ABCMdb

ABCB1 p.Ala354Cys

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Publications

PMID: 17696319 [PubMed] Storm J et al: "Residue G346 in transmembrane segment six is involved in inter-domain communication in P-glycoprotein."

No. Sentence Comment

66 Table 1: Mutagenic Oligonucleotide Primers Used to Generate TM6 Mutationsa mutation primer sequence 5'-3' diagnostic restriction digest S344C TTAATTGGGGCcTTTtGTGTTGGACAG + Eco 0109 I V345C TTAATTGGGGCaTTcAGTtgTGGACAGGCAT + Bsm I G346C F:GGGGCTTTTAGTGTTtGcCAGGCgTCTCCAAGCATTG +Bsa H I R:CAATGCTTGGAGAcGCCTGgCaAACACTAAAAGCCCC Q347C GCTTTTAGTGTTGGAtgcGCATCTCCAAG + Fsp I A348C GTTGGACAGtgcagcCCAAGCATTG + Bsg I S349C GGACAGGCATgcCCAAGTATTGAAGCA + Sph I A354C CAAGCATTGAAtgcTTTGCAAATG + Bsm I G360C CAAATGCAAGAtGcGCAGCTTATG + Fsp I 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
77 Mutants (G346C, Q347C, A348C, S349C, A354C, and G360C) in pBlueBac_4.5 (2 µg) were cotransfected into Spodoptera frugiperda (Sf9) cells with Bac-N-Blue DNA (0.25 µg) and Cellfectin (10 µg) in medium without FCS and antibiotics. Login to comment
187 The Vmax in the presence of nicardipine was also reduced for Q347C but increased for the A348C and A354C isoforms. Login to comment
189 For Q347C, the Km remained lower in the presence of nicardipine, and in the presence of both nicardipine and vinblastine, the Km value was higher for A354C. Login to comment
194 It was increased for S344C, A354C, and G360C, and decreased for Q347C, but the potency of stimulation was unchanged compared to that of cysteine-less P-gp. Login to comment
229 Table 2: Michaelis-Menten Parameters for ATPase Activity of P-gpa basal nicardipine vinblastine Vmax (µmol/min/mg) Km (mM) Vmax (µmol/min/mg) Km (mM) Vmax (µmol/min/mg) Km (mM) CYS- 0.48 ( 0.10 0.54 ( 0.05 1.37 ( 0.19 0.38 ( 0.03 0.98 ( 0.10 0.38 ( 0.02 S344C 0.30 ( 0.05 0.34 ( 0.05 1.71 ( 0.28 0.45 ( 0.07 0.84 ( 0.09 0.28 ( 0.03 V345C 0.43 ( 0.07 0.42 ( 0.06 1.69 ( 0.29 0.24 ( 0.01 0.82 ( 0.15 0.36 ( 0.04 G346C 0.06 ( 0.01* 0.21 ( 0.05* 0.15 ( 0.02* 0.24 ( 0.05 0.06 ( 0.02* 0.26 ( 0.09 Q347C 0.25 ( 0.03 0.21 ( 0.03* 0.47 ( 0.06* 0.13 ( 0.01* 0.39 ( 0.13 0.19 ( 0.02 A348C 0.79 ( 0.15 0.37 ( 0.03 2.90 ( 0.52* 0.40 ( 0.05 1.58 ( 0.30* 0.41 ( 0.06 S349C 0.38 ( 0.04 0.36 ( 0.06 1.00 ( 0.10 0.23 ( 0.03 0.45 ( 0.04 0.27 ( 0.03 A354C 0.47 ( 0.10 0.50 ( 0.10 2.21 ( 0.37* 0.59 ( 0.08* 1.29 ( 0.23 0.61 ( 0.15* G360C 0.35 ( 0.03 0.36 ( 0.02 1.88 ( 0.12 0.46 ( 0.08 1.00 ( 0.07 0.43 ( 0.02 a ATPase activity was plotted as a function of ATP concentration and the Vmax and Km parameters obtained by nonlinear regression of the Michaelis-Menten equation. Login to comment
231 Table 3: Potency and Degree of Drug Stimulation of ATP Hydrolysis by P-gpa nicardipine vinblastine EC50 (µM) fold-stimulation EC50 (µM) fold-stimulation CYS3.2 ( 0.3 3.4 ( 0.3 4.2 ( 0.7 2.4 ( 0.2 S344C 5.4 ( 0.3 5.9 ( 0.4* 12.2 ( 0.5* 2.9 ( 0.2 V345C 3.2 ( 0.1 3.9 ( 0.1 9.3 ( 1.1* 2.1 ( 0.1 G346C 5.5 ( 1.1 3.4 ( 0.3 ND 1.0 ( 0.1* Q347C 2.0 ( 0.6 2.0 ( 0.1* ND 1.3 ( 0.1* A348C 3.4 ( 0.4 3.9 ( 0.3 9.0 ( 2.1* 2.3 ( 0.2 S349C 2.3 ( 0.1 2.6 ( 0.1 ND 1.2 ( 0.1* A354C 3.5 ( 0.2 5.0 ( 0.3* 6.6 ( 0.5 2.5 ( 0.2 G360C 4.8 ( 0.5 5.5 ( 0.3* 5.9 ( 0.4 2.7 ( 0.1 a ATPase activity was plotted as a function of drug concentration and the potency and degree of stimulation obtained by nonlinear regression of the dose-response relationship equation. Login to comment
238 Table 4: Displacement of [125 I]-Iodo-aryl-azido-prazosin Binding to P-gp Isoformsa mutant nicardipine (30 µM) vinblastine (100 µM) rhodamine123 (100 µM) hoechst33342 (100 µM) CYS- 0.36 ( 0.06 0.38 ( 0.06 1.29 ( 0.34 0.27 ( 0.05 S344C 0.48 ( 0.03 0.40 ( 0.02 1.61 ( 0.47 0.12 ( 0.01 G346C 0.41 ( 0.06 0.30 ( 0.03 1.54 ( 0.29 0.16 ( 0.05 Q347C 0.56 ( 0.10 0.45 ( 0.10 1.27 ( 0.16 0.16 ( 0.09 A348C 0.40 ( 0.03 0.36 ( 0.06 1.25 ( 0.18 0.20 ( 0.04 S349C 0.39 ( 0.05 0.34 ( 0.05 2.18 ( 0.62 0.31 ( 0.13 A354C 0.43 ( 0.04 0.39 ( 0.07 1.39 ( 0.25 0.21 ( 0.06 G360C 0.52 ( 0.12 0.34 ( 0.01 1.40 ( 1.37 0.23 ( 0.10 a The fraction of [125 I]-IAAP labeled P-gp isoforms was determined in the presence of drug and was expressed as a proportion of the amount in the absence of drug. Login to comment

