Regulation of the sodium potassium adenosine triphosphatase (Na,K-ATPPase) by FXYD 2

The Na,K-ATPase, or Na+ pump is an integral membrane protein found in the cells of virtually all higher eukaryotes and is one of the most important systems in cellular energy transduction. Na,K-ATPase catalyzes the electrogenic exchange of three intracellular Na+ for two extracellular K+ ions coupled to the hydrolysis of one molecule of ATP. The research described in this thesis concerns the regulation of the Na,K-ATPase by FXYD 2, a member of the FXYD family of small single transmembrane proteins. FXYD 2, commonly known as the gamma modulator, is located primarily in the kidney and has a role in modulating the enzyme's apparent affinities for ligands. This study has addressed several aspects of gamma structure and function, namely its function in intact cells, the function of the gamma transmembrane domain, and delineation of regions of the enzyme's catalytic alpha subunit with which gamma interacts. Transport assays using intact transfected HeLa cells showed that the two gamma variants, gammaa and gammab, cause (i) an increase in K+/Na+ antagonism, seen as an increase in K'Na at high K+ concentration, and (ii) an increase in apparent ATP affinity seen as an increase in ouabain-sensitive K+ influx as a function of ATP concentration. These results are consistent with those obtained earlier with unsided membrane preparations. The present study also showed a gamma-mediated increase in steady-state intracellular Na+ concentration and, in contrast to assays using permeabilized membranes, a gamma-mediated increase in apparent affinity for extracellular K+. Experiments with synthetic gamma transmembrane (gamma-TM) peptides provided insight into the role of the TM region such that incubation of these peptides with membranes containing alphabeta pumps modulated K'Na similarly to transfected full-length gamma, indicating that the TM domain alone can cause an increase in K'Na at high K+ concentration. Results with gamma-TM bearing the Gly41→Arg missense mutation associated with familial renal hypomagnesemia provided direct evidence that this mutation prevents gamma association with alphabeta pumps. In a study aimed to identify regions of alpha critical for the functional effects of gamma, interactions of alpha1/alpha2 (and the reverse alpha2/alpha1) chimeras with gamma showed the importance of the carboxy terminus, particularly TM 9. The chimera data also indicate that interactions of transmembrane regions of the catalytic alpha subunit with FXYD proteins are not necessarily the sole determinants of the kinetic effects of gamma on Na+ affinity since the extramembranous L7/8 loop of a appears to modulate intramembranous alpha-gamma interactions that mediated the increase in K+/Na+ antagonism