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

Abstract

The ␥ 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 ␥ is present primarily in the kidney as two main splice variants, ␥ a and ␥ b , 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 ␥ a and ␥ b . At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat ␣1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E 1 7 E 2 conformational equilibrium and (ii) an increase in K ؉ antagonism of cytoplasmic Na ؉ activation. Antibodies against the C terminus common to both variants (anti-␥) abrogate the first effect but not the second. In contrast, ␥ a and ␥ b show differences in their localization along the kidney tubule. Using anti-␥ (C-terminal) and antibodies to the rat ␣ subunit as well as antibodies to identify cell types, double immunofluorescence showed ␥ in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both ␥ a and ␥ b . In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express ␥ but at lower levels. Antibodies specific for ␥ a and ␥ b showed differences in their cortical location; ␥ a is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, ␥ b but not ␥ a is present in the cortical TAL only. Thus, the importance of ␥ a and ␥ b may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.A small membrane protein, ␥, first described over 20 years ago in purified kidney Na,K-ATPase preparations (1, 2) associates, in approximately equimolar amounts, with the ␣ and ␤ subunits (3, 4). Molecular cloning of the ␥ subunits of rat, mouse, cow, and sheep indicated a molecular weight of ϳ6500 (5). Cloning and sequencing of the human (6) and Xenopus laevis (7) ␥ subunits have also been reported. Comparison of sequences shows ϳ75% homology among ␥ subunits of the aforementioned different species but is much higher (93%) for only mammalian sequences. Further structural analysis has shown that ␥ comprises a single transmembrane domain and has an N terminus-out, C terminus-in topology (7,8). In addition, two major forms have been recently identified at the molecular level as described below.On SDS-polyacrylamide gel electrophoresis, the ␥ subunit runs as a doublet (apparent molecular masses of ϳ8 and ϳ9 kDa) (5,8), and a doublet is observed following expression in tissue culture cells (8, 9) and in in vitro expression in the pre...

Allosteric Effect of ATP on Na+,K+-ATPase Conformational Kinetics

“…In contrast, the biphasic kinetic behavior that we have observed disappears on increasing the ATP concentration ( Figure 1A), that is, there is no enzyme heterogeneity at high ATP concentrations. Therefore, if one were to attribute the biphasic behavior to enzyme heterogeneity caused by the presence of FXYD proteins, this would be in contradiction to previous observations (35,36). Furthermore, in their recent review of FXYD protein interaction with the Na + ,K + -ATPase, Garty and Karlish (34) stated that the effect of FXYD proteins on the kinetic parameters of the enzyme are modest, usually only about 2-fold.…”

Regulation of Na,K-ATPase by PLMS, the Phospholemman-like Protein from Shark

“…In the combined presence of Na ϩ and K ϩ changes in the competition between Na ϩ and K ϩ at the cytoplasmic face will also change the apparent Na ϩ affinity. The latter was demonstrated to be the case for ␣1-HeLa cells transfected with ␥ a or ␥ b splice variants (11). If the Na ϩ activation curve depicted in Fig.…”

“…In other investigations the change in apparent affinity for Na ϩ could not be unambiguously assigned to such increased Na ϩ /K ϩ competition alone but also indicated ␥ induced changes in the intrinsic binding affinity for Na ϩ (59). Effects on the extracellular K ϩ affinity of both splice variants of ␥ have also been reported (11,59). Co-expression of the FXYD4 protein CHIF with rat ␣1 increased the apparent affinity for cytoplasmic Na ϩ , without affecting the K ϩ affinity or V max (23,66).…”

“…Thus, association of FXYD1 proteins with the Na,K-ATPase leads in both cases to a decreased Na ϩ affinity and inhibition of enzyme activity, whereas dissociation of the FXYD1 proteins results in stimulation of hydrolytic activity. Other FXYD proteins have different patterns of effects: association of ␥ (FXYD2) with Na,K-ATPase has been shown to increase the apparent ATP affinity by supporting the E 1 conformation of the enzyme and to decrease the apparent affinity for cytoplasmic Na ϩ , which apparently is an effect secondary to an increased antagonism of cytoplasmic K ϩ to activation by Na ϩ (10,11). In other investigations the change in apparent affinity for Na ϩ could not be unambiguously assigned to such increased Na ϩ /K ϩ competition alone but also indicated ␥ induced changes in the intrinsic binding affinity for Na ϩ (59).…”

“…The ␥-subunit has been shown to modulate Na,K-ATPase activity in the kidney by affecting the E 2 /E 1 equilibrium toward E 1 , thus regulating the affinity for ATP at its low affinity site and the cytoplasmic Na ϩ and K ϩ competition (11). The ␥-subunit has a highly distinct distribution along different parts of the nephron allowing differential regulation of ion transport along different nephron segments (12).…”