Abstract
Sodium and potassium-exchanging adenosine triphosphatase (Na,K-ATPase) in the kidney is associated with the gamma subunit (gamma, FXYD2), a single-span membrane protein that modulates ATPase properties. Rat and human gamma occur in two splice variants, gamma(a) and gamma(b), with different N termini. Here we investigated their structural heterogeneity and functional effects on Na,K-ATPase properties. Both forms were post-translationally modified during in vitro translation with microsomes, indicating that there are four possible forms of gamma. Site-directed mutagenesis revealed Thr(2) and Ser(5) as potential sites for post-translational modification. Similar modification can occur in cells, with consequences for Na,K-ATPase properties. We showed previously that stable transfection of gamma(a) into NRK-52E cells resulted in reduction of apparent affinities for Na(+) and K(+). Individual clones differed in gamma post-translational modification, however, and the effect on Na(+) affinity was absent in clones with full modification. Here, transfection of gamma(b) also resulted in clones with or without post-translational modification. Both groups showed a reduction in Na(+) affinity, but modification was required for the effect on K(+) affinity. There were minor increases in ATP affinity. The physiological importance of the reduction in Na(+) affinity was shown by the slower growth of gamma(a), gamma(b), and gamma(b') transfectants in culture. The differential influence of the four structural variants of gamma on affinities of the Na,K-ATPase for Na(+) and K(+), together with our previous finding of different distributions of gamma(a) and gamma(b) along the rat nephron, suggests a highly specific mode of regulation of sodium pump properties in kidney.
Highlights
The Na,K-ATPase,1 or sodium pump, is the principal enzyme in animal cells that maintains ionic gradients of Naϩ and Kϩ at the expense of ATP hydrolysis
We begin by examining the electrophoretic mobilities of ␥ forms found in kidney, and use in vitro translation of ␥a and ␥b to detect post-translational modification of both splice variants
The blot was stained with the RCT-G1 antibody that recognizes the C terminus of ␥, a site that is highly conserved between species and identical in the splice variants. ␥ was seen as a clear doublet in both species, the resolution was significantly better for mouse
Summary
Na,K-ATPase, sodium and potassiumexchanging adenosine triphosphatase; HEK, human embryonic kidney; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; FXYD, gene nomenclature based on a conserved motif, Phe-X-Tyr-Asp, pronounced like “fix-it.”. Functional differences in intrinsic properties of Na,KATPase have been reported in the kidney; the affinity for Naϩ varies significantly along the rat and rabbit nephron (4 –7). The first clear effect of ␥ on Na,K-ATPase transport properties was detected by electrophysiological measurements in Xenopus oocytes It influenced the apparent affinity of the Na,KATPase pump current for extracellular Kϩ [28]. Others have reported that ␥a and ␥b have similar effects on intrinsic properties of the pump Both splice variants decreased apparent affinity for Naϩ without significant alteration of Kϩ affinity in HeLa transfected cells [21] and in Xenopus oocytes [32], and increased Km for ATP in HeLa transfectants [21]. Preliminary reports have been presented [38, 39]
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