PMID: 17696319

Storm J, O'Mara ML, Crowley EH, Peall J, Tieleman DP, Kerr ID, Callaghan R
Residue G346 in transmembrane segment six is involved in inter-domain communication in P-glycoprotein.
Biochemistry. 2007 Sep 4;46(35):9899-910. Epub 2007 Aug 14., 2007-09-04 [PubMed]
Sentences
No. Mutations Sentence Comment
66 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:66:324
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:66:229
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 17696319:66:136
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 17696319:66:183
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 17696319:66:489
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 17696319:66:450
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 17696319:66:408
status: NEW
view ABCB1 p.Ser349Cys details
ABCB1 p.Ala348Cys
X
ABCB1 p.Ala348Cys 17696319:66:368
status: NEW
view ABCB1 p.Ala348Cys details
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 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:77:16
status: NEW
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ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:77:9
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 17696319:77:48
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 17696319:77:37
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 17696319:77:30
status: NEW
view ABCB1 p.Ser349Cys details
ABCB1 p.Ala348Cys
X
ABCB1 p.Ala348Cys 17696319:77:23
status: NEW
view ABCB1 p.Ala348Cys details
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
79 ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 17696319:79:32
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 17696319:79:42
status: NEW
view ABCB1 p.Val345Cys details
Alternatively, mutant isoforms (S344C and V345C) in pFastBac-1 were transformed into DH10Bac E. coli. Login to comment
117 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:117:45
status: NEW
view ABCB1 p.Gly346Cys details
The native P-gp model underwent an in silico G346C mutation and was further energy minimized to refine Transport actiVity ) cells in lower right quandrant cells in the lower and upper right quadrants × 100% the structure. Login to comment
118 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:118:35
status: NEW
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The quality of the native P-gp and G346C mutant P-gp models was assessed using PROCHECK (41) and WHATIF (42). Login to comment
133 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:133:35
status: NEW
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In addition, the data obtained for G346C displayed a shift from the lower right quadrant toward the upper right quadrant. Login to comment
135 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:135:194
status: NEW
view ABCB1 p.Gly346Cys details
The relative expression levels for the various mutants did not show any consistent trend; for example, the dot plot on Figure 2a suggests higher expression of cysteine-less protein, compared to G346C protein whereas the immuno-blot in Figure 1a indicates the opposite. Login to comment
138 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:138:169
status: NEW
view ABCB1 p.Gly346Cys details
The striking observation from the data in Figure 2b was that only one of the mutations caused a significant reduction in the transport function of P-gp, namely, isoform G346C. Login to comment
164 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:164:108
status: NEW
view ABCB1 p.Gly346Cys details
Figure 4 demonstrates a representative curve for the ATPase activity of purified cysteine-less P-gp and the G346C isoform in the absence or presence of the modulator nicardipine (30 µM). Login to comment
170 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:170:68
status: NEW
view ABCB1 p.Gly346Cys details
The most dramatic effects on ATPase activity were observed with the G346C isoform (bold, italic text), which is consistent with the altered rhodamine123 transport data (Figure 2). Login to comment
172 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:172:48
status: NEW
view ABCB1 p.Gly346Cys details
(a) Quadrant analysis for the cysteine-less and G346C isoforms obtained from the flow cytometry assay. Login to comment
177 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:177:29
status: NEW
view ABCB1 p.Gly346Cys details
Panel 3 was obtained for the G346C isoform in the absence of inhibitor. Login to comment
183 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:183:66
status: NEW
view ABCB1 p.Gly346Cys details
approximately 9-fold to 0.056 ( 0.007 µmol min-1 mg-1 by the G346C mutation. Login to comment
185 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:185:79
status: NEW
view ABCB1 p.Gly346Cys details
In contrast, unlike the control cysteine-less P-gp, the ATPase activity of the G346C mutant was not stimulated by the presence of 30 µM vinblastine. Login to comment
187 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:187:61
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 17696319:187:99
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ala348Cys
X
ABCB1 p.Ala348Cys 17696319:187:89
status: NEW
view ABCB1 p.Ala348Cys details
The Vmax in the presence of nicardipine was also reduced for Q347C but increased for the A348C and A354C isoforms. Login to comment
188 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:188:245
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:188:235
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Ala348Cys
X
ABCB1 p.Ala348Cys 17696319:188:0
status: NEW
view ABCB1 p.Ala348Cys details
A348C also showed increased Vmax in the presence of vinblastine, but the overall fold stimulation for both substrates was comparable to that of cysteine-less P-gp. Overall, the Km of ATP was not changed for most P-gp isoforms, but for G346C and Q347C, it was reduced in the basal state when compared to that of cysteine-less P-gp. Login to comment
