Transfer of Metabolites through Lens Gap Junctions

描述(申请人提供)：晶状体细胞需要缝隙连接蛋白来相互沟通和正常发育。特别是，Cx43(Cx43)、Cx46和Cx5O是正常晶状体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会导致老鼠和人类的白内障。Cx43、Cx46和Cx5O形成具有不同渗透特性的通道。我们假设晶状体发育所需的跨连接分子通过晶状体缝隙连接，而白内障可能是由于细胞无法分享这些信号所致。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。这一新的研究计划将引入一种特别创新的技术方法来研究缝隙连接在晶状体中的作用。 在具体目标1中，我们将确定单个通道的大小、总电导和哺乳动物细胞表达的功能性缝隙连接通道的数量，这些连接蛋白在晶体中表达。这将通过标准的双电压钳位全电池记录方法来完成。 在特定的目标2中，我们将识别、定量和比较内源性代谢物通过晶状体中表达的连接蛋白形成的通道的转移。这将通过一种新的策略来实现，以识别和量化在给定时间框架内通过细胞间缝隙连接的内源分子的转移。将在几个时间点进行测量，以测量和比较特定代谢物通过由Cx43、Cx46和Cx50形成的缝隙连接通道的转移率。这些结果将与从目标1获得的数据相结合，以计算这些缝隙连接对每个通道代谢物的渗透选择性。我们假设晶状体连接蛋白形成了对特定代谢物具有选择性通透性的通道，而且这种连接蛋白的通透性不能仅仅根据外在物质的大小和电荷来预测。 我们将开始确定导致晶状体发育、透明度和疾病的渗透性特征和实际的跨连接分子。这项工作应该有助于更好地理解，并最终治疗由异常缝隙连接通讯引起的眼部疾病。

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.