POST-TRANSCRIPTIONAL CONTROL OF Na,K-ATPase ACTIVITY AND CELL GROWTH BY A SPLICE VARIANT OF FXYD2 WITH MODIFIED mRNA

In kidney, FXYD proteins regulate Na,KATPase 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 C to T in FXYD2b, but not FXYD2a, from both untreated and hypertonicity-treated NRK-52E cells. The C172T sequence change exposed a cryptic KKXX ER retrieval signal via a premature stop codon. The truncation affected trafficking of FXYD2b, 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. C172T 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. Na,K-ATPase is an enzyme in eukaryotic cells that provides non-equilibrium distribution of Na and K ions across the plasma membrane. Besides its obligatory  and  subunits, the complex contains a so-called “FXYD” subunit (1), a short single-span membrane protein involved in regulation of the kinetic properties of Na,KATPase. In mammals, there are 7 different FXYD genes that are expressed in a tissueand cellspecific manner. Remarkably, association of the Na,K-ATPase with each of them leads to specific changes in kinetic parameters of the pump either at the level of K0.5 for the substrates or Vmax [reviewed in (2)]. FXYD2, also called the gamma subunit, was the first accessory FXYD protein discovered in connection with the Na,K-ATPase (3). It was identified as a proteolipid in membranes from dog kidney that was labeled with a photoaffinity derivative of the specific inhibitor of the pump, ouabain, along with the  and  subunits (4). Expression of the FXYD2 protein is mostly in kidney (5), although the EST database suggests some other tissues as potential sources for FXYD2 (for instance, pancreas and mammary glands). There are two splice variants of FXYD2, FXYD2a and FXYD2b, that are identical in structure except for the N-terminal segment comprising the first exon (6,7). Based on results from different heterologous expression systems (Xenopus oocytes or mammalian cell transfectants), FXYD2 reduces the activity of the pump by increasing the K0.5 for Na as well as for K (8-11). We also demonstrated that both splice variants can be post-translationally modified when expressed in epithelial NRK-52E http://www.jbc. org/cgi/doi/10.1074/jbc.M111.241901 The latest version is at JBC Papers in Press. Published on April 1, 2011 as Manuscript M111.241901