Sarcolipin regulates the activity of SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase
Sarcolipin regulates the activity of SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase
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DOI：
10.1074/jbc.273.20.12360
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发表时间：
1998-05-15
影响因子：
4.8
通讯作者：
MacLennan, DH
中科院分区：
文献类型：
--
作者：
Odermatt, A;Becker, S;MacLennan, DH
在HEK-293 t细胞中，人类和家兔SLN以及在其N端（NF-SLN）或C端（SLN- fc）具有FLAG表位的家兔SLN的构建物与SERCA1共表达。免疫组织化学证实了NF-SLN和SERCA1在内质网膜上的共定位，并证实了SLN N N末端的胞质取向。结果表明，家兔SLN或NF-SLN与SERCA1的共表达降低了SERCA1对Ca2+的表观亲和力，但刺激了最大Ca2+摄取率（V-max）。SLN的N端在物种间的保守性不强，N端FLAG表位的加入并未改变SLN的功能。抗flag抗体逆转了低Ca2+浓度下NF-SLN对Ca2+摄取的抑制作用和NF-SLN对V-max的刺激作用。在高度保守的C端添加FLAG表位，相对于天然SLN降低了SERCA1对Ca2+的表观亲和力，并显著降低了V-max。c端结构域的突变表明该序列对SLN功能至关重要。跨膜螺旋的突变分析，以及SLN和磷蛋白（PLN）与SERCA1共表达的加性调节作用，为SLN和PLN与SERCA分子相互作用的不同机制提供了证据。从兔快速收缩肌（胫骨前肌）中分离出来的肌浆网囊泡中Ca2+的摄取率在慢性低频刺激下，在3天和4天的刺激肌肉中减少了大约40%，SERCA1对Ca2+的表观亲和力有边际增加。刺激后小鼠SERCA1 mRNA和蛋白水平未发生变化。相比之下，SLN mRNA减少15%，SLN蛋白减少40%。SLN表达减少可以解释在这些肌肉中观察到的SERCA1活性下降，并可能代表对慢性低频刺激的早期功能适应。
The 31-amino acid proteolipid, sarcolipin (SLN), is associated with the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA1), Constructs of human and rabbit SLN and of rabbit SLN with the FLAG epitope at its N terminus (NF-SLN) or its C terminus (SLN-FC) were coexpressed with SERCA1 in HEK-293 T-cells, Immunohistochemistry was used to demonstrate colocalization of NF-SLN and SERCA1 in the endoplasmic reticulum membrane and to demonstrate the cytosolic orientation of the N terminus of SLN, Coexpression of native rabbit SLN or NF-SLN with SERCA1 decreased the apparent affinity of SERCA1 for Ca2+ but stimulated maximal Ca2+ uptake rates (V-max). The N terminus of SLN is not well conserved among species, and the addition of an N-terminal FLAG epitope did not alter SLN function. Anti-FLAG antibody reversed both the inhibition of Ca2+ uptake by NF-SLN at low Ca2+ concentrations and the stimulatory effect of NF-SLN on V-max. Addition of the FLAG epitope to the highly conserved C terminus decreased the apparent affinity of SERCA1 for Ca2+ relative to native SLN and decreased V-max significantly. Mutations in the C-terminal domain showed that this sequence is critical for SLN function. Mutational analysis of the transmembrane helix, together with the additive regulatory effects of coexpression of both SLN and phospholamban (PLN) with SERCA1, provided evidence for different mechanisms of interaction of SLN and PLN with SERCA molecules. Ca2+ uptake rates in sarcoplasmic reticulum vesicles, isolated from rabbit fast-twitch muscle (tibialis anterior) subjected to chronic low frequency stimulation, were reduced by approximately 40% in 3- and 4 day stimulated muscle, with a marginal increase in apparent affinity of SERCA1 for Ca2+. SERCA1 mRNA and protein levels were unaltered after stimulation. In contrast, SLN mRNA was decreased by 15%, and SLN protein was reduced by 40%. Reduced SLN expression could explain the decrease in SERCA1 activity observed in these muscles and might represent an early functional adaptation to chronic low frequency stimulation.