Plasticity of Electrical Synapses

描述（由申请人提供）：本提案的目的是研究间隙连接介导的电突触在听觉系统中的作用和特性。在金鱼毛纳细胞上以大型混合（电和化学）突触终端结束的听觉传入是这些研究的理想选择，因为与哺乳动物的电突触不同，实验的可及性使得量化在不同生理条件下发生的连接电导的体内变化成为可能，并将它们与解剖、超微结构和分子分析相关联。引人注目的是，这些模型电突触的电导受到神经元活动的精细调节控制。电传递是由连接蛋白35 （Cx35）介导的，它是哺乳动物连接蛋白36 （Cx36）的鱼类同源物，存在于听觉系统中，这表明哺乳动物的听觉电突触可能受到类似的调节。本建议通过关注缝隙连接通道的调节贩运的作用，来理解这些终端连接电导双向控制的分子机制。目的1是通过结合超微结构和药理学方法，研究体内天然电突触中间隙连接通道运输的存在。我们的初步结果表明，间隙连接通道的活跃周转存在，这构成了天然突触中这种现象的第一个证据。目标2是确定受管制的贩运对活动依赖性电传输增强的贡献。它将测试是否需要胞吐机制来表达增强，以及是否需要与调节激酶CaM-KII和支架蛋白ZO-1直接相互作用。相反，目标3将调查受管制的贩运对活动依赖的电力传输抑制的可能贡献。使用类似的方法，我们将研究是否需要胞吞作用的机制来抑制活动依赖性的连接电导，以及直接的蛋白质-蛋白质相互作用的潜在作用。此外，CaM-KII和ZO-1通过Cx35和Cx36的保守区域的直接相互作用表明，其功能可能是电传递的基本和广泛特性的基础，也与哺乳动物的电突触有关。因此，提出的研究解决了一个新概念，即电突触的强度是通过动态调节间隙连接通道的运输来实现的。由于电突触已被证明促进协调的神经元活动，这种调节的功能障碍可能具有深刻的病理意义，有助于听力障碍，癫痫和认知（精神）和发育障碍

DESCRIPTION (provided by applicant): The goal of this proposal is to investigate the role and properties of gap junction-mediated electrical synapses in the auditory system. Auditory afferents terminating as large mixed (electrical and chemical) synaptic terminals on the goldfish Mauthner cell are ideally suited for these studies since unlike mammalian electrical synapses, the experimental accessibility makes it possible to quantify in vivo changes in junctional conductance that occur under different physiological conditions and to correlate them with anatomical, ultrastructural and molecular analysis. Strikingly, the conductance of these model electrical synapses is under the fine regulatory control of neuronal activity. Electrical transmission is mediated by connexin 35 (Cx35), the fish ortholog of the mammalian connexin 36 (Cx36) which is present in the auditory system, suggesting that mammalian auditory electrical synapses could be similarly regulated. This proposal deals on understanding the molecular mechanisms underlying the bi-directional control of junctional conductance at these terminals by focusing in the role of regulated trafficking of gap junction channels. Aim 1 is to investigate the existence of trafficking of gap junction channels at native electrical synapses, in vivo, by combining ultrastructural and pharmacological approaches. Our preliminary results suggest the existence of an active turnover of gap junction channels, which constitute the first evidence of this phenomenon in a native synapse. Aim 2 is to determine the contribution of regulated trafficking to activity-dependent potentiation of electrical transmission. It will test if mechanisms of exocytosis are required for the expression of the potentiation and if it requires of direct interactions with the regulatory kinase CaM-KII and the scaffold protein ZO-1. Conversely, Aim 3 will investigate the possible contribution of regulated trafficking to activity-dependent depression of electrical transmission. Using similar approaches, we will investigate if mechanisms of endocytosis are required for activity-dependent depression of junctional conductance, as well the potential roles of direct protein-protein interactions. Furthermore, the direct interactions of CaM-KII and ZO-1 through conserved regions of both Cx35 and Cx36 suggests that its function might underlie a fundamental and widespread property of electrical transmission, also relevant to mammalian electrical synapses. Thus, the proposed research addresses the novel concept that the strength of electrical synapses is achieved by dynamically regulating the trafficking of gap junction channels. Because electrical synapses have been shown to promote coordinated neuronal activity, dysfunction of this regulation could have profound pathological implications, contributing to auditory impairment, epilepsy and cognitive (psychiatric) and developmental disorders PUBLIC HEALTH RELEVANCE: The proposal explores the molecular mechanisms involved in the regulation of gap junction-mediated electrical synapses. Further, the proposal explores the role of regulated trafficking of gap junction channels as their underlying mechanism. This process has been shown involved in the regulation of chemical synapses, but no evidence suggests its participation in electrical synapses. Thus, the proposal investigates the possibility that both chemical and electrical synapses share common regulatory mechanisms. Because electrical synapses have been shown to promote coordinated neuronal activity, dysfunction of their regulation could have profound pathological implications, contributing to epilepsy and to cognitive (psychiatric) and developmental disorders.