Molecular Mechanisms of Hv1 Voltage-Gated Proton Channel Function

描述(申请人提供)：细胞内pH跨膜质子梯度的控制对所有形式的生命都是必不可少的。正常和病理生理过程可导致细胞酸化，酸化直接或间接调节多种细胞蛋白和信号通路的功能。电压门控质子通道的中心功能是为过剩的H+提供外流途径。2006年，我们和其他人确定了Hvcn1基因，并描述了编码的Hv1电压门控质子通道的功能。当在异源系统中表达时，HV1足以重建天然电压门控H+电导的标志性生物物理性质。在白细胞中，电压门控H+电流的表达也需要HV1。Hv1基因的发现有助于对质子通道结构和作用机制等重要问题的研究。与含有同源电压传感器结构域(VSD)的蛋白如电压门控的钙、钾、钠通道和电压敏感的磷酸酶一样，Hv1被膜去极化激活。然而，与其他VSD蛋白不同的是，Hv1通道的开放也受跨膜pH梯度的控制(即细胞内净酸化使电压依赖的激活向负电位转变)。打开时，Hv1选择性地允许质子沿其电化学梯度流下，以驱散任何现有的向外定向的pH梯度，从而促进细胞内的净碱化。目前尚缺乏对电压和pH梯度控制Hv1通道开放的分子机制的了解。同样，Hv1 VSD中形成Hv1中水合‘水线’H+渗透途径所需的结构决定因素也是未知的。我们将利用定点突变、电压钳电生理学和荧光测定法以及计算机辅助建模和模拟Hv1蛋白结构来回答关于Hv1质子通道的分子机制的基本问题。对Hv1功能特性的更详细了解将为开发新的药物和遗传疗法铺平道路，这些药物和遗传疗法旨在治疗由Hv1质子通道活动异常控制引起的疾病。 公共卫生相关性：Hv1电压门控质子通道是先天性免疫细胞有效清除细菌和B细胞增殖所必需的，但Hv1‘S专门化功能的分子机制尚不清楚。我们提出了一系列实验来阐明Hv1通道的开放如何受膜电压和pH的控制，以及Hv1结构如何维持水溶液中的质子选择渗透途径。对Hv1功能的详细了解将提供对质子转运和离子通道门控机制的基本见解，并最终促进治疗Hv1功能障碍引起的疾病的治疗方法的发展。

DESCRIPTION (provided by applicant): Control of intracellular pH transmembrane proton gradients is essential for all forms of life. Normal and pathophysiological processes may lead to cellular acidification, which both directly and indirectly modulates the function of a wide variety of cellular proteins and signaling pathways. A central function of voltage-gated proton channels is to provide an efflux pathway for excess H+. In 2006, we and others identified the Hvcn1 gene and described the function of the encoded Hv1 voltage-gated proton channel. Hv1 is sufficient to reconstitute the hallmark biophysical properties of the native voltage-gated H+ conductance when expressed in heterologous systems. Hv1 is also required for expression of voltage-gated H+ currents in leukocytes. The identification of Hv1 facilitates the investigation of fundamentally important questions of proton channel structure and mechanism of action. Like homologous voltage sensor domain (VSD)-containing proteins such as voltage-gated Ca2+, K+ and Na+ channels and voltage-sensitive phosphatases, Hv1 is activated by membrane depolarization. However, in contrast to other VSD proteins, the opening of Hv1 channels is also controlled by the transmembrane pH gradient (i.e., net intracellular acidification shifts voltage-dependent activation toward negative potentials). When open, Hv1 selectively allows protons to flow down their electrochemical gradient to dissipate any existing outwardly-directed pH gradient and thereby promote net intracellular alkalinization. A molecular understanding of the mechanisms that are responsible for the control of Hv1 channel opening in response to voltage and pH gradients is lacking. Likewise, the structural determinants in the Hv1 VSD that are required to form the aqueous 'water-wire' H+ permeation pathway in Hv1 are not known. We will utilize a combination of site-directed mutagenesis, voltage clamp electrophysiology and fluorimetry, and computer- aided modeling and simulation of the Hv1 protein structure to answer fundamental questions about molecular mechanisms in the Hv1 proton channel. A more detailed understanding of Hv1 functional properties will pave the way for the development of novel pharmacological and genetic therapies designed to treat diseases that are caused by aberrant control of Hv1 proton channel activity. PUBLIC HEALTH RELEVANCE: Hv1 voltage-gated proton channels are necessary for efficient bacterial clearance by innate immune cells and B-cell proliferation, but the molecular mechanism underlying Hv1's specialized function is poorly understood. We propose a series of experiments to elucidate how Hv1 channel opening is controlled by membrane voltage and pH, and how the Hv1 structure may sustain an aqueous proton-selective permeation pathway. Understanding Hv1 function in detail will provide fundamental insights into the mechanisms of proton transport and ion channel gating, and ultimately facilitate the development of therapies to cure diseases that result from Hv1 dysfunction.