Hockerman G H, Johnson B D, Scheuer T, Catterall W A
Department of Pharmacology, University of Washington, Seattle 98195-7280, USA.
J Biol Chem. 1995 Sep 22;270(38):22119-22. doi: 10.1074/jbc.270.38.22119.
The high affinity phenylalkylamine (-)D888 blocks ion currents through L-type Ca2+ channels containing the alpha 1C subunit with an apparent Kd of 50 nM, but N-type Ca2+ channels in the pheochromocytoma cell line PC12 are blocked with a 100-fold higher Kd value of 5 microM. L-type Ca2+ channels containing alpha 1C subunits with the site-directed mutations Y1463A, A1467S, or I1470A in the putative transmembrane segment S6 in domain IV (IVS6) were 6-12 times less sensitive to block by (-)D888 than control alpha 1C. Ca2+ channels containing paired combinations of these mutations were even less sensitive to block by (-)D888 than the single mutants, and channels containing all three mutations were > 100 times less sensitive to (-)D888 block, similar to N-type Ca2+ channels. In addition, the Y1463A mutant and all combination mutants including the Y1463A mutation had altered ion selectivity, suggesting that Tyr-1463 faces the pore and is involved in ion permeation. Since these three critical amino acid residues are aligned on the same face of the putative IVS6 alpha-helix, we propose that they contribute to a receptor site in the pore that confers a high affinity block of L-type channels by (-)D888.
高亲和力的苯烷基胺（-）D888可阻断通过含有α1C亚基的L型Ca2+通道的离子电流，其表观解离常数（Kd）为50 nM，但在嗜铬细胞瘤细胞系PC12中，N型Ca2+通道被阻断时的Kd值要高100倍，为5 μM。在结构域IV（IVS6）的假定跨膜片段S6中具有定点突变Y1463A、A1467S或I1470A的含有α1C亚基的L型Ca2+通道，对（-）D888阻断的敏感性比对照α1C低6至12倍。含有这些突变的配对组合的Ca2+通道对（-）D888阻断的敏感性甚至比单突变体更低，而含有所有三种突变的通道对（-）D888阻断的敏感性降低超过100倍，类似于N型Ca2+通道。此外，Y1463A突变体以及包括Y1463A突变在内的所有组合突变体都改变了离子选择性，这表明Tyr-1463面向孔道并参与离子通透。由于这三个关键氨基酸残基在假定的IVS6α螺旋的同一面上排列，我们提出它们有助于形成孔道中的一个受体位点，该位点赋予（-）D888对L型通道的高亲和力阻断作用。
