Deutsch C, Price M, Lee S, King V F, Garcia M L
Department of Physiology, University of Pennsylvania, Philadelphia 19104.
J Biol Chem. 1991 Feb 25;266(6):3668-74.
Charybdotoxin (ChTX) inhibits with high affinity a voltage-gated K+ channel that is present in human T lymphocytes. In this system, 125I-ChTX binds specifically and reversibly to a single class of sites which display a Kd of 8-14 pM, as measured by either equilibrium or kinetic binding protocols. The maximum density of sites, 542 sites/cell, correlates well with the density of K+ channel as determined by electrophysiological experiments. Binding of 125I-ChTX is modulated by the ionic strength of the incubation media and by Ca2+. Increasing concentrations of either K+, Na+, or Ca2+ cause inhibition of toxin binding. Inhibition of binding by Ca2+ is due, primarily, to an effect on toxin dissociation rates. Increasing the pH of the external media from 6.8 to 8.5 enhances toxin binding, due to an increase in affinity with no significant effect on the maximum density of receptor sites. Different agents that block the voltage-gated K+ channel in human T lymphocytes, inhibit toxin binding. Mitogen-stimulated T cells display 2.5-3-fold increase in toxin binding as compared with unstimulated control cells. These data, taken together, suggest that 125I-ChTX binding sites identified in this study, represent the predominant voltage-gated K+ channel present in peripheral human T lymphocytes. Therefore, 125I-ChTX is a useful probe for elucidating the physiological role of this type of K+ channel.
刺尾鱼毒素（ChTX）以高亲和力抑制人T淋巴细胞中存在的一种电压门控钾通道。在该系统中，通过平衡或动力学结合实验测定，125I-ChTX特异性且可逆地结合到一类位点上，其解离常数（Kd）为8 - 14 pM。位点的最大密度为542个位点/细胞，与通过电生理实验确定的钾通道密度密切相关。125I-ChTX的结合受孵育介质的离子强度和Ca2+的调节。K+、Na+或Ca2+浓度的增加都会导致毒素结合受到抑制。Ca2+对结合的抑制主要是由于对毒素解离速率的影响。将外部介质的pH从6.8提高到8.5会增强毒素结合，这是由于亲和力增加，而对受体位点的最大密度没有显著影响。在人T淋巴细胞中阻断电压门控钾通道的不同试剂会抑制毒素结合。与未刺激的对照细胞相比，有丝分裂原刺激的T细胞的毒素结合增加了2.5 - 3倍。综上所述，这些数据表明本研究中鉴定的125I-ChTX结合位点代表了外周人T淋巴细胞中主要的电压门控钾通道。因此，125I-ChTX是阐明这类钾通道生理作用的有用探针。
