SELECTIVE PERMEABILITY & PORE STRUCTURE OF GAP JUNCTION

选择通透性

• 批准号: 2023999
• 负责人: BRUCE J NICHOLSON
• 金额: $ 19.71万
• 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/2023999
• 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.

从本质上讲，缝隙连接起着电耦合的关键作用。 肌肉纤维，以确保快速传播的同步收缩。 然而，心脏中多个连接蛋白的鉴定(即， Cx37、40、43、45、46)清楚地表明了更大的复杂性。我们和其他人 已经证明了细胞内的通道由其中一些组成 连接蛋白具有独特的门控特性和导电性。 连接蛋白之间的异型相互作用可以进一步改变这些 属性。心脏中这种连接蛋白的多样性可以赋予 微妙的(可能是区域性的)电导调节或 可以反映其他角色的专业化认证，例如代谢 联轴器或第二信使传输。我们建议分析 这些不同的属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，这些不同的连接蛋白的分布将 通过原位杂交和高度特异的 核苷酸探针和抗体从推导出的多肽 大鼠和牛心脏切片的互补研究中的序列。 使用非洲爪哇卵母细胞表达系统的并行分析将是 旨在定义细胞间信道的特定属性 由不同的连接蛋白组成。利用非洲爪哇卵母细胞配对注射 对于感兴趣的连接蛋白的CRNAs，两组主要的实验 都被提出了。首先，不同连接蛋白的门控特性 将使用双电池来确定对电压、pH和Ca++的响应 电压钳。连接蛋白和连接蛋白之间可能的异构体相互作用 还将研究它们对通道特性的影响。关联 这些对连接蛋白在体内分布的结果可能表明 缝隙连接调节心脏中电流流动的方式。 第二系列研究将调查更大的 分子通过连接点。这些研究对第二次开发具有重要意义。 信使在心里回应。在大卫·特里格尔博士的实验室里， 我们将开发一系列分级探测器，使 确定包括的结点的排除限度和选择性 每一种心脏连接蛋白及其潜在的杂交形式。通过 根据表面电荷和表面电荷修饰这些探针 疏水性，并加入可光激活的交联基， 排列在通道内的残留物的性质可能有助于 还将研究其特殊性。通道的定点突变 残基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。这些研究将定义细胞间的特性 心脏在分子水平上的沟通，因此 电导中的连接蛋白及其作为细胞外信号调节因子的作用 兴奋性是可以解决的。