Department of Biological Sciences, SUNY College of Optometry, New York, NY 10036, USA.
J Gen Physiol. 2012 Jan;139(1):69-82. doi: 10.1085/jgp.201110678.
The anti-malarial drug quinine and its quaternary derivative N-benzylquininium (BQ(+)) have been shown to inhibit gap junction (GJ) channels with specificity for Cx50 over its closely related homologue Cx46. Here, we examined the mechanism of BQ(+) action using undocked Cx46 and Cx50 hemichannels, which are more amenable to analyses at the single-channel level. We found that BQ(+) (300 µM-1 mM) robustly inhibited Cx50, but not Cx46, hemichannel currents, indicating that the Cx selectivity of BQ(+) is preserved in both hemichannel and GJ channel configurations. BQ(+) reduced Cx50 hemichannel open probability (P(o)) without appreciably altering unitary conductance of the fully open state and was effective when added from either extracellular or cytoplasmic sides. The reductions in P(o) were dependent on BQ(+) concentration with a Hill coefficient of 1.8, suggesting binding of at least two BQ(+) molecules. Inhibition by BQ(+) was voltage dependent, promoted by hyperpolarization from the extracellular side and conversely by depolarization from the cytoplasmic side. These results are consistent with binding of BQ(+) in the pore. Substitution of the N-terminal (NT) domain of Cx46 into Cx50 significantly impaired inhibition by BQ(+). The NT domain contributes to the formation of the wide cytoplasmic vestibule of the pore and, thus, may contribute to the binding of BQ(+). Single-channel analyses showed that BQ(+) induced transitions that did not resemble pore block, but rather transitions indistinguishable from the intrinsic gating events ascribed to loop gating, one of two mechanisms that gate Cx channels. Moreover, BQ(+) decreased mean open time and increased mean closed time, indicating that inhibition consists of an increase in hemichannel closing rate as well as a stabilization of the closed state. Collectively, these data suggest a mechanism of action for BQ(+) that involves modulation loop gating rather than channel block as a result of binding in the NT domain.
抗疟药物奎宁及其季铵衍生物 N-苄基奎宁鎓（BQ(+)）已被证明能特异性抑制缝隙连接（GJ）通道，对 Cx50 的抑制作用强于其密切相关的同源物 Cx46。在这里，我们使用未对接的 Cx46 和 Cx50 半通道研究了 BQ(+)的作用机制，这对半通道水平的分析更为适用。我们发现，BQ(+)（300µM-1mM）强烈抑制 Cx50，但不抑制 Cx46 半通道电流，表明 BQ(+)对 Cx 的选择性在半通道和 GJ 通道构型中都得到了保留。BQ(+)降低 Cx50 半通道开放概率（P(o)），而不明显改变完全开放状态的单位电导，并且当从细胞外或细胞质侧添加时都有效。P(o)的降低依赖于 BQ(+)的浓度，Hill 系数为 1.8，表明至少结合了两个 BQ(+)分子。抑制作用随 BQ(+)浓度而变化，对细胞外侧超极化有促进作用，对细胞质侧去极化有相反的作用。这些结果与 BQ(+)在孔中的结合一致。Cx46 的 N 端（NT）结构域的取代显著削弱了 BQ(+)的抑制作用。NT 结构域有助于形成孔的宽细胞质前庭，因此可能有助于 BQ(+)的结合。单通道分析表明，BQ(+)诱导的转变不同于孔阻塞，而是类似于归因于环门控的固有门控事件的转变，这是两种使 Cx 通道门控的机制之一。此外，BQ(+)降低了平均开放时间，增加了平均关闭时间，表明抑制作用包括半通道关闭率的增加以及关闭状态的稳定。总的来说，这些数据表明 BQ(+)的作用机制涉及调制环门控，而不是由于结合在 NT 结构域而导致的通道阻塞。
