Inhibition of gap junction hemichannels by chloride channel blockers.
Electrophysiological methods were used to assess the effect of chloride-channel blockers on the macroscopic and microscopic currents of mouse connexin50 (Cx50) and rat connexin46 (Cx46) hemichannels expressed in Xenopus laevis oocytes. Oocytes were voltage-clamped at -50 mV and hemichannel currents (ICx50 or ICx46) were activated by lowering the extracellular Ca2+ concentration ([Ca2+]o) from 5 mM to 10 microM. Ion-replacement experiments suggested that ICx50 is carried primarily (>95%) by monovalent cations (PK : PNa : PCl = 1.0 : 0.74 : 0.05). ICx50 was inhibited by 18beta-glycyrrhetinic acid (apparent Ki, 2 microM), gadolinium (3 microM), flufenamic acid (3 microM), niflumic acid (11 microM), NPPB (15 microM), diphenyl-2-carboxylate (26 microM), and octanol (177 microM). With the exception of octanol, niflumic acid, and diphenyl-2-carboxylate, the above agents also inhibited ICx46. Anthracene-9-carboxylate, furosemide, DIDS, SITS, IAA-94, and tamoxifen had no inhibitory effect on either ICx50 or ICx46. The kinetics of ICx50 inhibition were not altered at widely different [Ca2+]o (10-500 microM), suggesting that drug-hemichannel interaction does not involve the Ca2+ binding site. In excised membrane patches, application of flufenamic acid or octanol to the extracellular surface of Cx50 hemichannels reduced single channel-open probability without altering the single-channel conductance, but application to the cytoplasmic surface had no effect on the channels. We conclude that some chloride-channel blockers inhibit lens-connexin hemichannels by acting on a site accessible only from the extracellular space, and that drug-hemichannel interaction involves a high-affinity site other than the Ca2+ binding site.
Eskandari
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Department of Physiology, School of Medicine, University of California, LosAngeles 90095-1751, USA. seskandari@csupomona.edu
Zampighi
G A
GA
Leung
D W
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Wright
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EM
Loo
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D000818:Animals; D002463:Cell Membrane Permeability; D018118:Chloride Channels / Q000037:antagonists & inhibitors*; D017630:Connexins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D004106:Dilatation; D005136:Eye Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D017629:Gap Junctions / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology; D051379:Mice; D000442:Octanols / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D018075:RNA, Complementary / Q000235:genetics; D051381:Rats; D014982:Xenopus laevis; D000099264:Connexin 50
D018118:Chloride Channels; D017630:Connexins; D005136:Eye Proteins; D000442:Octanols; D018075:RNA, Complementary; D000099264:Connexin 50; C082917:GJA3 protein, human