Physiological function of voltage-dependent Na^+ channel β-subunit as an adhesion molecule

电压依赖性钠离子通道β-亚基含有一个结构类似于细胞黏附分子的胞外Ig样结构域。在本项目中，我们研究了β亚基的表达和细胞内转运的调控机制以及它们在细胞分化中的作用。在培养的牛肾上腺嗜铬细胞中，胰岛素和胰岛素样生长因子-1增加了细胞表面Na~+通道的表达。胰岛素和胰岛素样生长因子-1可增加Na~+通道α亚单位基因水平，但不能增加β亚单位基因水平。在大鼠嗜铬细胞瘤PC 12细胞中，经神经生长因子诱导分化后，β_1亚单位基因表达水平升高，而β_3亚单位基因表达水平无明显变化，提示α亚单位和β亚单位基因表达的调控机制不同。β_2-亚单位-绿色荧光蛋白主要在细胞表面表达，这些细胞延伸出许多微绒毛。β_3-亚基-绿色荧光蛋白在细胞膜和胞内球状膜结构处有较强的表达，这些细胞还延伸出微绒毛，说明β-亚基在细胞内的转运和生理功能不同。此外，即使在没有神经生长因子的情况下，表达绿色荧光蛋白各亚基的细胞也能延长神经炎的发生，且β_2-绿色荧光蛋白或β_3-绿色荧光蛋白的作用强于β_1-绿色荧光蛋白。神经生长因子在各β-亚单位-绿色荧光蛋白表达细胞中的突起延伸作用比在对照细胞中更明显，这说明β-亚单位在细胞分化中起着调节作用，β-亚单位在细胞内的转运机制及其表达调控机制是不同的。

Voltage-dependent Na^+ channel β-subunits contain a single extracellular Ig-like domain with structural similarity to the cell adhesion molecules. In this project, we have studied the regulatory mechanisms of expression and intracellular trafficking of the β-subunits as well as their role in cell differentiation.In cultured bovine adrenal chromaffin cells, insulin and IGF-1 increased cell surface expression of Na^+ channels. Insulin and IGF-1 increased Na^+ channel α-subunit mRNA level but not (β-subunit mRNA level. In rat pheochromocytoma PC 12 cells, neuronal differentiation by nerve growth factor increased β_1-subunit mRNA level but not β_3-subunit mRNA level, indicating that the mechanisms regulating the expression of a-subunit and β-subunits are different.When HEK293 cells were transfected with GFP tagged β-subunit constructs, β_1-Subunit-GFP was expressed strongly at the cell surface and weakly in endoplasmic reticulum. β_2-Subunit-GFP was expressed predominantly at cell surface, and these cells extended many microvilli. β_3-Subunit-GFP was expressed strongly at cell membrane and at intracellular spherical membrane structure, and these cells also extended microvilli, indicating that the intracellular trafficking and the physiological function of the β-subunits are different. In addition, PC12 cells expressing each subunit tagged with GFP extended neuritis even in the absence of nerve growth factor, and the effects of β_2-GFP or β_3-GFP were stronger than that of β_1-GFP. The neurite extending effects of nerve growth factor were more pronounced in each β-subunit-GFP expressing cells than in the control cells.These results indicate that β-subunits play a role in the regulation of cell differentiation, and that the intracellular trafficking mechanisms of the β-subunits and their regulatory mechanisms of expression are different.