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PubMed1988

Lindane but not deltamethrin blocks a component of GABA-activated chloride channels.

Effects of deltamethrin, a pyrethroid insecticide, and lindane, the gamma isomer of hexachlorocyclohexane, on the gamma-aminobutyric acid (GABA)-activated ion channels were studied using cultured neurons isolated from the newborn rat dorsal root ganglia. The neuron was voltage clamped using the whole cell patch clamp technique. Two components of inward chloride currents were generated in response to bath application of GABA. One of the components was selectively blocked by 1 x 10(-5)M lindane in a stereospecific manner, but neither component was affected by 1 x 10(-5)M deltamethrin, which drastically prolonged the voltage-activated sodium channel current. Thus, the action of lindane to stimulate the nervous system is partly because of an interaction with the GABA receptor-channel complex. The target site of deltamethrin is not the GABA receptor-channel complex but the sodium channel. The results suggest a multiplicity of the GABAA receptor-chloride channel complex.

Animals↗
PubMed2000

Effects of chloride channel inhibitors on H(2)O(2)-induced renal epithelial cell injury.

Oxidative stress contributes to renal epithelial cell injury in certain settings. Chloride influx has also been proposed as an important component of acute renal epithelial cell injury. The present studies examined the role of Cl(-) in H(2)O(2)-induced injury to LLC-PK(1) renal epithelial cells. Exposure of LLC-PK(1) cells to 1 mM H(2)O(2) resulted in the following: depletion of intracellular ATP content; DNA damage; lipid peroxidation; and a loss of membrane integrity to both small molecules, e.g., trypan blue, and macromolecules, e.g., lactate dehydrogenase (LDH), and cell death. Substitution of Cl(-) by isethionate or the inclusion of certain Cl(-) channel blockers, e.g., diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino). benzoate (NPPB), and niflumic acid, prevented the H(2)O(2)-induced loss of membrane integrity to LDH. In addition, the H(2)O(2)-induced loss of membrane integrity was prevented by raising the osmolality of the extracellular solutions, by depletion of cell ATP, and by inhibitors of volume-sensitive Cl(-) channels. However, these maneuvers did not prevent the H(2)O(2)-induced permeability to small molecules or H(2)O(2)-induced ATP depletion, DNA damage, lipid peroxidation, or cell death. These results support the view that volume-sensitive Cl(-) channels play a role in the progressive loss of cell membrane integrity during injury.

Adenosine Triphosphate↗
PubMed1988

Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium.

Chloride (Cl-) secretion by the airway epithelium regulates, in part, the quantity and composition of the respiratory tract fluid, thereby facilitating mucociliary clearance. The rate of Cl- secretion is controlled by apical membrane Cl- channels. Apical Cl- channels are opened and Cl- secretion is stimulated by a variety of hormones and neurotransmitters that increase intracellular levels of cyclic AMP (cAMP). In cystic fibrosis (CF), a common lethal genetic disease of Caucasians, airway, sweat-gland duct, secretory-coil and possibly other epithelia are anion impermeable. This abnormality may explain several of the clinical manifestations of the disease. The Cl- impermeability in CF-airway epithelia has been localized to the apical cell membrane, where regulation of Cl- channels is abnormal: hormonal secretagogues stimulate cAMP accumulation appropriately but Cl- channels fail to open. Here we report that the purified catalytic subunit of cAMP-dependent protein kinase plus ATP opens Cl- channels in excised, cell-free patches of membrane from normal cells, but fails to open Cl- channels in CF cells. These results indicate that in normal cells, the cAMP-dependent protein kinase phosphorylates the Cl- channel or an associated regulatory protein, causing the channel to open. The failure of CF Cl- channels to open suggests a defect either in the channel or in such an associated regulatory protein.

Adenosine Triphosphate↗
PubMed1995

Evidence that pH-titratable groups control the activity of a large epithelial chloride channel.

