Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength.
Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength.
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DOI：
10.1085/jgp.91.3.317
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发表时间：
1988-03
影响因子：
3.8
通讯作者：
Miller, C
中科院分区：
文献类型：
--
作者：
Anderson, C S;MacKinnon, R;Smith, C;Miller, C
河豚毒素(CTX)是蝎子毒液中的一种小的碱性蛋白，它通过高电导、钙激活的K+通道强烈地抑制K离子的传导。通过在不带电荷的平面磷脂双层膜上插入单个通道，研究了环磷酰胺与大鼠骨骼肌质膜钙激活钾通道的相互作用。CTX通过与通道外侧结合来阻断K+传导，在生理离子强度下，其表观解离常数约为10 nM。阻塞态和非阻塞态的驻留时间均为单指数分布。毒素结合率与CTX浓度呈线性关系，解离率与CTX浓度无关。CTX能够阻断开放和关闭的通道；开放通道的结合速率快七倍，而两种通道构象的解离速率相同。膜去极化使CTX解离速率增加e倍/28 mV；如果通道的开放概率随电压变化而保持恒定，则毒素结合率不受电压影响。将外液离子强度从20 mM增加到300 mM(含K+、Na+或精氨酸+)可使缔合率降低两个数量级，对解离率几乎没有影响。我们得出结论，CTX与钙激活的K+通道的结合是一个双分子过程，并且CTX相互作用既可以感知电压，也可以感知通道的构象状态。我们进一步提出在通道的CTX结合部位附近存在固定负电荷的区域。
Charybdotoxin (CTX), a small, basic protein from scorpion venom, strongly inhibits the conduction of K ions through high-conductance, Ca2+-activated K+ channels. The interaction of CTX with Ca2+-activated K+ channels from rat skeletal muscle plasma membranes was studied by inserting single channels into uncharged planar phospholipid bilayers. CTX blocks K+ conduction by binding to the external side of the channel, with an apparent dissociation constant of approximately 10 nM at physiological ionic strength. The dwell-time distributions of both blocked and unblocked states are single-exponential. The toxin association rate varies linearly with the CTX concentration, and the dissociation rate is independent of it. CTX is competent to block both open and closed channels; the association rate is sevenfold faster for the open channel, while the dissociation rate is the same for both channel conformations. Membrane depolarization enhances the CTX dissociation rate e-fold/28 mV; if the channel's open probability is maintained constant as voltage varies, then the toxin association rate is voltage independent. Increasing the external solution ionic strength from 20 to 300 mM (with K+, Na+, or arginine+) reduces the association rate by two orders of magnitude, with little effect on the dissociation rate. We conclude that CTX binding to the Ca2+-activated K+ channel is a bimolecular process, and that the CTX interaction senses both voltage and the channel's conformational state. We further propose that a region of fixed negative charge exists near the channel's CTX-binding site.