title: Glutamate 90 at the luminal ion gate of sarcoplasmic reticulum Ca2+-ATPase is critical for Ca(2+) binding on both sides of the membrane.
abstract: The roles of Ser(72), Glu(90), and Lys(297) at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca(2+)-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca(2+) concentration of phosphorylation by P(i) ("Ca(2+) gradient-dependent E2P formation") showed a reduction of the apparent affinity for luminal Ca(2+) in mutants with alanine or leucine replacement of Glu(90), whereas arginine replacement of Glu(90) or Ser(72) allowed E2P formation from P(i) even at luminal Ca(2+) concentrations much too small to support phosphorylation in wild type. The latter mutants further displayed a blocked dephosphorylation of E2P and an increased rate of conversion of the ADP-sensitive E1P phosphoenzyme intermediate to ADP-insensitive E2P as well as insensitivity of the E2.BeF(3)(-) complex to luminal Ca(2+). Altogether, these findings, supported by structural modeling, indicate that the E2P intermediate is stabilized in the mutants with arginine replacement of Glu(90) or Ser(72), because the positive charge of the arginine side chain mimics Ca(2+) occupying a luminally exposed low affinity Ca(2+) site of E2P, thus identifying an essential locus (a "leaving site") on the luminal Ca(2+) exit pathway. Mutants with alanine or leucine replacement of Glu(90) further displayed a marked slowing of the Ca(2+) binding transition as well as slowing of the dissociation of Ca(2+) from Ca(2)E1 back toward the cytoplasm, thus demonstrating that Glu(90) is also critical for the function of the cytoplasmically exposed Ca(2+) sites on the opposite side of the membrane relative to where Glu(90) is located.
authors.affiliation: Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Department of Physiology and Biophysics, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Johannes D
authors.initials: JD
authors.lastname: Clausen
authors.forename: Jens Peter
authors.initials: JP
authors.lastname: Andersen
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D030562:Databases, Protein; D018698:Glutamic Acid / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D018698:Glutamic Acid; D012694:Serine; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D008239:Lysine; D002118:Calcium