SELECTIVE PERMEABILITY & PORE STRUCTURE OF GAP JUNCTION

选择通透性

• 批准号: 2857284
• 负责人: BRUCE J NICHOLSON
• 金额: $ 20.61万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2857284
• 关键词: Xenopus oocyte; chickens; complementary RNA; fluorescent dye /probe; gap junctions; heart conduction system; heart electrical activity; heart function; histochemistry /cytochemistry; immunochemistry; in situ hybridization; laboratory mouse; laboratory rabbit; membrane channels; nucleic acid probes; protein structure function; second messengers; site directed mutagenesis; voltage gated channel; western blottings

In heart, gap junctions serve the crucial role of electrically coupling muscle fibers to ensure rapid propagation of synchronous contractions. However, the identification of multiple connexins in heart (i.e., Cx37,40,43,45,46) clearly indicates a greater complexity. We and others have demonstrated that intracellular channels comprised of some of these connexins show distinctive gating properties and conductance. Heterotypic interactions between connexins can further modify these properties. This diversity of connexins in heart could then confer subtle (and possibly regional) regulation of electrical conductance or could reflect specializations for additional roles such as metabolic coupling or second messenger transmission. We propose to analyze the nature of the specialized properties conferred by these different connexins and their potential role in cardiac function. Initially, the distribution of these different junctional proteins will be established using both in situ hybridization with highly specific nucleotide probes and antibodies raised to peptides from the deduced sequences in complementary studies on rat and bovine heart sections. Concurrent analyses using the Xenopus oocyte expression system will be aimed at defining the specific properties of intercellular channels comprised of different connexins. Using paired Xenopus oocytes injected with cRNAs of the connexins of interest, two major groups of experiments are proposed. First, the gating properties of the various connexins in response to voltage, pH and Ca++ will be determined using dual cell voltage clamps. Possible heteromeric interactions between connexins and their effects on channel properties will also be investigated. Relating these results to the in vivo distribution of connexins may indicate the manner in which gap junctions modulate the flow of current in the heart. A second series of studies will investigate the passage of larger molecules through junctions. These studies have significance for second messenger responses in heart. In the laboratory of Dr. David Triggle, we will develop a graded series of probes which will allow the determination of exclusion limits and selectivity of junctions comprised of each of the cardiac connexins and their potential hybrid forms. By modifying these probes with respect to surface charge and hydrophobicity, and incorporating photoactivatable cross-linking groups, the nature of the residues lining the channel which may contribute to specificity will also be studied. Site-directed mutagenesis of channel residues will further refine our understanding of the structure of these channels and the molecular basis of any selectivity filters that may be detected. These studies will define the properties of intercellular communication in the heart at a molecular level, so that the roles of junctional proteins in electrical conductance and as regulators of excitability can be addressed.

在心脏中，缝隙连接起着电耦合的关键作用 肌肉纤维，以确保同步收缩的快速传播。 然而，心脏中多种连接蛋白的鉴定（即， Cx37，40，43，45，46）清楚地表明了更大的复杂性。 我们和其他人 已经证明，细胞内通道包括其中一些 连接蛋白显示出独特的门控特性和电导。 连接蛋白之间的异型相互作用可以进一步修饰这些 特性. 心脏中连接蛋白的多样性 微妙的（可能是区域性的）电导调节， 可以反映其他角色的专业化，例如代谢 耦合或第二信使传输。 我们建议分析 这些不同的特性所赋予的特殊属性的性质 连接蛋白及其在心脏功能中的潜在作用。 最初，这些不同连接蛋白的分布将 使用原位杂交和高度特异性 核苷酸探针和抗体产生的肽从推导的 在大鼠和牛心脏切片的互补研究中， 使用非洲爪蟾卵母细胞表达系统的并行分析将是 旨在确定细胞间通道的具体特性 由不同的连接蛋白组成。 使用成对的非洲爪蟾卵母细胞注射 利用感兴趣的连接蛋白的cRNA，两组主要的实验 被提议。 第一，不同连接蛋白的门控特性， 将使用双电池测定对电压、pH和Ca++的响应 电压箝位。 连接蛋白和连接蛋白之间可能的异聚体相互作用 还将研究它们对沟道特性的影响。 有关 连接蛋白在体内分布的这些结果可能表明 缝隙连接调节心脏电流的方式。 第二个系列的研究将调查通过更大的 分子通过连接。 这些研究对第二次世界大战的爆发具有重要意义。 信使在心中回应。 在大卫特里格博士的实验室里， 我们将开发一个分级系列的探针， 排除限制和组成的连接的选择性的确定 每种心脏连接蛋白及其潜在的杂交形式。 通过 相对于表面电荷修改这些探针， 疏水性，并引入可光活化的交联基团， 可能导致通道内残留物的性质 还将研究特异性。 通道定点突变 残留物将进一步完善我们对这些结构的理解， 通道和任何选择性过滤器的分子基础， 检测到 这些研究将确定细胞间的性质， 在分子水平上的心脏通讯， 连接蛋白在电导和调节 可以解决兴奋性问题。