Na,K-ATPase from Mice Lacking the γ Subunit (FXYD2) Exhibits Altered Na+ Affinity and Decreased Thermal Stability

D Holstead Jones,Tony Y Li,Elena Arystarkhova,Kevin J Barr,Randall K Wetzel,Jun Peng,Kathryn Markham,Kathleen J Sweadner,Guo-Hua Fong,Gerald M Kidder

doi:10.1074/jbc.m500697200

Abstract

The gamma subunit of the Na,K-ATPase, a 7-kDa single-span membrane protein, is a member of the FXYD gene family. Several FXYD proteins have been shown to bind to Na,K-ATPase and modulate its properties, and each FXYD protein appears to alter enzyme kinetics differently. Different results have sometimes been obtained with different experimental systems, however. To test for effects of gamma in a native tissue environment, mice lacking a functional gamma subunit gene (Fxyd2) were generated. These mice were viable and without observable pathology. Prior work in the mouse embryo showed that gamma is expressed at the blastocyst stage. However, there was no delay in blastocele formation, and the expected Mendelian ratios of offspring were obtained even with Fxyd2-/- dams. In adult Fxyd2-/- mouse kidney, splice variants of gamma that have different nephron segment-specific expression patterns were absent. Purified gamma-deficient renal Na,K-ATPase displayed higher apparent affinity for Na+ without significant change in apparent affinity for K+. Affinity for ATP, which was expected to be decreased, was instead slightly increased. The results suggest that regulation of Na+ sensitivity is a major functional role for this protein, whereas regulation of ATP affinity may be context-specific. Most importantly, this implies that gamma and other FXYD proteins have their effects by local and not global conformation change. Na,K-ATPase lacking the gamma subunit had increased thermal lability. Combined with other evidence that gamma participates in an early step of thermal denaturation, this indicates that FXYD proteins may play an important structural role in the enzyme complex.

Highlights

CHIF has no effect on the intrinsic affinity for Kϩ; it greatly increases its sensitivity to competition by extracellular Naϩ, with the consequence that apparent Kϩ affinity is reduced in physiological conditions [15, 20]
Production of Mice Lacking ␥ Subunits—The ␥ subunit is encoded by a single gene belonging to the FXYD family [1]
Alterations of Na,K-ATPase Properties—The increase in apparent affinity for Naϩ for the Na,K-ATPase isolated from Fxyd2Ϫ/Ϫ mice is consistent with observations both from mammalian cell transfectants and from expression of ␥ subunit in Xenopus oocytes

Summary

EXPERIMENTAL PROCEDURES

Cytology—For staining of ␤-galactosidase activity expressed in the knock-out mice, tissues were fixed overnight at 4 °C in 0.2% paraformaldehyde in 0.1 M PIPES buffer, pH 6.9, containing 2 mM MgCl2 and 5 mM EGTA They were cryoprotected in 30% sucrose in PBS overnight at 4 °C and embedded in frozen section medium (Stephens Scientific, Riverdale, NJ). Thermostability Assay—Purified enzyme from kidney of either Fxyd2Ϫ/Ϫ or wild type mice was diluted to a concentration of 5 ␮g/ml in a buffer (ATPase assay reaction mixture without ATP) containing 100 mM NaCl, 20 mM KCl, 4 mM MgCl2, and 30 mM histidine, pH 7.4, and incubated for 60 min at three different sets of temperatures as follows: 4, 37, and 41 °C. Na,K-ATPase activity was defined as the ouabain-sensitive difference in Pi release/mg of protein/h

RESULTS

No mice

DISCUSSION