Structure and Function of Na+ Channel B Subunits in Adrenal Chromaffin Cells

电压敏感钠通道负责可兴奋细胞的动作电位上升阶段，包括中枢和外周神经元、心脏、骨骼肌和神经内分泌细胞。脑内钠通道是由α、β -1和β -2亚基组成的异三聚体复合物，而心脏和骨骼肌内钠通道是由α和β -1样亚基组成的异二聚体。神经内分泌细胞中钠离子通道的结构尚未确定。该研究员之前的工作已经导致了大鼠脑钠通道β -1亚基的分子克隆和功能表达。克隆的β -1亚基与α亚基在非洲爪蟾卵母细胞中的共表达导致峰值电流增加，失活加速，以及失活的电压依赖性向负膜电位的转变。假设可兴奋细胞中钠离子通道的表达水平和功能特性不仅取决于α亚基的表达和特性，还取决于β亚基的表达和功能相互作用。这些研究的目的是通过与已知α和β亚基的杂交鉴定在肾上腺髓质染色质细胞中表达的钠通道α和β亚基，通过分子克隆确定染色质细胞β亚基的结构，评估β亚基在染色质细胞钠通道功能表达中的作用。包括刺激-分泌耦合。可采用的方法包括：1)染色质细胞钠通道β亚基的分子克隆，2)通过杂交研究鉴定染色质细胞钠通道α亚基，3)通过反义RNA或反义寡核苷酸在体外和通过基因破坏实验在体内阻断染色质细胞钠通道β亚基的表达，4)体外转染和体内转基因表达对嗜铬细胞钠通道β亚基的过表达。电压敏感钠通道是可兴奋细胞响应和传递信号机制的重要组成部分。可兴奋细胞的例子包括神经细胞、肌肉细胞和神经内分泌细胞。钠离子通道由亚基组成。钠通道的亚基组成在不同的细胞类型中是不同的。神经内分泌细胞（如肾上腺髓质的染色质细胞）中钠离子通道的亚基结构尚未确定。这些研究将使用分子生物学技术来鉴定在肾上腺髓质染色质细胞中表达的钠通道亚基，并评估它们在染色质细胞功能中的作用。

Voltage-sensitive sodium channels are responsible for the rising phase of the action potential in excitable cells, including central and peripheral neurons, heart, skeletal muscle and neuroendocrine cells. Sodium channels in brain are heterotrimeric complexes composed of alpha, beta-1 and beta-2 subunits, while sodium channels in heart and skeletal muscle are heterodimeric, composed of alpha and beta-1-like subunits. The structure of sodium c channels in neuroendocrine cells has not been identified. Previous work by this investigator has resulted in the molecular cloning and functional expression of the beta-1 subunit of the rat brain sodium channel. Co-expression of cloned beta-1 subunits with alpha subunits in Xenopus oocytes results in increased peak current, acceleration of inactivation, and a shift in the voltage dependence of inactivation to more negative membrane potentials. It is hypothesized that the level of expression and functional properties of sodium channels in excitable cells depends not only on the expression and properties of alpha subunits but also on the expression and functional interaction with beta subunits. The goal of these studies is to identify the sodium channel alpha and beta subunits that are expressed in adrenal medullary chromaffin cells by hybridization to known alpha and beta subunits, determine the structure of chromaffin cell beta subunits by molecular cloning and assess the role of beta subunits in chromaffin cell sodium channel functional expression, including stimulus-secretion coupling . Methods to be employed include: 1) molecular cloning of chromaffin cell sodium channel beta subunits, 2) identification of chromaffin cell sodium channel alpha subunits by hybridization studies, 3) blockade of chromaffin cell sodium channel beta subunit expression in vitro through the use of antisense RNA or antisense oligonucleotides and in vivo through the use of gene disruption experiments, and 4) overexpression of chromaffin cell sodium channel beta subunits by transfection in vitro and by transgenic expression in vivo. %%% Voltage-sensitive sodium channels are an important component of the mechanism by which excitable cells respond to and transmit signals. Examples of excitable cells include nerve cells, muscle cells and neuroendocrine cells. Sodium channels are composed of subunits. The subunit composition of sodium channels differs in different cell types. The subunit structure of sodium channels in neuroendocrine cells, such as chromaffin cells of the adrenal medulla, has not been determined. These studies will use techniques of molecular biology to identify the sodium channel subunits that are expressed in adrenal medullary chromaffin cells and assess their role in chromaffin cell function.