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PubMed1996

A microplate assay measuring chloride ion channel activity.

A microplate chloride ion channel assay, using N-(6-methoxyquinolyl) acetoethyl ester (MQAE) fluorescence changes has been developed. Forskolin stimulation of T84 cells caused cAMP-dependent, increased Cl- loss. Forskolin responses after 6 min gave an EC50 of 0.27 +/- 0.05 microM, illustrating the reproducibility of the assay. Forskolin exposure of CFPAC-1 cells, containing delta F508 CFTR, and CFPAC-1 PLJ4.7 cells, transfected with WT-CFTR stimulated Cl- secretion only from the latter, showing that the assay can be used to measure CFTR function. Stimulation of CFPAC-1 cells with ionomycin increased Cl- efflux, demonstrating functional Ca(2+)-mediated Cl- secretion in these cells. Ionomycin also induced a dose-responsive Cl- efflux from T84 cells that, unlike the forskolin response, was Ca2+ dependent. Removal of Na+ ions severely inhibited basal and stimulated Cl- efflux from T84 cells. However, furosemide did not affect forskolin-stimulated JCl, although the magnitude of the Cl- loss was reduced. The Stern-Volmer constant for MQAE fluorescence in T84 cells was calculated as 28.3 +/- 0.9 M-1 and the [Cl-]i in untreated T84 cells was determined as 52.4 +/- 0.6 mM. Stimulation of T84 cells with ionomycin and forskolin before inducing Cl- efflux allowed calculation of initial efflux rates without interference by second messenger generation and ion channel activation kinetics.

Cell Line↗
PubMed2006

Expression and cellular localisation of chloride intracellular channel 3 in human placenta and fetal membranes.

Chloride channels regulate the movement of a major cellular anion and are involved in fundamental processes that are critical for cell viability. Regulation of intracellular chloride is achieved by multiple classes of channel proteins. One class of putative channels are the chloride intracellular channel (CLIC) family. Evidence suggests that several CLICs are expressed in human placenta, although their roles in this tissue are not certain. Northern blot analysis has shown that CLIC3 is highly expressed in placenta relative to other human tissues; however, its cellular distribution is not known. This study used microarray expression profiling to clarify which CLICs are expressed in human placenta and RT-PCR, Western blot and immunohistochemistry to determine the expression pattern of CLIC3 in human placenta and fetal membranes. Placentas and fetal membranes were obtained from term pregnancies after delivery and placental tissue was obtained from first trimester following either chorionic villous sampling or elective pregnancy termination. Trophoblast cells were isolated from first trimester and term placentas and placental endothelial cells were isolated from term placentas. Microarray expression profiling identified high expression of mRNA for CLICs 1, 3 and 4 in the isolated first trimester and term trophoblast cells. High mRNA expression in the isolated endothelial cells was also found for CLICs 1 and 4, but not CLIC3. Low expression was found for CLIC5 in all three types of isolated cells. RT-PCR confirmed that CLIC3 mRNA was expressed in trophoblast cells at both gestational ages, but was not present in endothelial cells. CLIC3 mRNA was also identified in whole placental extracts at both gestational ages and in term amnion and choriodecidua. Immunohistochemistry using a chicken anti-human CLIC3 antibody localised strong CLIC3-specific staining to the syncytiotrophoblast and villous cytotrophoblast cells in both first trimester and term placentas, and weaker staining in extravillous trophoblast cells in first trimester. In fetal membranes at term strong CLIC3-specific staining was localised to chorionic trophoblast cells, with weaker staining in amniotic epithelial and decidual cells. It was previously shown that chloride uptake was increased into cells that had been transfected with CLIC3. CLIC3 may facilitate chloride ion movement and the regulation of cellular processes associated with the movement of chloride in the placental and fetal membrane cells in which it is expressed.

Chloride Channels↗
PubMed2003

Structure-activity and interaction effects of 14 different pyrethroids on voltage-gated chloride ion channels.

