Structure-Function Releationships of Voltage-Gated Proton Channels

描述（由申请人提供）：电压门控质子通道是在许多细胞膜中发现的蛋白质，允许质子以高度调节的方式穿过膜。二十多年来，这些分子在细胞中使用电测量（电压钳）进行了研究。2006年，编码这些分子的基因被鉴定出来，这使得人们有可能以前所未有的细节来研究分子结构。在这个项目中，质子通道基因将产生特定的突变，突变的通道将在细胞系中表达。这些突变对分子行为的影响将使用膜片钳测量进行评估。一个目标是确定质子通道如何感知pHo和pHi，细胞膜两侧的质子浓度，这对它们执行功能的能力至关重要。我们假设，一个或几个氨基酸残基，可访问的解决方案结合质子，从而作为pH传感器。第二个目标是确定分子的哪些部分感知膜电位。因为质子通道分子类似于其他离子通道的电压感应域，所以第一次尝试将是确定是否相同的残基感应电压。接下来，将检查位于跨膜区域的其他带电氨基酸。第三，质子通过分子的路径的位置将被确定。测试的第一个假设将基于与其他通道的相应域的相似性。质子通道的独特方面是质子跳跃（Grotthuss样）的传导机制，其可以发生在可滴定的驻留处。接下来，通过在特定位置插入半胱氨酸残基，然后确定它们可以被化学修饰的速率（“半胱氨酸扫描”），系统地检查分子中特定位置的氨基酸残基的外部或内部溶液的可及性。最后，在2008年发现质子通道是一个同源二聚体提出的问题，将得到解决。我们将测试抑制剂Zn 2+是否以高亲和力结合在两个单体之间。这些和其他实验将表明这两个单体是否独立发挥作用，或者它们是否相互作用。 公共卫生相关性：质子通道是白色血细胞杀死细菌和其他微生物入侵者所需的蛋白质。该项目将确定质子通道分子的工作部分，以便开发调节质子通道功能的药物。

DESCRIPTION (provided by applicant): Voltage gated proton channels are proteins found in many cell membranes that allow protons to cross the membrane in a highly regulated manner. For over two decades, these molecules were studied in cells using electrical measurements (voltage-clamp). The identification in 2006 of the gene that codes for these molecules makes it possible to study the molecular structure in unprecedented detail. In this project, specific mutations in the proton channel gene will be produced and the mutant channels will be expressed in cell lines. The effects these mutations have on molecular behavior will be evaluated using patch-clamp measurements. One goal is to determine how proton channels sense pHo and pHi, the proton concentrations on either side of the cell membrane, which is crucial to their ability to carry out their functions. We hypothesize that one or a few amino acid residues that are accessible to the solution bind protons and thus act as pH sensors. The second goal is to determine which parts of the molecule sense membrane potential. Because the proton channel molecule resembles the voltage-sensing domains of other ion channels, the first attempt will be to determine if the same residues sense voltage. Next, other charged amino acids located in membrane-spanning regions will be examined. Third, the location of the pathway by which protons pass through the molecule will be determined. The first hypothesis tested will be based on similarities to corresponding domains of other channels. The unique aspect of proton channels is a conduction mechanism of proton hopping (Grotthuss-like) that could occur at a titratable reside. Next the accessibility to external or internal solutions of amino acid residues at specific locations in the molecule will be examined systematically, by inserting cysteine residues at specific locations and then determining the rate at which they can be modified chemically ("cysteine scanning"). Finally, the discovery in 2008 that proton channel is a homodimer raises questions that will be addressed. We will test whether the inhibitor Zn2+ binds with high affinity between the two monomers. These and other experiments will indicate whether the two monomers function independently, or if they interact. PUBLIC HEALTH RELEVANCE: Proton channels are proteins that white blood cells need in order to kill bacteria and other microbial invaders. This project will identify the working parts of the proton channel molecule so that drugs to regulate proton channel function can be developed.