Xu M, Akabas M H
Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
J Gen Physiol. 1996 Feb;107(2):195-205. doi: 10.1085/jgp.107.2.195.
The gamma-aminobutyric acid type A (GABA(A)) receptors are the major inhibitory, postsynaptic, neurotransmitter receptors in the central nervous system. The binding of gamma-aminobutyric acid (GABA) to the GABA(A) receptors induces the opening of an anion-selective channel that remains open for tens of milliseconds before it closes. To understand how the structure of the GABA(A) receptor determines the functional properties such as ion conduction, ion selectivity and gating we sought to identify the amino acid residues that line the ion conducting channel. To accomplish this we mutated 26 consecutive residues (250-275), one at a time, in and flanking the M2 membrane-spanning segment of the rat alpha1 subunit to cysteine. We expressed the mutant alpha1 subunit with wild-type beta1 and gamma2 subunits in Xenopus oocytes. We probed the accessibility of the engineered cysteine to covalent modification by charged, sulfhydryl-specific reagents added extracellularly. We assume that among residues in membrane-spanning segments, only those lining the channel would be susceptible to modification by polar reagents and that such modification would irreversibly alter conduction through the channel. We infer that nine of the residues, alpha1 Val257, alpha1 Thr26l, alpha1 Thr262, alpha1 Leu264, alpha1 Thr265, alpha1 Thr268, alpha1 Ile27l, alpha1 Ser272 and alpha1 Asn275 are exposed in the channel. On a helical wheel plot, the exposed residues, except alpha1 Thr262, lie on one side of the helix in an arc of 120 degrees. We infer that the M2 segment forms an alpha helix that is interrupted in the region of alpha1 Thr262. The modification of residues as cytoplasmic as alpha1 Val257 in the closed state of the channel suggests that the gate is at least as cytoplasmic as alpha1 Val257. The ability of the positively charged reagent methanethiosulfonate ethylammonium to reach the level of alpha1 Thr261 suggests that the charge-selectivity filter is at least as cytoplasmic as this residue.
γ-氨基丁酸A型（GABA(A)）受体是中枢神经系统中主要的抑制性突触后神经递质受体。γ-氨基丁酸（GABA）与GABA(A)受体结合会诱导一个阴离子选择性通道打开，该通道在关闭前会保持开放数十毫秒。为了了解GABA(A)受体的结构如何决定诸如离子传导、离子选择性和门控等功能特性，我们试图确定构成离子传导通道的氨基酸残基。为了实现这一目标，我们将大鼠α1亚基M2跨膜片段及其侧翼的26个连续残基（250 - 275）逐个突变为半胱氨酸。我们在非洲爪蟾卵母细胞中表达了带有野生型β1和γ₂亚基的突变α1亚基。我们通过细胞外添加的带电荷的、巯基特异性试剂来探测工程化半胱氨酸对共价修饰的可及性。我们假设在跨膜片段中的残基中，只有那些构成通道壁的残基才会易受极性试剂的修饰，并且这种修饰会不可逆地改变通过通道的传导。我们推断其中九个残基，即α1 Val257、α1 Thr261、α1 Thr262、α1 Leu264、α1 Thr265、α1 Thr268、α1 Ile271、α1 Ser272和α1 Asn275在通道中是暴露的。在螺旋轮图上，除α1 Thr262外，暴露的残基位于螺旋的一侧，呈120度的弧形。我们推断M2片段形成一个α螺旋，在α1 Thr262区域中断。在通道关闭状态下，像α1 Val257这样位于胞质侧的残基的修饰表明门控至少与α1 Val257一样位于胞质侧。带正电荷的试剂甲硫基磺酸乙酯铵能够到达α1 Thr261的水平，这表明电荷选择性过滤器至少与这个残基一样位于胞质侧。
