Jin Ping, Weiger Thomas M, Levitan Irwin B
Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
J Biol Chem. 2002 Nov 15;277(46):43724-9. doi: 10.1074/jbc.M205795200. Epub 2002 Sep 9.
Large conductance Ca(2+)-dependent potassium (K(Ca) or maxi K) channels are composed of a pore-forming alpha subunit and an auxiliary beta subunit. We have shown that the brain-specific beta4 subunit modulates the voltage dependence, activation kinetics, and toxin sensitivity of the hSlo channel (Weiger, T. M., Holmqvist, M. H., Levitan, I. B., Clark, F. T., Sprague, S., Huang, W. J., Ge, P., Wang, C., Lawson, D., Jurman, M. E., Glucksmann, M. A., Silos-Santiago, I., DiStefano, P. S., and Curtis, R. (2000) J. Neurosci. 20, 3563-3570). We investigated here the N-linked glycosylation of the beta4 subunit and its effect on the modulation of the hSlo alpha subunit. When expressed alone in HEK293 cells, the beta4 subunit runs as a single molecular weight band on an SDS gel. However, when coexpressed with the hSlo alpha subunit, the beta4 subunit appears as two different molecular weight bands. Enzymatic deglycosylation or mutation of the N-linked glycosylation residues in beta4 converts it to a single lower molecular weight band, even in the presence of the hSlo alpha subunit, suggesting that the beta4 subunit can be present as an immature, core glycosylated form and a mature, highly glycosylated form. Blockage of protein transport from the endoplasmic reticulum to the Golgi compartment with brefeldin A abolishes the mature, highly glycosylated beta4 band. Glycosylation of the beta4 subunit is not required for its binding to the hSlo channel alpha subunit. It also is not necessary for cell membrane targeting of the beta4 subunit, as demonstrated by surface biotinylation experiments. However, the double glycosylation site mutant beta4 (beta4 N53A/N90A) protects the channel less against toxin blockade, as compared with the hSlo channel coexpressed with wild type beta4 subunit. Taken together, these data show that the pore-forming alpha subunit of the hSlo channel promotes N-linked glycosylation of its auxiliary beta4 subunit, and this in turn influences the modulation of the channel by the beta4 subunit.
大电导钙依赖性钾通道（K(Ca)或大电导钾通道）由一个形成孔道的α亚基和一个辅助性β亚基组成。我们已经表明，脑特异性β4亚基可调节hSlo通道的电压依赖性、激活动力学和毒素敏感性（韦格，T.M.，霍尔姆奎斯特，M.H.，莱维坦，I.B.，克拉克，F.T.，斯普拉格，S.，黄，W.J.，葛，P.，王，C.，劳森，D.，朱尔曼，M.E.，格鲁克斯曼，M.A.，西洛斯 - 圣地亚哥，I.，迪斯泰法诺，P.S.，和柯蒂斯，R.（2000年）《神经科学杂志》20，3563 - 3570）。我们在此研究了β4亚基的N - 糖基化及其对hSloα亚基调节作用的影响。当单独在HEK293细胞中表达时，β4亚基在SDS凝胶上呈现为单一分子量条带。然而，当与hSloα亚基共表达时，β4亚基呈现为两条不同分子量的条带。对β4中N - 糖基化残基进行酶促去糖基化或突变，即使在存在hSloα亚基的情况下，也会将其转化为单一的较低分子量条带，这表明β4亚基可以以未成熟的核心糖基化形式和成熟的高度糖基化形式存在。用布雷菲德菌素 A 阻断从内质网到高尔基体区室的蛋白质转运可消除成熟的、高度糖基化的β4条带。β4亚基的糖基化对于其与hSlo通道α亚基的结合并非必需。如表面生物素化实验所示，对于β4亚基靶向细胞膜也不是必需的。然而，与野生型β4亚基共表达的hSlo通道相比，双糖基化位点突变体β4（β4 N53A/N90A）对通道的毒素阻断保护作用较小。综上所述，这些数据表明，hSlo通道的形成孔道的α亚基促进其辅助性β4亚基的N - 糖基化，而这反过来又影响β4亚基对通道的调节作用。