The effects of pH changes were examined on the properties of a large, voltage dependent, epithelial Cl- channel from bovine tracheal cells. Alkaline solutions in the range pH = 7.4-9.2 had no detectable effects on channel conductance or gating. However, acid solutions significantly reduced channel open probability, raising the voltage required to open the channel. Analysis of channel activity in the acidic pH range suggested that at least one charged group on the channel with an apparent pK = 6.09, is responsible for its voltage dependence. Neutralization of this charge does not eliminate the voltage dependence, but changes the energy difference between the closed and open states. The absence of any change in channel conductance over this wide pH range suggests that the protonation site is far removed from the channel permeation pathway.

Animals↗
PubMed1993

Chloride channels with reduced single-channel conductance in recessive myotonia congenita.

Myoballs were cultured from muscle biopsies of patients having Becker-type recessive generalized myotonia (RGM). Single-channel recordings showed that the conductance of the "intermediate" Cl- channel, the Cl- channel having the highest membrane density and accounting for most of the Cl- conductance in myoballs, was reduced to about 50%. All other channel parameters tested, such as gating properties, ion selectivity, and regulatory pathways, were not different from controls. Two other Cl- channel types were only rarely observed and did not seem different from controls. Also, the macroscopic Cl- conductance measured with tight-seal whole-cell recording was smaller than controls, particularly in the positive membrane potential domain. These results might explain myotonia in RGM on the single-channel level.

Cell Membrane↗
PubMed1994

Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes.

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.

Algorithms↗
PubMed1997

Inward rectification in ClC-0 chloride channels caused by mutations in several protein regions.

Several cloned ClC-type Cl- channels open and close in a voltage-dependent manner. The Torpedo electric organ Cl- channel, ClC-0, is the best studied member of this gene family. ClC-0 is gated by a fast and a slow gating mechanism of opposite voltage direction. Fast gating is dependent on voltage and on the external and internal Cl- concentration, and it has been proposed that the permeant anion serves as the gating charge in ClC-0 (Pusch, M., U. Ludewig, A. Rehfeldt, and T.J. Jentsch. 1995. Nature (Lond.). 373:527-531). The deactivation at negative voltages of the muscular ClC-1 channel is similar but not identical to ClC-0. Different from the extrinsic voltage dependence suggested for ClC-0, an intrinsic voltage sensor had been proposed to underlie the voltage dependence in ClC-1 (Fahlke, C., R. Rüdel, N. Mitrovic, M. Zhou, and A.L. George. 1995. Neuron. 15:463-472; Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695-706). The gating model for ClC-1 was partially based on the properties of a point-mutation found in recessice myotonia (D136G). Here we investigate the functional effects of mutating the corresponding residue in ClC-0 (D70). Both the corresponding charge neutralization (D70G) and a charge conserving mutation (D70E) led to an inwardly rectifying phenotype resembling that of ClC-1 (D136G). Several other mutations at very different positions in ClC-0 (K165R, H472K, S475T, E482D, T484S, T484Q), however, also led to a similar phenotype. In one of these mutants (T484S) the typical wild-type gating, characterized by a deactivation at negative voltages, can be partially restored by using external perchlorate (ClO4-) solutions. We conclude that gating in ClC-0 and ClC-1 is due to similar mechanisms. The negative charge at position 70 in ClC-0 does not specifically confer the voltage sensitivity in ClC-channels, and there is no need to postulate an intrinsic voltage sensor in ClC-channels.

Amino Acid Sequence↗
PubMed1997

Mechanism of ion permeation in skeletal muscle chloride channels.

Voltage-gated Cl- channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl- channel (hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I- and related anions. Extracellular and intracellular I- exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br-) and polyatomic (SCN-, NO3-, CH3SO3-) anions. In addition, I- block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I- causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I- slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined KD values for I- block in three distinct kinetic states and found that binding of I- to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I- binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.

