ABCMdb

PMID: 9685357

Menguy T, Corre F, Bouneau L, Deschamps S, Moller JV, Champeil P, le Maire M, Falson P
The cytoplasmic loop located between transmembrane segments 6 and 7 controls activation by Ca2+ of sarcoplasmic reticulum Ca2+-ATPase.
J Biol Chem. 1998 Aug 7;273(32):20134-43., [PubMed]

Sentences

No. Mutations Sentence Comment

27 ABCC8 p.Asp818Ala ABCC8 p.Asp818Ala 1 The abbreviations used are: SR, sarcoplasmic reticulum; SERCA, sarco(endo)plasmic reticulum Ca2ϩ -ATPase; p95, C-terminal proteolytic fragment of SR Ca2ϩ -ATPase starting at residue Lys-120; p83C, C-terminal proteolytic fragment of SR Ca2ϩ -ATPase starting at residue Glu-243; p28N, N-terminal proteolytic fragment of SR Ca2ϩ -ATPase ending at residue Thr-242; p20C or p20, C-terminal proteolytic fragment of SR Ca2ϩ -ATPase starting at residue Gly-808; p19C or p19, C-terminal proteolytic fragment of SR Ca2ϩ -ATPase starting at residue Asp-818; C12E8, octaethylene glycol monododecyl ether; Tes, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; Ab, Antibody; WT, wild type; ADA, aspartate mutant D813A/D818A; AAA aspartate mutant D813A/ D815A/D818A; PVDF, polyvinylidine difluoride; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; E1, conformational state of Ca2ϩ -ATPase of high affinity for calcium; E2, conformational state of Ca2ϩ -ATPase of low affinity for calcium. Login to comment 43 ABCC8 p.Asp818Ala ABCC8 p.Asp818Ala The N-terminal part of the loop contains three aspartic residues (Asp-813, -815, and -818), which were mutated in a cluster, D813A/D818A and D813A/D815A/D818A. Login to comment 47 ABCC8 p.Asp818Ala ABCC8 p.Asp818Ala The experiments were designed to characterize the functional consequences of mutations of aspartic residues, D813A/D818A and D813A/D815A/D818A, as well as those of mutations of the proline residues in the same loop, Pro-811, -812, -820, and -821. Login to comment 52 ABCC8 p.Asp818Ala ABCC8 p.Asp818Ala MATERIALS AND METHODS Mutation and Expression of Ca2ϩ -ATPase in Yeast-The single mutants E309Q and E771Q and the cluster mutants D813A/D818A (referred to later as ADA) and D813A/D815A/D818A (referred to later as AAA) were obtained as in Ref. 32. Login to comment 92 ABCC8 p.Asp818Ala ABCC8 p.Asp818Ala Therefore, these residues were mutated to alanine residues to give the following mutants: D813A/D818A (referred to as ADA), D813A/ D815A/D818A (referred to as AAA), P811A/P812A, P812A, P820A/P821A, and P821A. Login to comment 211 ABCC8 p.Asp818Ala An important outcome of our investigation is that cluster mutations of the aspartic residues shifted the Ca2ϩ -ATPase affinity for Ca2ϩ to much higher concentrations than those required to activate wild type ATPase; the K0.5 for Ca2ϩ activation of phosphorylation from ATP of the D813A/D818A mutated ATPase was shifted from the micromolar to the millimolar range, while the maximal level of phos- FIG. 6. Login to comment 219 ABCC8 p.Asp818Ala ABCC8 p.Asp818Ala In agreement with this, the double and triple mutants D813A/D818A and D813A/D815A/D818A only displayed a very low ATPase activity when they were tested at pCa 4, (Fig. 2). Login to comment 283 ABCC8 p.Asp818Gly Thus, as an alternative model, the L6-7 loop could be a direct contributor in the control of Ca2ϩ binding to Ca2ϩ - ATPase, as deduced from (a) our previous demonstration that Ca2ϩ has some affinity for the Gly-808-Gly-994 peptide but not for the shorter Asp-818-Gly-994 peptide (p20 and p19, respectively; Ref. 32) and (b) the present data that show that the removal of some of the oxygen atoms (Asp 3 Ala) located in the region from Gly-808 to Asp-818 of the L6-7 loop decreases the affinity for calcium by almost 3 orders of magnitude. Login to comment