Importance of transmembrane segment M3 of the sarcoplasmic reticulum Ca2+-ATPase for control of the gateway to the Ca2+ sites

The specific functional roles of various parts of the third transmembrane segment (M3) of the sarcoplasmic reticulum Ca2+-ATPase were examined by functionally characterizing a series of mutants with multiple or single substitutions of M3 residues. Steady-state and transient kinetic measurements, assisted by computer simulation of the time and Ca2+ dependences of the phosphorylation level, were used to study the partial reaction steps of the enzyme cycle, including the binding and dissociation of Ca2+ at the high affinity cytoplasmically facing sites. The mutation Lys-Leu-Asp-Glu(255) --> Glu-ne-Glu-Ris resulted in a conspicuous increase in the rate of Ca2+ dissociation as well as a displacement of the major conformational equilibria of the phosphoenzyme and dephosphoenzyme forms. The point mutant Phe(256) --> Ala also showed an increased rate of Ca2+ dissociation, whereas a conspicuous decrease both in the rate of Ca2+ dissociation and in the rate of Ca2+ binding was found for the mutant Gly-Glu-Gln-Leu(260) --> Ile-His-Leu-Ile. These findings suggest that the NH2-terminal half of M3 is involved in control of the gateway to the Ca2+ sites. The main effect of two mutations to the COOH-terminal half of M3, Ser-Lys-Val-Ile-Ser(265) --> Thr-Gly-Val-Ala-Val and Leu-Ile-Cys-Val-Ala-Val-Trp-Leu-Ile(274) --> Phe-Leu-Gly-Val-Ser-Phe-Phe-Ile-Leu, was a block of the dephosphorylation.