Transfer of Metabolites through Lens Gap Junctions

描述（由申请方提供）：透镜细胞需要间隙连接蛋白相互沟通并正常发育。特别是，连接蛋白43（Cx43）、Cx46和Cx50是正常透镜发育和功能所必需的。 编码这些连接蛋白的基因的敲除或突变分别在小鼠和人类中引起白内障。Cx43、Cx46和Cx5O形成具有不同渗透性特征的通道。我们假设透镜发育所需的跨连接分子通过透镜间隙连接，白内障可能是由于细胞无法共享这些信号。我们建议识别和比较通过这些通道的内源性代谢物的转移。这项新的研究计划将引入一种特别创新的技术方法来研究间隙连接在透镜中的作用。 在具体目标1中，我们将确定单个通道的大小，总电导，和表达的哺乳动物细胞表达的功能性间隙连接通道的数量与连接蛋白表达的透镜。这将通过标准双电压钳全细胞记录方法完成。 在具体目标2中，我们将鉴定、定量和比较内源性代谢物的转移，这些代谢物通过透镜中表达的连接蛋白形成的通道。这将通过一种新的策略来实现，以识别和定量在给定的时间范围内通过细胞之间的间隙连接的内源性分子的转移。将在几个时间点进行测量，以测量和比较特定代谢物通过Cx43、Cx46和Cx5O形成的间隙连接通道的转移速率。这些结果将与目标1中获得的数据相结合，以计算每个通道上这些间隙连接对代谢物的选择透过性。我们假设，透镜连接蛋白形成通道，显示选择性渗透特定的代谢物，这种连接蛋白渗透选择性不能预测的基础上的大小和电荷的渗透。 我们将开始鉴定渗透性特征和负责透镜发育、透明度和疾病的实际跨接分子。这项工作应该导致更好的理解，并最终，治疗眼睛疾病，导致异常缝隙连接通讯。

DESCRIPTION (provided by applicant): Lens cells require gap junction proteins to communicate with each other and develop properly. In particular, connexin43 (Cx43), Cx46, and Cx5O are needed for normal lens development and function. Knockout or mutations of genes encoding these connexins cause cataracts in mice and humans, respectively. Cx43, Cx46, and Cx5O form channels with different permeability characteristics. We hypothesize that transjunctional molecules needed for lens development pass through lens gap junctions, and that cataracts can result from an inability of cells to share these signals. We propose to identify and compare the transfer of endogenous metabolites that pass through these channels. This new research program will introduce a particularly innovative technological approach to investigate the role of gap junctions in the lens. In Specific Aim 1 we will determine the single channel size, total conductance, and number of functional gap junction channels expressed by mammalian cells transfected with connexins that are expressed in the lens. This will be done by standard dual voltage-clamp whole-cell recording methods. In Specific Aim 2 we will identify, quantitate, and compare the transfer of endogenous metabolites that pass through channels formed by connexins expressed in the lens. This will be accomplished with a novel strategy to identify and quantitate the transfer of endogenous molecules that pass through gap junctions between cells within a given time frame. Measurement will be taken at several time points to measure and compare the rates of transfer of specific metabolites through gap junction channels formed by Cx43, Cx46, and Cx5O. These results will be combined with data obtained from Aim 1 to calculate the permselectivity of these gap junctions to metabolites on a per channel basis. We hypothesize that lens connexins form channels that display selective permeabilities to specific metabolites, and that this connexin permselectivity can not be predicted solely on the basis of size and charge of the permeant. We will begin to identify the permeability characteristics and actual transjunctional molecules that are responsible for lens development, transparency, and disease. This work should lead to a greater understanding, and, ultimately, treatments, of eye diseases that result from aberrant gap junctional communication.