Laboratory of Molecular Physiology & Biophysics, Faculty of Medicine, Kagawa University, Miki-cho, Kagawa, Japan.
RIKEN SPring-8 Center, Sayo-cho, Hyogo, Japan.
J Physiol. 2023 Sep;601(18):4073-4089. doi: 10.1113/JP284175. Epub 2023 Aug 9.
ATP is an important molecule implicated in diverse biochemical processes, including the modulation of ion channel and transporter activity. The voltage-gated proton channel (Hv1) controls proton flow through the transmembrane pathway in response to membrane potential, and various molecules regulate its activity. Although it is believed that ATP is not essential for Hv1 activity, a report has indicated that cytosolic ATP may modulate Hv1. However, the detailed molecular mechanism underlying the effect of ATP on Hv1 is unknown, and whether ATP is involved in the physiological regulation of Hv1 activity remains unclear. Here, we report that cytosolic ATP is required to maintain Hv1 activity. To gain insight into the underlying mechanism, we analysed the effects of ATP on the mouse Hv1 channel (mHv1) using electrophysiological and microscale thermophoresis (MST) methods. Intracellular ATP accelerated the activation kinetics of mHv1, thereby increasing the amplitude of the proton current within the physiological concentration range. The increase in proton current was reproduced with a non-hydrolysable ATP analogue, indicating that ATP directly influences Hv1 activity without an enzymatic reaction. The direct molecular interaction between the purified mHv1 protein and ATP was analysed and demonstrated through MST. In addition, ATP facilitation was observed for the endogenous proton current flowing through Hv1 in the physiological concentration range of ATP. These results suggest that ATP influences Hv1 activity via direct molecular interactions and is required for the physiological function of Hv1. KEY POINTS: We found that ATP is required to maintain the activity of voltage-gated proton channels (Hv1) and investigated the underlying molecular mechanism. Application of intracellular ATP increased the amplitude of the proton current flowing through Hv1, accompanied by an acceleration of activation kinetics. The direct interaction between purified Hv1 protein and ATP was quantitatively analysed using microscale thermophoresis. ATP enhanced endogenous proton currents in breast cancer cell lines. These results suggest that ATP influences Hv1 activity via direct molecular interactions and that its functional characteristics are required for the physiological activity of Hv1.
ATP 是一种参与多种生化过程的重要分子，包括调节离子通道和转运体活性。电压门控质子通道（Hv1）控制质子通过跨膜途径的流动，响应膜电位，并且有各种分子调节其活性。尽管人们认为 ATP 对于 Hv1 活性不是必需的，但有报道表明细胞质 ATP 可能调节 Hv1。然而，ATP 对 Hv1 影响的详细分子机制尚不清楚，并且 ATP 是否参与 Hv1 活性的生理调节仍不清楚。在这里，我们报告细胞质 ATP 是维持 Hv1 活性所必需的。为了深入了解潜在的机制，我们使用电生理学和微尺度热泳（MST）方法分析了 ATP 对小鼠 Hv1 通道（mHv1）的影响。细胞内 ATP 加速了 mHv1 的激活动力学，从而在生理浓度范围内增加了质子电流的幅度。非水解型 ATP 类似物增加了质子电流，表明 ATP 直接影响 Hv1 活性而无需酶反应。通过 MST 分析和证明了纯化的 mHv1 蛋白与 ATP 之间的直接分子相互作用。此外，在生理浓度范围内的 ATP 中观察到了对通过 Hv1 流动的内源性质子电流的促进作用。这些结果表明，ATP 通过直接分子相互作用影响 Hv1 活性，并且是 Hv1 生理功能所必需的。关键点：我们发现 ATP 是维持电压门控质子通道（Hv1）活性所必需的，并研究了潜在的分子机制。细胞内 ATP 的应用增加了通过 Hv1 流动的质子电流的幅度，同时加速了激活动力学。使用微尺度热泳对纯化的 Hv1 蛋白和 ATP 之间的直接相互作用进行了定量分析。在乳腺癌细胞系中，ATP 增强了内源性质子电流。这些结果表明，ATP 通过直接分子相互作用影响 Hv1 活性，并且其功能特性是 Hv1 生理活性所必需的。
