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PubMed1994

Actin-based cytoskeleton regulates a chloride channel and cell volume in a renal cortical collecting duct cell line.

The regulatory volume decrease (RVD) of a renal cortical collecting duct cell line (RCCT-28A) exposed to a hypotonic solution was studied using electronic cell sizing to measure cell volume and the patch clamp technique to measure Cl- channel activity. Results demonstrate that RVD was mediated in part by KCl loss through separate K+ and Cl- channels. The Cl- channel had a conductance of 305 pS and was activated by cell swelling, membrane stretch, and disruption of F-actin by dihydrocytochalasins. In contrast, stabilizing F-actin with phalloidin prevented swelling and stretch activation of the Cl- channel and inhibited the RVD. Thus, the state of actin polymerization regulates the probability of the 305 pS Cl- channel being open. Short actin filaments activate whereas long actin filaments inactivate the channel. Taken together, our studies suggest that RVD in this renal collecting duct cell line cell is mediated in part by a 305 pS Cl- channel, which is activated, during cell swelling, by a signaling pathway that includes disruption of F-actin.

Actins↗
PubMed1996

Endothelin receptor coupling to potassium and chloride channels in isolated rat pulmonary arterial myocytes.

Endothelin (endothelin)-1 may in important role in the control of pulmonary arterial tone, part of its action being due to a putative ability to regulate membrane ion channel activity. Consequently, we have examined its effects on membrane currents in pulmonary arterial myocytes by using the patchclamp recording technique. Endothelin-1 (0.1-50 nM) activated an oscillatory inward current and caused a brief enhancement, followed by inhibition of the outward K+ current. Pharmacological and anion substitution experiments revealed that the oscillations of inward current were due to ET(A) receptor stimulation and subsequent activation of Ca(+2)-activated Cl- channels (EC50 > or = 16 nM). Enhancement of K+ current was inhibited by FR139317 (an ET(A) receptor antagonist) and by buffering intracellular Ca+2, which suggests that ET(A) receptor stimulation also activates Ca(+2)-activated K+ channels. Inhibition of K+ current (IC50 approximately 0.5 nM) by endothelin-1 was not dependent on phosphorylation or intracellular Ca+2 and was unaffected by FR139317 but was mimicked by ET(B) receptor agonists. It appears, therefore, that ET(A) and ET(B) receptors in pulmonary arterial myocytes are differentially coupled to Cl- and K+ channels. Modulation of these channels by endothelin-1 may prove to play an important role in regulating pulmonary arterial smooth muscle tone under both physiological and pathological conditions.

Animals↗
PubMed1999

Hydrogen peroxide inhibits chloride channels of the sarcoplasmic reticulum of skeletal muscle.

