MOLECULAR ANALYSIS OF NA+ CHANNEL BETA1 SUBUNIT FUNCTION

大鼠脑钠（Na+）通道由糖蛋白亚基组成， 大的α亚基和辅助的β 1和β 2。虽然 α亚基足以表达功能性Na+通道， β 1亚基与α亚基同种型在细胞中的共表达 非洲爪蟾卵母细胞表达系统导致通道加速 失活，稳态电压依赖性的转变 失活到更负的膜电位，以及增加 峰值钠电流的幅度。我们的实验室和其他 强烈表明β 1蛋白的胞外NH 2-末端结构域 亚基是调节α亚基功能所必需的。的 该提议的最初目标是定位β 1的区域， 与α亚基相互作用并引起观察到的 Na+电活动的变化。我们建议进行丙氨酸扫描 β细胞外结构域的诱变调查，以确定 潜在的重要区域，然后是更详细的现场指导 为了实现这一目标，进行了诱变实验。最终 本研究的目的是描述 α和β亚基。α和β 1亚基的区域， 参与亚基间相互作用将在体外鉴定， 基因融合、免疫化学和诱变技术。的结果 这项研究将产生一个细胞外结构域的功能图， 大鼠大脑Na+通道的β亚基，并将提供进一步的 深入了解通道门控的机制。

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.