Cation selectivity of gastric H,K-ATPase and Na,K-ATPase chimeras

Chimeras of the catalytic subunits of the gastric H,K-ATPase and Na,K-ATPas e were constructed and expressed in LLC-PK1 cells. The chimeras included th e following: (i) a control, H85N (the first 85 residues comprising the cyto plasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-A TPase); (ii) H85N/H356 -519N (the N-terminal half of the cytoplasmic M4-M5 loop also replaced); and (iii) H519N (the entire front half replaced). The latter two replacements confer a decrease in apparent affinity for extracel lular K+. The 356-519 domain and, to a greater extent, the H519N replacemen t confer increased apparent selectivity for protons relative to Na+ at cyto plasmic sites as shown by the persistence of K+ influx when the proton conc entration is increased and the Naf concentration decreased. The pH and Kf d ependence of ouabain-inhibitable ATPase of membranes derived from the trans fected cells indicate that the H519N and, to a lesser extent, the H356-519N substitution decrease the effectiveness of K+ to compete for protons at pu tative cytoplasmic H+ activation sites, Notable pH-independent behavior of H85N/H356-519N at low Na+ suggests that as pH is decreased, Na+/K+. exchang e is replaced largely by (Na+ + H+)/K+ exchange. With H519N, the pH and Naf dependence of pump and ATPase activities suggest relatively active H+/K+ e xchange even at neutral pH. Overall, this study provides evidence for impor tant roles in cation selectivity for both the N-terminal half of the M4-M5 loop and the adjacent transmembrane helice(s).