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 (I-Cx50 or I-Cx46) were activated by lowering the extracellular Ca2+ concentration ([Ca2+](o)) from 5 mm to 10 mum. Ion-replacement experiments suggested that I-Cx50 is carried primarily (> 95%) by monovalent cations (P-K : P-Na : P-Cl = 1.0 : 0.74 : 0.05). I-Cx50 was inhibited by 18beta-glycyrrhetinic acid (apparent K-i, 2 mum), gadolinium (3 mum), flufenamic acid (3 mum), niflumic acid (11 mum), NPPB (15 mum), diphenyl-2-carboxylate (26 mum), and octanol (177 mum). With the exception of octanol, niflumic acid, and diphenyl-2-carboxylate, the above agents also inhibited I-Cx46. Anthracene-9-carboxylate, furosemide, DIDS, SITS, IAA-94, and tamoxifen had no inhibitory effect on either I-Cx50 or I-Cx46. The kinetics of I-Cx50 inhibition were not altered at widely different [Ca2+](o) (10-500 mum), 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.