Data obtained with the lipid bilayer technique indicate that cis (cytoplasmic) concentration of 4.4-22 mm hydrogen peroxide (H(2)O(2)), is a water-soluble oxidant. [H(2)O(2)](cis) (n = 26) reversibly inhibits the multisubconductance SCl channel of the sarcoplasmic reticulum vesicles from rabbit skeletal muscle. At -40 mV, the mean values of the current amplitude (I) and the probability of the SCl channel being open (P(o)) were reduced significantly (n = 8) from -6.14 +/- 0.42 pA and 0.69 +/- 0.06 (for all conductance levels) in control 0.0 mm [H(2)O(2)](cis) to -1.10 +/- 0.51 pA and 0. 13 +/- 0.04 (for the intermediate subconductance states) in 8.8 mm [H(2)O(2)](cis), respectively. The [H(2)O(2)](cis)-induced decrease in P(o) is mainly due to a decrease in the mean open time T(o). The mechanism of [H(2)O(2)](cis) effects on the multiconductance SCl channel is characterized by a mode shift in the channel state from the main conductance state to the low subconductance states. The estimated concentration of the [H(2)O(2)](cis) for the half inhibitory constant, K(i), was 11.78 mm, higher than the estimated 8. 0 and 8.1 mm for the parameters P(o) and T(o), respectively, indicating that the conductance of the SCl channel is less sensitive than the gating kinetics of the channel. After a lag period of between 30 to 60 sec, the lipophilic SH-oxidizing agent 4, 4'-dithiodipyridine (4,4'-DTDP) added to the cis side at 1.0 mm removed the inhibitory effects of 8.8 mm [H(2)O(2)](cis). The 4, 4'-DTDP-enhanced SCl channel activity was blocked after the addition of 0.5 mm ATP to the cis side of the channel. The addition of 1.0 mm 4,4'-DTDP to the cis or trans solutions facing an SCl channel already subjected to 0.5 mm [ATP](cis) or [ATP](trans) failed to activate the ATP-inhibited SCl channel. These findings suggest that 4,4'-DTDP is not preventing the binding of ATP to its binding site on the channel protein. The interaction of H(2)O(2) with the SCl channel proteins is consistent with a thiol-disulfide redox state model for regulating ion transport, where SH groups can directly modify the function of the channel and/or the availability of regulatory sites on the channel proteins. The H(2)O(2) effects on the Ca(2+) countercurrent through the SCl channel are also consistent with H(2)O(2)-modification of the mechanisms involved in the Ca(2+) regulation, which underlies excitation-contraction coupling in skeletal muscle.

Adenosine Triphosphate↗
PubMed1999

Cation permeability and cation-anion interactions in a mutant GABA-gated chloride channel from Drosophila.

To investigate the structural basis of anion selectivity of Drosophila GABA-gated Cl(-) channels, the permeation properties of wild-type and mutant channels were studied in Xenopus oocytes. This work focused on asparagine 319, which by homology is one amino acid away from a putative extracellular ring of charge that regulates cation permeation in nicotinic receptors. Mutation of this residue to aspartate reduced channel conductance, and mutation to lysine or arginine increased channel conductance. These results are consistent with an electrostatic interaction between this site and permeating anions. The lysine mutant, but not the arginine mutant, formed a channel that is permeable to cations, and this cannot be explained in terms of electrostatics. The lysine mutant had a 25-mV reversal potential in solutions with symmetrical Cl(-) and asymmetrical cations. The permeability ratio of K(+) to Cl(-) was determined as 0. 33 from reversal potential measurements in KCl gradients. Experiments with large organic cations and anions showed that cation permeation can only be seen in the presence of Cl(-), but Cl(-) permeation can be seen in the absence of permeant cations. Measurements of permeability ratios of organic anions indicated that the lysine mutant has an increased pore size. The cation permeability of the lysine-containing mutant channel cannot be accounted for by a simple electrostatic interaction with permeating ions. It is likely that lysine substitution causes a structural change that extends beyond this one residue to influence the positions of other channel-forming residues. Thus protein conformation plays an important role in enabling ion channels to distinguish between anions and cations.

Amino Acid Sequence↗
PubMed1995

Volume-sensitive chloride channel activity does not depend on endogenous P-glycoprotein.

To determine whether endogenous P-glycoprotein, the MDR1 gene product that functions as a drug transport pump, is a volume-sensitive Cl- channel molecule or a protein kinase C-mediated regulator of the Cl- channel, whole-cell patch-clamp and molecular biological experiments were carried out in a human small intestinal epithelial cell line. Endogenous expression of P-glycoprotein was confirmed by Northern blot analysis, reverse transcription-polymerase chain reaction, Western blot analysis, and immunostaining. The P-glycoprotein expression was abolished by the antisense (but not sense) oligonucleotide for the MDR1 gene, whereas the magnitude of the Cl- current activated by osmotic swelling was not distinguishable between both antisense- and sense-treated cells. The volume-sensitive Cl- currents were not specifically affected by the anti-P-glycoprotein monoclonal antibodies, MRK16, C219, and UIC2. An inhibitor of P-glycoprotein-mediated pump activity, verapamil, was found to never affect the Cl- current. A substrate for the P-glycoprotein-mediated drug pump, vincristine or daunomycin, did not prevent swelling-induced activation of the Cl- current. Furthermore, the Cl- current was not affected by an activator of protein kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol). Thus, it is concluded that the endogenous P-glycoprotein molecule is not itself a volume-sensitive Cl- channel nor a protein kinase C-mediated regulator of the channel in the human epithelial cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Functional and structural conservation of CBS domains from CLC chloride channels.

