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.
