ABCMdb

ABCB1 p.Gly984Cys

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Publications

PMID: 16492138 [PubMed] Loo TW et al: "Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket."

No. Sentence Comment

41 A series of double cysteine mutants containing L65C in TM1 with another cysteine in TMD2 (C-terminal TMD containing TM7-TM12) predicted to line the drug-binding pocket [34] (i.e. F942C or T945C in TM11 and L975C, V981C, V982C, G984C or A985C in TM12) were also constructed for cross-linking analysis. Login to comment
60 Disulphide cross-linking analysis Mutants L65C, F942C, T945C, L975C, V981C, V982C, G984C, A985C, L65C/F942C, L65C/T945C, L65C/975C, L65C/V981C, L65C/V982C, L65C/G984C and L65C/A985C were transiently expressed in HEK-293 cells. Login to comment
160 Accordingly, Figure 6 Disulphide cross-linking of P-gp mutants (A) Membranes were prepared from HEK-293 cells (A) expressing mutants L65C, L65C/T945C, L65C/V982C, L65C/G984C or L65C/A985C. Login to comment

PMID: 19456124 [PubMed] Crowley E et al: "Transmembrane helix 12 modulates progression of the ATP catalytic cycle in ABCB1."

No. Sentence Comment

67 This necessitated the centrifugation (100000g for 30 min) of 500 μL Table 1: Mutagenic Oligonucleotide Primers Used To Generate TM12 Mutationsa mutation primer sequence (50 -30 ) diagnostic restriction digest L976C GAGGATGTTCTAtgtGTATTTTCAGCTGTTG -SpeI F978C GTTCTACTAGTATgTTCtGCaGTTGTCTTTGGTG +PstI A980C CTACTAGTATTTTCAtgcGTTGTCTTTGGTGCCATGGCC -PvuII V982C CTAGTATTTTCAGCgGTTtgCTTTGGTGCCATGGCC -PvuII G984C GCTGTTGTCTTTtGTGCtATGGCCGTGG -NcoI M986C GTATTTGGTGCttgtGCtGTGGGGCAAGTC -NcoI V988C GGTGCCATGGCCtgtGGGCAAGTCAGTTC -BstXI G989C CTTTGGTGCCATGGCCGTGtGcCAAGTCAGTTCATTTGC +BstXI Q990C GGCCGTGGGGtgtGTCtcTTCATTTGCTCC +EarI a Primer sequences contain an introduced cysteine residue (bold) and additional silent mutations (lower case), with respect to the coding sequence that generates or removes the indicated restriction site. Login to comment
151 There was, however, a drug-dependent difference on the stimulation of ATPase activity observed with the G984C and FIGURE 4: Maximal ATPase activity of TM12 mutant isoforms. Login to comment
155 Table 2: Potency and Degree of Drug Stimulation of ATP Hydrolysis by ABCB1a nicardipine vinblastine EC50 (μM) fold stimulation EC50 (μM) fold stimulation Cys-less 4.1 ( 1.1 4.0 ( 0.6 5.91 ( 2.9 2.2 ( 0.2 L976C 5.2 ( 0.2 7.4 ( 1.4 10.0 ( 0.0 3.5 ( 0.6 F978C 24.1 ( 2.3b 9.5 ( 1.4 42.9 ( 4.3b 2.3 ( 0.5 A980C 3.4 ( 0.3 5.1 ( 0.9 12.3 ( 1.8 3.2 ( 0.8 V982C 5.8 ( 0.9 4.2 ( 0.5 2.0 ( 0.7 1.8 ( 0.2 G984C 37.6 ( 11.2b 16.2 ( 6.6b 6.7 ( 1.7 6.2 ( 2.3 M986C 9.2 ( 0.8 4.7 ( 1.1 15.0 ( 2.0b 2.8 ( 0.7 V988C 3.9 ( 0.6 3.1 ( 0.1 7.3 ( 2.3 1.9 ( 0.2 G989C 13.6 ( 1.5 5.1 ( 1.6 4.9 ( 0.9 2.4 ( 0.3 Q990C 6.9 ( 1.1 3.7 ( 1.0 NDc NDc S992C 4.9 ( 0.5 4.2 ( 0.6 7.1 ( 2.6 2.3 ( 0.4 F994C 1.7 ( 0.4 3.2 ( 0.8 5.9 ( 2.5 1.6 ( 0.3 a ATPase activity was plotted as a function of the drug concentration and potency (EC50) and degree of stimulation obtained by nonlinear regression of the dose-response relationship equation. Login to comment
160 For G984C, nicardipine was associated with a reduction in potency and degree of stimulation, whereas the influence of vinblastine was similar to that in cysteine-less ABCB1. Login to comment

PMID: 20731718 [PubMed] Crowley E et al: "Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1."