We have proposed that since the type II pyrethroids deltamethrin and cypermethrin, but not the type I pyrethroid cismethrin act on chloride channels, this could contribute to the bimodal nature of pyrethroid poisoning syndromes. We now examine a wider range of pyrethroid structures on the activity of these calcium-independent voltage-gated maxi-chloride channels. Excised inside-out membrane patches from differentiated mouse neuroblastoma cells were used, and mean channel open probabilities calculated. For single dosing at 10 microM, bioallethrin, beta-cyfluthrin, cypermethrin, deltamethrin, and fenpropathrin were all found to significantly decrease open channel probability (p < 0.05). Bifenthrin, bioresmethrin, cispermethrin, cisresmethrin, cyfluthrin isomers 2 and 4, lambda-cyhalothrin, esfenvalerate, and tefluthrin, did not significantly alter open channel probability (p > 0.05). Since the type II pyrethroids, esfenvalerate, and lambda-cyhalothrin were ineffective, we must conclude that actions at the chloride ion channel target cannot in themselves account for the differences between the two types of poisoning syndrome. Sequential dosing with type II pyrethroids caused no further chloride ion channel closure. The type I pyrethroid cisresmethrin did however prevent a subsequent effect by the mixed type pyrethroid fenpropathrin. In contrast, the type I pyrethroid cispermethrin did not prevent a subsequent effect due to the type II pyrethroid deltamethrin. The difference in effect may be the result of differences in potency, as deltamethrin had a greater effect than fenpropathrin. It therefore appears clear that in some combinations the type I and type II pyrethroids can compete and may bind to the same chloride channel target site.

Animals↗
PubMed2000

Physiology of apoptosis.

Ion fluxes and volume changes of the whole cell as well as of organelles belong to the hallmarks of apoptosis; however, the molecular mechanism regulating these changes is only poorly characterized. Several ion channels in the plasma membrane, in particular the N-type K(+) channel, the chloride channel cystic fibrosis conductance regulator, and an outward rectifying chloride channel, as well as the mitochondrial permeability transition pore, have been implicated to be involved in signal transduction cascades regulating apoptosis. Furthermore, Bcl-2-like proteins have been suggested to function, at least in part, as ion channels, because they display some homology to bacterial pore-forming toxins. In contrast to the demonstration of the involvement of these different ion channels in apoptosis, the molecular consequences regulated by these ion channels, and finally triggering apoptosis, are almost completely unknown.

Animals↗
PubMed1994

Isolation and partial characterization of calcium-activated chloride ion channels from thylakoids.

The goal of the present work was to characterize the thylakoid anion channel. Toward this purpose a new, more sensitive, assay was developed to measure chloride channel activity of solubilized and partially purified thylakoid membrane proteins. In this assay potassium channel function was reduced or eliminated by the use of cesium as the coion of chloride. Under these conditions calcium but not magnesium stimulated anion uptake, suggesting the presence of a calcium-activated anion channel in isolated thylakoid membrane proteins. A polyclonal antibody raised against an anion channel isolated from epithelial membranes immunoreacted with three thylakoid membrane proteins; a doublet in the 62 to 66-kDa range and a single band in the 57 to 59-kDa region suggesting that the thylakoid anion channel may be homologous to the mammalian chloride channel. Furthermore, the cDNA which encodes the epithelial chloride channel hybridized on Northern blots prepared from corn total RNA with three mRNA bands.

Biological Transport↗
PubMed1994

Effects of Cl channel blockers on Ca-activated chloride and potassium currents in smooth muscle cells from rabbit portal vein.

