ABCMdb

PMID: 11801592

Menguy T, Corre F, Juul B, Bouneau L, Lafitte D, Derrick PJ, Sharma PS, Falson P, Levine BA, Moller JV, le Maire M
Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase.
J Biol Chem. 2002 Apr 12;277(15):13016-28. Epub 2002 Jan 18., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC8 p.Asp818Ala 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 ABCC8 p.Asp818Ala ABCC8 p.Asp818Ala 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 ABCC8 p.Asp818Ala ABCC8 p.Asp818Ala 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 ABCC8 p.Asp818Ala 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 ABCC8 p.Asp818Ala 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 ABCC8 p.Asp818Ala 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 ABCC8 p.Asp818Ala ABCC8 p.Asp818Ala 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 351 ABCC8 p.Leu814Ala Similarly, Asahi et al. (27) report that the site-directed mutants L814A, I816A, or M817A have Ca2ϩ transport activity so low that no functional interaction with phospholamban could be evaluated. Login to comment