Structure-Function Releationships of Voltage-Gated Proton Channels

描述(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，它允许质子以高度调控的方式穿过细胞膜。二十多年来，人们在细胞中使用电学测量(电压钳)来研究这些分子。2006年发现了编码这些分子的基因，使得以前所未有的细节研究分子结构成为可能。在这个项目中，将产生质子通道基因的特定突变，并在细胞系中表达突变的通道。这些突变对分子行为的影响将通过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，即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。我们假设溶液中可以接触到的一个或几个氨基酸残基与质子结合，从而起到pH传感器的作用。第二个目标是确定分子的哪些部分感受膜电位。由于质子通道分子类似于其他离子通道的电压敏感结构域，第一次尝试将确定是否相同的残基感知电压。接下来，我们将研究位于跨膜区域的其他带电氨基酸。第三，将确定质子通过分子的路径的位置。检验的第一个假设将基于与其他渠道的相应领域的相似性。质子通道的独特之处在于质子跳跃的传导机制(类似格洛特斯)，这种机制可以在可滴定的溶液中发生。下一步，将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对外部或内部溶液的可及性。最后，2008年发现的质子通道是同源二聚体提出了一些问题，这些问题将得到解决。我们将测试抑制剂锌是否与两个单体之间的高亲和力结合。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。 与公共健康相关：质子通道是白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，以便开发出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.