concentration depolarized the membrane, suggesting that Cl(-) conductance is a major contributor for establishing the resting membrane potential. Membrane currents recorded by whole-cell voltage clamp showed outward rectification and confirmed that a major component of these embryonic currents are carried by Cl(-) ions. A Cl(-) channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suppressed the outward rectifier current in a voltage- and concentration-dependent manner. Other Cl(-) channel blockers (5-nitro-2-[3-phenylpropyl-amino] benzoic acid and 2-[3-(trifluoromethyl)-anilino] nicotinic acid [niflumic acid]) similarly inhibited this current. Simultaneous application of niflumic acid with DIDS further suppressed the outward rectifier current. Under high osmotic condition, niflumic acid, but not DIDS, inhibited the Cl(-)current, suggesting the presence of two types of Cl(-) channels: a DIDS-sensitive (swelling-activated) channel, and a DIDS-insensitive (niflumic acid-sensitive) Cl(-) channel. Anion permeability of the DIDS-insensitive Cl(-) current differed from that of the compound Cl(-) current: Rank order of anion permeability of the DIDS-sensitive Cl(-) channels was I(-) = Br(-) > Cl(-) > gluconate(-), whereas that of the DIDS-insensitive Cl(-) channel was I(-) = Br(-) > Cl(-) >> gluconate(-). These results indicate that early mouse embryos have a Cl(-) channel that is highly permeable to amino acids, which may regulate intracellular amino acid concentration.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Single channel characteristics of a high conductance anion channel in "sarcoballs".

Previously undescribed high conductance single anion channels from frog skeletal muscle sarcoplasmic reticulum (SR) were studied in native membrane using the "sarcoball" technique (Stein and Palade, 1988). Excised inside-out patches recorded in symmetrical 200 mM TrisCl show the conductance of the channel's predominant state was 505 +/- 25 pS (n = 35). From reversal potentials, the Pcl/PK ratio was 45. The slope conductance vs. Cl- ion concentration curve saturates at 617 pS, with K0.5 estimated at 77 mM. The steady-state open probability (Po) vs. holding potential relationship produces a bell-shaped curve, with Po values reaching a maximum near 1.0 at 0 mV, and falling off to 0.05 at +/- 25 mV. Kinetic analysis of the voltage dependence reveals that while open time constants are decreased somewhat by increases in potential, the largest effect is an increase in long closed times. Despite the channel's high conductance, it maintains a moderate selectivity for smaller anions, but will not pass larger anions such as gluconate, as determined by reversal-potential shifts. At least two substates different from the main open level are distinguishable. These properties are unlike those described for mitochondrial voltage-dependent anion channels or skeletal muscle surface membrane Cl channels and since SR Ca channels are present in equally high density in sarcoball patches, we propose these sarcoball anion channels originate from the SR. Preliminary experiments recording currents from frog SR anion channels fused into liposomes indicate that either biochemical isolation and/or alterations in lipid environment greatly decrease the channel's voltage sensitivity. These results help underline the potential significance of using sarcoballs to study SR channels. The steep voltage sensitivity of the sarcoball anion channel suggests that it could be more actively involved in the regulation of Ca2+ transport by the SR.

Animals↗
PubMed1993

Single-channel characteristics of the large-conductance anion channel in rat cortical astrocytes in primary culture.

Cultured rat cortical astrocytes, in addition to a variety of voltage-sensitive potassium channels, also express anion channels. However, the behavior and regulation of these anion channels have been far less studied. This paper describes a patch-clamp study on a voltage-sensitive 200-300 pS high-conductance single-channel anion current, which seems to possess at least five different open sublevels or, alternatively, be formed from five or more small-conductance ion channels linked together. This channel is voltage dependent, showing a bell-shaped open probability curve with highest open probability close to the reversal potential (zero-current). Although potassium channels are commonly detected in astrocytes in cell-attached and excised patches with both normal osmolarity and hypoosmotic solutions, the occurrence of the anion channel is clearly increased in isolated patches when hypoosmotic bath solutions are used. Also, cell aging in culture and the preparation of secondary cell cultures by trypsinization seem to increase the rate of occurrence of the anion channel. Though this channel is more routinely seen when a membrane patch is excised from the cell, occasionally cell-attached configurations with instant channel activity can be formed. While the modulation of this anion channel was being studied, it was found to be blocked by an anion transport inhibitor, L-644,711, reported to affect cell volume regulation in astrocytes.

