Hering S, Savchenko A, Strübing C, Lakitsch M, Striessnig J
Institut für Biochemische Pharmakologie, Universität Innsbruck, Austria.
Mol Pharmacol. 1993 May;43(5):820-6.
To determine which side of L-type Ca2+ channels forms the benzothiazepine binding domain, we tested the effects of a membrane-impermeable, diltiazem-like, Ca2+ antagonist, SQ32,428 [(cis)-1,3,4,5-tetrahydro-4-(4-methoxyphenyl)-3-methyl-6- (trifluoromethyl)-1-[2-trimethylammonio)ethyl]-2H-1-benzazepin-2-o ne], on Ca2+ channels in smooth muscle-like cells (A7r5 cells) and skeletal muscle-like cells (differentiated BC3H1 cells). This permanently charged, quaternary benzazepine bound to the benzothiazepine-selective domain of skeletal muscle Ca2+ channels with a Ki of 1.2 +/- 0.1 microM. Extracellular application of SQ32,428 reversibly blocked whole-cell barium currents through L-type Ca2+ channels in A7r5 and BC3H1 cells with similar potencies (A7r5, IC50 = 86 microM; BC3H1, IC50 = microM). Block was fully reversible, was independent of stimulation frequency, and did not affect steady state inactivation of the channel in A7r5 cells. Intracellular dialysis of the cells with 100 microM SQ32,428 was without effect, but the same concentration of the quaternary phenylalkylamine D890 blocked channel activity from the cytoplasmic side. Our data demonstrate that the benzothiazepine binding domain of L-type Ca2+ channels binds diltiazem-like benzazepine Ca2+ antagonists and is formed by amino acid residues exposed to the extracellular channel surface.
为了确定L型钙通道的哪一侧形成苯并噻氮䓬结合域，我们测试了一种膜不可渗透的、地尔硫䓬样的钙拮抗剂SQ32428[（顺式）-1,3,4,5-四氢-4-（4-甲氧基苯基）-3-甲基-6-（三氟甲基）-1-[2-（三甲基铵）乙基]-2H-1-苯并氮杂䓬-2-酮]对平滑肌样细胞（A7r5细胞）和骨骼肌样细胞（分化的BC3H1细胞）中钙通道的影响。这种带永久电荷的季铵苯并氮杂䓬以1.2±0.1μM的Ki值与骨骼肌钙通道的苯并噻氮䓬选择性域结合。细胞外应用SQ32428以相似的效力可逆地阻断了A7r5和BC3H1细胞中通过L型钙通道的全细胞钡电流（A7r5，IC50 = 86μM；BC3H1，IC50 = μM）。阻断是完全可逆的，与刺激频率无关，并且不影响A7r5细胞中通道的稳态失活。用100μM SQ32428对细胞进行细胞内透析没有效果，但相同浓度的季铵苯烷基胺D890从细胞质侧阻断了通道活性。我们的数据表明，L型钙通道的苯并噻氮䓬结合域结合地尔硫䓬样苯并氮杂䓬钙拮抗剂，并且由暴露于细胞外通道表面的氨基酸残基形成。
