TY - JOUR
T1 - Inhibition of gap junction hemichannels by chloride channel blockers
AU - Eskandari, S.
AU - Zampighi, G. A.
AU - Leung, D. W.
AU - Wright, E. M.
AU - Loo, D. D.F.
PY - 2002/1/15
Y1 - 2002/1/15
N2 - 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 μM. 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 18β-glycyrrhetinic acid (apparent Ki, 2 μM), gadolinium (3 μM), flufenamic acid (3 μM), niflumic acid (11 μM), NPPB (15 μM), diphenyl-2-carboxylate (26 μM), and octanol (177 μM). 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 μM), 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.
AB - 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 μM. 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 18β-glycyrrhetinic acid (apparent Ki, 2 μM), gadolinium (3 μM), flufenamic acid (3 μM), niflumic acid (11 μM), NPPB (15 μM), diphenyl-2-carboxylate (26 μM), and octanol (177 μM). 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 μM), 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.
KW - Chloride-Channel Blockers
KW - Connexin46
KW - Connexin50
KW - Connexins
KW - Hemichannels
KW - Pharmacology
UR - http://www.scopus.com/inward/record.url?scp=0037080849&partnerID=8YFLogxK
U2 - 10.1007/s00232-001-0115-0
DO - 10.1007/s00232-001-0115-0
M3 - Article
C2 - 11891568
AN - SCOPUS:0037080849
SN - 0022-2631
VL - 185
SP - 93
EP - 102
JO - Journal of Membrane Biology
JF - Journal of Membrane Biology
IS - 2
ER -