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β1和β2。尽管 α亚基足以表达功能性Na+通道， β1亚基与α亚基异构体在大肠杆菌中的共表达 非洲爪哇卵母细胞表达系统导致通道加速 失活，稳态电压依赖关系的变化 失活到更负的膜电位，并增加 峰值钠电流的幅度。我们实验室和其他机构提供的证据 强烈提示Beta1的胞外NH2-末端结构域 亚基是调节α亚基功能所必需的。这个 这项提议的最初目标是将Beta1的区域本地化 与α亚基相互作用的多肽，并导致观察到的 Na+电活动的变化。我们建议进行丙氨酸扫描 β胞外区的诱变检测以鉴定 潜在的重要区域，然后是更详细的站点定向 为了实现这一目标，进行了诱变实验。终极的 这项研究的目标是描述两个人之间相互作用的地点 α亚基和β亚基。α和β1亚基的区域 参与亚基间相互作用将在体外被鉴定为 基因融合、免疫化学和诱变技术。结果是 这项研究将产生一个胞外结构域的功能图谱 大鼠脑Na+通道的β亚基，并将进一步提供 深入了解通道选通的机制。

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.