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PubMed1994

Cyanotriphenylborate: subtype-specific blocker of glycine receptor chloride channels.

The inhibitory glycine receptor is a ligand-gated ion-channel protein existing in different homo- and heterooligomeric isoforms. Here we show that the chloride channel of the recombinant alpha 1-subunit homooligomeric glycine receptor is efficiently blocked by cyanotriphenylborate (CTB) with a concentration effecting 50% inhibition (IC50) of 1.3 microM in the presence of 50 microM glycine. The antagonistic effect of CTB is noncompetitive, use dependent, and more pronounced at positive membrane potentials, suggesting open-channel block. In contrast to alpha 1-subunit receptors, alpha 2-subunit homooligomers are resistant to CTB (IC50 >> 20 microM). By exchanging the channel-lining transmembrane segment M2 of the alpha 1 polypeptide by that of the alpha 2 polypeptide, we could transfer this resistance to alpha 1 channels, indicating that a single glycine residue at position 254 of the alpha 1 subunit is critical for CTB sensitivity. The blocker did not affect the cation-selective channel of the nicotinic acetylcholine receptor. Thus, CTB may prove useful as a tool to probe the subunit structure of native glycine receptors in mammalian neurons.

Amino Acid Sequence↗
PubMed2001

Interaction of hydrophobic anions with the rat skeletal muscle chloride channel ClC-1: effects on permeation and gating.

Permeation of a range of hydrophobic anions through the rat skeletal muscle chloride channel, rClC-1, expressed in Sf-9 (a Spodoptera frugiperda insect cell line) cells has been studied using the whole-cell patch-clamp technique. Bi-ionic reversal potentials measured with external application of foreign anions gave the following permeability sequence: Cl- (1) > benzoate (0.15) > hexanoate (0.12) > butyrate (0.09) > propionate (0.047) approximately formate (0.046). Anions with larger hydrophobic moieties were more permeant, which suggested that ClC-1 selectivity for hydrophobic anions is dominated by their interaction with a hydrophobic region in the external mouth of the pore. All anions studied when applied from outside show an apparently paradoxical voltage-dependent block of inward currents; this voltage-dependent block could be qualitatively described by a discrete-state permeation model with two binding sites and three barriers. Effects of the external anions with aliphatic side-chains on the apparent open probability (Po) suggested that they are unable to gate the channel, but can modulate ClC-1 gating, probably, by changing Cl- affinity to the gating site. Effects of internal application of benzoate, hexanoate or propionate mimicked those of increasing internal pH, and similarly depended on the channel protonation from the external side. Results for internal benzoate support the concept of a negatively charged cytoplasmic particle being involved in the ClC-1 gating mechanism sensitive to the internal pH.

Animals↗
PubMed1995

Selectivity of maxi chloride channels in the L6 rat muscle cell line.

A method is described for the determination of reversal potentials (EREV) from variance of single-channel currents vs. membrane potential in ramp pulse mode. The variance-voltage relationship is represented by a parabola with a minimum of the best fit curve corresponding to the value of EREV. The reversal potential of the maxi-Cl channels changes according to the activity of Cl ions at the internal side of the excised (inside-out) patch membrane as expressed by the Goldman-Hodgkin-Katz equation with PA/PCl = 0.15, indicating the anion nature of the channel. The relative permeabilities (PA/PCl found for halide anions, were 1.15 for iodide and 1.18 for bromide. The relative permeabilities measured (PA/PCl) for other anions were 1.13 for nitrate, 0.59 for bicarbonate, 0.60 for methanesulfonate, 0.40 for SO42-, 0.44 for propionate, and 0.10 for glutamate. No significant differences in PA/PCl of the investigated anions were observed between proliferating myoballs and quiescent myoblasts. This may mean that the newly formed channels possess full-grown selective filters. A close correlation (r = 0.89) was found between the calculated Stokes diameters of the anions under investigation and their relative permeabilities (PA/PCl). The intercept of the best fit line with the abscissa is 7.2 A (7.2 x 10(-10)m), which may correspond to the diameter of the selectivity filter of the maxi-chloride channel in L6 myoblasts. A similar value of the channel size was obtained from the relationship between the minimum cross-sectional areas of the anions and their relative permeabilities, PA/PCl. The best fit line intercepts the abscissa at 27.5 A2, indicating a pore size approximately 6 A. The minimum areas were obtained by computer from molecular models of the anions.

Animals↗
PubMed1990

Benzodiazepine agonist and inverse agonist actions on GABAA receptor-operated chloride channels. I. Acute effects of ethanol.

