Wu G, Raines D E, Miller K W
Department of Anesthesia, Massachusetts General Hospital, Boston.
Biochemistry. 1994 Dec 27;33(51):15375-81. doi: 10.1021/bi00255a018.
3-(Trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) has recently been found to be a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAcChoR) by both photolabeling and flux assays (White et al., 1991). However, these experiments were done when TID was in equilibrium with the nAcChoR, and thus only its interactions with the resting and the desensitized states of the nAcChoR were studied. In this work we characterized the interaction between TID and nAcChoR in the open and resting states using a flux assay. When TID and acetylcholine were simultaneously mixed with the nAcChoR in native Torpedo vesicles, TID did not inhibit agonist-induced 86Rb+ flux. However, following prolonged preincubation (4 min) of TID with nAcChoR, complete inhibition was observed with a half-inhibition constant of 0.4 microM TID and a Hill coefficient of 0.9. This suggested that TID might act either on the resting or the desensitized state in preference to the open state of the nAcChoR. Preincubation of nAcChoR with TID, followed by a 7 ms agonist-induced flux assay, showed that the flux response declined exponentially with preincubation time. Assuming a pseudo-first-order process, analysis revealed the rate constant for the onset of inhibition of nAcChoR in the resting state to be in the range of (1.2-3.4) x 10(6) M-1 s-1. To test if fast desensitization was enhanced by TID under these conditions, we used a fluorescent analog of acetylcholine. Stopped-flow fluorimetry showed that the fraction of nAcChoR in the predesensitized state did not increase during preincubation with TID.(ABSTRACT TRUNCATED AT 250 WORDS)
3-（三氟甲基）-3-（间碘苯基）二氮丙啶（TID）最近通过光标记和通量测定被发现是烟碱型乙酰胆碱受体（nAcChoR）的非竞争性抑制剂（怀特等人，1991年）。然而，这些实验是在TID与nAcChoR处于平衡状态时进行的，因此仅研究了其与nAcChoR的静息态和脱敏态的相互作用。在这项工作中，我们使用通量测定法表征了TID与处于开放态和静息态的nAcChoR之间的相互作用。当TID和乙酰胆碱同时与天然电鳐囊泡中的nAcChoR混合时，TID不抑制激动剂诱导的86Rb+通量。然而，在TID与nAcChoR长时间预孵育（4分钟）后，观察到完全抑制，半抑制常数为0.4微摩尔TID，希尔系数为0.9。这表明TID可能优先作用于nAcChoR的静息态或脱敏态而非开放态。用TID对nAcChoR进行预孵育，随后进行7毫秒激动剂诱导的通量测定，结果表明通量响应随预孵育时间呈指数下降。假设为假一级过程，分析显示静息态nAcChoR抑制开始的速率常数在（1.2 - 3.4）×10（6）M-1 s-1范围内。为了测试在这些条件下TID是否增强快速脱敏，我们使用了乙酰胆碱的荧光类似物。停流荧光法表明，在与TID预孵育期间，预脱敏态nAcChoR的比例没有增加。（摘要截短于250字）
