Degtiar V E, Aczél S, Döring F, Timin E N, Berjukow S, Kimball D, Mitterdorfer J, Hering S
Institut für Biochemische Pharmakologie, Innsbruck, Austria.
Biophys J. 1997 Jul;73(1):157-67. doi: 10.1016/S0006-3495(97)78056-1.
The pore-forming alpha 1 subunit of L-type calcium (Ca2+) channels is the molecular target of Ca2+ channel blockers such as phenylalkylamines (PAAs). Association and dissociation rates of (-)devapamil were compared for a highly PAA-sensitive L-type Ca2+ channel chimera (Lh) and various class A Ca2+ channel mutants. These mutants carry the high-affinity determinants of the PAA receptor site in a class A sequence environment. Apparent drug association and dissociation rate constants were significantly affected by the sequence environment (class A or L-type) of the PAA receptor site. Single point mutations affecting the high-affinity determinants in segments IVS6 of the PAA receptor site, introduced into a class A environment, reduced the apparent drug association rates. Mutation I1811M in transmembrane segment IVS6 (mutant AL25/-I) had the highest impact and decreased the apparent association rate for (-)devapamil by approximately 30-fold, suggesting that this pore-lining isoleucine in transmembrane segment IVS6 plays a key role in the formation of the PAA receptor site. In contrast, apparent drug dissociation rates of Ca2+ channels in the resting state were almost unaffected by point mutations of the PAA receptor site.
L型钙(Ca2+)通道的成孔α1亚基是诸如苯烷基胺(PAA)等Ca2+通道阻滞剂的分子靶点。比较了(-)地尔硫䓬与高度PAA敏感的L型Ca2+通道嵌合体(Lh)及各种A类Ca2+通道突变体的结合和解离速率。这些突变体在A类序列环境中携带PAA受体位点的高亲和力决定簇。PAA受体位点的序列环境(A类或L型)对药物的表观结合和解离速率常数有显著影响。引入A类环境中的影响PAA受体位点IVS6片段高亲和力决定簇的单点突变降低了药物的表观结合速率。跨膜片段IVS6中的I1811M突变(突变体AL25/-I)影响最大,使(-)地尔硫䓬的表观结合速率降低约30倍,表明跨膜片段IVS6中这个位于孔道内衬的异亮氨酸在PAA受体位点的形成中起关键作用。相比之下,静息状态下Ca2+通道的药物表观解离速率几乎不受PAA受体位点点突变的影响。