189 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:189:4
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 17696319:189:150
status: NEW
view ABCB1 p.Ala354Cys details
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 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:194:64
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 17696319:194:21
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 17696319:194:39
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 17696319:194:28
status: NEW
view ABCB1 p.Ala354Cys details
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
196 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:196:30
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:196:23
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 17696319:196:41
status: NEW
view ABCB1 p.Ser349Cys details
In contrast, mutations G346C, Q347C, and S349C abrogated the stimulation of ATP hydrolysis by vinblastine. Login to comment
199 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:199:81
status: NEW
view ABCB1 p.Gly346Cys details
The SDS-PAGE gel (8%) was silver-stained and demonstrates the retention of P-gp (G346C) on the column through the washing steps (up to 30 mM imidazole) and its elution in 120 mM imidazole. Login to comment
201 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:201:50
status: NEW
view ABCB1 p.Gly346Cys details
(b) Following reconstitution, an aliquot of P-gp (G346C) was subjected to sucrose density centrifugation. Login to comment
206 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:206:71
status: NEW
view ABCB1 p.Gly346Cys details
FIGURE 4: ATPase activity of purified, reconstituted cysteine-less and G346C isoforms of P-gp. Login to comment
207 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:207:104
status: NEW
view ABCB1 p.Gly346Cys details
ATPase activity was determined as a function of ATP concentration for cysteine-less P-gp (O, b) and the G346C mutant (0, 9) in the absence (open symbols) or presence (closed symbols) of 30 µM nicardipine. Login to comment
218 ABCB1 p.Ala348Cys
X
ABCB1 p.Ala348Cys 17696319:218:135
status: NEW
view ABCB1 p.Ala348Cys details
Figure 5 provides representative autoradiograms for the specific photolabeling of the substrate [125 I]- IAAP to the cysteine-less and A348C isoforms of P-gp. Login to comment
224 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:224:74
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:224:106
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 17696319:224:81
status: NEW
view ABCB1 p.Ser349Cys details
Consequently, the lack of any effect of vinblastine on ATPase activity in Q347C, S349C, and in particular G346C (Tables 2 and 3) is not due to major disruption of the drug binding site for this substrate by the TM6 mutation. Login to comment
225 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:225:38
status: NEW
view ABCB1 p.Gly346Cys details
Nucleotide Binding Characteristics of G346C. Login to comment
227 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:227:37
status: NEW
view ABCB1 p.Gly346Cys details
To further examine the impact of the G346C mutation on P-gp function, the binding of nucleotide was examined using the photoactive ATP analogue, [γ-32 P]-8-azido-ATP. Login to comment
228 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:228:105
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:228:106
status: NEW
view ABCB1 p.Gly346Cys details
Figure 6a demonstrates the dose-dependent binding of [γ-32 P]-8-azido-ATP to the cysteine-less and G346C isoforms of P-gp. Login to comment
229 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:229:507
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:229:425
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 17696319:229:269
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 17696319:229:347
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 17696319:229:827
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 17696319:229:746
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 17696319:229:668
status: NEW
view ABCB1 p.Ser349Cys details
ABCB1 p.Ala348Cys
X
ABCB1 p.Ala348Cys 17696319:229:588
status: NEW
view ABCB1 p.Ala348Cys details
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 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:231:342
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:231:344
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:231:302
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:231:304
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 17696319:231:206
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 17696319:231:208
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 17696319:231:255
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Val345Cys
X
ABCB1 p.Val345Cys 17696319:231:257
status: NEW
view ABCB1 p.Val345Cys details
ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 17696319:231:517
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 17696319:231:519
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 17696319:231:470
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 17696319:231:472
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 17696319:231:430
status: NEW
view ABCB1 p.Ser349Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 17696319:231:432
status: NEW
view ABCB1 p.Ser349Cys details
ABCB1 p.Ala348Cys
X
ABCB1 p.Ala348Cys 17696319:231:383
status: NEW
view ABCB1 p.Ala348Cys details
ABCB1 p.Ala348Cys
X
ABCB1 p.Ala348Cys 17696319:231:385
status: NEW
view ABCB1 p.Ala348Cys details
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
234 ABCB1 p.Ala348Cys
X
ABCB1 p.Ala348Cys 17696319:234:126
status: NEW
view ABCB1 p.Ala348Cys details