Acute exposure to ethanol was found to enhance the ability of a benzodiazepine (BZ) inverse agonist, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), to reduce muscimol-activated 36Cl- uptake by membranes isolated from mouse cerebral cortex. Pretreatment in vivo with a hypnotic dose of ethanol (but not a subhypnotic dose), or exposure to a corresponding concentration in vitro, was effective. This increase in sensitivity of gamma-aminobutyric acid receptor-operated chloride channels to the actions of DMCM was due to an increase in both the potency and efficacy of DMCM. Sensitization to DMCM was reversible and was not observed 24 hr after a single injection of ethanol. Pretreatment with ethanol (10, 50 and 100 mM) in vitro produced sensitization to DMCM in a concentration-dependent manner, similar to that produced by in vivo exposure; this increase in sensitivity did not develop if the membranes were pretreated with ethanol at 0 degrees C. Similarly, in vitro exposure to pentobarbital (200 microM) or flunitrazepam (1 microM) enhanced the actions of the inverse agonist Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]BZ-3- carboxylate). Acute ethanol exposure did not alter low-affinity gamma-aminobutyric acidA receptor binding or muscimol action, or the ability of a BZ agonist, flunitrazepam, to augment muscimol-activated chloride flux. Ethanol exposure did not alter [3H]flumazenil (Ro15-1788) binding to central BZ receptors, its displacement by DMCM or allosteric modulation of DMCM binding by muscimol (muscimol-shift).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

mRNA expression of kidney-specific ClC-K1 chloride channel in single-cell reverse transcription-polymerase chain reaction analysis of outer hair cells of rat cochlea.

Outer hair cells (OHCs) of cochlea have been suggested to have Cl(-) channels sensitive to an ototoxic diuretic, furosemide. We therefore examined whether kidney-specific chloride channels (ClC-K1 and ClC-K2) and ClC-5 are also expressed in OHCs of rat cochlea, assuming that these channels might be the targets of oto-nephrotoxic drugs, by single-cell reverse transcription-polymerase chain reaction (RT-PCR) technique. Single-cell RT-PCR revealed the presence of transcripts of ClC-K1 in OHCs which was verified by DNA sequencing, while ClC-K2 and ClC-5 were not detected. The possible roles of ClC-K1 in OHCs are discussed.

Animals↗
PubMed2006

Parasitoid wasp sting: a cocktail of GABA, taurine, and beta-alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host.

The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal-giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI-TOF-MS revealed high levels of GABA (25 mM), and its receptor agonists beta-alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal-giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin-sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host.

Animals↗
PubMed1996

Purification of the CIC-0 chloride channel from Torpedo california electroplax identification of a phosphorylation site for cAMP-dependent protein kinase.

The voltage-gated chloride channel (CIC-0) from the electric organ of Torpedo californica was purified by immunoaffinity chromatography. A polyclonal antibody was shown to specifically recognize the CIC-0 channel (M(r) 85,000-90,000) in a Western blot of total membrane proteins. As monitored by immunoprecipitation, the formation of antibody-antigen complexes in solution strongly depends on the detergent composition. The highest yield of precipitated CIC-0 was obtained from an incubation mixture containing both an anionic detergent, cholate or lauryl sulfate, and the zwitterionic detergent CHAPS. In contrast, immuno-precipitation of CIC-0 was largely reduced when cholate was exchanged for the nonionic detergent Triton x-100, suggesting that the efficient formation of immuno-complexes is favored by negatively charged detergent. In initial immunopurification experiments, in addition to CIC-0 a major contaminating polypeptide of M(r) approximately 115,000 was copurified, which represents the SITS-binding protein [Jentsch et al. (1989) Biochem. J. 261, 155]. Purification of CIC-0 could be increased from about 35% up to 60% homogeneity when immunoaffinity chromatography was performed in the presence of N-acetylglucosamine. Therefore the highly glycosylated SITS-binding protein most likely interacts with the CIC-0 protein via its carbohydrate parts. The purified CIC-0 channel was found to be phosphorylated by PKA in vitro to a level of 0.35-0.4 mol of phosphate incorporated per mol of CIC-0. Proteolytic digestion with endoproteinase GluC and HPLC-separation revealed two major phosphopeptides, which could be identified by amino acid sequence analysis as different size fragments of the same consensus phosphorylation site. Comparison of the peptide sequences with the deduced protein sequence of CIC-0 [Jentsch et al. (1990) Nature 348, 510; O'Neill et al. (1991) Biochem. Biophys. Acta 1129, 131] indicates serine 600 as the phosphorylated residue. Therefore, our results provide strong evidence that CIC-0 is phosphorylated at this single site by PKA in vitro.

Animals↗
PubMed2005

Gene expression and immunolocalisation of a calcium-activated chloride channel during the stratification of cultivated and developing corneal epithelium.

The spatial and temporal localisation of a calcium-activated chloride channel (CLCA) and its mRNA was investigated, during the in vivo and in vitro development of stratified epithelia, by fluorescence immunohistochemistry and quantitative polymerase chain reaction in embryonic chicken corneas and the expansion of excised human corneal stem cells on amniotic membrane. Single-layered human epithelial cultures on amniotic membrane and early day embryonic chicken corneas expressed relatively little human CLCA2 or its chicken homologue. However, as the epithelium in both models matured and the number of cell-layers increased, the gene expression level and protein staining intensity increased, primarily within the basal cells of both the cultured and embryonic tissues. These results demonstrate that human CLCA2 protein and mRNA expression are elevated during epithelial stratification, suggesting that this protein plays a role in the growth of multi-layered corneal epithelia during both natural development and tissue cultivation.

Animals↗
PubMed2004

Purifying selection and demographic expansion affect sequence diversity of the ligand-binding domain of a glutamate-gated chloride channel gene of Haemonchus placei.

Ninety-five genomic sequences of the ligand-binding domain of glutamate-gated chloride channel genes of three populations of the parasitic nematode H. placei were evaluated for patterns of diversity, demography, and selection. These genes code for subunits of ion channels, which are involved in the mode of action of the most commonly used antiparasitic drugs, the macrocyclic lactones. An extremely high frequency of unique segregating sites in exons and introns was observed, with significantly negative neutrality tests in each population for noncoding, synonymous, and nonsynonymous sites. Several tests indicated that support for balancing selection, positive selection, and hitchhiking was lacking. McDonald-Kreitman tests using H. contortus or C. elegans as an outgroup revealed an extreme excess of replacement polymorphism, consistent with weak purifying selection. Although these tests agree that negative selection may explain the excess of replacement changes, an alternative interpretation is required for the significantly negative Fu and Li's D statistics based on silent and noncoding sites. These include homogeneous forces such as background selection and demographic expansion. The lack of population subdivision and the negative values of Tajima's D for this outbreeding parasitic nematode render background selection less likely than demographic expansion. Comparison of D statistics based on different site types using neutral coalescent simulations supported this interpretation. Although this statistic was more negative for nonsynonymous sites than for synonymous sites, most comparisons of the D statistic were not significantly different between mutation classes. A few significant site comparisons were also consistent with demographic expansion, because the observed test statistic ( D(neutral) - D(selected)) were low relative to the neutral expectations. Finally, previous mitochondrial studies also identified a demographic expansion of this parasitic nematode species, which lends further support to a scenario involving both demographic and purifying forces in the ligand-binding domain of H. placei.

Amino Acid Sequence↗
PubMed2006

Autosomal dominant osteopetrosis: clinical severity and natural history of 94 subjects with a chloride channel 7 gene mutation.

CONTEXT: Autosomal dominant osteopetrosis (ADO) is a sclerosing bone disorder caused by heterozygous mutations in the chloride channel 7 (ClCN7) gene. The clinical manifestations of this disease have not been well characterized since the discovery of the genetic basis of ADO. OBJECTIVES: The primary objectives were to improve our understanding of ADO clinical characteristics and to study the natural history of the disease in the largest series of patients reported to date. DESIGN AND SETTING: This study was primarily a retrospective cross-sectional analysis of individuals with a ClCN7 mutation that was conducted over a 4-yr period at a tertiary referral center and through family reunions. Longitudinal data on a subset of subjects were also studied. PATIENTS AND INTERVENTIONS: We studied 311 subjects from 11 ADO families, including 62 individuals with ADO (patients with the classic clinical phenotype based on radiographs and/or biochemistry), 32 unaffected gene carriers (subjects with the gene mutation but no radiographic and/or biochemical phenotype), and 217 controls who did not harbor a ClCN7 gene mutation. Clinical data were collected through patient interviews and examinations, medical records, and/or self-reported responses on a questionnaire that was completed by all subjects. MAIN OUTCOME MEASURES: The prevalence of fracture, osteomyelitis, visual loss, and bone marrow failure was determined. Differences in clinical manifestations were analyzed according to affected vs. carrier status, age, and underlying genotype. RESULTS: Ninety-two percent of ADO subjects had at least one sequela of the disease. Gene carriers did not have an increased risk of disease manifestations, although they were found to have significant increases in bone mineral density (P < 0.05). Compared with controls, subjects with ADO had a significantly increased prevalence of fracture (84 vs. 36%; P < 0.0001) and osteomyelitis (16 vs. 0.9%; P < 0.0001). Severe fractures (defined as > or =10 fractures of any type and/or greater than one hip/femur fracture) were identified only in ADO subjects, and osteomyelitis typically occurred in the maxilla or mandible in older adults. Visual loss, which typically had its onset in childhood, and bone marrow failure occurred in 19 and 3% of ADO subjects, respectively. Adults were more likely to manifest an ADO clinical characteristic, but no definitive genotype-phenotype relationship could be concluded. Longitudinal data suggest that the ADO clinical phenotype worsens over time. CONCLUSIONS: ADO caused by mutations in the CLCN7 gene is a frequently symptomatic disease manifested by a high rate of fracture, osteomyelitis, visual loss, and occasional bone marrow failure. The sequelae of ADO, which can be identified as early as infancy, appear to worsen over time. Fracture is the most prevalent consequence of ADO, although other more severe manifestations of disease can occur and should not be confused with recessive forms of osteopetrosis, particularly when identified in early childhood.

