State-dependent accessibility and electrostatic potential in the channel of the acetylcholine receptor. Inferences from rates of reaction of thiosulfonates with substituted cysteines in the M2 segment of the alpha subunit.
State-dependent accessibility and electrostatic potential in the channel of the acetylcholine receptor. Inferences from rates of reaction of thiosulfonates with substituted cysteines in the M2 segment of the alpha subunit.
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DOI：
10.1085/jgp.111.6.717
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发表时间：
1998-06
期刊：
影响因子：
--
通讯作者：
Karlin A
中科院分区：
文献类型：
--
作者：
Pascual JM;Karlin A
离子通道的功能取决于通道内腔排列的残基的化学和物理性质以及空间排列，并取决于内腔内的静电势。我们已经使用小的，巯基特异性的硫代磺酸盐试剂，带正电和中性的，探测乙酰胆碱（ACh）受体通道内的环境。用半胱氨酸取代α亚基第二跨膜段（M2）中9个暴露的残基，测定其反应的速率常数。在存在和不存在ACh的情况下，最大的速率常数是在通道的细胞内末端附近用半胱氨酸取代α Thr 244的反应。在通道的开放状态下，但不是在关闭状态下，带电试剂与几个取代的半胱氨酸的反应的速率常数取决于跨膜静电势，并且这些半胱氨酸的电距离从M2的细胞外端到细胞内端增加。即使在零跨膜电位下，三种带正电荷的试剂与αT244C、αL251C和αL258C反应的速率常数与不带电荷的试剂反应的速率常数之比在开放状态下也比在封闭状态下大得多。速率常数对试剂电荷的这种依赖性与通道中的固有静电势一致，该静电势在打开状态下比在关闭状态下负得多。ACh对取代Cys沿着αM2长度反应的速率常数的影响、对速率常数对跨膜电位的依赖性的影响以及对内在电位的影响支持了比α Thr 244更胞内的门的位置。
Ion channel function depends on the chemical and physical properties and spatial arrangement of the residues that line the channel lumen and on the electrostatic potential within the lumen. We have used small, sulfhydryl-specific thiosulfonate reagents, both positively charged and neutral, to probe the environment within the acetylcholine (ACh) receptor channel. Rate constants were determined for their reactions with cysteines substituted for nine exposed residues in the second membrane-spanning segment (M2) of the α subunit. The largest rate constants, both in the presence and absence of ACh, were for the reactions with the cysteine substituted for αThr244, near the intracellular end of the channel. In the open state of the channel, but not in the closed state, the rate constants for the reactions of the charged reagents with several substituted cysteines depended on the transmembrane electrostatic potential, and the electrical distance of these cysteines increased from the extracellular to the intracellular end of M2. Even at zero transmembrane potential, the ratios of the rate constants for the reactions of three positively charged reagents with αT244C, αL251C, and αL258C to the rate constant for the reaction of an uncharged reagent were much greater in the open than in the closed state. This dependence of the rate constants on reagent charge is consistent with an intrinsic electrostatic potential in the channel that is considerably more negative in the open state than in the closed state. The effects of ACh on the rate constants for the reactions of substituted Cys along the length of αM2, on the dependence of the rate constants on the transmembrane potential, and on the intrinsic potential support a location of a gate more intracellular than αThr244.