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.
Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
Guiyan
G
Xu
David J
DJ
Kane
Larry D
LD
Faller
Robert A
RA
Farley
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