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

本研究旨在探讨钙处理相关蛋白肌磷脂（sarcolipin，SLN）对细胞内钙离子浓度的调节作用。肌浆网（SR）（SERCA 2a）受受磷蛋白（PLB）和SLN的调节，SLN与SERCA 2a偶联。非磷酸化的-PLB和-SLN均降低SR的Ca^2+摄取速率。我们在小鼠心室肌中过表达SLN（SLN-TG），并研究了SLN如何影响SERCA 2a。我们用水母发光蛋白法测定了小鼠完整左心室乳头肌的Ca^<2+>瞬变和收缩。此外，我们还用皂苷处理的薄骨小梁研究Ca^2+摄取、Ca^2+释放和Ca^2+渗漏。SLN-TG的颤搐收缩频率、Ca^2+瞬变峰和收缩峰均低于对照组。Ca^<2+>瞬变和张力的下降速度比对照组慢。在去皮纤维中，在ATP和Ca^<2+>（pCa 6.2）存在的情况下，SR中的Ca^<2+>被负载不同的时间，并且SR中的Ca^<2+>被咖啡因释放。我们用fluo-3测量了释放的Ca^<2+>。钙负荷时间越短，SLN-TG的钙摄取速率越慢，而钙负荷时间越长，两者之间无明显差异。稳态时，TG和非TG心肌SR的最大Ca^<2+>含量没有差异。钙诱导的钙释放（CICR）在SLN-TG和non-TG中没有差异。通过测量不含ATP的溶液中泄漏的Ca和Ca^<2+>负载后的Ca^<2 +>来估计Ca^<2 +>泄漏。SLN-TG组与非TG组的Ca^<2+>渗漏无明显差异。因此，SLN可降低SR对Ca^2+的摄取，从而影响细胞内Ca^2+浓度。SLN在搏动收缩中的作用是显著的，但在稳态下SLN不起显著作用。

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.