We have reported that the reduction in plasma membrane cholesterol could decrease cellular Na/K-ATPase α1 expression through a Src-dependent pathway. However, it is unclear whether cholesterol could regulate other Na/K-ATPase α isoforms and the molecular mechanisms of this regulation are not fully understood. Here we used cells expressing different Na/K-ATPase α isoforms and found that membrane cholesterol reduction by U18666A decreased expression of the α1 isoform, but not the α2 or α3 isoform. Imaging analyses showed the cellular redistribution of α1 and α3, but not α2. Moreover, U18666A led to redistribution of α1 to late endosomes/lysosomes, while the proteasome inhibitor blocked α1 reduction by U18666A. These results suggest that the regulation of the Na/K-ATPase α subunit by cholesterol is isoform-specific and α1 is unique in this regulation through the endocytosis-proteasome pathway. Mechanistically, loss-of-Src binding mutation of A425P in α1 lost its capacity for regulation by cholesterol. Meanwhile, gain-of-Src binding mutations in α2 partially restored the regulation. Furthermore, through studies in caveolin-1 knockdown cells, as well as subcellular distribution studies in cell lines with different α isoforms, we found that Na/K-ATPase, Src and caveolin-1 worked together for the cholesterol regulation. Taken together, these new findings reveal that the putative Src binding domain and the intact Na/K-ATPase/Src/caveolin-1 complex is indispensable for the isoform-specific regulation of Na/K-ATPase by cholesterol.

中文翻译：

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胆固醇对 Na/K-ATPase 表达的调节：异构体特异性和分子机制

我们已经报道了质膜胆固醇的降低可以通过 Src 依赖性途径降低细胞 Na/K-ATPase α1 的表达。然而，尚不清楚胆固醇是否可以调节其他 Na/K-ATPase α 异构体，并且这种调节的分子机制尚不完全清楚。在这里，我们使用表达不同 Na/K-ATPase α 异构体的细胞，发现 U18666A 降低膜胆固醇会降低 α1 异构体的表达，但不会降低 α2 或 α3 异构体的表达。成像分析显示 α1 和 α3 的细胞重新分布，而不是 α2。此外，U18666A 导致 α1 重新分配到晚期内体/溶酶体，而蛋白酶体抑制剂通过 U18666A 阻止了 α1 减少。这些结果表明，胆固醇对 Na/K-ATPase α 亚基的调节是异构体特异性的，而 α1 通过内吞作用-蛋白酶体途径在该调节中是独一无二的。从机制上讲，α1 中 A425P 的 Src 缺失结合突变失去了胆固醇调节的能力。同时，α2 中的 Src 结合突变部分恢复了调节。此外，通过对caveolin-1 敲低细胞的研究，以及在具有不同α 亚型的细胞系中的亚细胞分布研究，我们发现Na/K-ATPase、Src 和caveolin-1 共同调节胆固醇。总之，这些新发现表明，假定的 Src 结合域和完整的 Na/K-ATPase/Src/caveolin-1 复合物对于胆固醇对 Na/K-ATPase 的异构体特异性调节是必不可少的。