Lester H A, Krouse M E, Nass M M, Wassermann N H, Erlanger B F
J Gen Physiol. 1980 Feb;75(2):207-32. doi: 10.1085/jgp.75.2.207.
After disulphide bonds are reduced with dithiothreitol, trans-3- (alpha-bromomethyl)-3'-[alpha- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this "tethered agonist" shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 muM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to "open-channel blockade" bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3',bis-[alpha-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel's activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.
用二硫苏糖醇还原二硫键后，反式-3-（α-溴甲基）-3'-[α-（三甲基铵）甲基]偶氮苯（反式-QBr）使受体上的巯基烷基化。这种“拴系激动剂”诱导的膜电导与可逆激动剂诱导的膜电导具有许多共同特性。随着膜电位变得更负，平衡电导增加；电压敏感性类似于50μM卡巴胆碱的情况。电压跃变弛豫遵循指数时间进程；速率常数约为50μM卡巴胆碱时的两倍，并且具有相同的电压和温度敏感性。对于可逆激动剂，通道开放速率随激动剂-受体碰撞频率增加；对于拴系的反式-QBr，该速率仅取决于分子内事件。与可逆激动剂诱导的电导相比，QBr诱导的电导对筒箭毒碱竞争性阻断的敏感性至少低10倍，对QX-222的“开放通道阻断”敏感性大致相同。用拴系QBr进行的闪光实验类似于用可逆光异构化激动剂3,3',双-[α-（三甲基铵）甲基]偶氮苯（双-Q）进行的实验：顺式→反式光异构化使电导增加，反式→顺式光异构化使电导降低。与双-Q一样，闪光弛豫的速率常数与电压跃变弛豫相同。具有拴系反式异构体的受体。通过比较激动剂诱导的电导与顺式/反式比率，我们得出结论，每个通道的激活由单个拴系QBr分子的构型决定。QBr诱导的电导显示出缓慢下降（时间常数为几百毫秒），闪光可部分逆转这种下降。这些相似性表明，相同的限速步骤控制着可逆激动剂和拴系激动剂作用下通道的开放和关闭。因此，这一步骤可能不是激动剂与受体分子的初始相遇。