1. The effects of some chloride channel antagonists were studied on the calcium-activated chloride current (ICl(Ca)) in smooth muscle cells from the rabbit portal vein with the perforated patch technique. 2. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) reduced the amplitude of spontaneous transient inward currents (STICs, calcium-activated chloride currents) in a concentration-dependent manner. The concentrations required to reduce the amplitude by 50% (IC50) of STICs were 2.1 x 10(-4) M and 6.4 x 10(-4) M for DIDS and SITS, respectively. This effect was not voltage-dependent. 3. The time constant of decay of STICs (tau), which is voltage-dependent, was increased by about 30% by SITS and decreased by about 20% by DIDS. The effect of DIDS and SITS on tau was similar at holding potentials of -50 and +50 mV. 4. These compounds did not modify the characteristics of spontaneous transient outward currents (STOCs, calcium-activated potassium currents). 5. DIDS and SITS decreased the amplitude of ICl(Ca) evoked by noradrenaline and caffeine less potently than STICs with IC50 values of 7.5 x 10(-4) M and 1.8 x 10(-3) M, respectively. 6. DIDS and SITS increased the calcium-activated potassium current (IK(Ca) evoked by noradrenaline and caffeine by 3-6 fold. 7. Anthracene-9-carboxylic acid (A-9-C) inhibited STICs in a voltage-dependent fashion and was about 3 fold more active at +50 mV than at -50 mV. A-9-C increased STIC tau and this effect was enhanced by depolarization. 8. A-9-C also inhibited caffeine-evoked IC1(ca) but less potently than STICs and also increased the evoked IK(ca) without altering spontaneous IK(Ca).9. The results from the present work are compared with the pharmacology of other chloride conductances and the mechanism of action of the chloride channel antagonists in vascular smooth muscle is discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2006

FXYD3 is overexpressed in pancreatic ductal adenocarcinoma and influences pancreatic cancer cell growth.

The expression and localization of FXYD domain containing ion transport regulator 3 (FXYD3), a transmembrane protein that acts as a chloride channel or chloride channel regulator, was analyzed in pancreatic tissues derived from donors and patients suffering from chronic pancreatitis (CP) or pancreatic ductal adenocarcinoma (PDAC) as well as in pancreatic cancer cells using QRT-PCR, laser-capture microdissection and microarray analysis, in situ hybridization and immunohistochemistry. FXYD3 antisense expressing T3M4 pancreatic cancer cells were generated and compared to control cells using anchorage-dependent and independent growth assays, and xenotransplantation into nude mice. FXYD3 mRNA levels were 3.4-fold increased in PDAC tissues compared to donor specimens (p = 0.006), and 3.9-fold increased in microdissected cancer cells compared to normal pancreatic ductal cells (p = 0.02). FXYD3 was localized in the tubular complexes and PanIN lesions of both CP and PDAC, as well as in pancreatic cancer cells. Downregulation of FXYD3 by stable antisense transfection increased significantly the doubling time of T3M4 pancreatic cancer cells from 44 +/- 2 hr to 55 +/- 12 hr (p = 0.02). Nude mice transplanted with antisense transfected cells displayed a significant increase in tumor doubling time from 3.3 days +/- 1.0 to 4.3 days +/- 0.43 (p = 0.058). Anchorage-independent growth and sensitivity to 5-fluorouracil, gemcitabine and cisplatin as well as to MgCl(2) were not dependent on the level of FXYD3 expression. In conclusion, overexpression of FXYD3 in pancreatic cancer may contribute to the proliferative activity of this malignancy.

Adenocarcinoma↗
PubMed1994

Calcium-activated chloride current in rabbit coronary artery myocytes.

