ABCMdb

ABCC8 p.Asp818Ala

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Publications

PMID: 11801592 [PubMed] Menguy T et al: "Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase."

No. Sentence Comment

2 We found that the D813A/D818A mutant that displays markedly low calcium affinity was capable of occluding Ca2؉ to the same extent as wild type ATPase. Login to comment
39 This part of the loop contains three conserved aspartic residues (Asp813 , Asp815 , and Asp818 ) whose cluster mutation generated the D813A/D818A and D813A/ D815A/D818A mutants that we found to display a marked reduction in the apparent affinity with which Ca2ϩ controls ATPase phosphorylation and turn-over (12). Login to comment
49 EXPERIMENTAL PROCEDURES Mutation and Expression of Ca2ϩ -ATPase in Yeast-The single mutants E309Q and E771Q and the cluster mutants D813A/D818A and D813A/D815A/D818A (referred to as ADA and AAA mutants, respectively) were obtained as previously described in Refs. 12 and 25. Login to comment
150 In Fig. 2B, it is seen that with the D813A/D818A mutant in the L6-7 loop, the radioactivity profile is slightly lower than that observed for the wild type ATPase, but the Ca2ϩ -ATPase content is also lower (the reason for this difference probably is unrelated to differences in the level of expression, see below). Login to comment
180 Comparison of the Ca2؉ -occlusion reaction in presence of Cr⅐ATP for the wild type, L6-7 loop ADA mutated Ca2؉ -ATPase (D813A/ D818A), E309Q mutated Ca2؉ -ATPase and SR ؉ control membrane using gel filtration coupled to Western blot. Login to comment
181 The calcium occlusion induced by Cr⅐ATP is tested on light membrane fractions containing wild type Ca2ϩ -ATPase (chromatogram A), the ADA mutant (D813A/ D818A) in the L6-7 loop (chromatogram B) and the E309Q mutant (chromatogram D) Data for control membranes alone (Control Mb; chromatogram A, B, and C) or in the presence of SR Ca2ϩ -ATPase (SR ϩ Control Mb; chromatogram C) are also shown. Login to comment
212 Nanomole of occluded Ca2ϩ per mg of Ca2ϩ -ATPase Aggregation state of the Ca2ϩ -ATPaseLower estimates Upper estimates SR ϩ control membrane 5.6 Ϯ 0.2 11 Ϯ 0.18 Monomeric Wild type 5.4 Ϯ 0.9 9.8 Ϯ 0.9 Monomeric D813A-D818A (ADA) 4.2 Ϯ 0.9 9.6 Ϯ 0.9 Monomeric E309Q 0 0 Aggregated or oligomeric E771Q Not measurable Not measurable Not solubilized D813A-D815A-D818A (AAA) Not measurable Not measurable Not solubilized helical turn involving residues 816-819 (4). Login to comment

PMID: 12763779 [PubMed] Corre F et al: "Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase."

No. Sentence Comment

6 Bioquímica y Biología Molecular y Genética, E.U. Enfermería y T.O., Universidad de Extremadura, Cáceres, Spain cSchool of Biosciences, University of Birmingham, B15 2TT United Kingdom dDepartment of Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark ABSTRACT: We have found that despite a markedly low calcium affinity the D813A/D818A mutant is capable, after complexation with Cr.ATP, of occluding Ca2+ to the same extent (1-2 Ca2+ per ATPase monomer) as wild-type ATPase. Login to comment
13 lemairem@dsvidf.cea.fr part of the L6-7 loop (p808-818) interacted with Ca2+.1 Cluster mutation Ca2+- ATPase constructs, D813A-D818A (later called ADA) and D813A-D815A-D818A, displayed a marked reduction in the apparent affinity with which Ca2+ controls ATPase phosphorylation and turnover.2,3 We have rationalized our findings in terms of involvement of the conserved aspartic residues of the L6-7 loop in the interaction with calcium ions during the initial steps of the Ca2+ binding process.4 The crystal structures of SERCA 1a,5,6 however, show the acidic residues of the L6-7 loop in a conformation unsuitable for multiple coordination by Ca2+. Login to comment
20 Solubilization by C12E8 of Various Cr.ATP-reacted Ca2+-ATPase Species as a Function of Calcium In similar experiments we found that, as previously reported,7 the E309Q mutant does not occlude Ca2+, but several attempts to measure Cr.ATP-induced Ca2+ occlusion by the E771Q (previously also reported to display no calcium occlusion7) and the D813A-D815A-D818A mutants failed, because we were unable to recover any Ca2+-ATPase after elution from the HPLC column. Login to comment

PMID: 9685357 [PubMed] Menguy T et al: "The cytoplasmic loop located between transmembrane segments 6 and 7 controls activation by Ca2+ of sarcoplasmic reticulum Ca2+-ATPase."

No. Sentence Comment

27 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 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 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 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 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 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 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

PMID: 9211861 [PubMed] Falson P et al: "The cytoplasmic loop between putative transmembrane segments 6 and 7 in sarcoplasmic reticulum Ca2+-ATPase binds Ca2+ and is functionally important."

No. Sentence Comment

4 Two cluster mutants of Ca2؉ -ATPase, D813A/D818A and D813A/D815A/D818A, expressed in the yeast Saccharomyces cerevisiae, were found to have a very low Ca2؉ -ATPase activity. Login to comment
46 One single mutation E309Q, one double mutation D813A/D818A (referred to later as ADA), and one triple mutation D813A/D815A/D818A (AAA) were introduced. Login to comment
121 Preliminary attempts with molecular modeling of the Ca2ϩ -ATPase 808-818 sequence by energy minimization according to the ␣-lactalbumin Ca2ϩ binding site suggested that the side chains of Asp-813 and Asp-818, together with the peptide carbonyl group between Asp-815 and Ile-816 could be involved in Ca2ϩ binding to the L6-7 loop.2 We then took advantage of the functional expression of Ca2ϩ -ATPase in yeast (53) to test the activity of two cluster mutants in this region, i.e. D813A/D818A (ADA) and D813A/D815A/D818A (AAA). Login to comment
142 The reaction medium contained 50 ␮g/ml yeast membranes with wild type or mutant Ca2ϩ -ATPases: D813A/D818A (ADA), D813A/D815A/D818A (AAA), or E309Q. Login to comment