Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation
Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation
复制标题
乙酰胆碱受体 α 亚基 M1 结构域的突变降低了激动剂解离速率
DOI：
--
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
S. Sine
中科院分区：
文献类型：
--
作者：
Hai;A. Auerbach;N. Bren;K. Ohno;A. Engel;S. Sine
我们描述了导致慢通道先天性肌无力综合征（SCCMS）的乙酰胆碱受体(AChR) α亚基M1结构域突变N217K的动力学后果。我们之前的研究表明，293 HEK细胞中表达的含有αN217K的受体在长时间的激活事件中开放，与在SCCMS端板上观察到的结果惊人地相似。在这里，我们使用单通道动力学分析来表明，延长的激活事件主要是由于乙酰胆碱（ACh）从结合位点解离的速度减慢。通道打开和关闭的速率常数也减慢了，但幅度要小得多。动力学分析得出的速率常数也描述了受体激活的浓度依赖性，揭示了αN217K的EC50向较低的激动剂浓度变化了20倍。αN217K对乙酰胆碱结合的表观亲和性（与125I-α-班加罗毒素结合率的竞争）也提高了20倍。乙酰胆碱解离的减缓和表观亲和力的增强都是赖氨酸取代所特有的，而谷氨酰胺和谷氨酸取代对乙酰胆碱解离没有影响。用赖氨酸取代β、ε或δ亚基中的等效天冬酰胺不会影响受体激活的动力学或明显的激动剂亲和力。结果表明，M1结构域氨基末端的突变会产生局部扰动，从而稳定与AChR静息状态结合的激动剂。
We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC50 to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of 125I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.
登录
查看更多内容
乙酰胆碱、氨甲酰胆碱和四甲基铵激活重组小鼠乙酰胆碱受体。
DOI：
10.1113/jphysiol.1995.sp020802
发表时间：
1995
期刊：
The Journal of physiology
影响因子：
--
作者：
Zhang,Y;Chen,J;Auerbach,A
通讯作者：
Auerbach,A
影响因子：
2.9
作者：
BLANTON, MP;COHEN, JB
通讯作者：
COHEN, JB
影响因子：
3.4
作者：
SIGWORTH, FJ;SINE, SM
通讯作者：
SINE, SM
通过成纤维细胞中的表达表征成人肌肉乙酰胆碱受体亚基。
DOI：
--
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Blount,P;Merlie,JP
通讯作者：
Merlie,JP