Whole-cell patch-clamp techniques were used to study enzymatically dispersed epicardial coronary artery smooth muscle cells. Depolarizing voltage pulses of 500-millisecond duration from -60 mV (118 mmol/L CsCl, 22 mmol/L tetraethylammonium chloride, and 5 mmol/L EGTA pipette solution) elicited inward L-type calcium currents (ICa). When EGTA was omitted from the pipette solution, an outward current was superimposed on the calcium current, and repolarizing voltage steps produced an inward tail current (IT). The amplitude of these inward currents was proportional to the ICa amplitude from -30 to +50 mV. The time course of decay of the current was well fit by a single exponential equation. The time constant (tau) of this equation did not change with the size of IT but was clearly voltage dependent (shorter at more negative potentials). Changing the chloride reversal potential from -1.3 to -39.7 mV by anion substitution using methanesulfonate as the chloride replacement in the pipette solution shifted the zero current level of IT from 0.9 +/- 0.56 to -33.1 +/- 0.85 mV. The tail current was blocked by nifedipine (10(-6) mol/L) and by isosmolar calcium substitution with barium in the bath solution and was enhanced by the dihydropyridine agonist Bay K 8644 (10(-6) mol/L). IT was also blocked by the chloride channel blockers DIDS (10(-4) mol/L) and niflumic acid (10(-5) mol/L). Caffeine (10(-2) mol/L), which releases intracellular calcium stores, caused an inward current at holding potentials (-60 mV), which was inhibited by DIDS. Caffeine also inhibited subsequent attempts to elicit IT by depolarizing pulses (88% reduction in IT).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Recent advances in osteoclast biology and pathological bone resorption.

The osteoclast is a bone-degrading polykaryon. Recent studies have clarified the differentiation of this cell and the biochemical mechanisms it uses to resorb bone. The osteoclast derives from a monocyte/macrophage precursor. Osteoclast formation requires permissive concentrations of M-CSF and is driven by contact with mesenchymal cells in bone that bear the TNF-family ligand RANKL. Osteoclast precursors express RANK, and the interaction between RANKL and RANK (which is inhibited by OPG) is the major determinant of osteoclast formation. Hormones, such as PTH/PTHrP, glucocorticoids and 1,25(OH)2D3, and humoral factors, including TNFalpha, interleukin-1, TGFss and prostaglandins, influence osteoclast formation by altering expression of these molecular factors. TNFalpha, IL-6 and IL-11 have also been shown to promote osteoclast formation by RANKL-independent processes. RANKL-dependent/independent osteoclast formation is likely to play an important role in conditions where there is pathological bone resorption such as inflammatory arthritis and malignant bone resorption. Osteoclast functional defects cause sclerotic bone disorders, many of which have recently been identified as specific genetic defects. Osteoclasts express specialized proteins including a vacuolar-type H+-ATPase that drives HCl secretion for dissolution of bone mineral. One v-ATPase component, the 116 kD V0 subunit, has several isoforms. Only one isoform, TCIRG1, is up-regulated in osteoclasts. Defects in TCIRG1 are common causes of osteopetrosis. HCl secretion is dependent on chloride channels; a chloride channel homologue, CLCN7, is another common defect in osteopetrosis. Humans who are deficient in carbonic anhydrase II or who have defects in phagocytosis also have variable defects in bone remodelling. Organic bone matrix is degraded by thiol proteinases, principally cathepsin K, and abnormalities in cathepsin K cause another sclerotic bone disorder, pycnodysostosis. Thus, bone turnover in normal subjects depends on relative expression of key cytokines, and defects in osteoclastic turnover usually reflect defects in specific ion transporters or enzymes that play essential roles in bone degradation.

Animals↗
PubMed1991

Substrate regulation of single potassium and chloride ion channels in Arabidopsis plasma membrane.

Patch clamp measurements of excised inside-out patches of Arabidopsis thaliana plasma membrane reveal at least two ion channels which conduct either potassium or chloride. The conductance of the potassium channel ranged from 5 to 70 picosiemens depending on KCl concentration. The conductance increased linearly with increasing cytoplasmic-side [KCl]; the extent of this dependence declined as extracytoplasmic-side [KCl] was increased. This indicates that substrate regulation of the potassium channel is a consequence of the molecular architecture of the channel: in particular, multi-ion binding sites within the channel pore. The chloride channel conductance (ranging from 5-40 picosiemens) was independent of cytoplasmic-side [KCl] until a threshold concentration of about 300 millimolar was reached. Such behavior is expected only if the channel is allosterically regulated by cytoplasmic-side K(+) and/or Cl(-). The median open times of either channel (about 200 milliseconds for the potassium channel and 20 milliseconds for the chloride channel) were unaffected by substrate concentrations.