No. Sentence Comment

82 The rate of labelling (i.e. t1 / 2) was divided into fast (L986C- G992C, average t1 / 2 $ 8 min) and slow (L976C- G984C, average t1 / 2 $ 25 min) kinetics between the carboxy-half and the amino-half, respectively. Login to comment
92 However, two residues (G984C and M986C) in the central region of TM12 did display labelling above background, albeit with Lext values of approximately 20%. Login to comment
139 Mutant CM BM FM Lext (%) t1 / 2 (min) Lext (%) t1 / 2 (min) Lext (%) t1 / 2 (min) L976C 38 ± 5 29 ± 12 66 ± 14 29 ± 18 - - A980C 53 ± 6 34 ± 1 54 ± 8 20 ± 9 - - V982C 98 ± 14 15 ± 6 164 ± 50 27 ± 17 - - G984C 73 ± 14 29 ± 6 84 ± 24 22 ± 7 13 ± 10 ND M986C 89 ± 30 25 ± 10 51 ± 5 3 ± 2 21 ± 2 ND V988C 53 ± 6 37 ± 18 221 ± 63 18 ± 12 - - G989C 64 ± 7 15 ± 6 21 ± 3 9 ± 2 - - S992C 55 ± 4 22 ± 6 51 ± 5 4 ± 1 32 ± 3 25 ± 5 F994C 51 ± 10 11 ± 9 111 ± 35 13 ± 10 129 ± 24 8 ± 3 Conformational changes - central region Two of the residues examined in the central region (G984C and M986C) of TM12 have been shown to accommodate partial labelling with FM, suggestive of aqueous accessibility in the basal state. At M986C, the extent of labelling with the hydrophilic probe was increased following the addition of nonhydrolysable nucleotide. Login to comment
143 G984C underwent a broadly similar shift in topography as M986C, although the degree of alteration was somewhat less striking. Login to comment
164 ABCB1 isoform Catalytic intermediate CM BM FM L976C Basal ++ +++ ) AMP-PNP +++ ++ ) Vi trapped +++ +++ ) A980C Basal ++ ++ ) AMP-PNP +++ + ) Vi trapped +++ +++ ) V982C Basal +++ +++ ) AMP-PNP +++ +++ ) Vi trapped +++ +++ ) G984C Basal +++ +++ + AMP-PNP +++ +++ + Vi trapped +++ ++ ) M986C Basal +++ ++ + AMP-PNP ++ +++ ++ Vi trapped +++ ++ ) V988C Basal ++ +++ ) AMP-PNP +++ +++ ) Vi trapped +++ +++ ) G989C Basal ++ + ) AMP-PNP ++ ++ ) Vi trapped ++ + ) S992C Basal ++ ++ + AMP-PNP +++ +++ ++ Vi trapped ++ ++ + F994C Basal ++ +++ +++ AMP-PNP ++ +++ ++ Vi trapped +++ +++ + reflect localization at the membrane-solute interface. Login to comment
183 The homology models predict that both V982C and G984C, located within the centre of the helix, experience little change in molecular environment upon ATP binding, which is in agreement with the biochemical data. Login to comment
196 Surprisingly, although FM labels G984C, the homology model suggests that this residue faces into the lipid bilayer. Login to comment
197 However, G984C is not in a very densely packed region, and it may be possible for FM to gain access to the residue via the translocation pore. Login to comment
198 In addition, the loss of labelling of G984C with FM following progression to a vanadate-trapped state suggests that labelling is not optimal and therefore is very sensitive to even minor environmental changes. Login to comment
228 The structures are shown in the panel as viewed from the translocation pore; the relative environments of V982C (cyan) and G984C (blue) are unaltered by nucleotide binding. Login to comment

PMID: 9405384 [PubMed] Loo TW et al: "Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate."

No. Sentence Comment

83 There was no detectable activity with mutants S344C, G341C, and G984C, whereas mutants A342C, G346C, Q347C, A985C, G989C, and Q990C had much reduced activity (10-40%). Login to comment
87 Mutants G341C and G984C, however, appeared to be degraded quite rapidly. Login to comment
96 Mutants G341C, S344C, G346C, G984C, and G989C were not assayed because of their low or defective expression (Fig. 2B). Login to comment

PMID: 16545467 [PubMed] Shilling RA et al: "New light on multidrug binding by an ATP-binding-cassette transporter."

No. Sentence Comment

78 Single-cysteine mutants in human P-glycoprotein that are protected from cross-linking to cysteine-reactive MTS substrate analogues by the non-reactive substratea P-glycoprotein residueb Corresponding residue in V. cholera MsbA Cysteine-reactive substrate I340C (6) G293 MTS-rhodamine A841C (9) A151 MTS-rhodamine L975C (12) T285 MTS-rhodamine V981C (12) M291 MTS-rhodamine V982C (12) F292 MTS-rhodamine S222C (4) A175 MTS-verapamil L339C (6) M291 MTS-verapamil A342C (6) M295 MTS-verapamil I868C (10) F180 MTS-verapamil F942C (11) Q256 MTS-verapamil T945C (11) A259 MTS-verapamil G984C (12) L294 MTS-verapamil a Data adapted from [24,2]. Login to comment
76 Single-cysteine mutants in human P-glycoprotein that are protected from cross-linking to cysteine-reactive MTS substrate analogues by the non-reactive substratea P-glycoprotein residueb Corresponding residue in V. cholera MsbA Cysteine-reactive substrate I340C (6) G293 MTS-rhodamine A841C (9) A151 MTS-rhodamine L975C (12) T285 MTS-rhodamine V981C (12) M291 MTS-rhodamine V982C (12) F292 MTS-rhodamine S222C (4) A175 MTS-verapamil L339C (6) M291 MTS-verapamil A342C (6) M295 MTS-verapamil I868C (10) F180 MTS-verapamil F942C (11) Q256 MTS-verapamil T945C (11) A259 MTS-verapamil G984C (12) L294 MTS-verapamil a Data adapted from [24,25]. Login to comment