CONFORMATIONAL ALTERATIONS RESULTING FROM MUTATIONS IN CYTOPLASMIC DOMAINS OF THE ALPHA-SUBUNIT OF THE NA,K-ATPASE

This paper summarizes experiments concerned with the functional conseq uences of mutations in cytoplasmic regions of the ctl subunit of the N a,K-ATPase, in particular the amino terminus, the first cytoplasmic lo op between transmembrane segments M2 and M3, and the major cytoplasmic loop between M4 and M5. In the first mutation (alpha 1M32), 32 residu es were removed from the N-terminus. The second mutation (E233K) was i n the putative beta strand of M2-M3 loop and the third, comprised the replacement of the amino terminal half of loop M4-M5 of the Na,K-ATPas e with the homologous segment (residues 356-519) of the gastric H,K-AT Pase. The first two mutations, either separately or in combination (al pha 1M32E233K), shift the equilibrium between the major conformational states of the enzyme, E-1 and E-2, in favor of E-1 as manifested by i ncreased apparent affinity for ATP, lower catalytic turnover, and decr eased sensitivity to inhibition by vanadate. The striking changes obse rved with alpha 1M32E233K suggests interactions between the N-terminus , the beta-strand in the M2-M3 loop and the catalytic phosphorylation site. The behavior of these mutants contrasts with that of least one m utant involving substitution of a residue in the putative cation bindi ng pocket, nam S775A in the fifth transmembrane segment (Arguello, J. M., & Lingrel, J. B. J. Biol. em. 270: 22764-22771, 1995). Although it s K+/ATP antagonism resembles that of the foregoing cytoplasmic mutant s, its vanadate sensitivity is unaltered suggesting that changes in ap parent affinity for Am are secondary to changes in K+ ligation. The qu estion of cation selectivity, in particular that of Na+ versus protons , has been addressed in structure/function analysis of a cytoplasmic c himera involving the M4-M5 loop. Transport studies performed in the pr esence or absence of Na+ and at low versus high pH indicate a marked a lteration in cation affinity and/or selectivity. This results suggests coupling of an alteration in the large M4-M5 cytoplasmic domain to ca tion binding in, presumably, the juxtapositioned transmembrane domain.