PMID: 18303860 [PubMed] Storm J et al: "Cytosolic region of TM6 in P-glycoprotein: topographical analysis and functional perturbation by site directed labeling."

No. Sentence Comment

52 Single cysteine containing mutant isoforms of ABCB1 (S344C, V345C, G346C, Q347C, S349C, A354C, and G360C) were constructed as previously described (44) using site directed mutagenesis with the Altered Sites II (Promega) or the QuickChange (Stratagene) systems. Login to comment
118 A complete graphical representation of similar labeling time-courses of two selected isoforms (V345C and A354C) is shown in Figure 2. Login to comment
124 By contrast, isoform A354C (Figure 2) displays conformation dependent labeling of probes. Login to comment
128 BM labeled the A354C isoform equally well in the basal (Lext ) 99 ( 15%) and nucleotide bound conformations (Lext ) 130 ( 11%). Login to comment
141 The maximal extent of labeling (Lext) was ascertained for the (a) V345C and (b) A354C mutant isoforms of ABCB1 using the three maleimide containing probes, FM, BM and CM. Login to comment
151 Similarly, large changes in accessibility are observed with A354C (Figure 2). Login to comment
161 While there were no effects on the basal ATPase activity for any of the mutant isoforms, except for a 2-fold increase in the basal ATPase activity of A354C following CM labeling (Table 2), there were numerous distinct effects on the maximal extent of drug stimulated ATPase activity. Login to comment
166 A more dramatic effect was observed in this isoform following CM modification, namely, that vinblastine could no longer stimulate the basal ATPase Table 1: Summary of Relative Accessibilities of TM6 Residuesa ABCB1 isoform catalytic intermediate FM BM CM S344C basal - +++ +++ AMPPNP + +++ +++ vanadate + +++ +++ V345C basal - + +++ AMPPNP + ++ +++ vanadate + + +++ G346C basal - + ++ AMPPNP - ++ ++ vanadate + + ++ Q347C basal + + +++ AMPPNP + + ++ vanadate + + +++ S349C basal - + + AMPPNP + ++ + vanadate - ++ +++ A354C basal + +++ +++ AMPPNP ++ +++ +++ vanadate + + +++ G360C basal +++ +++ ++ AMPPNP +++ +++ ++ vanadate + ++ +++ a The accessibility of each introduced cysteine residue was determined using fluorescein-maleimide (FM), BODIPY-maleimide (BM) or coumarin-maleimide (CM). Login to comment
170 Table 2: Effects of Covalent Modification by CM on ATPase Activity of Mutant ABCB1 TM6 Isoformsa basal Vmax (nmol Pi min-1 mg-1) stimulated Vmax (nmol Pi min-1 mg-1) (-)CM (+) CM % change (-)CM (+) CM % change S344C 188 ( 62 192 ( 56 857 ( 216 317 ( 93* -63 V345C 563 ( 85 388 ( 41 -31 1173 ( 355 937 ( 292 -20 G346C 101 ( 18 102 ( 12 268 ( 47 186 ( 59 -30 Q347C 66 ( 6 41 ( 15 -37 161 ( 33 61 ( 4* -62 A354C 126 ( 10 229 ( 27* +81 535 ( 72 353 ( 18* -34 G360C 396 ( 82 508 ( 134 +28 1244 ( 252 810 ( 108 -34 a The ABCB1 isoforms containing mutations within TM6 were examined for ATPase activity prior to and following reaction with the hydrophobic maleimide probe CM. Login to comment
175 Table 3: Effects of Coumarin Labeling on the Potency of Drugs to Stimulate ATP Hydrolysis by Mutant ABCB1 TM6 Isoformsa EC50 nicardipine (µM) EC50 vinblastine (µM) (-) CM (+) CM (-) CM (+) CM S344C 3.0 ( 0.4 0.5 ( 0.1* 12.1 ( 0.5 na V345C 2.3 ( 0.4 3.9 ( 0.6 4.9 ( 1.4 8.5 ( 1.9 G346C 4.8 ( 1.8 7.1 ( 1.7 na na Q347C 2.7 ( 0.4 1.9 ( 0.8 na na A354C 2.4 ( 0.2 1.6 ( 0.2 2.5 ( 0.2 na G360C 2.7 ( 0.4 5.1 ( 0.2* 5.7 ( 0.1 8.6 ( 1.2 a The potency of nicardipine and vinblastine to stimulate ATP hydrolysis was examined for a wide range of drug concentrations (10-9-10-4 M). Login to comment
