The volume-sensitive chloride channel in mouse cardiac myocytes
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Objective:To characterize the volume_sensitive chloride channel (I Cl,Vol ) in isolated cardiac myocytes of mouse. Methods:The cardiac myocytes were isolated from mouse heart and the whole_cell patch clamp was employed.Results:Affer exposure of the cardiac myocytes to a hypotonic solution,a volume_sensitive chloride current was activated. Time_dependent inactivation was observed at large positive potentials. The current_voltage relationship showed that the reversal potential of the hypotonic_activated current (_34.5±0.8?mV) was close to the calculated equilibrium potential for Cl _(E Cl =_38.6?mV). When the extracellular Cl _ concentration changed, the reversal potential (E rev ) of the current also shifted. The E rev shifted per 10_fold of o was 43.9?mV . The activation of the current depended on intracellular ATP. The anion permeability order of volume_sensitive Cl _ current was I _Br _ Cl _. A relative permeability of I _ and Br _ to Cl _ was 2.32∶1.28∶1.Conclusions:In mouse cardiac myocytes , the volume_sensitive Cl _ current that is dependent on intracellular ATP has an outward rectification and time_dependent inactivation at positive potential.[Keywords:
Reversal potential
Chloride channel
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We studied the contribution of the Cl- channel as well as K+ channel in the regulation of Ca2+ signalling in fura-2-loaded cultured human aortic endothelial cells. Low Cl- (20 mM) superfusion did not affect the ATP (10 microM)-induced [Ca2+]i increase at the initial peak (control 309 +/- 30 nM (mean +/- SD, n = 17) versus 20 mM [Cl-]o 308 +/- 40 nM (n = 8)) but depressed it at the sustained phase (at 5 min, 170 +/- 29 nM versus 85 +/- 10 nM). Similar selective depression of the sustained phase occurred also in Ca(2+)-free and 140 mM K+ solutions and in the presence of niflumic acid (300 microM), a blocker of the Cl- channel and Ca2+ permeable cation channel. After application of ATP, the increase of [Cl-]o from 20 to 146 mM resulted in a Ca2+ overshoot. Both Cl- and K+ channels play an important role in the regulation of Ca2+ influx presumably by controlling the membrane potential in vascular endothelial cells.
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Ionic channels regulated by extracellular Ca2+ concentration ([Ca2+]o) were examined in freshly isolated rabbit osteoclasts. K+ current was suppressed by intracellular and extracellular Cs+ ions. In this condition, high [Ca2+]o evoked an outwardly rectifying current with a reversal potential of about -25 mV. When the concentration of extracellular Cl ions was altered, the reversal potential of the outwardly rectifying current shifted as predicted by the Nernst equation. 4',4-diisothiocyanostilbene-2' 2-disulphonic acid (DIDS) inhibited the outwardly rectifying current. These results indicated that this current was carried through Cl- channels. Cd2+ or Ni2+ caused a transient activation of the Cl- current in contrast to the sustained activation elicited by Ca2+. Intracellular 20 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited the divalent cation-induced Cl- current. Either when the osmolarity of extracellular medium was increased, or when 100 microM cAMP was dissolved in the patch pipette solution, high [Ca2+]o still elicited the Cl- current, indicating that the divalent cation-induced Cl- current was carried through Ca(2+)-activated Cl- channels. Under perforated whole cell clamp extracellular divalent cations evoked the Cl- current, indicating that the activation of Cl- current did not arise from possible leakage of divalent cations from the extracellular medium under the whole cell clamp condition. This experiment further excluded a possible activation of volume-sensitive Cl- channels under whole cell clamp. Intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) activated the Cl current and it was inhibited by intracellular 20 mM EGTA, suggesting that the activation of Cl current was mediated through a G protein, and that an increase in [Ca2+]i was critical for the activation of Cl-channels. A protein phosphatase inhibitor, okadaic acid (100 nM), caused an irreversible activation of the Cl current, suggesting that protein phosphatase 1 or 2A was involved in the regulation of Ca(2+)-activated Cl- channels.
