Regions of the catalytic alpha subunit of Na,K-ATPase important for functional interactions with FXYD 2

The gamma modulator (FXYD 2) is a member of the FXYD family of single transmembrane proteins that modulate the kinetic behavior of Na,K-ATPase. This study concerns the identification of regions in the alpha subunit that are important for its functional interaction with gamma. An important effect of gamma is to increase K+ antagonism of cytoplasmic Na+ activation apparent as an increase in KNa' at high [K+]. We show that although gamma associates with alpha1, alpha2, and alpha3 isoforms, it increases the KNa' of alpha1 and alpha3 but not alpha2. Accordingly, chimeras of alpha1 and alpha2 were used to identify regions of alpha critical for the increased KNa'. As with alpha1 and alpha2, all chimeras associate with gamma. Kinetic analysis of alpha2front/alpha1back chimeras indicate that the C-terminal (Lys907-Tyr1018) region of alpha1, which includes transmembrane (TM)9 close to gamma, is important for the increase in KNa'. However, similar experiments with alpha1front/alpha2back chimeras indicate a modulatory role of the loop between TMs 7 and 8. Thus, as long as the alpha1 L7/8 loop is present, replacement of TM9 of alpha1 with that of alpha2 does not abrogate the gamma effect on KNa'. In contrast, as long as TM9 is that of alpha1, replacement of L7/8 of alpha1 with that of alpha2 does not abolish the effect. It is suggested that structural association of the TM regions of alpha and FXYD 2 is not the sole determinant of this effect of FXYD on KNa' but is subject to long range modulation by the extramembranous L7/8 loop of alpha.