Ortiz-Miranda S I, Lasalde J A, Pappone P A, McNamee M G
Section of Neurobiology, Physiology and Behavior, Division of Biological Sciences, University of California, Davis, CA 95616, USA.
J Membr Biol. 1997 Jul 1;158(1):17-30. doi: 10.1007/s002329900240.
We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type alpha418Cys residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold, respectively. The mutation of a homologous cysteine in the beta subunit (beta447Trp) had similar but smaller effects on mean open time. Coexpression of alpha418Trp and beta447Trp had the largest effect on channel open time, increasing mean open time 58-fold. No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested. However, the coexpression of the alpha418Trp and beta447Trp mutated subunits also produced channels with at least two additional conductance levels. Block by acetylcholine was apparent in the current records from alpha418Trp mutants. Burst analysis of the alpha418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the alpha- and beta subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating, and that mutations in multiple subunits act additively to increase channel open time.
我们在爪蟾卵母细胞中表达的加州电鳐烟碱型乙酰胆碱受体（nAChRs）的假定M4跨膜结构域，在单通道水平上研究了单个氨基酸替换的功能效应。在低乙酰胆碱浓度和低温条件下，用大的疏水性氨基酸色氨酸或苯丙氨酸替换野生型α418Cys残基，分别使平均开放时间增加了26倍和3倍。β亚基中同源半胱氨酸（β447Trp）的突变对平均开放时间有类似但较小的影响。α418Trp和β447Trp共表达对通道开放时间的影响最大，平均开放时间增加了58倍。对于所测试的任何单个亚基氨基酸替换，均未检测到电导或离子选择性的变化。然而，α418Trp和β447Trp突变亚基的共表达也产生了至少具有两种额外电导水平的通道。在α418Trp突变体的电流记录中，乙酰胆碱的阻断是明显的。对α418Trp突变的爆发分析表明，通道开放概率增加，这是由于明显的通道关闭速率降低以及有效开放速率可能增加。我们的结果表明，假定位于脂质 - 蛋白质界面的α和β亚基M4结构域一级结构的修饰可显著改变通道门控，并且多个亚基中的突变具有累加作用以增加通道开放时间。
