The Role of Loop 6/7 in Folding and Functional Performance of Na,K-ATPase

• Published in: The Journal of biological chemistry 2004-10, Vol.279 (44), p.45594-45602
• Main Authors: Xu, Guiyan, Kane, David J, Faller, Larry D, Farley, Robert A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Humans; Lanthanoid Series Elements - pharmacology; Molecular Sequence Data; Ouabain - metabolism; Phosphorylation; Potassium - metabolism; Protein Folding; Sodium - metabolism; Sodium-Potassium-Exchanging ATPase - chemistry; Sodium-Potassium-Exchanging ATPase - physiology; Structure-Activity Relationship
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M408147200

Alanine substitutions were made for 15 amino acids in the cytoplasmic loop between transmembrane helices 6 and 7 (L6/7) of the human α 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 (Hoving, S., Bar-Shimon, M., Tijmes, J. J., Goldschleger, R., Tal, D. M., and Karlish, S. J. D. (1995) J. Biol. Chem . 270, 29788–29793), 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 μ m . 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.