Na+-K+-ATPase from mice lacking the γ subunit exhibits decreased thermal stability. Phospholamban (PLN) and sarcolipin (SLN) are small homologous proteins that regulate sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) with properties similar to the γ subunit, through physical interactions with SERCAs. Here, we tested the hypothesis that PLN and SLN may protect against thermal inactivation of SERCAs. HEK-293 cells were co-transfected with different combinations of cDNAs encoding SERCA2a, PLN, a PLN mutant (N34A) that cannot bind to SERCA2a, and SLN. One-half of the cells were heat stressed at 40°C for 1 h (HS), and one-half were maintained at 37°C (CTL) before harvesting the cells and isolating microsomes. Compared with CTL, maximal SERCA activity was reduced by 25–35% following HS in cells that expressed either SERCA2a alone or SERCA2a and mutant PLN (N34A) whereas no change in maximal SERCA2a activity was observed in cells that co-expressed SERCA2a and either PLN or SLN following HS. Increases in SERCA2a carbonyl group content and nitrotyrosine levels that were detected following HS in cells that expressed SERCA2a alone were prevented in cells co-expressing SERCA2a with PLN or SLN, whereas co-expression of SERCA2a with mutant PLN (N34A) only prevented carbonyl group formation. In other experiments using knock-out mice, we found that thermal inactivation of SERCA was increased in cardiac left ventricle samples from Pln-null mice and in diaphragm samples from Sln-null mice, compared with WT littermates. Our results show that both PLN and SLN form a protective interaction with SERCA pumps during HS, preventing nitrosylation and oxidation of SERCA and thus preserving its maximal activity.

中文翻译：

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磷脂酰肌醇和肌钙蛋白可防止肌浆网（内质网）Ca2 + -ATPases热失活

来自缺乏γ亚基的小鼠的Na + -K + -ATP酶表现出降低的热稳定性。磷脂酰肌醇（PLN）和肌钙蛋白（SLN）是小的同源蛋白，通过与SERCA的物理相互作用来调节具有类似γ亚基特性的肌浆网（内质网）Ca2 + -ATPases（SERCA）。在这里，我们测试了PLN和SLN可以防止SERCA热失活的假设。将HEK-293细胞与编码SERCA2a，PLN，不能与SERCA2a和SLN结合的PLN突变体（N34A）的cDNA的不同组合共转染。在收获细胞并分离微粒体之前，将一半的细胞在40°C加热1小时（HS），并在37°C（CTL）保持一半。与CTL相比，HS单独表达SERCA2a或SERCA2a和突变PLN（N34A）的细胞在HS后最大SERCA活性降低了25-35％，而在HS后共表达SERCA2a和PLN或SLN的细胞中未观察到最大SERCA2a活性的变化。 。共表达SERCA2a与PLN或SLN的细胞中，仅表达​​SERCA2a的细胞在HS后检测到的SERCA2a羰基含量和硝基酪氨酸水平增加，而与突变PLN（N34A）共表达SERCA2a只能阻止羰基形成。 。在其他使用基因敲除小鼠的实验中，我们发现与WT同窝仔相比，SERN的热失活在Pln无小鼠的心脏左心室样本和Sln无小鼠的隔膜样本中有所增加。