187 A354C, which was avidly labeled by CM, displayed a both small reduction in the Vmax for nicardipine stimulation of ATPase activity due to a reduced degree of stimulation by this compound. Login to comment
194 The isoforms S344C, Q347C, and A354C displayed significant reductions in drug stimulated ATP hydrolysis following CM labeling, and this may be attributed to altered drug binding. Login to comment
204 Table 4: Effects of Coumarin Labeling on Drug Binding in Mutant ABCB1 TM6 Isoformsa vinblastine nicardipine S344C Q347C A354C S344C Q347C A354C (-) CM 0.34 ( 0.09 0.39 ( 0.06 0.61 ( 0.11 0.35 ( 0.02 0.40 ( 0.03 0.64 ( 0.09 (+) CM 0.46 ( 0.06 0.34 ( 0.09 0.39 ( 0.21 0.27 ( 0.03 0.41 ( 0.06 0.57 ( 0.16 a [125I]-IAAP photoaffinity labeling was undertaken in the S344C, Q347C and A354C mutant TM6 isoforms prior to and following labeling with coumarin maleimide. Login to comment
212 Following labeling, three of the residues we have studied (S344C, Q347C, and A354C) were associated with a reduction in the magnitude to which drug substrates stimulate ATP hydrolysis. Login to comment
213 In addition, an alteration in the potency of the stimulation, or abrogation of ATPase activity, was observed for a number of residues (S344C, V345C, S349C, A354C, and G360C). Login to comment
234 The observations were also undertaken for two residues (A354C and G360C) within the extension of TM6 that links the helix to the N-terminal NBD. Login to comment
254 Similar analysis helps rationalize the A354C data and provides further evidence that our homology models are a valid reflection of TM helix packing. Login to comment
256 A354C forms part of the TM3-TM6 contact interface with the sulfur atom being accessible to the translocation pore, thus enabling covalent modification by the hydrophilic FM and zwitterionic BM probes. Login to comment
257 Further examination of the residues adjacent to A354C revealed a number of charged and polar residues in its immediate vicinity, in particular, K181, E184, and D188 from TM3, and the adjacent E353 from TM6, creating a favorable local environment for FM and BM. Login to comment
258 Comparison of the AMP-PNP state model with the basal state model shows a disruption of the TM3-TM6 interface around A354C (Figure 4c). Login to comment
259 In the basal conformation homology model, the side chain of A354C was angled away from the pore, while charged residues in the local vicinity faced into the translocation pore (Figure 4d), decreasing the surface accessibility of A354C to the translocation pore and thus the accessibility to FM. Login to comment
260 The position of A354C at the interface of TM3 and TM6, which both have a lipid exposed face, allowed the hydrophobic CM probe to access A354C through the bilayer. Login to comment
261 On the basis of the observations in the present investigation and from previous studies (42-44), it appears that TM6 FIGURE 4: Molecular modeling insight into the V345C and A354C mutations in ABCB1. Login to comment
267 (c) A view from the top of the pore shows the AMP-PNP configuration: A354C (orange) hydrogen bonds to its nearest neighbour, D188 (cyan). Login to comment
269 Adjacent charged residues from TM3 (cyan) and TM6 (purple) face the translocation pore, while A354C is situated in a cleft between TM3 and TM6. Login to comment