Vázquez J, Feigenbaum P, King V F, Kaczorowski G J, Garcia M L
Department of Membrane Biochemistry and Biophysics, Merck Institute for Therapeutic Research, Rahway, New Jersey 07065.
J Biol Chem. 1990 Sep 15;265(26):15564-71.
Charybdotoxin (ChTX), a potent peptidyl inhibitor of several types of K+ channels, binds to sites in vascular smooth muscle sarcolemma (Vázquez, J., Feigenbaum, P., Katz, G. M., King, V. F., Reuben, J. P., Roy-Contancin, L., Slaughter, R. S., Kaczorowski, G. J., and Garcia, M. L. (1989) J. Biol. Chem. 265, 20902-20909) which are functionally associated with a high conductance Ca2(+)-activated K+ channel (PK,Ca). 125I-ChTX also binds specifically and reversibly to a single class of sites in plasma membranes prepared from rat brain synaptosomes. These sites exhibit a Kd of 25-30 pM, as measured by either equilibrium or kinetic binding protocols and display a maximum density of about 0.3-0.5 pmol/mg of protein. Competition studies with native ChTX yield a Ki of 8 pM for the noniodinated toxin. The highest density of ChTX sites exists in vesicle fractions of plasma membrane origin. Binding of 125I-ChTX is modulated by metal ions that interact with K+ channels: Ba2+, Ca2+, and Cs+ cause inhibition of ChTX binding; Na+ and K+ stimulate binding at low concentration before producing complete inhibition as their concentration is increased. Stimulation of binding is due to an allosteric interaction that decreases Kd whereas inhibition results from an ionic strength effect. Tetraethylammonium ion has no effect on binding, but tetrabutylammonium ion blocks binding with a Ki of 2.5 mM. Different toxins (i.e. alpha-dendrotoxin, noxiustoxin) that inhibit an inactivating, voltage-dependent K+ channel (PK,V) block 125I-ChTX binding in brain. In marked contrast, iberiotoxin, a selective inhibitor of PK,Ca, has no effect on ChTX binding in this preparation. Inhibition of ChTX binding by alpha-dendrotoxin and noxiustoxin results from an allosteric interaction between separate binding sites for these agents and the ChTX receptor. Taken together, these results suggest that the ChTX sites present in brain are associated with PK,V rather than with PK,Ca. Therefore, 125I-ChTX is a useful probe for elucidating the biochemical properties of a number of different types of K+ channels.
章鱼毒素（ChTX）是几种类型钾通道的强效肽类抑制剂，它与血管平滑肌肌膜上的位点结合（巴斯克斯，J.，费根鲍姆，P.，卡茨，G.M.，金，V.F.，鲁本，J.P.，罗伊 - 孔坦辛，L.，斯劳特，R.S.，卡佐罗夫斯基，G.J.，以及加西亚，M.L.（1989年）《生物化学杂志》265卷，20902 - 20909页），这些位点在功能上与高电导钙激活钾通道（PK，Ca）相关。125I - ChTX也能特异性且可逆地结合从大鼠脑突触体制备的质膜中的一类单一的位点。通过平衡或动力学结合实验测定，这些位点的解离常数（Kd）为25 - 30皮摩尔，并且显示出最大密度约为0.3 - 0.5皮摩尔/毫克蛋白质。用天然ChTX进行的竞争研究得出非碘化毒素的抑制常数（Ki）为8皮摩尔。ChTX位点的最高密度存在于源自质膜的囊泡部分。125I - ChTX的结合受到与钾通道相互作用的金属离子的调节：Ba2 +、Ca2 +和Cs +会抑制ChTX的结合；Na +和K +在低浓度时刺激结合，随着浓度增加则产生完全抑制。结合的刺激是由于变构相互作用降低了Kd，而抑制是由离子强度效应导致的。四乙铵离子对结合没有影响，但四丁铵离子以2.5毫摩尔的Ki阻断结合。抑制失活的电压依赖性钾通道（PK，V）的不同毒素（即α - 树眼镜蛇毒素、诺西毒素）会阻断脑中125I - ChTX的结合。与之形成显著对比的是，PK，Ca的选择性抑制剂iberiotoxin对该制剂中ChTX的结合没有影响。α - 树眼镜蛇毒素和诺西毒素对ChTX结合的抑制是由于这些药物与ChTX受体的不同结合位点之间的变构相互作用。综上所述，这些结果表明脑中存在的ChTX位点与PK，V相关，而非与PK，Ca相关。因此，125I - ChTX是阐明多种不同类型钾通道生化特性的有用探针。
