Glutamate 90 at the Luminal Ion Gate of Sarcoplasmic Reticulum Ca2+-ATPase Is Critical for Ca2+ Binding on Both Sides of the Membrane

The roles of Ser(72), Glu(90), and Lys(297) at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca2+-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca2+ concentration of phosphorylation by P-i (Ca2+ gradient-dependent E2P formation) showed a reduction of the apparent affinity for luminal Ca2+ 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 Ca2+ 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.BeF3- complex to luminal Ca2+. 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 Ca2+ occupying a luminally exposed low affinity Ca2+ site of E2P, thus identifying an essential locus (a leaving site) on the luminal Ca2+ exit pathway. Mutants with alanine or leucine replacement of Glu(90) further displayed a marked slowing of the Ca2+ binding transition as well as slowing of the dissociation of Ca2+ from Ca(2)E1 back toward the cytoplasm, thus demonstrating that Glu(90) is also critical for the function of the cytoplasmically exposed Ca2+ sites on the opposite side of the membrane relative to where Glu(90) is located.