Animals↗
PubMed2000

Permeation through the CFTR chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein forms a Cl(-) channel found in the plasma membranes of many epithelial cells, including those of the kidney, gut and conducting airways. Mutation of the gene encoding CFTR is the primary defect in cystic fibrosis, a disease that affects approximately 30 000 individuals in the United States alone. Alteration of CFTR function also plays an important role in the pathophysiology of secretory diarrhea and polycystic kidney disease. The basic mechanisms of permeation in this channel are not well understood. It is not known which portions of the protein contribute to forming the pore or which amino acid residues in those domains are involved in the biophysical processes of ion permeation. In this review, I will discuss (i) the present understanding of ion transport processes in the wild-type CFTR channel, (ii) the experimental approaches currently being applied to investigate the pore, and (iii) a proposed structure that takes into account the present data on mechanisms of ion selectivity in the CFTR channel and on blockade of the pore by open-channel blockers.

Amino Acid Sequence↗
PubMed1999

[A cytoprotective chloride channel in gastric parietal cells].

This review summarizes the regulatory mechanisms and physiological functions of the novel sub-pS Cl- channel (0.3 pS) that is present abundantly in the basolateral membrane of rabbit gastric parietal cells. The sub-pS Cl- channel is voltage-independent and inhibited by NPPB, a Cl- channel blocker. We found that this gastric Cl- channel is linked to three important physiological roles. First, the sub-pS Cl- channel has a housekeeping role through dominating the cell membrane potential. Although several types of cation channels are present, they do not significantly contribute to the membrane potential in the parietal cells. Second, the Cl- channel is activated by prostaglandin E2 via the EP3 receptor/Ca2-/nitric oxide (NO)/cGMP pathway. A vasodilator ecabapide also activates the channel by increasing the intracellular cGMP content. The NO/cGMP pathway-mediated opening of the sub-pS Cl- channel is essential for cytoprotection against ethanol-induced damage in the gastric parietal cells. The NO/cGMP-elicited cytoprotection is abolished by NPPB. To our knowledge, this Cl- channel is the first identified target for the cytoprotective NO/cGMP pathway. Third, the sub-pS Cl- channel is inhibited by the GTP-binding protein-mediated intracellular production of superoxide anion. Hydrogen peroxide and hydroxyl radicals have no effect on the channel activity. The intracellular superoxide anion acts as a messenger in the negative regulatory mechanism of the sub-pS Cl- channel. The similar sub-pS Cl- channel is also found in rat gastric parietal cells.

Animals↗
PubMed2005

Regulation of cardiac volume-sensitive chloride channel by focal adhesion kinase and Src kinase.

The volume-sensitive chloride current (ICl,swell) is found in the mammalian myocardium and is activated by osmotic swelling. The goal of this study was to examine the importance of the tyrosine kinases focal adhesion kinase (FAK) and Src kinase in cardiac ICl,swell regulation. Neonatal rat ventricular myocytes were cultured on collagen membranes and infected with adenovirus expressing beta-galactosidase (AdLacZ), FAK, or FAK-related nonkinase. FAK-related nonkinase (FRNK) is an endogenous cardiac protein, which functions as an inhibitor of FAK. Whole cell patch-clamp recordings demonstrated that osmotic swelling was associated with the activation of an outward rectifying current in uninfected and AdLacZ-infected cells. Consistent with the properties of ICl,swell, this current displayed a reversal potential close to the equilibrium potential for Cl-; was inhibited by the Cl- channel blockers 4,4'-dinitrostilbene-2,2'-disulfonic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and tamoxifen; and was eliminated in hypertonic solution. In addition to activating ICl,swell, hypotonic swelling enhanced the tyrosine phosphorylation of multiple cardiac proteins including those in the range of 68-70 and 120-130 kDa. Pretreatment of the cells with the drug 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, an inhibitor of FAK and Src, diminished swelling-induced phosphorylation of these proteins but paradoxically increased ICl,swell. Furthermore, overexpression of FRNK but not FAK caused a twofold augmentation in I(Cl,swell) and increased the rate of current activation. Thus the tyrosine kinases FAK and Src contribute to the regulation of ICl,swell.

Animals↗