All eukaryotic CLC Cl(-) channel subunits possess a long cytoplasmic carboxy-terminus that contains two so-called CBS (cystathionine beta-synthase) domains. These domains are found in various unrelated proteins from all phylae. The crystal structure of the CBS domains of inosine monophosphate dehydrogenase (IMPDH) is known, but it is not known whether this structure is conserved in CLC channels. Working primarily with ClC-1, we used deletion scanning mutagenesis, coimmunoprecipitation and electrophysiology to demonstrate that its CBS domains interact. The replacement of CBS domains of ClC-1 with the corresponding CBS domains from other CLC channels and even human IMPDH yielded functional channels, indicating a high degree of structural conservation. Based on a homology model of the pair of CBS domains of CLC channels, we identified some residues that, when mutated, affected the common gate which acts on both pores of the dimeric channel. Thus, we propose that the structure of CBS domains from CLC channels is highly conserved and that they play a functional role in the common gate.

Amino Acid Sequence↗
PubMed2004

Inwardly rectifying chloride channel activity in intestinal pacemaker cells.

Cl(-) channels are proposed to play a role in gut pacemaker activity, but little is known about the characteristics of Cl(-) channels in interstitial cells of Cajal (ICC), the intestinal pacemaker cells. The objective of the present study was to identify whole cell Cl(-) currents in ICC associated with previously observed single-channel activity and to characterize its inward rectification. Whole cell patch-clamp studies showed that ICC express an inwardly rectifying Cl(-) current that was not sensitive to changes in cation composition of the extracellular solutions. Currents were not affected by replacing all cations with N-methyl-d-glucamine (NMDG(+)). Whole cell currents followed the Cl(-) equilibrium potential and were inhibited by DIDS and 9-anthracene carboxylic acid. Ramp protocols of single-channel activity showed that inward rectification was due to reduction in single-channel open probability, not a reduction in single-channel conductance. Single-channel data led to the hypothesis that strong cooperation exists between 30-pS channels that show less cooperation at potentials positive to the reversal potential. Hence, an inwardly rectifying Cl(-) channel plays a prominent role in determining pacemaker activity in the gut.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Effect of deletion mutations on the function of CFTR chloride channels.

Mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a unique R domain; CFTR functions as a Cl- channel regulated by phosphorylation and by nucleoside triphosphates. To study the domains of CFTR involved in Cl- channel function, we expressed mutants lacking various domains and assayed cAMP-stimulated Cl- channel activity using the halide-sensitive fluorophore, 6-methoxy-N-(3'-sulfopropyl)-quinolinium. We previously reported that deletion of part of the R domain (residues 708-835) produced Cl- channels that were constitutively open. Here we show that more extensive deletions within the R domain failed to generate functional CFTR Cl- channels; the portion of protein that could be deleted without destroying function corresponds to sequences that are not conserved in related proteins. In contrast, when we deleted the two NBDs (either alone, together, or in combination with the R domain), we did not observe functional Cl- channels. CFTR has a unique carboxyl terminus that is conserved across species. However, truncation of the carboxyl terminus (up to, but not including, NBD2) produced a regulated anion permeability similar to that of wild-type CFTR, suggesting that this region is not essential for channel function. Expression of two CF-associated nonsense mutants (G542X and W1316X) also failed to generate functional CFTR Cl- channels. These results help define structure:function relationships for CFTR and identify the domains that are required for Cl- channel function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Resolving three types of chloride channels in demyelinated Xenopus axons.

