Single streptomyces lividans K(+) channels: functional asymmetries and sidedness of proton activation.
Single streptomyces lividans K(+) channels: functional asymmetries and sidedness of proton activation.
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DOI：
10.1085/jgp.114.4.551
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发表时间：
1999-10
期刊：
影响因子：
--
通讯作者：
Miller C
中科院分区：
文献类型：
--
作者：
Heginbotham L;LeMasurier M;Kolmakova-Partensky L;Miller C
KcsA K+通道的基本电生理特性在平面脂质双层膜中进行了研究。该通道显示开放状态整流和弱电压依赖性门控。四乙基铵封闭亲和力取决于加入封闭剂的双层的侧面。此外，Na+的反式室的原因阻塞的开放通道电流，而除了顺式侧没有影响。最引人注目的是激活KcsA的质子;通道活动时，观察到只有在反式双层室是在低pH值。为了确定哪一侧的通道面临的腔室，结构上已知的位置的残基被映射到定义的两侧的双层。突变Y82，一个外部残基，导致在四乙基铵亲和力的变化完全从顺式侧。在外部暴露的Y82或内部暴露的Q119处具有半胱氨酸残基取代的通道分别被来自顺式或反式室的甲硫基磺酸盐试剂功能性修饰。已知结合到通道的外部口的Charybdotoxin的阻断仅在将毒素添加到突变为毒素敏感的通道的顺式侧时观察到。这些结果清楚地表明，连接到门控的质子化位点是在KcsA蛋白的细胞内部分。
Basic electrophysiological properties of the KcsA K+ channel were examined in planar lipid bilayer membranes. The channel displays open-state rectification and weakly voltage-dependent gating. Tetraethylammonium blocking affinity depends on the side of the bilayer to which the blocker is added. Addition of Na+ to the trans chamber causes block of open-channel current, while addition to the cis side has no effect. Most striking is the activation of KcsA by protons; channel activity is observed only when the trans bilayer chamber is at low pH. To ascertain which side of the channel faces which chamber, residues with structurally known locations were mapped to defined sides of the bilayer. Mutation of Y82, an external residue, results in changes in tetraethylammonium affinity exclusively from the cis side. Channels with cysteine residues substituted at externally exposed Y82 or internally exposed Q119 are functionally modified by methanethiosulfonate reagents from the cis or trans chambers, respectively. Block by charybdotoxin, known to bind to the channel's external mouth, is observed only when the toxin is added to the cis side of channels mutated to be toxin sensitive. These results demonstrate unambiguously that the protonation sites linked to gating are on the intracellular portion of the KcsA protein.