Journal Article↗
PubMed1989

Acetylcholine-gated and chloride conductance channel expression in rat muscle membrane.

1. During the differentiation of skeletal muscle, there is a synchronized expression of a number of muscle-specific proteins including the acetylcholine-gated ion channel (AChR). Another muscle-specific ion channel, responsible for chloride conductance, was shown to be expressed in an anticoordinate fashion to AChR. An organ culture system for rat lumbrical muscles was developed to manipulate the expression of these two ion channels. 2. Denervation induced a change in expression of both channels that was mimicked in culture and reversed by direct electrical stimulation. 3. The time course of the disappearance of both channels was similar and started immediately after denervation (chloride conductance) or stimulation (AChR). The time course of the appearance of AChR was delayed several days after denervation and culture but chloride conductance increased immediately upon stimulation. 4. The loss of chloride conductance in muscle cultured in cycloheximide exhibited first-order kinetics, providing an estimate of the half-life (2.3 days) for the chloride conductance channel. This resembled the disappearance of chloride conductance in normal medium, suggesting that synthesis of this channel ceases following denervation. The decrease in chloride conductance characteristic of denervated muscle was not halted by cycloheximide. 5. Changes in chloride conductance presumably alter the intracellular concentration of chloride. The possibility that chloride might regulate the expression of AChRs in skeletal muscle was tested by altering the intracellular concentration of chloride in muscles maintained in organ culture. 6. Denervated muscles, whose intracellular concentration of chloride is elevated, were cultured in medium containing 9 mM-chloride (low-Cl- medium). AChR expression was reduced by either low-Cl- medium or electrical stimulation. Together, low-Cl- medium and electrical stimulation reduced expression more than either treatment alone. 7. The loss of AChRs in low-Cl- medium was blocked when muscle fibrillation was halted by TTX. 8. When chloride conductance was blocked by 9AC (9-anthracene carboxylic acid) intracellular chloride was elevated to the levels seen in denervated muscle. The elevated levels of chloride did not prevent the reduction in AChR expression induced by electrical stimulation. 9. The uncoupling of AChR expression and the intracellular concentration of chloride showed that they were not rigidly linked. Chloride affects the expression of AChR indirectly, by altering the activity of muscle cells.

Acetylcholine↗
PubMed2006

Mapping functional domains of chloride intracellular channel (CLIC) proteins in vivo.

Chloride intracellular channel (CLIC) proteins are small proteins distantly related to the omega family of glutathione S-transferases (GSTs). CLIC proteins are expressed in a wide variety of tissues in multicellular organisms and are targeted to specific cellular membranes. Members of this family are capable in vitro of changing conformation from a globular, soluble state to a membrane-inserted state in which they provide chloride conductance. The structural basis for in vivo CLIC protein function, however, is not well understood. We have mapped the functional domains of CLIC family members using an in vivo assay for membrane localization and function of CLIC proteins in the nematode Caenorhabditis elegans. A<70 amino acid N-terminal domain is a key determinant of membrane localization and function of invertebrate CLIC proteins. This domain, which we term the ''PTM'' domain, named after an amphipathic putative transmembrane helix contained within it, directs distinct C. elegans CLIC homologs to distinct subcellular membranes. We find that within the PTM region, the cysteine residues required for GST-type activity are unnecessary for invertebrate CLIC function, but that specific residues within the proposed transmembrane helix are necessary for correct targeting and protein function. We find that among all tested invertebrate CLIC proteins, function appears to be completely conserved despite striking differences in the charged residues contained within the amphipathic helix. This indicates that these residues do not contribute to anion selectivity as previously suggested. We find that outside the PTM region, the remaining three-quarters of CLIC protein sequence is functionally equivalent not only among vertebrate and invertebrate CLIC proteins, but also among the more distantly related GST-omega and GST-sigma proteins. The PTM region thus provides both targeting information and CLIC functional specificity, possibly adapting GST-type proteins to function as ion channels.

