Desmond Patrick F, Muriel Joaquin, Markwardt Michele L, Rizzo Megan A, Bloch Robert J
From the Department of Physiology and Program in Biochemistry and Molecular Biology, University of Maryland, Baltimore, Maryland 21230.
From the Department of Physiology and.
J Biol Chem. 2015 Nov 13;290(46):27854-67. doi: 10.1074/jbc.M115.676585. Epub 2015 Sep 24.
Small ankyrin 1 (sAnk1) is a 17-kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum in skeletal muscle. We report that sAnk1 shares homology in its TM amino acid sequence with sarcolipin, a small protein inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Here we investigate whether sAnk1 and SERCA1 interact. Our results indicate that sAnk1 interacts specifically with SERCA1 in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle, and in COS7 cells transfected to express these proteins. This interaction was demonstrated by co-immunoprecipitation and an anisotropy-based FRET method. Binding was reduced ~2-fold by the replacement of all of the TM amino acids of sAnk1 with leucines by mutagenesis. This suggests that, like sarcolipin, sAnk1 interacts with SERCA1 at least in part via its TM domain. Binding of the cytoplasmic domain of sAnk1 to SERCA1 was also detected in vitro. ATPase activity assays show that co-expression of sAnk1 with SERCA1 leads to a reduction of the apparent Ca(2+) affinity of SERCA1 but that the effect of sAnk1 is less than that of sarcolipin. The sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM and cytoplasmic domains to regulate SERCA1 activity and modulate sequestration of Ca(2+) in the sarcoplasmic reticulum lumen. The identification of sAnk1 as a novel regulator of SERCA1 has significant implications for muscle physiology and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca(2+) misregulation.
小锚蛋白1（sAnk1）是一种17 kDa的跨膜（TM）蛋白，它与细胞骨架蛋白 obscurin 结合，并稳定骨骼肌中的肌浆网网络。我们报告称，sAnk1在其TM氨基酸序列上与肌脂蛋白具有同源性，肌脂蛋白是肌浆网（内质网）Ca(2+) -ATP酶（SERCA）的一种小蛋白抑制剂。在这里，我们研究sAnk1和SERCA1是否相互作用。我们的结果表明，sAnk1在从兔骨骼肌分离的肌浆网囊泡以及转染以表达这些蛋白的COS7细胞中与SERCA1特异性相互作用。这种相互作用通过共免疫沉淀和基于各向异性的FRET方法得以证明。通过诱变将sAnk1的所有TM氨基酸替换为亮氨酸后，结合减少了约2倍。这表明，与肌脂蛋白一样，sAnk1至少部分通过其TM结构域与SERCA1相互作用。在体外也检测到了sAnk1的胞质结构域与SERCA1的结合。ATP酶活性测定表明，sAnk1与SERCA1共表达会导致SERCA1的表观Ca(2+)亲和力降低，但sAnk1的作用小于肌脂蛋白。sAnk1 TM突变体对SERCA1活性没有影响。我们的结果表明，sAnk1通过其TM和胞质结构域与SERCA1相互作用，以调节SERCA1活性并调节肌浆网腔中Ca(2+)的隔离。将sAnk1鉴定为SERCA1的新型调节剂对肌肉生理学以及治疗与Ca(2+)调节异常相关的心力衰竭和肌肉萎缩症的治疗方法的开发具有重要意义。
