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 Ca(2+) dissociation from the high-affinity Ca(2+) sites of the dephosphoenzyme (i.e., from the E(1)Ca(2) form toward the cytoplasmic side) as well as the rate of Ca(2+) binding with associated conformational changes (E(2) --> E(1)Ca(2) transition) in the wild type and mutants of the sarcoplasmic reticulum Ca(2+)-ATPase expressed in mammalian cells. Cluster mutations as well as single mutations in transmembrane segment M3 resulted in conspicuous effects on the rate of Ca(2+) migration. Furthermore, mutation of Asp(59) in transmembrane segment M1 to arginine exerted a profound effect on Ca(2+) interaction. The data demonstrate an important role for M3 residues in control of the Ca(2+) 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 E(2) structure. In addition, rapid kinetic measurements demonstrated that the hydrogen bond network involving Asp(813) of loop L6-7 and Lys(758) of M5 is important for the E(2) --> E(1)Ca(2) transition.
Andersen
Jens Peter
JP
Department of Physiology, University of Aarhus, Denmark. jpa@fi.au.dk
Clausen
Johannes D
JD
Einholm
Anja Pernille
AP
Vilsen
Bente
B
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
D010446:Peptide Fragments; D011994:Recombinant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium