Regulation of Inositol 1,4,5-Trisphosphate Receptor-mediated Calcium Release by the Na/K-ATPase in Cultured Renal Epithelial Cells^*

Abstract

It is known that the Na/K-ATPase α1 subunit interacts directly with inositol 1,4,5-triphosphate (IP₃) receptors. In this study we tested whether this interaction is required for extracellular stimuli to efficiently regulate endoplasmic reticulum (ER) Ca²⁺ release. Using cultured pig kidney LLC-PK1 cells as a model, we demonstrated that graded knockdown of the cellular Na/K-ATPase α1 subunit resulted in a parallel attenuation of ATP-induced ER Ca²⁺ release. When the knockdown cells were rescued by knocking in a rat α1, the expression of rat α1 restored not only the cellular Na/K-ATPase but also ATP-induced ER Ca²⁺ release. Mechanistically, this defect in ATP-induced ER Ca²⁺ release was neither due to the changes in the amount or the function of cellular IP₃ and P2Y receptors nor the ER Ca²⁺ content. However, the α1 knockdown did redistribute cellular IP₃ receptors. The pool of IP₃ receptors that resided close to the plasma membrane was abolished. Because changes in the plasma membrane proximity could reduce the efficiency of signal transmission from P2Y receptors to the ER, we further determined the dose-dependent effects of ATP on protein kinase Cϵ activation and ER Ca²⁺ release. The data showed that the α1 knockdown de-sensitized the ATP-induced ER Ca²⁺ release but not PKCϵ activation. Moreover, expression of the N terminus of Na/K-ATPase α1 subunit not only disrupted the formation of the Na/K-ATPase-IP₃ receptor complex but also abolished the ATP-induced Ca²⁺ release. Finally, we observed that the α1 knockdown was also effective in attenuating ER Ca²⁺ release provoked by angiotensin II and epidermal growth factor.