Vázquez J, Feigenbaum P, Katz G, King V F, Reuben J P, Roy-Contancin L, Slaughter R S, Kaczorowski G J, Garcia M L
Department of Membrane Biochemistry and Biophysics, Merck Institute for Therapeutic Research, New Jersey 07065.
J Biol Chem. 1989 Dec 15;264(35):20902-9.
Charybdotoxin (ChTX), a peptidyl inhibitor of the high conductance Ca2+-activated K+ channel (PK,Ca), has been radiolabeled to high specific activity with 125I, and resulting derivatives have been well separated. The monoiodotyrosine adduct blocks PK,Ca in vascular smooth muscle with slightly reduced potency compared with the native peptide under defined experimental conditions. [125I]ChTX, representing this derivative, binds specifically and reversibly to a single class of sites in sarcolemmal membrane vesicles prepared from bovine aortic smooth muscle. These sites display a Kd of 100 pM for the iodinated toxin, as determined by either equilibrium or kinetic binding analyses. Binding site density is about 500 fmol/mg of protein in isolated membranes. The addition of low digitonin concentrations to disrupt the vesicle permeability barrier increases the maximum receptor concentration to 1.5 pmol/mg of protein, correlating with the observations that ChTX binds only at the external pore of PK,Ca and that the membrane preparation is of mixed polarity. Competition studies with ChTX yield a Ki of about 20 pM for native toxin. Binding of [125I]ChTX is modulated by ionic strength as well as by metal ions that are known to interact with PK,Ca. Moreover, tetraethylammonium ion, which blocks PK,Ca with moderately high affinity when applied at the external membrane surface, inhibits [125I]ChTX binding in an apparently competitive fashion with a Ki similar to that found for channel inhibition. In marked contrast, agents that do not inhibit PK,Ca in smooth muscle (e.g. tetrabutylammonium ion, other toxins homologous with ChTX, and pharmacological agents that modulate the activity of dissimilar ion channels) have no effect on [125I]ChTX binding in this tissue. Taken together, these results suggest that the binding sites for ChTX which are present in vascular smooth muscle are directly associated with PK,Ca, thus identifying [125I]ChTX as a useful probe for elucidating the biochemical properties of these channels.
刺尾鱼毒素（ChTX）是一种高电导钙激活钾通道（PK,Ca）的肽类抑制剂，已用125I标记至高比活度，所得衍生物已得到很好的分离。在特定实验条件下，单碘酪氨酸加合物阻断血管平滑肌中的PK,Ca，其效力与天然肽相比略有降低。代表该衍生物的[125I]ChTX特异性且可逆地结合于从牛主动脉平滑肌制备的肌膜囊泡中的单一类位点。通过平衡或动力学结合分析确定，这些位点对碘化毒素的Kd为100 pM。在分离的膜中，结合位点密度约为500 fmol/mg蛋白质。添加低浓度洋地黄皂苷以破坏囊泡通透性屏障可将最大受体浓度提高至1.5 pmol/mg蛋白质，这与ChTX仅在PK,Ca的外部孔道结合以及膜制剂具有混合极性的观察结果相关。用ChTX进行的竞争研究得出天然毒素的Ki约为20 pM。[125I]ChTX的结合受离子强度以及已知与PK,Ca相互作用的金属离子调节。此外，四乙铵离子在施加于外膜表面时以中等高亲和力阻断PK,Ca，以明显竞争性方式抑制[125I]ChTX结合，其Ki与通道抑制的Ki相似。与之形成鲜明对比的是，在平滑肌中不抑制PK,Ca的试剂（例如四丁铵离子、与ChTX同源的其他毒素以及调节不同离子通道活性的药理试剂）对该组织中[125I]ChTX的结合没有影响。综上所述，这些结果表明血管平滑肌中存在的ChTX结合位点与PK,Ca直接相关，从而确定[125I]ChTX是阐明这些通道生化特性的有用探针。
