FUNCTIONAL CONSEQUENCES OF ALTERATIONS TO HYDROPHOBIC AMINO-ACIDS LOCATED IN THE M(4) TRANSMEMBRANE SECTOR OF THE CA2-ATPASE OF SARCOPLASMIC-RETICULUM()

Those hydrophobic residues between Ile298 and Ile315 in transmembrane segment M4 of the Ca2+-ATPase of sarcoplasmic reticulum, not previousl y mutated, were mutated systematically in ways that would alter their size or polarity, and functional consequences were measured. Fourteen residues in this sequence are organized as juxtapositions of large, hy drophobic (Val, Leu, Ile) and small (Ala, Gly) residues, and these wer e altered so that large residues were substituted for small and vice v ersa. Several mutants exhibited diminished Ca2+ transport, but mutants A305V and A306V lost all Ca2+ transport function. In both cases, the mutants were phosphorylated with ATP in the presence of Ca2+ and with inorganic phosphate only in the absence of Ca2+, indicating that the C a2+-binding sites were intact. Reduced Ca2+ affinity, as measured by C a2+ dependence of phosphorylation from ATP, was observed for mutant A3 05V. In both mutants, the ADP-insensitive phosphoenzyme intermediate ( E2P) decayed slowly relative to the wild-type enzyme, suggesting that the E2P to E2 conformational transition was impaired, slowing the rate of the phosphatase reaction. Double mutants which reversed the order of Val304 and Ala305 and Ala306 and Ile307, resulted in the same pheno type as the single Ala mutations. These results, combined with our pre vious demonstration that Glu309 is a Ca2+ binding residue, that Pro312 is involved in E1P to E2P conformational changes, and that Gly310 is involved in E2P to E2 conformational changes, support the hypothesis t hat transmembrane segment M4 plays a key role in the Ca2+ transport fu nction of the Ca2+-ATPase through its involvement in both the binding of Ca2+ and the subsequent conformational changes which bring about th e translocation of Ca2+ to the lumen of the membrane.