Xu M, Covey D F, Akabas M H
Center for Molecular Recognition, Columbia University, New York, New York 10032, USA.
Biophys J. 1995 Nov;69(5):1858-67. doi: 10.1016/S0006-3495(95)80056-1.
We used the substituted-cysteine-accessibility method to identify the channel-lining residues in a region (257-261) near the putative cytoplasmic end of the M2 membrane-spanning segment of the rat gamma-aminobutyric acid type A (GABAA) receptor alpha 1 subunit. The residues alpha 1Val257 and alpha 1Thr261 were accessible to charged, sulfhydryl-specific reagents applied extracellularly in both the open and closed states. The accessibility of alpha 1V257C and alpha 1T261C in the closed state implies that the gate must be at least as close to the cytoplasmic end of the channel as alpha 1Val257. Also, the positively charged reagent methanethiosulfonate ethylammonium penetrated from the extracellular end of the channel to alpha 1T261C, with which it reacted, indicating that the anion-selectivity filter is closer to the cytoplasmic end of the channel than this residue is. Co-application of picrotoxin prevented the sulfhydryl reagents from reacting with alpha 1V257C but did not prevent reaction with the more extracellular residue alpha 1T261C. Picrotoxin protection of alpha 1V257C may be due to steric block by picrotoxin bound in the channel at the level of alpha 1Val257; however, if this protection is allosteric, it is not due to the induction of the resting closed state in which alpha 1V257C was accessible to sulfhydryl reagent.
我们采用半胱氨酸替代可及性方法，来鉴定大鼠γ-氨基丁酸A型（GABAA）受体α1亚基M2跨膜片段假定胞质端附近区域（257-261）中的通道内衬残基。在开放和关闭状态下，胞外施加的带电荷的、对巯基具有特异性的试剂，均可接触到α1Val257和α1Thr261残基。α1V257C和α1T261C在关闭状态下的可及性表明，门控结构必定至少与通道的胞质端一样靠近α1Val257。此外，带正电荷的试剂甲硫基磺酸盐乙铵从通道的胞外端穿透至α1T261C并与其发生反应，这表明阴离子选择性过滤器比该残基更靠近通道的胞质端。共同施加印防己毒素可阻止巯基试剂与α1V257C发生反应，但不能阻止其与更靠胞外的残基α1T261C发生反应。印防己毒素对α1V257C的保护作用可能是由于结合在α1Val257水平通道中的印防己毒素产生的空间位阻；然而，如果这种保护是变构性的，那么它并非是由于诱导了α1V257C可被巯基试剂接触到的静息关闭状态。
