Specific sites in the cytoplasmic N terminus modulate conformational transitions of the Na,K-ATPase.
The cytoplasmic N terminus of the Na,K-ATPase is a highly charged and flexible structure that comprises three predicted helical regions including H1 spanning residues 27 to 33 and H2 spanning residues 42 to 50. Previous deletion mutagenesis experiments showed that deletion of residues up to and including most of H2 shifts the E(1)/E(2) conformational equilibrium toward E(1). The present study describes a clustered charge-to-alanine mutagenesis approach designed to delineate specific sites within the N terminus that modulate the steady-state E(1) <--> E(2) and E(1)P <--> E(2)P poise. Criteria to assess shifts in poise include (i) sensitivity to inhibition by inorganic orthovanadate to assess overall poise; (ii) K(+)-sensitivity of Na-ATPase measured at micromolar ATP to assess changes in the E(2)(K) + ATP --> E(1) x ATP + K(+) rate; (iii) K'(ATP) for low-affinity ATP binding at the latter step; (iv) overall catalytic turnover, and (v) the E(1)P --> E(2)P transition. The results of alanine replacements in H1 (31KKE) suggest that this site stabilizes E(2)P and to a lesser extent E(2). In H2, residues within 47HRK have a role in stabilizing E(2) but not E(2)P as revealed with double mutants 31KKE --> AAA/47H --> A and 31KKE --> AAA/47HRK --> AAA. Taken together, these observations suggest that sites 31KKE in H1 and 47HRK in H2 have distinct roles in modulating the enzyme's conformational transitions during the catalytic cycle of the enzyme.
Scanzano
Rosemarie
R
Departments of Biochemistry and Medicine, McGill University, Montreal, Quebec H3G 1A4, Canada.
Segall
Laura
L
Departments of Biochemistry and Medicine, McGill University, Montreal, Quebec H3G 1A4, Canada.
Blostein
Rhoda
R
Departments of Biochemistry and Medicine, McGill University, Montreal, Quebec H3G 1A4, Canada. Electronic address: Rhoda.Blostein@mcgill.ca.
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011188:Potassium / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D014638:Vanadates / Q000737:chemistry
D014638:Vanadates; D000254:Sodium-Potassium-Exchanging ATPase; D000409:Alanine; D011188:Potassium