Cheung Joseph Y, Rothblum Lawrence I, Moorman J Randall, Tucker Amy L, Song Jianliang, Ahlers Belinda A, Carl Lois L, Wang JuFang, Zhang Xue-Qian
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Ann N Y Acad Sci. 2007 Mar;1099:119-34. doi: 10.1196/annals.1387.004.
Phospholemman (PLM) is the first sequenced member of the FXYD family of regulators of ion transport. The mature protein has 72 amino acids and consists of an extracellular N terminus containing the signature FXYD motif, a single transmembrane (TM) domain, and a cytoplasmic C-terminal domain containing four potential sites for phosphorylation. PLM and other members of the FXYD family are known to regulate Na+-K+-ATPase. Using adenovirus-mediated gene transfer into adult rat cardiac myocytes, we showed that changes in contractility and intracellular Ca2+ homeostasis associated with PLM overexpression or downregulation are not consistent with the effects expected from inhibition of Na+-K+-ATPase by PLM. Additional studies with heterologous expression of PLM and cardiac Na+/Ca2+ exchanger 1 (NCX1) in HEK293 cells and cardiac myocytes isolated from PLM-deficient mice demonstrated by co-localization, co-immunoprecipitation, and electrophysiological and radioactive tracer uptake techniques that PLM associates with NCX1 in the sarcolemma and transverse tubules and that PLM inhibits NCX1, independent of its effects on Na+-K+-ATPase. Mutational analysis indicates that the cytoplasmic domain of PLM is required for its regulation of NCX1. In addition, experiments using phosphomimetic and phospho-deficient PLM mutants, as well as activators of protein kinases A and C, indicate that PLM phosphorylated at serine68 is the active form that inhibits NCX1. This is in sharp contrast to the finding that the unphosphorylated PLM form inhibits Na+-K+-ATPase. We conclude that PLM regulates cardiac contractility by modulating the activities of NCX and Na+-K+-ATPase.
磷膜蛋白（PLM）是离子转运调节因子FXYD家族中第一个被测序的成员。成熟蛋白有72个氨基酸，由一个含有标志性FXYD基序的细胞外N端、一个单一跨膜（TM）结构域和一个含有四个潜在磷酸化位点的细胞质C端结构域组成。已知PLM和FXYD家族的其他成员可调节钠钾ATP酶。通过腺病毒介导的基因转移到成年大鼠心肌细胞中，我们发现与PLM过表达或下调相关的收缩性和细胞内钙稳态变化与PLM抑制钠钾ATP酶所预期的效应不一致。在HEK293细胞和从PLM缺陷小鼠分离的心肌细胞中对PLM和心脏钠钙交换蛋白1（NCX1）进行异源表达的进一步研究，通过共定位、免疫共沉淀、电生理和放射性示踪剂摄取技术表明，PLM在肌膜和横管中与NCX1结合，并且PLM抑制NCX1，这与其对钠钾ATP酶的作用无关。突变分析表明，PLM的细胞质结构域是其调节NCX1所必需的。此外，使用模拟磷酸化和磷酸化缺陷的PLM突变体以及蛋白激酶A和C的激活剂进行的实验表明，在丝氨酸68处磷酸化的PLM是抑制NCX1的活性形式。这与未磷酸化的PLM形式抑制钠钾ATP酶的发现形成鲜明对比。我们得出结论，PLM通过调节NCX和钠钾ATP酶的活性来调节心脏收缩性。