Animals↗
PubMed1993

Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial membrane channel, the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane is a small abundant protein found in all eukaryotic kingdoms which forms a voltage-gated pore when incorporated into planar lipid bilayers. VDAC is also the site of binding of the metabolic enzymes hexokinase and glycerol kinase to the mitochondrion in what may be a significant metabolic regulatory interaction. Recently, there has been speculation that there may be multiple forms of VDAC in mammals which differ in their localization in the outer mitochondrial membrane and in their physiological function. In this report, we describe the identification and characterization of two human cDNAs encoding VDAC homologs (HVDAC1 and HVDAC2). To confirm VDAC function, each human protein has been expressed in yeast lacking the endogenous VDAC gene. Human proteins isolated from yeast mitochondria formed channels with the characteristics expected of VDAC when incorporated into planar lipid bilayers. In addition, expression of the human proteins in such strains can complement phenotypic defects associated with elimination of the endogenous yeast VDAC gene. Since VDAC is the site of binding of hexokinase to the outer mitochondrial membrane, the binding capacity of each VDAC isoform expressed in yeast mitochondria was assessed. When compared with the binding of hexokinase to mitochondria lacking VDAC, the results show that mitochondria expressing HVDAC1 are capable of specifically binding hexokinase, whereas mitochondria expressing HVDAC2 only bind hexokinase at background levels. The expression of each human cDNA has been assessed by Northern blot and polymerase chain reaction techniques. With one exception, each is expressed in all human cell lines and tissues examined.

Amino Acid Sequence↗
PubMed1996

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.

Animals↗
PubMed2005

Genetic demonstration that the plasma membrane maxianion channel and voltage-dependent anion channels are unrelated proteins.

The maxianion channel is widely expressed in many cell types, where it fulfills a general physiological function as an ATP-conductive gate for cell-to-cell purinergic signaling. Establishing the molecular identity of this channel is crucial to understanding the mechanisms of regulated ATP release. A mitochondrial porin (voltage-dependent anion channel (VDAC)) located in the plasma membrane has long been considered as the molecule underlying the maxianion channel activity, based upon similarities in the biophysical properties of these two channels and the purported presence of VDAC protein in the plasma membrane. We have deleted each of the three genes encoding the VDAC isoforms individually and collectively and demonstrate that maxianion channel (approximately 400 picosiemens) activity in VDAC-deficient mouse fibroblasts is unaltered. The channel activity is similar in VDAC1/VDAC3-double-deficient cells and in double-deficient cells with the VDAC2 protein depleted by RNA interference. VDAC deletion slightly down-regulated, but never abolished, the swelling-induced ATP release. The lack of correlation between VDAC protein expression and maxianion channel activity strongly argues against the long held hypothesis of plasmalemmal VDAC being the maxianion channel.

Adenosine Triphosphate↗
PubMed2002

Anion channels influence ECC by modulating L-type Ca(2+) channel in ventricular myocytes.

