Structural dynamics underlying voltage and pH gating of the human proton channel

项目摘要/摘要 电压门控质子(Hv)通道通过膜携带强健的质子电流，并由 电压和跨膜pH梯度(∆pH)。它们通常作为质子挤出器来维持PH值 代谢活跃细胞的动态平衡。在吞噬细胞中，人类Hv1(HHv1)通道补偿电荷 呼吸爆发过程中NADPH氧化酶和pH失衡促进活性氧的产生 用于病原菌防御的物种(ROS)。精子的hHv1通道触发细胞内的烷基化 获能。HHv1通道也与肿瘤侵袭性和缺血神经细胞高度相关 死亡。电压和∆pH门控是决定质子动力学的两个基本生物物理性质 电流通过Hv通道，而Hv通道反过来又构成了它们在 上面提到的细胞。在家长奖的资助下，我们研究了纯化后的构象动力学 用单分子FRET(荧光共振能量转移)检测脂质体中的hHv1蛋白我们有 首次展示了hHv1电压传感器中的实时构象转变，并表明 电压和pH都通过改变电压传感器的构象景观来门控hHv1通道。 我们还建立了一个动力学模型来解释电压pH相互作用如何决定hHv1通道门控。至 最大限度地发挥已经取得的令人兴奋的发现的影响，我们需要获得准确的速率常数 由动力学模型描述的构象转变，它可以提供对 其他电压门控阳离子通道中的电压传感和选通。《行政副刊》 附件将升级现有的TIRF(全内反射荧光)显微镜 分子FRET成像，这将提供关键的技术力量，以最大限度地发挥 家长奖。该附件包含用于电生理记录的膜片钳模块和 单分子FRET成像模块，包含高速sCMOS(科学互补金属- 氧化物半导体)相机的时间分辨率接近1毫秒。有了这个配件， 现有的TIRF显微镜将升级为进行单分子FRET成像和电生理 同时进行录音。因此，我们将能够控制施加到hHv1通道的电压和pH 更准确地说，为了获得准确的hHv1通道中由 PH值和电压。此外，我们将能够通过膜片钳记录来检查通道门控动力学 和构象动力学同时使用单分子FRET成像，从而定义结构 和函数关系直接相关。

Project Summary/Abstract Voltage-gated proton (Hv) channels carry robust proton currents across membranes and are gated by both voltage and transmembrane pH gradient (∆pH). They normally serve as proton extruders to maintain the pH homeostasis of metabolically active cells. In phagocytes, human Hv1 (hHv1) channels compensate for charge and pH imbalance during the respiratory burst of NADPH oxidase to promote the production of reactive oxygen species (ROS) for pathogen defense. The sperm hHv1 channels trigger intracellular alkylation essential for capacitation. The hHv1 channel also highly correlates with cancer invasiveness and ischemic neuronal cell death. Voltage and ∆pH gating are two fundamental biophysical properties determining the dynamics of proton currents through Hv channels, which in turn underlie their physiological and pathophysiological roles in the cells mentioned above. Funded by the parent award, we examined the conformational dynamics of the purified hHv1 proteins in liposomes using single molecule FRET (Fluorescence Resonance Energy Transfer). We have provided the first glimpse of real-time conformational transitions in the hHv1 voltage sensor and showed that both voltage and pH gate the hHv1 channel by modifying the conformational landscapes of the voltage sensor. We also generated a kinetic model to explain how voltage pH interplay determines hHv1 channel gating. To maximize the impacts of the exciting findings that have been made, we need to obtain accurate rate constants of conformational transitions described by the kinetic model, which can provide key mechanistic insights into the voltage sensing and gating in other voltage-gated cation channels. The administrative supplement for the accessory will upgrade the existing TIRF (Total Internal Reflection Fluorescence) microscope for single molecule FRET imaging, which will provide the critical technical strength to maximize the scientific impacts of the parent award. The accessory contains the patch-clamp module for electrophysiological recording and the single molecule FRET imaging module containing a high-speed sCMOS (scientific Complementary Metal- Oxide-Semiconductor) camera reaching a time resolution close to 1 millisecond. With the accessory, the existing TIRF microscope will be upgraded to perform single molecule FRET imaging and electrophysiological recordings simultaneously. As a result, we will be able to control the voltage and pH applied to hHv1 channels more precisely to get accurate rate constants of the conformational transitions in the hHv1 channel induced by pH and voltage. In addition, we will be able to examine the channel gating dynamics by patch-clamp recordings and conformational dynamics using single molecule FRET imaging simultaneously, thus defining the structure and function relationship directly.