Axons from Xenopus sciatic nerve were demyelinated by intraneural injection of lysolecithin rendering the entire internodal axolema accessible to a patch electrode. In this region, three types of anion selective pores were found and characterized at the single-channel level. These included outwardly rectifying, inwardly rectifying, and maxi Cl- channels. The outwardly rectifying Cl- channels (24 pS) are activated by depolarization with a weak voltage dependence of 42 mV per e-fold change in open probability. The inwardly rectifying Cl- channels (27 pS) are insensitive to voltage, but can be blocked by internal application of 100 microM SITS or DIDS. The I-V curves of rectifying channels are S-shaped and can be fitted by a kinetic model with a single free energy barrier. The rectification may be related to the location of this barrier. The maxi Cl- channel (335 pS) is often open at the resting potential, but is inactivated by a large depolarization. The rectification, voltage dependence, and inactivation of these channels may contribute to the regulation of axonal Cl- balance and resting potential.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Actions of cyclic esters, S-esters, and amides of phenyl- and phenylthiophosphonic acids on mammalian and insect GABA-gated chloride channels.

Cyclic esters, S-esters, and amides of phenyl(thio)phosphonic acid were synthesized to probe the interaction between noncompetitive antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors and their binding site. Some of these compounds competitively inhibited the specific binding of [3H]EBOB, a noncompetitive GABA antagonist, to rat-brain and housefly-head membranes. The trans isomer of the ester bearing a tert-butyl group at the 5-position and a bromine atom at the p-position (5t) was most potent in rat receptors with an IC50 value of 40 nM, while the trans isomer of the S-ester bearing the same substituents (10t) was most potent in housefly receptors with an IC50 value of 55 nM. In both cases, the corresponding amide analogue (12t) was less potent. The potencies of 5t and 12t tended to decrease in the presence of GABA, particularly in housefly receptors, while that of 10t remained unchanged. The rank order of activity in inhibiting [3H]EBOB binding to housefly-head membranes in the presence of GABA (10t > 5t > 12t) was in accord with that of insecticidal activity. S-Ester 10t depressed 10 microM and 300 microM GABA-induced 36Cl- influx into mouse cerebral synaptoneurosomes, whereas ester 5t depressed 10 microM GABA-induced 36Cl- influx but not 300 microM GABA-induced flux. Amide 12t was inactive at both GABA concentrations. These findings indicate that six-membered cyclic phenylthiophosphonic acid derivatives act as noncompetitive antagonists of GABA receptors and suggest that 10t is able to bind to the receptor in the open, desensitized, and closed states, whereas the affinity of 5t and 12t is lower in the open and desensitized states than in the closed state. The derivatives have similar structures except for the heteroatoms at the 1- and 3-positions, so that the heteroatoms may play a unique role when antagonists bring the open state of the GABA-gated channel to the desensitized or closed state.

Affinity Labels↗
PubMed2005

Chloride channel activity in human lung fibroblasts and myofibroblasts.

It is well established that transforming growth factor (TGF)-beta stimulates human lung fibroblasts (HLF) to differentiate into myofibroblasts. We characterized lysophosphatidic acid (LPA)-activated Cl- channel current (I(Cl-LPA)) in cultured human lung fibroblasts and myofibroblasts and investigated the influence of I(Cl-LPA) on fibroblast-to-myofibroblast differentiation. We recorded I(Cl-LPA) using the amphotericin perforated-patch technique. We activated I(Cl-LPA) using LPA or sphingosine-1-phosphate. We determined phenotype by Western blotting and immunohistochemistry using an anti-alpha-smooth muscle actin (SMA) antibody. RT-PCR was performed to determine which phospholipid growth factor receptors are present in HLF. We found that HLF cultured in TGF-beta (myofibroblasts) had significantly elevated alpha-SMA levels and I(Cl-LPA) current density compared with control fibroblasts. I(Cl-LPA) activation was blocked by DIDS, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and the LPA receptor-specific antagonist dioctyl-glycerol pyrophosphate (1 microM). DIDS and NPPB, in a dose-dependent manner, significantly reduced alpha-SMA levels in HLF stimulated with TGF-beta. These results demonstrate the receptor-mediated activation of I(Cl-LPA) by LPA and sphingosine-1-phosphate in cultured human lung myofibroblasts, with only minimal I(Cl-LPA) activity in fibroblasts. This Cl- channel activity appears to play a critical role in the differentiation of human lung fibroblasts to myofibroblasts.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2002