Anion channels are extensively expressed in the heart, but their roles in cardiac excitation-contraction coupling (ECC) are poorly understood. We, therefore, investigated the effects of anion channels on cardiac ventricular ECC. Edge detection, fura 2 fluorescence measurements, and whole cell patch-clamp techniques were used to measure cell shortening, the intracellular Ca(2+) transient, and the L-type Ca(2+) current (I(Ca,L)) in single rat ventricular myocytes. The anion channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid reversibly inhibited the Ca(2+) transients and cell shortening in a dose-dependent manner. Comparable results were observed when the majority of the extracellular Cl(-) was replaced with the relatively impermeant anions glutamate (Glt(-)) and aspartate (Asp(-)). NPPB and niflumic acid or the Cl(-) substitutes did not affect the resting intracellular Ca(2+) concentration but significantly inhibited I(Ca,L). In contrast, replacement of extracellular Cl(-) with the permeant anions NO, SCN(-), and Br(-) supported the ECC and I(Ca,L), which were still sensitive to blockade by NPPB. Exposure of cardiac ventricular myocytes to a hypotonic bath solution enhanced the amplitude of cell shortening and supported I(Ca,L), whereas hypertonic stress depressed the contraction and I(Ca,L). Moreover, cardiac contraction was completely abolished by NPPB (50 microM) under hypotonic conditions. It is concluded that a swelling-activated anion channel may be involved in the regulation of cardiac ECC through modulating L-type Ca(2+) channel activity.

Animals↗
PubMed1996

Modulation of frog skeletal muscle Ca2+ release channel gating by anion channel blockers.

Effects of niflumic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on frog skeletal muscle ryanodine receptors have been studied by incorporating sarcoplasmic reticulum vesicles into planar lipid bilayers. Niflumic acid increased the mean open probability (Po) at 10 microM and decreased Po at 100 microM with no change in open time constants, unitary conductance, and reversal potential. The Po was augmented by DIDS at 5-200 microM without affecting either unitary conductance or reversal potential. DIDS induced a new third open time constant, probably contributing to a long-lived open state. Channels modified by niflumic acid or DIDS still responded to Ca2+ release channel modulators. These results provide evidence that niflumic acid and DIDS modify the gating mechanism of ryanodine receptors without affecting binding sites to the modulators and the physical pathway of the conducting pore. p-Chloromercuriphenyl sulfonic acid (pCMPS) transiently increased the Po. The channel modified by DIDS responded to pCMPS, whereas that by ryanodine did not. The long open state of the channel induced by DIDS is produced by a quite different mechanism(s) from that by ryanodine. Contrary to cardiac ryanodine receptors, Po of skeletal muscle channels was independent of voltage after DIDS modification.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

A new scheme of symbiosis: ligand- and voltage-gated anion channels in plants and animals.

Anion channels in the plasma membrane of both plant and animal cells participate in a number of important cellular functions such as volume regulation, trans-epithelial transport, stabilization of the membrane potential and excitability. Only very recently attention has turned to the presence of anion channels in higher plant cells. A dominant theme among recent discoveries is the role of Ca2+ in activating or modulating channel current involved in signal transduction. The major anion channel of stomatal guard cell protoplasts is a 32-40 pS channel which is highly selective for anions, in particular NO3-, Cl- and malate. These channels are characterized by a steep voltage dependence. Anion release is elicited upon depolarization and restricted to a narrow voltage span of -100 mV to the reversal potential of anions. During prolonged activation the current slowly inactivates. A rise in cytoplasmic calcium in the presence of nucleotides evokes activation of the anion channels. Following activation they catalyse anion currents 10-20 times higher than in the inactivated state thereby shifting the resting potential of the guard cell from a K(+)-conducting to an anion-conducting state. Patch-clamp studies have also revealed that growth hormones directly affect voltage-dependent activity of the anion channel in a dose-dependent manner. Auxin binding resulted in a shift of the activation potential towards the resting potential. Auxin-dependent gating of the anion channel is side- and hormone-specific. Its action is also channel-specific as K+ channels coexisting in the same membrane patch were insensitive to this ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Cation and anion channels in rat and human spermatozoa.

