Structure, Function and Regulation of Renal K Channels

我们的实验室专注于肾钾通道的研究，这是一组不同的完整的膜蛋白，促进钾在细胞膜上的被动移动(由K的电化学梯度驱动)。这项工作导致了两个新的K通道基因的发现，KCNA10(振荡器相关的电压门控K通道)，KCNK1(两孔通道)，并导致电压门控振荡器K通道KCNA3的功能增益突变。KCNA10编码一种电压门控的、环核苷酸调节的K-选择性通道，在肾脏、心脏和血管中表达。我们的主要目标是确定其分子结构并确定其在肾脏中的作用。初步研究表明，KCNA10是一个异多聚体复合体，鉴定了三种与KCNA10相互作用并改变其动力学性质和表达水平的蛋白质。我们建议研究这些相互作用背后的分子机制。此外，我们计划确定我们最近发现的KCNA10剪接异构体的动力学性质和肾脏表达。用兔抗KCNA10多克隆抗体进行免疫细胞化学染色，检测到KCNA10蛋白位于大鼠近端小管的管腔膜上。Western blotting也证实了KCNA10蛋白在大鼠皮质膜上的表达。在此基础上，我们假设KCNA10是一种调节近端肾小管膜电位的异构体、电压门控型尖端钾通道。因此，我们建议使用膜片钳证实KCNA10在近端小管的顶膜上的表达。KCNA10基因敲除的小鼠模型将被建立并充分鉴定，并特别关注其在近端小管功能中的作用。这些研究应该提供关于KCNA10的分子结构及其在肾脏溶质动态平衡中的作用的重要信息。

Our laboratory is focused on the study of renal potassium channels, a diverse group of integral membrane proteins that facilitate the passive (driven by the electrochemical gradient for K) movement of K across the cell membranes. This work has led to the discovery of two novel K channel genes, KCNA10 (Shaker-related, voltage-gated K channel), KCNK1 (a two-pore channel) and to a gain-of-function mutation in the voltage-gated Shaker K channel, KCNA3. KCNA10, the subject of the present proposal, encodes a voltage-gated, cyclic nucleotide regulated, K-selective channel expressed in kidney, heart and blood vessels. Our main goals are to determine its molecular structure and establish its role in kidney. Preliminary studies suggest that KCNA10 is a heteromultimeric complex as evidenced by the identification of three proteins that interact with KCNA10 and modify its kinetic properties and expression level. We propose to investigate the molecular mechanisms underlying these interactions. In addition, we plan to determine the kinetic properties and renal expression of a KCNA10 splice isoform we recently identified. KCNA10 protein was detected at the luminal membrane of rat proximal tubule by immunocytochemistry using a polyclonal antibody generated in rabbit. Western blotting also confirmed KCNA10 protein expression in rat cortical membrane. Based on the foregoing we hypothesize KCNA10 is a heteromultimeric, voltage-gated, apical K channel that modulates proximal tubular membrane potential. We therefore, propose to confirm the expression of KCNA10 at the apical membrane of proximal tubules using patch clamp.A knockout mouse model for KCNA10 will be generated and characterized fully, with particular attention paid to its role on proximal tubular function. These studies should provide important information regarding the molecular structure of KCNA10 and its role in renal solute homeostasis.