Adolescent↗
PubMed1999

The regulation of epithelial cell cAMP- and calcium-dependent chloride channels.

This chapter has focused on two types of chloride conductance found in epithelial cells. The leap from the Ussing chamber to patch-clamp studies has identified yet other conductances present which have also been electrophysiologically characterized. In the case of the swelling activated wholecell chloride current, a physiological function is apparent and a single-channel basis found, but its genetic identity remains unknown (see reviews by Frizzell and Morris, 1994; and Strange et al., 1996). The outwardly rectified chloride channel has been the subject of considerable electrophysiological interest over the past 10 years and is well characterized at the single-channel level, but its physiological function remains controversial (reviewed by Frizzell and Morris, 1994; Devidas and Guggino, 1997). Yet other conductances related to the CLC gene family also appear to be present in epithelial cells of the kidney (reviewed by Jentsch, 1996; Jentsch and Gunter, 1997) where physiological functions for some isoforms are emerging. Clearly, there remain many unknowns. Chief among these is the molecular basis of GCa2+Cl and many of other the conductances. As sequences become available it is expected that the wealth of information gained by investigation into CFTR function will provide a conceptual blueprint for similar studies in these later channel clones.

Animals↗
PubMed1990

A large, multiple-conductance chloride channel in normal human T lymphocytes.

Chloride (Cl) channels have been proposed to play roles in lymphocyte functions including volume regulation and cellular cytotoxicity; however, direct studies of such channels in normal human lymphocytes are lacking. In the present study we describe a large conductance Cl channel observed in about 50% of excised, inside-out patches from normal human peripheral T lymphocytes. The channel has multiple conductance states with linear single-channel current-versus-voltage relationships in symmetrical Cl solutions. The most prevalent state is the largest, which has a conductance of about 365 pS. The channel closes in a voltage-dependent manner at both negative and positive potentials, but does not show voltage-dependent inactivation. The probability of opening is maximal between -15 mV and +15 mV and the voltage dependence is well described by two Boltzmann equations with half-maximal probabilities at -22.8 mV and +18.0 mV. The slopes of the voltage dependence suggest two gates in series with 5.7 and 9.6 equivalent charges. The channel was about 30 times more selective for Cl- than for Na+ or K+ under balanced osmolarity but less selective (approx. 11:1) under a large osmotic gradient. The single-channel conductance increased with Cl concentration with an apparent saturation at about 581 pS and a Michaelis-Menten constant of about 120 mM. The selectivity sequence among anions, determined from changes in reversal potential was: I- greater than NO3- greater than Br-, Cl- greater than F-, isethionate, HCO3- greater than SO4(2-) greater than gluconate, propionate greater than aspartate much greater than Na+, K+ and was apparently the same for subconductance states.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1996

Concentration and pH dependence of skeletal muscle chloride channel ClC-1.

