MUTATIONAL ANALYSIS OF TRANS-MEMBRANE HELICES M3, M4, M5 AND M7 OF THE FAST-TWITCH CA2-ATPASE()

Mutational analysis of trans-membrane helices M3, M4, M5 and M7 of the Ca2+-ATPase revealed a novel phenotypic variant, M4 [Y295A (the one-l etter symbols are used for amino acid residues throughout)], displayin g an increased affinity for P-i and decreased affinity for MgATP, whil e retaining the ability to translocate Ca2+ ions across the endoplasmi c reticulum membrane. The properties of this mutant suggest that the E 1-E2 equilibrium is shifted towards E2, and indicate a key role for th is aromatic residue (Y295) at the end of trans-membrane helix M4. A mu tant containing three amino acid residue substitutions at the end of t he seventh trans-membrane helix, M7 (F834A, F835A, T837F), showed a co mplete loss of ATPase activity and a reduced ability to phosphorylate with Pi, although MgATP-initiated phosphorylation was unaffected. The observation that single mutations in this cluster of residues had no e ffect on Ca2+ transport suggests that correct anchoring of the helix a t the lipid-water interface by these aromatic residues is important in the functioning of the ATPase. Mutation of polar residues in helix M3 did not affect inhibition of the ATPase by thapsigargin, thapsivillos in A or t-butyl hydroquinone, suggesting that hydrogen-bonding partner s for the essential -OH groups on these inhibitors lie elsewhere in th e ATPase.