Distribution and oligomeric association of splice forms of Na⁺-K⁺-ATPase regulatory γ-subunit in rat kidney

Abstract

Renal Na(+)-K(+)-ATPase is associated with the gamma-subunit (FXYD2), a single-span membrane protein that modifies ATPase properties. There are two splice variants with different amino termini, gamma(a) and gamma(b). Both were found in the inner stripe of the outer medulla in the thick ascending limb. Coimmunoprecipitation with each other and the alpha-subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect Na(+)-K(+)-ATPase conformation. In the cortex, the proportion of the gamma(b)-subunit was markedly lower, and the gamma(a)-subunit predominated in isolated proximal tubule cells. By immunofluorescence, the gamma(b)-subunit was detected in the superficial cortex only in the distal convoluted tubule and connecting tubule, which are rich in Na(+)-K(+)-ATPase but comprise a minor fraction of cortex mass. In the outer stripe of the outer medulla and for a short distance in the deep cortex, the thick ascending limb predominantly expressed the gamma(b)-subunit. Because different mechanisms maintain and regulate Na(+) homeostasis in different nephron segments, the splice forms of the gamma-subunit may have evolved to control the renal Na(+) pump through pump properties, gene expression, or both.

Interaction of SDS with Na+/K+-ATPase

“…6 indicate the existence of ␣,␥-association in the native enzyme, and show that this association is also retained but altered in the TDS-solubilized state. The cross-linking evidence in support of ␣,␥-association confirms previous evidence for such association obtained in thermal denaturation experiments (11) and coimmunoprecipitation studies (36,37).…”

“…The kidney enzyme preparation used here contains a third subunit, ␥ (ϳ7 kDa), that regulates its function (3,4,36,37,48). To see whether ␥ interaction with the other subunits could be detected by cross-linking in the native and the TDS-solubilized enzymes, experiments of Fig.…”

Stress-induced Expression of the γ Subunit (FXYD2) Modulates Na,K-ATPase Activity and Cell Growth

“…The RCT-G1 polyclonal antibody raised against the C-terminal peptide shared by ␥a and ␥b was used to detect both splice variants of ␥ (20). The RNGB antibody (specific for the N terminus of ␥b) was used for ␥b detection (14). Relative band intensities were determined by densitometry using a Amersham Biosciences 300A analyzer.…”

“…Three of them have now been identified as constitutively expressed in kidney. ␥ colocalizes with the Na,K-ATPase in proximal tubules, distal convoluted tubules, medullary thick ascending limb, and deep inner medulla (13)(14)(15). CHIF colocalizes with the pump in the collecting duct (10).…”

“…Interestingly, expression of splice variants of ␥ is also segment-specific (14,19). Both ␥a and ␥b are found in medullary thick ascending limb and in inner medulla (i.e.…”