Mutagenesis of Residues Involved in Control of the Ca2+ Entry Pathway and Conformational Changes Associated with Ca2+ Binding in the SR Ca2+-ATPase
Rapid kinetic measurements were used to study the rate of Ca2+ dissociation from the high-affinity Ca2+ sites of the dephosphoenzyme (i.e., from the E1Ca2 form toward the cytoplasmic side) as well as the rate of Ca2+ binding with associated conformational changes (E2E1Ca2 transition) in the wild type and mutants of the sarcoplasmic reticulum Ca2+-ATPase expressed in mammalian cells. Cluster mutations as well as single mutations in transmembrane segment M3 resulted in conspicuous effects on the rate of Ca2+ migration. Furthermore, mutation of Asp59 in transmembrane segment M1 to arginine exerted a profound effect on Ca2+ interaction. The data demonstrate an important role for M3 residues in control of the Ca2+ entry pathway and provide functional evidence in support of a close relationship between this pathway and the water-accessible channel leading between transmembrane segments M1 and M3 in the thapsigargin stabilized E2 structure. In addition, rapid kinetic measurements demonstrated that the hydrogen bond network involving Asp813 of loop L6-7 and Lys758 of M5 is important for the E2E1Ca2 transition.
mutagenesis ca2+ entry ca2+-atpase rapid kinetics ca2+ dissociation sarcoplasmic reticulum m3