Molecular mechanisms of Ca^<2+> uptake by sarcolipin in cardiac sarcoplasmic reticulum.

本研究旨在探讨Ca^<2+>处理相关蛋白肌磷脂（sarcolipin， SLN）在细胞内Ca^<2+>浓度调节中的作用。Sarcoplasmic reticulum （SERCA2a）受phospholamban （PLB）和SLN调控，它们与SERCA2a偶联。非磷酸化- plb和-SLN均可降低sr对Ca^<2+>的摄取率。我们在小鼠心室肌中过表达SLN (SLN- tg)，并研究SLN对SERCA2a的影响。我们用aequorin法测量了小鼠完整左心室乳头肌Ca^<2+>瞬态和收缩。此外，我们使用皂素处理的薄小梁来研究Ca^<2+>摄取，Ca^<2+>释放和Ca^<2+>泄漏。在抽动收缩Hz中，SLN-TG组Ca^<2+>瞬态和收缩峰均低于对照组。Ca^<2+>瞬态和张力的下降速度比对照慢。在剥皮纤维中，在ATP和Ca^<2+> (pCa 6.2)存在的情况下，Ca^<2+>在不同时期被加载到SR中，SR中的Ca^<2+>被咖啡因释放。我们用fluo-3测量了释放的Ca^<2+>。当Ca^<2+>加载时间较短时，SLN-TG对Ca^<2+>的摄取速率较慢，而加载时间较长时则无差异。TG和非TG心肌稳态时SR的最大Ca^<2+>含量没有差异。Ca^<2+>诱导的Ca^<2+>释放（CICR）在SLN-TG中与非tg中没有差异。Ca^<2+>泄漏量通过测量无ATP溶液中泄漏的Ca和Ca^<2+>加载后的Ca^<2+>来估计。SLN-TG组Ca^<2+>渗漏与非tg组无明显差异。因此，SLN降低SR中Ca^<2+>的摄取并影响细胞内Ca^<2+>的浓度。在搏动-搏动收缩中，SLN的作用显著，但在稳态时，SLN的作用不显著。

The aim of the study was to explore the role of Ca^<2+> handling-related protein, sarcolipin (SLN) for the regulation of intracellular Ca^<2+> concentration. Sarcoplasmic reticulum (SR)(SERCA2a) is regulated by phospholamban (PLB) and SLN which couple with SERCA2a. Both non-phosphorylated-PLB and -SLN decrease the Ca^<2+> uptake rate of SR. We overexpressed SLN in ventricular muscles of mouse (SLN-TG) and investigated how SERCA2a is influenced by SLN. We measured the Ca^<2+> transients and contraction in intact left ventricular papillary muscles of mouse using the aequorin method. Also, we used saponin-treated thin trabeculae to investigate Ca^<2+> uptake, Ca^<2+> release and Ca^<2+> leakage. In twitch contraction, Hz, the peaks of the Ca^<2+> transient and contraction in SLN-TG were lower than those of the control. The rate of decline of the Ca^<2+> transient and tension was slower than that of the control. In the skinned fiber, Ca^<2+> was loaded in the SR in the presence of ATP and Ca^<2+> (pCa 6.2) for various periods, and Ca^<2+> in the SR was released by caffeine. We measured the released Ca^<2+> with fluo-3. The rate of Ca^<2+> uptake in SLN-TG was slower when the Ca^<2+> loading time was short but no difference was observed when the loading time was longer. The maximal Ca^<2+> content of SR at a steady state in both TG and non-TG cardiac muscles did not differ. Ca^<2+>-induced Ca^<2+> release (CICR) in SLN-TG did not differ from that in non-TG. Ca^<2+> leakage was estimated by measuring the leaked Ca in the solution without ATP and Ca^<2+> after Ca^<2+> loading. Ca^<2+> leakage in SLN-TG was not different from that in non-TG. Thus, SLN decreases the Ca^<2+> uptake in SR and influences intracellular Ca^<2+> concentration. The role of SLN is significant in beat-to-beat contraction, but SLN does not play a significant role at a steady state.