1. The influence of Cl- concentration and pH on gating of the skeletal muscle Cl- channel, ClC-1, has been assessed using the voltage-clamp technique and the Sf-9 insect cell and Xenopus oocyte expression systems. 2. Hyperpolarization induces deactivating inward currents comprising a steady-state component and two exponentially decaying components, of which the faster is weakly voltage dependent and the slower strongly voltage dependent. 3. Open probability (Po) and kinetics depend on external but not internal Cl- concentration. 4. A point mutation, K585E, in human ClC-1, equivalent to a previously described mutation in the Torpedo electroplaque chloride channel, ClC-0, alters the I-V relationship and kinetics, but retains external Cl- dependence. 5. When external pH is reduced, the deactivating inward currents of ClC-1 are diminished without change in time constants while the steady-state component is enhanced. 6. In contrast, reduced internal pH slows deactivating current kinetics as its most immediately obvious action and the Po curve is shifted in the hyperpolarizing direction. Addition of internal benzoate at low internal pH counteracts both these effects. 7. A current activated by hyperpolarization can be revealed at an external pH of 5.5 in ClC-1, which in some ways resembles currents due to the slow gates of ClC-0. 8. Gating appears to be controlled by a Cl(-)-binding site accessible only from the exterior and, possibly, by modification of this site by external protonation. Intracellular hydroxyl ions strongly affect gating either allosterically or by direct binding and blocking of the pore, an action mimicked by intracellular benzoate.

Animals↗
PubMed1995

Missense mutation (G480C) in the CFTR gene associated with protein mislocalization but normal chloride channel activity.

We have identified a novel CFTR missense mutation associated with a protein trafficking defect in mammalian cells but normal chloride channel properties in a Xenopus oocyte assay. The mutation, a cysteine for glycine substitution at residue 480 (G480C), was detected in a pancreatic insufficient, African-American, cystic fibrosis (CF) patient. G480C was found on one additional CF chromosome and on none of 220 normal chromosomes, including 160 chromosomes from normal African-American individuals. Western blot analysis and immunofluorescence studies revealed that, in 293T cells, the encoded mutant protein was not fully glycosylated and failed to reach the plasma membrane, suggesting that the G480C protein was subject to defective intracellular processing. However, in Xenopus oocytes, a system in which mutant CFTR proteins are less likely to experience an intracellular processing/trafficking deficit, expression of G480C CFTR was associated with a chloride conductance that exhibited a sensitivity to activation by forskolin and 3-isobutyl-1-methylxanthine (IBMX) that was similar to that of wild-type CFTR. This appears to be the first identification of a CFTR mutant with a single amino acid substitution in which the sole basis for disease is mislocalization of the protein.

Animals↗
PubMed1999

Disruption of clh-1, a chloride channel gene, results in a wider body of Caenorhabditis elegans.

We cloned the clh-1 gene coding for a putative ClC chloride channel in Caenorhabditis elegans. The gene product exhibited a high degree of homology with human ClC-1 and ClC-2. The clh-1 gene was predominantly expressed in the hypodermis, including seam cells. Null mutations of clh-1 caused a significantly wider body and an abnormal alae structure. High osmolarity in the culture medium restored the normal body width of the clh-1 mutants. These results suggest that the clh-1 gene contributes to maintenance of the body width through regulation of osmolarity.

Amino Acid Sequence↗
PubMed2001

Chloride channel function is linked to epithelium-dependent airway relaxation.

We previously reported that substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of mouse tracheal smooth muscle. Since both SP and ATP are known to evoke transepithelial Cl- secretion across epithelial monolayers, we tested the hypothesis that epithelium-dependent relaxation of mouse trachea depends on Cl- channel function. In perfused mouse tracheas, the responses to SP and ATP were both inhibited by the Cl- channel inhibitors diphenylamine-2-carboxylate and 5-nitro-2-(3-phenylpropylamino)benzoate. Relaxation to ATP or SP was unaffected by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), and relaxation to SP was unaffected by either DIDS or DNDS. Replacing Cl- in the buffer solutions with the impermeable anion gluconate on both sides of the trachea inhibited relaxation to SP or ATP. In contrast, increasing the gradient for Cl- secretion using Cl- free medium only in the tracheal lumen enhanced the relaxation to SP or ATP. We conclude that Cl- channel function is linked to receptor-mediated, epithelium-dependent relaxation. The finding that relaxation to SP was not blocked by DIDS suggested the involvement of a DIDS-insensitive Cl- channel, potentially the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. To test this hypothesis, we evaluated tracheas from CFTR-deficient mice and found that the peak relaxation to SP or ATP was not significantly different from those responses in wild-type littermates. This suggests that a DIDS-insensitive Cl- channel other than CFTR is active in the SP response. This work introduces a possible role for Cl- pathways in the modulation of airway smooth muscle function and may have implications for fundamental studies of airway function as well as therapeutic approaches to pulmonary disease.

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