Ionic fluxes are thought to be important in the initiating process of gamete interaction such as acrosome reaction. Different populations of ion channels in rat and human spermatozoa were investigated using the planar lipid bilayer technique. Membrane proteins were isolated from rat and human sperm and inserted into lipid bilayer via fusion. We observed K(+) selective and Na(+)-selective channels, as well as divalent permeable cation channels in membrane preparations from rat sperm K+ channels, which were sensitive to the K+ channel blocker, tetraethylammonium (0.1 mM), exhibited a mean single channel conductance of 24 pS. Whereas, larger conductance, 109 pS, was found to be associated with Na+ channels. Low conductance anion channel, 15 pS, was also observed when permeant cations in the bathing solutions were substituted with N-methyl-D-glucamine leaving Cl- as the major permeant ion species. This channel exhibited a slower channel open and closed kinetics when compared to other cation channels. Both cation and anion channels with characteristics similar to that found in rat sperm were also observed in preparations from human sperm. The variety in the types of ion channels observed in rat and human spermatozoa suggests that ion channels may play different roles in sperm physiology and gamete interaction.

Animals↗
PubMedDate not supplied

Challenging accepted ion channel biology: p64 and the CLIC family of putative intracellular anion channel proteins (Review).

Parchorin, p64 and the related chloride intracellular channel (CLIC) proteins are widely expressed in multicellular organisms and have emerged as candidates for novel, auto-inserting, self-assembling intracellular anion channels involved in a wide variety of fundamental cellular events including regulated secretion, cell division and apoptosis. Although the mammalian phosphoproteins p64 and parchorin (49 and 65K, respectively) have only been indirectly implicated in anion channel activity, two CLIC proteins (CLIC1 and CLIC4, 27 and 29K, respectively) appear to be essential molecular components of anion channels, and CLIC1 can form anion channels in planar lipid bilayers in the absence of other cellular proteins. However, these putative ion channel proteins are controversial because they exist in both soluble and membrane forms, with at least one transmembrane domain. Even more surprisingly, soluble CLICs share the same glutaredoxin fold as soluble omega class glutathione-S-transferases. Working out how these ubiquitous, soluble proteins unfold, insert into membranes and then refold to form integral membrane proteins, and how cells control this potentially dangerous process and make use of the associated ion channels, are challenging prospects. Critical to this future work is the need for better characterization of membrane topology, careful functional analysis of reconstituted and native channels, including their conductances and selectivities, and detailed structure/function studies including targeted mutagenesis to investigate the structure of the putative pore, the role of protein phosphorylation and the role of conserved cysteine residues.

Amino Acid Sequence↗
PubMed1988

Evidence for anion channels in secretory vesicles.

Secretory vesicles from bovine neurohypophysis were reconstituted into lipid bilayers. Electrical measurements on the lipid bilayers under voltage clamp demonstrated the presence of channels that are permeable to chloride ions, are blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and are slightly voltage dependent. When several different membrane fractions were used for the reconstitution, the probability of finding channels correlated with the fraction of secretory vesicle membrane in the membrane fraction, indicating that the secretory vesicles are the source of the channels. The observed anion channel can provide a pathway for the anion transport that has previously been described for secretory vesicles. The secretory vesicle anion channel may play a role in calcium-induced secretion.

Animals↗
PubMed1996

Anion channel blockers inhibit swelling-activated anion, cation, and nonelectrolyte transport in HeLa cells.

The effect of osmotic cell swelling on the permeability of HeLa cells to a range of structurally unrelated solutes including taurine, sorbitol, thymidine, choline, and K+ (96Rb+) was investigated. For each solute tested, reduction in the osmolality of the medium from 300 to 200 mosmol/kgH2O caused a significant increase in the unidirectional influx rate. In each case, the osmotically activated transport component was nonsaturable up to external substrate concentrations of 50 mM. Inhibitors of the swelling-activated anion channel of HeLa cells [quinine, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, niflumate, 1,9-dideoxyforskolin, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and tamoxifen] blocked the osmotically activated influx of each of the different substrates tested, as well as the osmotically activated efflux of taurine and I-. Tamoxifen and NPPB were similarly effective at blocking the osmotically activated efflux of 96Rb+. The simplest of several hypotheses consistent with the data is that the osmotically activated transport of the different solutes tested here is via a swelling-activated anion-selective channel that has a significant cation permeability and a minimum pore diameter of 8-9 A.

Anions↗