Inhibition of gap junction hemichannels by chloride channel blockers
Inhibition of gap junction hemichannels by chloride channel blockers
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DOI：
10.1007/s00232-001-0115-0
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发表时间：
2002-01-15
影响因子：
2.4
通讯作者：
Loo, DDF
中科院分区：
文献类型：
--
作者：
Eskandari, S;Zampighi, GA;Loo, DDF
用电生理学方法研究氯通道阻断剂对非洲爪哇卵母细胞表达的小鼠连接蛋白50(CX50)和大鼠连接蛋白46(Cx46)半通道宏观和微观电流的影响。以-50 mV电压钳制卵母细胞，通过将细胞外钙离子浓度([Ca~(2+)](O))从5 mM降至10微米，激活半通道电流(I-CX50或I-Cx46)。离子置换实验表明，I-CX50主要由一价阳离子(P-K：P-Na：P-Cl=1.0：0.74：0.05)携带(~gt；95%)。18β-甘草次酸(表观K-I，2um)、Gd(3um)、氟苯那酸(3um)、尼氟米酸(11um)、NPPB(15um)、2-二苯基-2-羧酸二苯酯(26um)和辛醇(177 Um)对I-CX50有抑制作用。除辛醇、尼氟米酸和2-羧酸二苯酯外，上述药物对I-Cx46均有抑制作用。9-羧酸菲、速尿、DIDS、SITS、IAA-94和他莫昔芬对I-CX50和I-Cx46均无抑制作用。在差异很大的[Ca~(2+)](O)(10-500微米)时，抑制I-CX50的动力学没有改变，这表明药物-半通道相互作用不涉及钙结合部位。在离体膜片上，在CX50半通道的胞外表面应用氟芬酸或辛醇，在不改变单通道电导的情况下，减少了单通道开放的几率，但在细胞质表面应用对通道没有影响。我们得出结论，一些氯通道阻滞剂通过作用于只能从细胞外空间进入的部位来抑制晶状体连接蛋白半通道，药物与半通道的相互作用涉及一个高亲和力的部位，而不是钙离子结合部位。
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.