Amino Acid Sequence↗
PubMed2003

Interaction of Sedlin with chloride intracellular channel proteins.

Sedlin is an evolutionarily conserved protein encoded by the causative gene SEDL for spondyloepiphyseal dysplasia tarda. Nevertheless, how Sedlin mutations cause the disease remains unknown. Here, the intracellular chloride channel protein CLIC1 was shown to associate with Sedlin by yeast two-hybrid screening. Green fluorescence protein-CLIC1 readily co-immunoprecipitated with FLAG-Sedlin. In addition, both proteins colocalized extensively in cytoplasmic vesicular/reticular structures in COS-7 cells, suggesting their interaction at intracellular membranous organelles. Sedlin also associated with CLIC2 in yeast two-hybrid assays. The link between Sedlin and the intracellular chloride channels is the first step to understand their functional interplays.

Animals↗
PubMed2003

Pharmacological effects of ivermectin, an antiparasitic agent for intestinal strongyloidiasis: its mode of action and clinical efficacy.

Ivermectin is an oral semi-synthetic lactone anthelmintic agent derived from avermectins isolated from fermentation products of Streptomyces avermitilis. Ivermectin showed a concentration-dependent inhibitory effect on motility of a free-living nematode, Caenorhabditis elegans (C. elegans). There exist specific binding sites having a high affinity for ivermectin in the membrane fraction of C. elegans, and a strong positive correlation was detected between the affinity for these binding sites and the suppressive effect on motility of C. elegans in several ivermectin-related substances. These results suggested that the binding to these binding sites is important for the nematocidal activity of ivermectin. In oocytes of Xenopus laevis injected with the Poly (A)(+) RNA of C. elegans, expression of a chloride channel, which is irreversibly activated by ivermectin, was recognized. The pharmacological properties of this channel suggest that the ivermectin-sensitive channel is a glutamate-activated chloride channel. As to the glutamate-activated chloride channel, two subtypes (GluCl-alpha and GluCl-beta) were cloned, suggesting these subtypes constitute the glutamate-activated chloride channel. These findings suggest that ivermectin binds to glutamate-activated chloride channels existing in nerve or muscle cells of nematode with a specific and high affinity, causing hyperpolarization of nerve or muscle cells by increasing permeability of chloride ion through the cell membrane, and as a result, the parasites are paralyzed to death. In experimental infections in sheep and cattle, ivermectin exhibited potent dose-dependent anthelmintic effects on Haemonchus, Ostertagia, Trichostrongylus, Cooperia, Oesphagostomum, and Dictyocaulus. Anthelmintic effects were reported also in dogs, horses, and humans infected with Strongyloides. In the clinical Phase III trial in Japan, 50 patients infected with Strongyloides stercoralis were administered approx. 200 microg/kg of ivermectin to be given orally twice at an interval of 2 weeks. As a result, the Strongyloides stercoralis-eradicating rate was 98.0% (49/50).

Animals↗
PubMed1992

Expression of cystic fibrosis transmembrane regulator Cl- channels in heart.

Cyclic AMP (cAMP)-dependent chloride channels modulate changes in resting membrane potential and action potential duration in response to autonomic stimulation in heart. A growing body of evidence suggests that there are marked similarities in the properties of the cAMP-dependent chloride channels in heart and cystic fibrosis transmembrane regulator (CFTR) chloride channels found in airway epithelia or in cells expressing the CFTR gene product. We isolated poly A+ mRNA from rabbit ventricle and converted it to cDNA for amplification using the polymerase chain reaction (PCR). A fragment corresponding to the nucleotide-binding domain 1 (NBD1) of the CFTR transcript was cloned. Comparison of the amino acid sequence of NBD1 of human CFTR with the deduced sequence of the rabbit heart PCR product indicated 98% identity. Northern blot analysis, using the heart amplification product as a cDNA probe, demonstrated expression of homologous transcripts in human atrium, guinea pig and rabbit ventricle, and dog pancreas. Xenopus oocytes injected with poly A+ mRNA extracted from rabbit and guinea pig ventricle or dog pancreas expressed robust time-independent chloride currents in response to an elevation of cAMP. We conclude that CFTR chloride channels are expressed in heart and are responsible for the observed cAMP-dependent chloride conductance.

