Post-transcriptional control of Na,K-ATPase activity and cell growth by a splice variant of FXYD2 protein with modified mRNA.

In kidney, FXYD proteins regulate Na,K-ATPase in a nephron segment-specific way. FXYD2 is the most abundant renal FXYD but is not expressed in most renal cell lines unless induced by hypertonicity. Expression by transfection of FXYD2a or FXYD2b splice variants in NRK-52E cells reduces the apparent Na(+) affinity of the Na,K-ATPase and slows the cell proliferation rate. Based on RT-PCR, mRNAs for both splice variants were expressed in wild type NRK-52E cells as low abundance species. DNA sequencing of the PCR products revealed a base alteration from C to T in FXYD2b but not FXYD2a from both untreated and hypertonicity-treated NRK-52E cells. The 172C→T sequence change exposed a cryptic KKXX endoplasmic reticulum retrieval signal via a premature stop codon. The truncation affected trafficking of FXYD2b and its association with Na,K-ATPase and blocked its effect on enzyme kinetics and cell growth. The data may be explained by altered splicing or selective RNA editing of FXYD2b, a supplementary process that would ensure that it was inactive even if transcribed and translated, in these cells that normally express only FXYD2a. 172C→T mutation was also identified after mutagenesis of FXYD2b by error-prone PCR coupled with a selection for cell proliferation. Furthermore, the error-prone PCR alone introduced the mutation with high frequency, implying a structural peculiarity. The data confirm truncation of FXYD2b as a potential mechanism to regulate the amount of FXYD2 at the cell surface to control activity of Na,K-ATPase and cell growth.

Stress-induced expression of the gamma subunit (FXYD2) modulates Na,K-ATPase activity and cell growth.

In kidney, the Na,K-ATPase is associated with a single span protein, the gamma subunit (FXYD2). Two splice variants are differentially expressed along the nephron and have a differential influence on Na,K-ATPase when stably expressed in mammalian cells in culture. Here we used a combination of gene induction and gene silencing techniques to test the functional impact of gamma by means other than transfection. NRK-52E cells (of proximal tubule origin) do not express gamma as a protein under regular tissue culture conditions. However, when they were exposed to hyperosmotic medium, induction of only the gammaa splice variant was observed, which was accompanied by a reduction in the rate of cell division. Kinetic analysis of stable enzyme properties from control (alpha1beta1) and hypertonicity-treated cultures (alpha1beta1gammaa) revealed a significant reduction (up to 60%) of Na,K-ATPase activity measured under V(max) conditions with little or no change in the amounts of alpha1beta1. This effect as well as the reduction in cell growth rate was practically abolished when gamma expression was knocked down using specific small interfering RNA duplexes. Surprisingly, a similar induction of endogenous gammaa because of hypertonicity was seen in rat cell lines of other than renal origin: C6 (glioma), PC12 (pheochromocytoma), and L6 (myoblasts). Furthermore, exposure of NRK-52E cells to other stress inducers such as heat shock, exogenous oxidation, and chemical stress also resulted in a selective induction of gammaa. Taken together, the data imply that induction of gammaa may have adaptive value by being a part of a general cellular response to genotoxic stress.