Kwak Y G, Hu N, Wei J, George A L, Grobaski T D, Tamkun M M, Murray K T
Department of Physiology and Biochemistry and Molecular Biology, Colorado State University, Ft. Collins, Colorado 80523, USA.
J Biol Chem. 1999 May 14;274(20):13928-32. doi: 10.1074/jbc.274.20.13928.
The human Kv1.5 potassium channel forms the IKur current in atrial myocytes and is functionally altered by coexpression with Kvbeta subunits. To explore the role of protein kinase A (PKA) phosphorylation in beta-subunit function, we examined the effect of PKA stimulation on Kv1.5 current following coexpression with either Kvbeta1.2 or Kvbeta1.3, both of which coassemble with Kv1.5 and induce fast inactivation. In Xenopus oocytes expressing Kv1.5 and Kvbeta1.3, activation of PKA reduced macroscopic inactivation with an increase in K+ current. Similar results were obtained using HEK 293 cells which lack endogenous K+ channel subunits. These effects did not occur when Kv1.5 was coexpressed with either Kvbeta1.2 or Kvbeta1.3 lacking the amino terminus, suggesting involvement of this region of Kvbeta1.3. Removal of a consensus PKA phosphorylation site on the Kvbeta1.3 NH2 terminus (serine 24), but not alternative sites in either Kvbeta1.3 or Kv1.5, resulted in loss of the functional effects of kinase activation. The effects of phosphorylation appeared to be electrostatic, as replacement of serine 24 with a negatively charged amino acid reduced beta-mediated inactivation, while substitution with a positively charged residue enhanced it. These results indicate that Kvbeta1.3-induced inactivation is reduced by PKA activation, and that phosphorylation of serine 24 in the subunit NH2 terminus is responsible.
人类Kv1.5钾通道在心房肌细胞中形成IKur电流,并且与Kvβ亚基共表达时其功能会发生改变。为了探究蛋白激酶A(PKA)磷酸化在β亚基功能中的作用,我们检测了PKA刺激对与Kvβ1.2或Kvβ1.3共表达后的Kv1.5电流的影响,这两种亚基均能与Kv1.5共同组装并诱导快速失活。在表达Kv1.5和Kvβ1.3的非洲爪蟾卵母细胞中,PKA的激活减少了宏观失活并增加了钾电流。使用缺乏内源性钾通道亚基的HEK 293细胞也获得了类似结果。当Kv1.5与缺乏氨基末端的Kvβ1.2或Kvβ1.3共表达时,这些效应并未出现,提示Kvβ1.3的该区域参与其中。去除Kvβ1.3氨基末端的一个一致性PKA磷酸化位点(丝氨酸24),而非Kvβ1.3或Kv1.5中的其他位点,导致激酶激活的功能效应丧失。磷酸化的效应似乎是静电性的,因为用带负电荷的氨基酸取代丝氨酸24会减少β介导的失活,而用带正电荷的残基取代则会增强失活。这些结果表明,PKA激活可减少Kvβ1.3诱导的失活,并且亚基氨基末端丝氨酸24的磷酸化是其原因。