title: Cation selectivity of gastric H,K-ATPase and Na,K-ATPase chimeras.
abstract: Chimeras of the catalytic subunits of the gastric H,K-ATPase and Na, K-ATPase were constructed and expressed in LLC-PK1 cells. The chimeras included the following: (i) a control, H85N (the first 85 residues comprising the cytoplasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-ATPase); (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 extracellular K+. The 356-519 domain and, to a greater extent, the H519N replacement confer increased apparent selectivity for protons relative to Na+ at cytoplasmic sites as shown by the persistence of K+ influx when the proton concentration is increased and the Na+ concentration decreased. The pH and K+ dependence of ouabain-inhibitable ATPase of membranes derived from the transfected cells indicate that the H519N and, to a lesser extent, the H356-519N substitution decrease the effectiveness of K+ to compete for protons at putative cytoplasmic H+ activation sites. Notable pH-independent behavior of H85N/H356-519N at low Na+ suggests that as pH is decreased, Na+/K+ exchange is replaced largely by (Na+ + H+)/K+ exchange. With H519N, the pH and Na+ dependence of pump and ATPase activities suggest relatively active H+/K+ exchange even at neutral pH. Overall, this study provides evidence for important roles in cation selectivity for both the N-terminal half of the M4-M5 loop and the adjacent transmembrane helice(s).
authors.affiliation: Departments of Medicine and Biochemistry, McGill University, Montreal, Quebec H3G 1A4 Canada.
authors.forename: R
authors.initials: R
authors.lastname: Blostein
authors.forename: L
authors.initials: L
authors.lastname: Dunbar
authors.forename: M
authors.initials: M
authors.lastname: Mense
authors.forename: R
authors.initials: R
authors.lastname: Scanzano
authors.forename: A
authors.initials: A
authors.lastname: Wilczynska
authors.forename: M J
authors.initials: MJ
authors.lastname: Caplan
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002412:Cations / Q000378:metabolism*; D002478:Cells, Cultured; D004285:Dogs; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014162:Transfection; D014981:Xenopus
chemical_list: D002412:Cations; D011993:Recombinant Fusion Proteins; D010042:Ouabain; D012964:Sodium; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011188:Potassium