Cyclic stretch and endothelin-1 mediated activation of chloride channels in cultured neonatal rat ventricular myocytes.

To date various types of Cl(-) currents have been recorded in cardiac myocytes from different regions of the heart and from different species. Most of these are silent under basal conditions, but are rapidly activated under the influence of various agonists or physical stress that, in the long term, also lead to development of hypertrophy. Previously, we identified three different Cl(-) channel activities in neonatal rat cardiomyocytes: (i) Ca(2+) regulated, (ii) cAMP regulated (cystic fibrosis transmembrane conductance regulator Cl(-) channels) and (iii) osmoregulated Cl(-) channels. In this study, we examined comparatively the effects of cyclic stretch and endothelin-1 (ET-1) on Cl(-) channel activity in primary cultures of neonatal rat ventricular myocytes using an (125)I-efflux assay. About 4 min after the start of the (125)I-efflux (mean basal rate amounts 6.3% of total (125)I incorporated/min), the addition of 10 nM ET-1 or the application of cyclic stretch rapidly and transiently increased (125)I-efflux by 3.8%/min and 0.8%/min respectively above the basal rate. The stretch induced (125)I-efflux rate could be blocked by 100 microM Gd(3+) but it had no effect on the ET-1 response. After 24 h stimulation by ET-1 or cyclic stretch the myocytes responded by hypertrophy which is detected by increases of (3)H-leucine incorporation into protein and protein/DNA ratio. In conclusion, cyclic stretch as well as ET rapidly and transiently activate Cl(-) channels in rat neonatal cardiomyocytes. The results suggest that the activation of distinct types of Cl(-) channels (co)transduce the stretch- and agonist-induced hypertrophic responses in these myocytes.

Animals↗
PubMed1997

Subunit stoichiometry of human muscle chloride channels.

Voltage-gated Cl- channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl- channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their native configurations exhibit similar sedimentation properties consistent with a multimeric complex having a molecular mass of a dimer. Expression of the heterodimeric channel in a mammalian cell line results in a homogenous population of Cl- channels exhibiting novel gating properties that are best explained by the formation of heteromultimeric channels with an even number of subunits. Heteromultimeric channels were not evident in cells cotransfected with homodimeric WT-WT and D136G-D136G constructs excluding the possibility that functional hClC-1 channels are assembled from more than two subunits. These results demonstrate that the functional hClC-1 unit consists of two subunits.

Animals↗
PubMed1999

High-level expression, functional reconstitution, and quaternary structure of a prokaryotic ClC-type chloride channel.

ClC-type anion-selective channels are widespread throughout eukaryotic organisms. BLAST homology searches reveal that many microbial genomes also contain members of the ClC family. An Escherichia coli-derived ClC Cl(-) channel homologue, "EriC," the product of the yadQ gene, was overexpressed in E. coli and purified in milligram quantities in a single-step procedure. Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels. Cross-linking studies argue that EriC is a homodimer in both detergent micelles and reconstituted liposomes, a conclusion corroborated by gel filtration and analytical sedimentation experiments.

Bacterial Proteins↗
PubMed1996

Three distinct chloride channels control anion movements in rat parotid acinar cells.