Animals↗
PubMed1996

Chloride ion channels are associated with adherence of lymphatic endothelial cells.

We have examined the role of chloride ion channels as part of the control mechanism for adherence and growth of lymphatic endothelial cells. The chloride channel inhibitor N-phenylanthranilic acid (0.1 mM) inhibited the initial adherence of previously nonadherent cells in the period up to 3 hr following seeding onto gelatin-coated culture dishes. The potassium channel inhibitor 4-aminopyridine (1 mM) had no effect on the rate of cell adherence. N-Phenylanthranilic acid had no effect when added to confluent monolayers of cells that had been growing for 3 days. This, and the observation that N-phenylanthranilic acid did not affect the ultrastructure of the cells, suggested that plasma membrane chloride channels are involved in the initial signaling cascade triggered when lymphatic endothelial cells begin to adhere to a substrate.

Animals↗
PubMed2005

Patch clamped single pancreatic zymogen granules: direct measurements of ion channel activities at the granule membrane.

BACKGROUND/AIM: Pancreatic acinar cells are involved in the secretion of digestive enzymes. Digestive enzymes in pancreatic acinar cells are stored in membrane-bound secretory vesicles called zymogen granules (ZGs). The swelling of ZGs is implicated in the regulation of the expulsion of intravesicular contents during secretion. The molecular mechanism of ZG swelling has been previously elucidated. It has been further demonstrated that the water channel aquaporin-1, the potassium channel IRK-8, and the chloride channel CLC-2, are present in the ZG membrane and involved in ZG swelling. However, a direct measurement of these ion channels at the ZG membrane in intact ZGs had not been performed. The aim of this study was to investigate the electrical activity of single ZGs and verify the types of channels found within their membrane. METHODS: ZGs from pancreatic acinar cells were isolated from the pancreas of Sprague-Dawley rats. Direct measurements of whole vesicle currents, in the presence and absence of ion channel blockers (quinine, glyburide and DIDS), were recorded following successful patching of single ZGs. CONCLUSION: In this study, we were able, for the first time, to patch single ZGs and study ion channels in their membrane. We were able to record currents across the ZG membrane and, utilizing ion channel blockers, confirm the presence of the chloride channels CLC-2 and the potassium channel IRK-8 (Kir6.1), and additionally demonstrate the presence of a second chloride channel CLC-3.

ATP-Binding Cassette Transporters↗
PubMed1987

gamma-Hydroxybutyric acid binding sites: evidence for coupling to a chloride anion channel.

The effect of eight anions, including chloride, on the binding of gamma-hydroxy[2,3-3H]butyric acid (GHB) to synaptosomal membranes of rat and human brain was ascertained, as was the effect of a number of other allosteric modulators of the GABA/benzodiazepine/picrotoxin complex. All ions which were active at the chloride ion channel, inhibited the binding of [3H]GHB in a dose-dependent manner, with maximum inhibition of binding being 60% of 300 mM concentration of anion. Inactive ions in this binding system included sulfate, acetate and fluoride, all impermeable to the chloride ion channel. The inhibition of binding was temperature-dependent, being abolished at 37 degrees C and was independent of the cation used. The binding of [3H]GHB was also enhanced by pentobarbital, picrotoxin and diazepam but unchanged in the presence of GABA, muscimol, bicuculline, baclofen or strychnine. These data raise the possibility that the epileptogenic effect of GHB may be modulated by an action on the chloride ion channel, that is tightly coupled to the GABA/benzodiazepine/picrotoxin and/or GHB receptor complex.

Adult↗