Two calcium-activated chloride conductances in Xenopus laevis oocytes permeabilized with the ionophore A23187.
1. Currents evoked by elevated intracellular free Ca2+ in Xenopus laevis oocytes were studied using the two-electrode voltage clamp technique. The elevation in Ca2+ concentration was achieved in three ways: by the use of the divalent cation ionophore A23187; by application of Ca2+-mobilizing neurotransmitters serotonin and acetylcholine (ACh); by the entry of Ca2+ through voltage-dependent channels. 2. In most experiments, the membrane was permeabilized to Ca2+ by a 15 min pretreatment with A23187 in a Ca2+-free solution. Exposure of the ionophore-treated oocytes to external Ca2+ elicited an inward current (at holding potentials of -40 to -60 mV). At external Ca2+ concentrations ([Ca2+]) between 0.1 and 1 mM, the current had a time-to-peak of at least 10 s, and slowly decayed over tens of seconds. At [Ca2+] greater than 2 mM, the inward current had two distinct kinetic components, a fast and transient one (Ifast) and a slow one (Islow). 3. The main carrier of the Ca2+-evoked inward current was Cl-. Several data indicate the existence of a tetraethylammonium (TEA)-sensitive K+ conductance. No evidence for a Na+ current was found. 4. The two components of the Ca2+-evoked inward current in ionophore-permeabilized oocytes, and the two components of the current evoked by ACh and serotonin (the latter in oocytes injected with rat brain RNA but untreated with A23187), were blocked by intracellular injection of the Ca2+ chelator, ethyleneglycolbis-(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA). The two components of these currents displayed different sensitivity to Ca2+ buffering; higher doses of EGTA were necessary to inhibit the slow component than the fast one. 5. One to two minutes of treatment with 2 mM-9-anthracene carboxylic acid (9-AC) fully blocked Ca2+-dependent Cl- current evoked by Ca2+ influx through voltage- dependent Ca2+ channels in intact (untreated with A23187) oocytes. In ionophore-treated oocytes, block of Ifast was observed at holding potentials at which the current was outward (i.e. due to Cl- influx); Islow was inhibited only partially. The block of Ca2+-evoked Cl- efflux by 9-AC developed much more slowly and was less potent. to explain these results, the existence of two sites of 9-AC action is proposed. 6. Exposure of the ionophore-permeabilized oocytes to 0.1-0.2 mM [Ca2+] strongly reduced the response to higher concentrations of Ca2+. Ifast displayed stronger Ca2+-dependent inactivation than Islow.(ABSTRACT TRUNCATED AT 400 WORDS)
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Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel.
Dascal
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Gillo
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Lass
Y
Y
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D005071:Evoked Potentials / Q000187:drug effects; D005260:Female; D008564:Membrane Potentials / Q000187:drug effects; D009865:Oocytes / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D014982:Xenopus laevis
D000873:Anthracenes; D002712:Chlorides; D013757:Tetraethylammonium Compounds; D012701:Serotonin; D000001:Calcimycin; D004533:Egtazic Acid; C010030:9-anthroic acid; D000109:Acetylcholine; D002118:Calcium