The two-pore channels (TPCs) are voltage-gated cation channels consisting of single polypeptides with two repeats of a canonical 6-transmembrane unit. TPCs are known to be regulated by various physiological signals such as membrane voltage and phosphoinositide (PI). The fourth helix in the second repeat (second S4) plays a major role in detecting membrane voltage, whereas the first repeat contains a PI binding site. Therefore, each of these stimuli is detected by a unique repeat to regulate the gating of the TPC central pore. How these various stimuli regulate the dynamic structural rearrangement of the TPC molecule remain unknown. Here, we found that PI binding to the first repeat in TPC3 regulates the movement of the distally located second S4 helix, showing that the PI-binding signal is not confined to the pore gate but also transmitted to the voltage sensor. Using voltage clamp fluorometry, measurement of gating charges, and Cys-accessibility analysis, we observed that PI binding significantly potentiates the voltage dependence of the movement of the second S4 helix. Notably, voltage clamp fluorometry analysis revealed that the voltage-dependent movement of the second S4 helix occurred in two phases, of which the second phase corresponds to the transfer of the gating charges. This movement was observed in the voltage range where gate-opening occurs and was potentiated by PI. In conclusion, this regulation of the second S4 helix by PI indicates a tight inter-repeat coupling within TPC3, a feature which might be conserved among TPC family members to integrate various physiological signals.

中文翻译：

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磷酸肌醇在双孔通道 3 的第二次重复中调节 S4 电压传感器的动态运动。

双孔通道 (TPC) 是电压门控阳离子通道，由具有两个重复的典型 6 跨膜单元的单个多肽组成。已知 TPC 受各种生理信号的调节，例如膜电压和磷酸肌醇 (PI)。第二个重复（第二个 S4）中的第四个螺旋在检测膜电压中起主要作用，而第一个重复包含一个 PI 结合位点。因此，这些刺激中的每一个都通过一个独特的重复来检测，以调节 TPC 中心孔的门控。这些不同的刺激如何调节 TPC 分子的动态结构重排仍然未知。在这里，我们发现 PI 与 TPC3 中第一个重复的结合调节了位于远端的第二个 S4 螺旋的运动，表明 PI 结合信号不仅限于孔门，还传输到电压传感器。使用电压钳荧光法、门控电荷测量和 Cys 可及性分析，我们观察到 PI 结合显着增强了第二个 S4 螺旋运动的电压依赖性。值得注意的是，电压钳荧光分析表明，第二个 S4 螺旋的电压依赖性运动发生在两个阶段，其中第二阶段对应于门控电荷的转移。这种运动在发生栅极打开的电压范围内观察到，并被 PI 增强。总之，PI 对第二个 S4 螺旋的这种调节表明 TPC3 内存在紧密的重复耦合，TPC 家族成员之间可能保留这一特征以整合各种生理信号。