Akk G, Zhou M, Auerbach A
Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214 USA.
Biophys J. 1999 Jan;76(1 Pt 1):207-18. doi: 10.1016/S0006-3495(99)77190-0.
Mutagenesis and single-channel kinetic analysis were used to investigate the roles of four acetylcholine receptor channel (AChR) residues that are candidates for interacting directly with the agonist. The EC50 of the ACh dose-response curve was increased following alpha-subunit mutations Y93F and Y198F and epsilon-subunit mutations D175N and E184Q. Single-channel kinetic modeling indicates that the increase was caused mainly by a reduced gating equilibrium constant (Theta) in alphaY198F and epsilonD175N, by an increase in the equilibrium dissociation constant for ACh (KD) and a reduction in Theta in alphaY93F, and only by a reduction in KD in epsilonE184Q. This mutation altered the affinity of only one of the two binding sites and was the only mutation that reduced competition by extracellular K+. Additional mutations of epsilonE184 showed that K+ competition was unaltered in epsilonE184D and was virtually eliminated in epsilonE184K, but that neither of these mutations altered the intrinsic affinity for ACh. Thus there is an apparent electrostatic interaction between the epsilonE184 side chain and K+ ( approximately 1.7kBT), but not ACh+. The results are discussed in terms of multisite and induced-fit models of ligand binding to the AChR.
采用诱变和单通道动力学分析方法，研究了四个乙酰胆碱受体通道（AChR）残基的作用，这些残基是与激动剂直接相互作用的候选残基。α亚基突变Y93F和Y198F以及ε亚基突变D175N和E184Q后，ACh剂量反应曲线的EC50增加。单通道动力学建模表明，这种增加主要是由于αY198F和εD175N中门控平衡常数（Theta）降低，αY93F中ACh平衡解离常数（KD）增加和Theta降低，而εE184Q中仅KD降低。该突变仅改变了两个结合位点之一的亲和力，并且是唯一减少细胞外K +竞争的突变。εE184的其他突变表明，εE184D中K +竞争未改变，εE184K中几乎消除，但这些突变均未改变对ACh的固有亲和力。因此，εE184侧链与K +之间存在明显的静电相互作用（约1.7kBT），但与ACh +之间不存在。根据配体与AChR结合的多位点和诱导契合模型对结果进行了讨论。
