Conotoxins as sensors of local pH and electrostatic potential in the outer vestibule of the sodium channel.
Conotoxins as sensors of local pH and electrostatic potential in the outer vestibule of the sodium channel.
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结合毒素是钠通道外侧前庭中局部pH和静电电位的传感器。
DOI：
10.1085/jgp.200308842
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发表时间：
2003-07
影响因子：
3.8
通讯作者：
French, Robert J
中科院分区：
文献类型：
--
作者：
Hui, Kwokyin;McIntyre, Deane;French, Robert J
我们研究了用组氨酸、谷氨酸或丙氨酸残基取代精氨酸-13的μ-芋螺毒素GIIIA(μCTX)的衍生物对电压依赖的大鼠骨骼肌钠通道的阻断作用。毒素结合和解离作为电流波动观察到，从单一的，巴曲霉毒素处理的钠通道在平面脂质双层。μCTX的R13X衍生物仅部分阻断了单通道电流，使我们能够直接监测不同条件下μCTX结合和非结合状态的性质。在结合状态下，毒素衍生物R13E和R13H的单位滴定曲线表明，通过结合通道的部分剩余电流随pH变化，反映了改变残基上电荷的影响。用R13A进行的实验提供了一个对照，反映了除毒素残基13以外的所有残基对毒素和通道的影响。用于滴定剩余电导的表观pKs分别从组氨酸和谷氨酸的标称pk值正移了2-3个pH单位，并从通过核磁共振测量在游离溶液中毒素分子中测定的这些特定残基的值移位。毒素亲和力也随着pH的变化而急剧变化，几乎完全是由于结合速率常数kon的变化。从静电角度解释，我们的结果表明，即使在结合的阳离子毒素存在的情况下，通道前庭也强烈有利于阳离子进入，在通道残基E403、E758、D1241和D1532形成的“外电荷环”水平上，等效的局部静电势比−100 mV更负。在毒素残基13的pk比结合态更接近溶液值的过渡态，缔合率明显受到限制。因此，这些独特的多肽的作用可以用来在单个分子转变和确定的构象状态期间感觉配体-受体复合体中的局部环境。
We examined the block of voltage-dependent rat skeletal muscle sodium channels by derivatives of μ-conotoxin GIIIA (μCTX) having either histidine, glutamate, or alanine residues substituted for arginine-13. Toxin binding and dissociation were observed as current fluctuations from single, batrachotoxin-treated sodium channels in planar lipid bilayers. R13X derivatives of μCTX only partially block the single-channel current, enabling us to directly monitor properties of both μCTX-bound and -unbound states under different conditions. The fractional residual current through the bound channel changes with pH according to a single-site titration curve for toxin derivatives R13E and R13H, reflecting the effect of changing the charge on residue 13, in the bound state. Experiments with R13A provided a control reflecting the effects of titration of all residues on toxin and channel other than toxin residue 13. The apparent pKs for the titration of residual conductance are shifted 2–3 pH units positive from the nominal pK values for histidine and glutamate, respectively, and from the values for these specific residues, determined in the toxin molecule in free solution by NMR measurements. Toxin affinity also changes dramatically as a function of pH, almost entirely due to changes in the association rate constant, kon. Interpreted electrostatically, our results suggest that, even in the presence of the bound cationic toxin, the channel vestibule strongly favors cation entry with an equivalent local electrostatic potential more negative than −100 mV at the level of the “outer charged ring” formed by channel residues E403, E758, D1241, and D1532. Association rates are apparently limited at a transition state where the pK of toxin residue 13 is closer to the solution value than in the bound state. The action of these unique peptides can thus be used to sense the local environment in the ligand-–receptor complex during individual molecular transitions and defined conformational states.