Molecular Mechanisms of Hv1 Voltage-Gated Proton Channel Function

描述（由申请人提供）：细胞内pH跨膜质子梯度的控制对于所有生命形式都是必不可少的。正常和病理生理过程可导致细胞酸化，其直接和间接地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量的H+提供外排途径。在2006年，我们和其他人鉴定了Hvcn1基因，并描述了编码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特性。Hv1也是白细胞中电压门控H+电流表达所必需的。Hv1的鉴定有助于研究质子通道结构和作用机制的根本性重要问题。与含有同源电压传感器结构域（VSD）的蛋白质（如电压门控Ca2+、K+和Na+通道和电压敏感性磷酸酶）一样，Hv1通过膜去极化激活。然而，与其他VSD蛋白相反，Hv1通道的开放也受跨膜pH梯度控制（即，净细胞内酸化使电压依赖性激活向负电位移动）。当打开时，Hv1选择性地允许质子沿其电化学梯度向下流动，以消散任何现有的外向pH梯度，从而促进净细胞内碱化。缺乏对负责控制Hv1通道开放响应电压和pH梯度的机制的分子理解。同样地，在Hv1 VSD中形成Hv1中的水性“水线”H+渗透途径所需的结构决定因素也是未知的。我们将利用定点诱变，电压钳电生理学和荧光分析，以及Hv1蛋白结构的计算机辅助建模和模拟的组合来回答有关Hv1质子通道分子机制的基本问题。更详细地了解Hv1的功能特性将为开发新的药理学和遗传疗法铺平道路，这些疗法旨在治疗由Hv1质子通道活性异常控制引起的疾病。

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.