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DIDS
EGTA
Reversal potential
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Tetrandrine
Chloride channel
Calcium in biology
Tetraethylammonium chloride
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Extracellular ATP and elevated cytosolic Ca2+ concentration ([Ca2+]i) are major secretagogues for Cl- in the goblet cell-like clone cl.16E derived from colonic HT-29 cells. The involvement of [Ca2+]i as a messenger for the purinergically stimulated Cl- secretion was investigated using whole cell patch-clamp and Ussing chamber techniques, as well as [Ca2+]i measurements using fura 2-loaded cells. Under voltage-clamp conditions, the whole cell current at +50 mV was 3 +/- 1 pA/pF under unstimulated conditions. Stimulation of purinergic receptors with 200 microM extracellular ATP increased the current at +50 mV to 41 +/- 10 pA/pF, with a half-maximal effective dose (ED50) of approximately 3 microM. The current was transient, usually lasting 1-2 min, and the current-voltage relationship was approximately linear between -70 and +50 mV. Evidence that the ATP-stimulated current was carried by Cl- included 1) the reversal potential of the current closely followed the Cl- equilibrium potential, and 2) the stimulated current was absent when Cl- was removed from both bath and pipette solutions. Exposure to ATP also increased [Ca2+]i, with an ED50 of approximately 1 microM and maximal changes (at 200 microM) from baseline (71 +/- 3 nM) to 459 +/- 50 nM. The ATP-dependent Cl- conductance increase was not diminished when [Ca2+]i was clamped at 100 nM using a Ca(2+)-1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering system. However, the ATP effect did require some basal level of Ca2+ because clamping [Ca2+]i at < 10 nM abolished activation of the Cl- conductance. The presence of the protein kinase A inhibitor H-89 or the protein kinase C inhibitor staurosprine did not change the ATP-activated Cl-conductance. These data demonstrate that the ATP-stimulated increase in Cl- current does not require an increase in [Ca2+]i, suggesting the involvement of either another signaling pathway or direct activation of Cl- channels by purinergic receptors.
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EGTA
Hyperpolarization
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Bumetanide
DIDS
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The perforated whole‐cell configuration of patch clamp and the pH fluorescent indicator SNARF were used to determine the electrogenicity of the Na + ‐HCO 3 − cotransport in isolated rat ventricular myocytes. Switching from Hepes buffer to HCO 3 − buffer at constant extracellular pH (pH o ) hyperpolarized the resting membrane potential (RMP) by 2.9 ± 0.4 mV ( n = 9 , P < 0.05 ). In the presence of HCO 3 − , the anion blocker SITS depolarized RMP by 2.6 ± 0.5 mV ( n = 5 , P < 0.05 ). No HCO 3 − ‐induced hyperpolarization was observed in the absence of extracellular Na + . The duration of the action potential measured at 50 % of repolarization time (APD 50 ) was 29.2 ± 6.1 % shorter in the presence of HCO 3 − than in its absence ( n = 6 , P < 0.05 ). Quasi‐steady‐state currents were evoked by voltage‐clamped ramps ranging from −130 to +30 mV, during 8 s. The development of a novel component of Na + ‐dependent and Cl − ‐independent steady‐state outward current was observed in the presence of HCO 3 − . The reversal potential ( E rev ) of the Na + ‐HCO 3 − cotransport current ( I Na,Bic ) was measured at four different levels of extracellular Na + . A HCO 3 − :Na + ratio compatible with a stoichiometry of 2:1 was detected. I Na,Bic was also studied in isolation in standard whole‐cell experiments. Under these conditions, I Na,Bic reversed at −96.4 ± 1.9 mV ( n = 5 ), being consistent with the influx of 2 HCO 3 − ions per Na + ion through the Na + ‐HCO 3 − cotransporter. In the presence of external HCO 3 − , after 10 min of depolarizing the membrane potential (E m ) with 45 m m extracellular K + , a significant intracellular alkalinization was detected (0.09 ± 0.03 pH units; n = 5 , P < 0.05 ). No changes in pH i were observed when the myocytes were pre‐treated with the anion blocker DIDS (0.001 ± 0.024 pH units; n = 5 , n.s.), or when exposed to Na + ‐free solutions (0.003 ± 0.037 pH units; n = 6 , n.s.). The above results allow us to conclude that the cardiac Na + ‐HCO 3 − cotransport is electrogenic and has an influence on RMP and APD of rat ventricular cells.
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Reversal potential
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1. Voltage‐independent whole‐cell Cl‐ currents were recorded from both single, isolated parietal cells and parietal cells within gastric glands obtained from the fundus of guinea‐pig stomach. 2. The Cl‐ currents were rapidly suppressed by a Cl‐ channel blocker, NPPB (5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate), added to the (basolateral) bathing solution in a concentration‐dependent manner with a half‐maximal inhibition concentration of 12 microM. 3. The selectivity sequence among anions was I‐ > Br‐ > Cl‐ > F‐, corresponding to Eisenman's sequence I. 4. The Cl‐ currents were independent of cytosolic Ca2+, cyclic AMP, cyclic GMP, GTP‐gamma‐S and cell volume, and were not affected by application of acid secretagogues, omeprazol, arachidonic acid or prostaglandin E2. 5. Reduction of pH in the (basolateral) bathing solution immediately inhibited the Cl‐ current with a pK (‐log of KD) of 6.3, whereas changes in intracellular pH had no effect. 6. The single‐channel conductance was estimated to be 0.46‐0.6 pS by variance noise analysis during inhibition of whole‐cell Cl‐ currents by NPPB or acidic pH. 7. It is concluded that pH‐sensitive ‘mini’ Cl‐ channels, with a sub‐picosiemens unitary conductance, exist in the basolateral membrane of guinea‐pig parietal cells.
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