The rat brain sodium (Na+) channel is composed of glycoprotein subunits, the large alpha subunit and the auxiliary beta1 and beta2. Although the alpha subunit is sufficient for the expression of functional Na+ channels, co-expression of the beta1 subunit with an alpha subunit isoform in a Xenopus oocyte expression system results in a acceleration in channel inactivation, a shift in the voltage dependance of steady state inactivation to more negative membrane potentials, and an increase in the amplitude of peak sodium current. Evidence from our laboratory and other strongly suggest that the extracellular NH2-terminal domain of the beta1 subunit is necessary for the modulation of alpha subunit function. The initial goal of this proposal is to localize regions of the beta1 polypeptide which interact with the alpha subunit and cause the observed changes in Na+ electrical activity. We propose an alanine-scanning mutagenesis survey of the beta extracellular domain to identify potentially important regions, followed by more detailed site-directed mutagenesis experiments in order to accomplish this goal. The ultimate goal of this study is to characterize the sites of interaction between the alpha and beta subunits. Regions of the alpha and beta1 subunits which participate in intersubunit interaction will be identified in vitro by gene fusion, immunochemical, and mutagenesis technique. The results of this study will generate a functional map of the extracellular domain of the beta subunit of the rat brain Na+ channel and will provide further insight into the mechanisms of channel gating.
