Song Jianliang, Zhang Xue-Qian, Ahlers Belinda A, Carl Lois L, Wang JuFang, Rothblum Lawrence I, Stahl Richard C, Mounsey J Paul, Tucker Amy L, Moorman J Randall, Cheung Joseph Y
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033, USA.
Am J Physiol Heart Circ Physiol. 2005 May;288(5):H2342-54. doi: 10.1152/ajpheart.01133.2004. Epub 2005 Jan 14.
Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis and inhibited Na+/Ca2+ exchanger (NCX1). In addition, PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [Ca2+]i transients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)-K(+)-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser62, Ser63, Ser68, and Thr69) were deleted, a partial COOH-terminal deletion mutant in which Ser68 and Thr69 were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser68 or Thr69 in mediating PLM's effect on cardiac contractility. Focusing on Ser68, the Ser68 to Glu mutant was fully effective, the Ser63 to Ala (leaving Ser68 intact) mutant was partially effective, and the Ser68 to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca2+]i transients, and NCX1 currents. Both the Ser63 to Ala and Ser68 to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser68 in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca2+]i transients, and NCX1 activity by PLM is critically dependent on Ser68. We suggest that PLM phosphorylation at Ser68 may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility.
在正常成年大鼠心肌细胞中，磷肌酸蛋白（PLM）的过表达改变了收缩功能和胞质钙离子浓度（[Ca2+]i）稳态，并抑制了钠钙交换体（NCX1）。此外，PLM在心肌细胞裂解物中与NCX1进行了共免疫沉淀并共定位。在本研究中，我们评估了PLM的胞质结构域在介导其对收缩性、[Ca2+]i瞬变和NCX1活性的影响方面是否至关重要。通过腺病毒介导的基因转移，将犬PLM或其衍生突变体在成年大鼠心肌细胞中过表达。使用犬特异性PLM抗体的共聚焦免疫荧光图像表明，外源性PLM或其突变体正确定位于肌膜、横管和闰盘，在细胞内区室中几乎未检测到。犬PLM或其突变体的过表达不影响成年大鼠心肌细胞中NCX1、肌质（内质）网钙ATP酶、钠钾ATP酶和肌集钙蛋白的表达。一个缺失所有四个潜在磷酸化位点（Ser62、Ser63、Ser68和Thr69）的COOH末端缺失突变体、一个缺失Ser68和Thr69的部分COOH末端缺失突变体以及一个将所有四个潜在磷酸化位点都变为丙氨酸的突变体都丧失了野生型PLM调节心肌细胞收缩性的能力。这些观察结果表明Ser68或Thr69在介导PLM对心肌收缩性的影响中具有重要作用。聚焦于Ser68，Ser68突变为Glu的突变体在调节心肌收缩性、[Ca2+]i瞬变和NCX1电流方面完全有效，Ser69突变为Ala（Ser68保持不变）的突变体部分有效，而Ser68突变为Ala的突变体则完全无效。Ser63突变为Ala和Ser68突变为Ala的突变体以及PLM都能够与NCX1进行共免疫沉淀。已知PLM中的Ser68可被蛋白激酶A和C磷酸化。我们得出结论，PLM对心肌收缩性、[Ca2+]i瞬变和NCX1活性的调节严重依赖于Ser68。我们认为，Ser68处的PLM磷酸化可能参与了cAMP和/或蛋白激酶C依赖性的心肌收缩性调节。
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