Structure-Function Releationships of Voltage-Gated Proton Channels

电压门控质子通道的结构-功能关系

• 批准号: 8066327
• 负责人: THOMAS E DECOURSEY
• 金额: $ 28.96万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8066327
• 关键词: Acidic Amino Acids; Address; Affinity; Amino Acids; Bacteria; Behavior; Binding; C-terminal; Cell Line; Cell membrane; Cells; Charge; Code; Cysteine; Dependence; Development; Experimental Designs; Genes; Goals; Histidine; Homology Modeling; Human; Humulus; Ion Channel; Kinetics; Leukocytes; Location; Measurement; Membrane; Membrane Potentials; Methods; Modeling; Molecular; Molecular Structure; Mutation; Pathway interactions; Phagocytes; Pharmaceutical Preparations; Phosphorylation; Potassium Channel; Property; Proteins; Protons; Research Personnel; Scanning; Side; Site-Directed Mutagenesis; Solutions; Structure; Temperature; Tertiary Protein Structure; Testing; Universities; Work; base; constriction; design; electrical measurement; gene discovery; inhibitor/antagonist; killings; microbial; monomer; mutant; patch clamp; public health relevance; research study; sensor; voltage; voltage clamp

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.

描述（由申请人提供）：电压门控质子通道是许多细胞膜中发现的蛋白质，使质子可以高度调节的方式越过膜。在过去的二十年中，使用电测量（电压钳）在细胞中研究了这些分子。 2006年编码这些分子的基因的鉴定使得可以以前所未有的细节研究分子结构。在该项目中，将产生质子通道基因中的特定突变，并将突变通道在细胞系中表达。这些突变对分子行为的影响将使用斑块钳测量进行评估。一个目标是确定质子通道如何感知pho和phi，这是细胞膜两侧的质子浓度，这对于它们执行功能的能力至关重要。我们假设溶液可访问的一个或几个氨基酸残基结合质子，从而充当pH传感器。第二个目标是确定分子感知膜电位的哪些部分。由于质子通道分子类似于其他离子通道的电压传感域，因此首次尝试是确定相同的残基是否感应电压。接下来，将检查位于跨膜区域的其他带电氨基酸。第三，将确定质子穿过分子的途径的位置。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（类似Grotthuss）的传导机制，可以在可遗留下发生。接下来，通过在特定位置插入半胱氨酸残基，然后确定可以化学修改它们的速率（“半胱氨酸扫描”）。最后，2008年发现质子频道的发现提出了将要解决的问题。我们将测试两个单体之间的抑制剂Zn2+是否具有高亲和力结合。这些和其他实验将指示两个单体是否独立起作用，还是它们是否相互作用。 公共卫生相关性：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位，以便可以开发用于调节质子通道功能的药物。