1. We used the whole-cell configuration of the patch clamp technique to examine the different macroscopic Cl- currents present in single rat parotid acinar cells. 2. Cell swelling produced by negative osmotic pressure (hypotonic bath solutions) induced a large outwardly rectifying Cl- current with little or no time and voltage dependence. In contrast, an increase in intracellular [Ca2+] induced by ionomycin activated Cl- currents with very different properties. Ca(2+)-activated Cl- currents showed outward rectification, relatively slow activation kinetics and marked voltage dependence. These results are consistent with the existence of two different outwardly rectifying Cl- channels in rat parotid cells. 3. In conditions designed to eliminate the activation of these two Cl- currents, a third type of current was observed. This third current was activated in a time-dependent manner by hyperpolarized potentials and was about equally permeant to Cl-, I- and Br-. 4. The properties of the hyperpolarization-activated current were similar to those of the cloned ClC-2 channel. Polymerase chain reaction-based methods and ribonuclease protection analyses indicated the presence in parotid gland of mRNA homologous to ClC-2. 5. Individual parotid acinar cells expressed all three types of Cl- channels. Each type of channel may contribute to Cl- efflux in distinct stages of the secretion process depending on the intracellular [Ca2+], cell volume and membrane potential.

Animals↗
PubMed2002

Functional transplantation of chloride channels from the human syncytiotrophoblast microvillous membrane to Xenopus oocytes.

The materno-fetal transfer of metabolites and nutrients requires the operation of specific transport mechanisms through syncytiotrophoblast membranes. Electrophysiological studies on these cells are scarce and, because of their syncytial nature, whole-cell current recordings have not been carried out. We have now studied whether or not ion channels from the human syncytiotrophoblast microvillous (hSM) membrane can be transplanted to Xenopus oocytes. Sixty-two percent of hSM-injected oocytes displayed lower resting potential and higher membrane conductance than uninjected cells. The increased membrane conductance was due to the incorporation of Cl(-) channels, because neither replacing Na(+) in the bathing solution by N-methyl- D-glucamine or K(+), nor withdrawing Ca(2+) had any significant effect on the currents elicited by voltage pulses. In contrast, substitution of Cl(-) by different anions markedly affected the membrane conductance, giving an anion selectivity sequence of I(-)>Br(-)>Cl(-)>methanosulfonate congruent with gluconate. In addition, disulfonic stilbenes and gluconate, but not anthracene-9-carboxylic acid, blocked the transplanted channels. These properties are compatible with those of placental Cl(-) "maxi" channels. It is concluded that functional Cl(-) channels from the hSM become effectively incorporated into the Xenopus oocyte membrane, where their function can be studied in detail.

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

Purification, reconstitution, and subunit composition of a voltage-gated chloride channel from Torpedo electroplax.

The voltage-gated Cl- channel from Torpedo electroplax was purified in functional form by an immunoaffinity procedure. Channel activity was assayed by 36Cl- uptake into reconstituted liposomes and by direct recording after insertion into planar lipid bilayers. The purified channel displays the same "double-barreled" gating kinetics observed with native membranes, as well as the correct single-channel permeation characteristics. Preparations of active channels consist of a 90-kDa polypeptide, as expected from the known cDNA sequence. No associated subunits are present in the purified material. Direct protein sequencing confirms the absence of a cleavable signal sequence and demonstrates an N-terminus at Ser-2 of the cDNA-derived sequence. This "ClC-0" protein is lightly glycosylated, losing only approximately 2 kDa of sugar upon treatment with endoglycosidase H or N-glycanase. Most if not all of this glycosylation is found on Asn-365. This result necessitates revision of current transmembrane topology proposals, which have placed this residue on the cytoplasmic side of the membrane. Sedimentation in sucrose density gradients under activity-preserving conditions suggests the ClC-0 channel is slightly larger than the Na/K-ATPase alpha/beta-protomer (approximately equal to 150 kDa) and substantially smaller than the reduced form of the nicotinic acetylcholine receptor (approximately equal to 300 kDa). The detergent-solubilized ClC-0 channel, which invariably displays two Cl- diffusion pores in the active complex, is therefore built most likely as a homodimer of the 90-kDa protein purified here.

Animals↗