Bossuyt Julie, Despa Sanda, Han Fei, Hou Zhanjia, Robia Seth L, Lingrel Jerry B, Bers Donald M
Department of Pharmacology, University of California, Davis, California 95616, USA.
J Biol Chem. 2009 Sep 25;284(39):26749-57. doi: 10.1074/jbc.M109.047357. Epub 2009 Jul 28.
Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-alpha1 or CFP-NKA-alpha2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 +/- 3.4% for NKA-alpha1 and 27.5 +/- 2.5% for NKA-alpha2). Donor fluorescence depended linearly on acceptor fluorescence, indicating a 1:1 PLM:NKA stoichiometry for both isoforms. PLM phosphorylation induced by cAMP-dependent protein kinase and protein kinase C activation drastically reduced the FRET with both NKA isoforms. However, submaximal cAMP-dependent protein kinase activation had less effect on PLM-NKA-alpha2 versus PLM-NKA-alpha1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLM-CFP also showed FRET to PLM-YFP, but the relationship during progressive photobleach was highly nonlinear, indicating oligomers involving >or=3 monomers. Using cardiac myocytes from wild-type mice and mice where NKA-alpha1 is ouabain-sensitive and NKA-alpha2 is ouabain-resistant, we assessed the effects of PLM phosphorylation on NKA-alpha1 and NKA-alpha2 function. Isoproterenol enhanced internal Na(+) affinity of both isoforms (K((1/2)) decreased from 18.1 +/- 2.0 to 11.5 +/- 1.9 mm for NKA-alpha1 and from 16.4 +/- 2.5 to 10.4 +/- 1.5 mm for NKA-alpha2) without altering maximum transport rate (V(max)). Protein kinase C activation also decreased K((1/2)) for both NKA-alpha1 and NKA-alpha2 (to 9.4 +/- 1.0 and 9.1 +/- 1.1 mm, respectively) but increased V(max) only for NKA-alpha2 (1.9 +/- 0.4 versus 1.2 +/- 0.5 mm/min). In conclusion, PLM associates with and modulates both NKA-alpha1 and NKA-alpha2 in a comparable but not identical manner.
在心脏肾上腺素能刺激过程中，磷膜蛋白（PLM）磷酸化介导钠钾-ATP酶（NKA）功能增强。存在多种NKA亚型，其功能/调节可能有所不同。我们结合荧光共振能量转移（FRET）和功能测量来研究NKA与PLM相互作用的亚型特异性。在HEK-293细胞中，随着受体（PLM-YFP）逐步光漂白，FRET通过供体荧光（CFP-NKA-α1或CFP-NKA-α2）的增加来测量。这两对都表现出强烈的FRET（NKA-α1的最大值为23.6±3.4%，NKA-α2的最大值为27.5±2.5%）。供体荧光与受体荧光呈线性相关，表明两种亚型的PLM:NKA化学计量比均为1:1。由环磷酸腺苷（cAMP）依赖性蛋白激酶和蛋白激酶C激活诱导的PLM磷酸化显著降低了与两种NKA亚型的FRET。然而，亚最大程度的cAMP依赖性蛋白激酶激活对PLM-NKA-α2的影响比对PLM-NKA-α1的影响小。令人惊讶的是，哇巴因几乎消除了NKA-PLM FRET，但仅部分降低了共免疫沉淀。PLM-CFP与PLM-YFP也显示出FRET，但在逐步光漂白过程中的关系高度非线性，表明涉及≥3个单体的寡聚体。使用来自野生型小鼠和NKA-α1对哇巴因敏感而NKA-α2对哇巴因耐药的小鼠的心肌细胞，我们评估了PLM磷酸化对NKA-α1和NKA-α2功能的影响。异丙肾上腺素增强了两种亚型的细胞内钠亲和力（NKA-α1的K（1/2）从18.1±2.0 mM降至11.5±1.9 mM，NKA-α2的K（1/2）从16.4±2.5 mM降至10.4±1.5 mM），而不改变最大转运速率（V（max））。蛋白激酶C激活也降低了NKA-α1和NKA-α2的K（1/2）（分别降至9.4±1.0 mM和9.1±1.1 mM），但仅增加了NKA-α2的V（max）（1.9±0.4与1.2±0.5 mM/min）。总之，PLM以可比但不完全相同的方式与NKA-α1和NKA-α2结合并调节它们。
