Functional Role and Immunocytochemical Localization of the γa and γb Forms of the Na,K-ATPase γ Subunit

The γ subunit of the Na,K-ATPase is a member of the FXYD family of type 2 transmembrane proteins that probably function as regulators of ion transport. Rat γ is present primarily in the kidney as two main splice variants, γa and γb, which differ only at their extracellular N termini (TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. D. (2000) J. Biol. Chem. 275, 18441–18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between γa and γb. At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat α1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E1 ↔E2 conformational equilibrium and (ii) an increase in K+ antagonism of cytoplasmic Na+activation. Antibodies against the C terminus common to both variants (anti-γ) abrogate the first effect but not the second. In contrast, γa and γb show differences in their localization along the kidney tubule. Using anti-γ (C-terminal) and antibodies to the rat α subunit as well as antibodies to identify cell types, double immunofluorescence showed γ in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both γa and γb. In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express γ but at lower levels. Antibodies specific for γa and γb showed differences in their cortical location; γa is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, γb but not γa is present in the cortical TAL only. Thus, the importance of γa and γb may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.