Anderson C S, MacKinnon R, Smith C, Miller C
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
J Gen Physiol. 1988 Mar;91(3):317-33. doi: 10.1085/jgp.91.3.317.
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.
卡律蝎毒素（CTX）是一种来自蝎毒的小碱性蛋白质，它能强烈抑制钾离子通过高电导、钙激活钾通道的传导。通过将单通道插入不带电的平面磷脂双分子层中，研究了CTX与大鼠骨骼肌质膜上钙激活钾通道的相互作用。CTX通过结合通道外侧来阻断钾离子传导，在生理离子强度下其表观解离常数约为10 nM。阻断态和未阻断态的驻留时间分布均为单指数分布。毒素结合速率随CTX浓度呈线性变化，而解离速率与之无关。CTX能够阻断开放和关闭的通道；开放通道的结合速率快7倍，而两种通道构象的解离速率相同。膜去极化使CTX解离速率以每28 mV增加e倍；如果通道的开放概率在电压变化时保持恒定，那么毒素结合速率与电压无关。将外部溶液离子强度从20 mM增加到300 mM（使用钾离子、钠离子或精氨酸离子）会使结合速率降低两个数量级，而对解离速率影响很小。我们得出结论，CTX与钙激活钾通道的结合是一个双分子过程，并且CTX的相互作用能感知电压和通道的构象状态。我们进一步提出，在通道的CTX结合位点附近存在一个固定负电荷区域。
