The role of loop 6/7 in folding and functional performance of Na,K-ATPase.
Alanine substitutions were made for 15 amino acids in the cytoplasmic loop between transmembrane helices 6 and 7 (L6/7) of the human alpha(1)-subunit of Na,K-ATPase. Most mutations reduced Na,K-ATPase activity by less than 50%; however, the mutations R834A, R837A, and R848A reduced Na,K-ATPase activity by 75, 89, and 66%, respectively. Steady-state phosphoenzyme formation from ATP was reduced in mutants R834A, R837A, and R848A, and R837A also had a faster E(2)P --> E(2) dephosphorylation rate compared with the wild-type enzyme. Effects of L6/7 mutations on the phosphorylation domain of the protein were also demonstrated by (18)O exchange, which showed that intrinsic rate constants for P(i) binding and/or reaction with the protein were altered. Although most L6/7 mutations had no effect on the interaction of Na(+) or K(+) with Na,K-ATPase, the E825A, E828A, R834A, and R837A mutations reduced the apparent affinity of the enzyme for both Na(+) and K(+) by 1.5-3-fold. 1-Bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU(3+)), a competitive antagonist of Rb(+) and Na(+) occlusion, was used to test whether charged residues in L6/7 are involved in binding monovalent cations and cation antagonists. Br-TITU(3+) inhibited ouabain binding to wild type Na,K-ATPase with an IC(50) of 30 microM. Ouabain binding to the E825A, E828A, R834A, or R837A mutants was still inhibited by Br-TITU(3+), indicating that Br-TITU(3+) does not bind to charged residues in L6/7. This observation makes it unlikely that L6/7 functions as a cytoplasmic cation binding site in Na,K-ATPase, and together with the effects of L6/7 mutations on phosphate interactions with the enzyme suggests that L6/7 is important in stabilizing the phosphorylation domain and its relationship to the ion binding sites of the protein.
Xu
Guiyan
G
Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
Kane
David J
DJ
Faller
Larry D
LD
Farley
Robert A
RA
D000595:Amino Acid Sequence; D006801:Humans; D028581:Lanthanoid Series Elements / Q000494:pharmacology; D008969:Molecular Sequence Data; D010042:Ouabain / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D017510:Protein Folding; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000502:physiology; D013329:Structure-Activity Relationship
D028581:Lanthanoid Series Elements; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium