ABCMdb

ABCB1 p.Ala980Cys

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Publications

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

No. Sentence Comment

7 In contrast, the A980C mutation within TM12 enhanced the rate of ATP hydrolysis; once again, this was due to modified basal activity. Login to 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
121 In complete contrast, the A980C mutation in TM12 caused an increase in the basal ATPase activity (Vmax=203 ( 40 nmol min-1 mg-1 ) compared to the cysteine-less isoform. Login to comment
142 Again, in contrast, the nicardipine-stimulated activity of the A980C isoform was almost 3-fold higher (Vmax =1352 ( 50 nmol min-1 mg-1 ) than observed for the cysteine-less control. 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
164 Excluding the A980C isoform, which displayed enhanced ATPase activity, the effects were primarily a reduction of ATP hydrolysis. Login to comment
172 Figure 5A presents a representative autoradiogram of [γ-32 P]-azido-ATP binding to the mutants L976C, F978C, A980C, V988C, G989C, and Q990C. Login to comment
174 The results indicate that at a concentration of 10 μM [γ-32 P]-azido-ATP there was a discernible difference between the binding of the ATP analogue to L976C, F978C, and A980C. Login to comment
175 Mutant isoforms L976C and F978C displayed a 4and 15-fold decrease in binding, whereas A980C showed a 1.8-fold increase in binding at the concentration of [γ-32 P]-azido-ATP used. Login to comment
179 Figure 5C shows the densitometric analysis of the dose-response curve for the cysteine-less, A980C, L976C, and F978C isoforms. Login to comment
180 At the range of [γ-32 P]-azido-ATP concentrations studied, there was no discernible difference between the labeling of cysteine-less (Bmax = 1.00, KD = 25 μM) and A980C (Bmax = 1.21, KD = 23 μM) isoforms of ABCB1. Login to comment
183 However, mutant A980C, which was characterized by a significantly higher ATPase activity, did not show any appreciable changes in nucleotide binding. Login to comment
187 To investigate whether such differences are a reflection of a physical asymmetry between the helices and their surroundings, we attempted to rationalize three of the functionally perturbed TM12 single cysteine mutants (i.e., F978C, A980C, and V988C) byreference toamolecularmodel ofABCB1. Login to comment
193 (C) Data obtained for the cysteine-less, L976C, F978C, and A980C isoforms were analyzed by densitometry, and the amount bound was plotted as a function of the [γ-32 P]-azido-ATP concentration. Login to comment
199 Of the six mutations, only A980C resulted in increased ATPase activity. Login to comment
220 The most dramatic effects in TM12 were observed in residues at the extracellular Table 3: Nucleotide Binding to ABCB1a ABCB1 isoform [32 P]-N3-ATP [32 P]-N3-ATP+ 1 mM ATP [32 P]-N3-ATP+ 1 mM ADP Cys-less 1.00 0.21 ( 0.05 0.23 ( 0.06 L976C 0.21 ( 0.05 0.10 ( 0.02 0.05 ( 0.03 F978C 0.07 ( 0.01 ND ND A980C 1.81 ( 0.71 0.45 ( 0.10 0.15 ( 0.08 V988C 0.53 ( 0.20 ND ND G989C 0.83 ( 0.04 0.10 ( 0.05 0.13 ( 0.06 Q990C 1.05 ( 0.30 0.19 ( 0.11 0.01 ( 0.01 a The ABCB1 isoforms were incubated with 10 μM [γ32 P]-azido-ATP in the presence or absence of excess unlabeled nucleotides (1 mM). Login to comment
229 (B) Mutation to A980C allows for hydrogen bonding to S850 (orange), increasing TM12-TM9 contacts. 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

123 A980C shifted to a low level of BM accessibility following nucleotide binding by ABCB1, and again, an opposite shift was seen for CM. 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
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