Jin Ping, Weiger Thomas M, Wu Yuying, Levitan Irwin B
Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2002 Mar 22;277(12):10014-20. doi: 10.1074/jbc.M107682200. Epub 2002 Jan 14.
The auxiliary beta4 subunit of the human slowpoke calciumdependent potassium (slo) channel is expressed predominantly in the brain. Co-expression of beta4 subunit with the slo channel alpha subunit in HEK293 and Chinese hamster ovary cells slows channel activation and deactivation and also shifts the voltage dependence of the channel to more depolarized potentials. We show here that the functional interaction between the hbeta4 subunit and the slo channel is influenced by the phosphorylation state of hbeta4. Treatment of cells with okadaic acid (OA) reduces the effect of hbeta4 on slo channel activation kinetics and voltage dependence but not on slo channel deactivation kinetics. The effect of OA can be blocked by mutating three putative serine/threonine phosphorylation sites in hbeta4 (Thr-11/Ser-17/Ser-210) to alanines, suggesting that OA potentiates phosphorylation of hbeta4 and thereby suppresses its functional coupling to the slo channel. Mutation of Ser-17 alone to a negatively charged residue (S17E) can mimic the effect of OA. Mutating all three phosphorylation sites in hbeta4 to negatively charged residues (T11D/S17E/S210E) not only suppresses the effect of hbeta4 on slo channel activation kinetics and voltage dependence, it also suppresses its effect on slo channel deactivation kinetics. Co-immunoprecipitation/Western blot experiments indicate that all of these hbeta4 mutants, as well as the wild-type hbeta4, bind to the slo channel. Taken together, these data suggest that phosphorylation of the beta4 subunit dynamically regulates the functional coupling between the beta4 subunit and the pore-forming alpha subunit of the slo channel. In addition, phosphorylation of different residues in hbeta4 differentially influences its effects on slo channel activation kinetics, deactivation kinetics, and voltage dependence.
人类慢波钙依赖性钾离子（slo）通道的辅助β4亚基主要在大脑中表达。在HEK293细胞和中国仓鼠卵巢细胞中，β4亚基与slo通道α亚基共表达会减缓通道的激活和失活，并且还会将通道的电压依赖性转移到更去极化的电位。我们在此表明，hβ4亚基与slo通道之间的功能相互作用受hβ4磷酸化状态的影响。用冈田酸（OA）处理细胞会降低hβ4对slo通道激活动力学和电压依赖性的影响，但不影响slo通道的失活动力学。OA的作用可通过将hβ4中的三个假定的丝氨酸/苏氨酸磷酸化位点（Thr-11/Ser-17/Ser-210）突变为丙氨酸来阻断，这表明OA增强了hβ4的磷酸化，从而抑制了其与slo通道的功能偶联。仅将Ser-17突变为带负电荷的残基（S17E）就可以模拟OA的作用。将hβ4中的所有三个磷酸化位点突变为带负电荷的残基（T11D/S17E/S210E）不仅会抑制hβ4对slo通道激活动力学和电压依赖性的影响，还会抑制其对slo通道失活动力学的影响。免疫共沉淀/蛋白质印迹实验表明，所有这些hβ4突变体以及野生型hβ4都与slo通道结合。综上所述，这些数据表明β4亚基的磷酸化动态调节β4亚基与slo通道的孔形成α亚基之间的功能偶联。此外，hβ4中不同残基的磷酸化对其对slo通道激活动力学、失活动力学和电压依赖性的影响也有所不同。