The binding of [125I]-IAAP was measured by autoradiography of photoaffinity labeled purified, reconstituted cysteine-less and A348C isoforms of P-gp in the presence or absence of various drug substrates (100 µM). Login to comment
238 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:238:357
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:238:303
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 17696319:238:249
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Gly360Cys
X
ABCB1 p.Gly360Cys 17696319:238:573
status: NEW
view ABCB1 p.Gly360Cys details
ABCB1 p.Ala354Cys
X
ABCB1 p.Ala354Cys 17696319:238:519
status: NEW
view ABCB1 p.Ala354Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 17696319:238:465
status: NEW
view ABCB1 p.Ser349Cys details
ABCB1 p.Ala348Cys
X
ABCB1 p.Ala348Cys 17696319:238:411
status: NEW
view ABCB1 p.Ala348Cys details
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
241 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:241:165
status: NEW
view ABCB1 p.Gly346Cys details
However, the results do indicate that at a concentration of 100 µM, there was no discernible difference in the binding of the ATP analogue to cysteine-less and G346C isoforms of P-gp. Login to comment
242 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:242:64
status: NEW
view ABCB1 p.Gly346Cys details
To confirm that the binding of nucleotide was unaffected by the G346C mutation, the ability of ATP to displace [γ-32P]-8-azido-ATP (100 µM) photolabeling was assessed. Login to comment
243 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:243:132
status: NEW
view ABCB1 p.Gly346Cys details
Figure 6b shows a representative autoradiogram for the dose-dependent displacement of [γ-32 P]-8-azido-ATP binding by ATP for G346C. Login to comment
244 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:244:157
status: NEW
view ABCB1 p.Gly346Cys details
The potency of displacement by ATP for the control cysteine-less isoform (IC50 ) 0.37 ( 0.03 mM) was not significantly different from that observed with the G346C (IC50 ) 0.21 ( 0.12 mM) isoform, suggesting that the affinity for ATP binding is likely to be similar. Login to comment
248 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:248:91
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:248:92
status: NEW
view ABCB1 p.Gly346Cys details
An increase in the potency of displacement of [γ-32 P]-8-azido-ATP binding by ADP in G346C would signify greater affinity of the dinucleotide, thereby suggesting a slower rate of release. Login to comment
249 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:249:175
status: NEW
view ABCB1 p.Gly346Cys details
Figure 6c demonstrates that the ADP-mediated displacement of [γ-32 P]-8-azido-ATP binding displayed similar potencies for the cysteine-less (IC50 ) 67 ( 16 µM) and G346C (IC50 ) 72 ( 12 µM) isoforms of the protein. Login to comment
250 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:250:62
status: NEW
view ABCB1 p.Gly346Cys details
In summary, the impaired drug transport rate displayed by the G346C mutant isoform of P-gp is characterized by a reduced basal ATPase activity and an altered communication between the TMDs and NBDs subsequent to vinblastine, but not nicardipine, binding. Login to comment
252 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:252:47
status: NEW
view ABCB1 p.Gly346Cys details
Homology Modeling of TM6 in P-gp Suggests that G346C Disrupts the TMD R-Helical Packing. Login to comment
253 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:253:90
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:253:138
status: NEW
view ABCB1 p.Gly346Cys details
To provide a possible structural explanation for the unexpected functional effects of the G346C mutation, we have analyzed the native and G346C P-gp homology models, which map over 90% of the P-gp primary sequence, including TM6 in its entirety and the TMD-NBD and NBD-NBD interfaces. Login to comment
255 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:255:82
status: NEW
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The backbone root-mean-square deviation (RMSD) between Sav1866 and the native and G346C P-gp models was 0.98 Å, while the RMSD between the two P-gp homology models was <0.01 Å, indicating that the backbone conformations are highly similar. Login to comment
262 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:262:33
status: NEW
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ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:262:282
status: NEW
view ABCB1 p.Gly346Cys details
The introduction of an in silico G346C mutation in the P-gp homology model shows that the 346C side chain adopts an energetically favorable conformation when it is coordinated by A342 (TM6), F303, FIGURE 6: Nucleotide binding properties of purified, reconstituted cysteine-less and G346C P-gp. Login to comment
263 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:263:119
status: NEW
view ABCB1 p.Gly346Cys details
(a) The autoradiogram shows the amount of [γ-32P]-8-azido-ATP bound to purified, reconstituted cysteine-less and G346C isoforms of P-gp. Login to comment
266 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:266:46
status: NEW
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(b) Purified, reconstituted cysteine-less and G346C isoforms of P-gp were labeled with [γ-32P]-8-azido-ATP (100 µM) in the presence or absence of varying concentrations of ATP. Login to comment
269 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:269:46
status: NEW
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(c) Purified, reconstituted cysteine-less and G346C isoforms of P-gp were labeled with [γ-32P]-8-azido-ATP (10 µM) in the presence or absence of varying concentrations of ADP. Login to comment
286 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:286:116
status: NEW
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For example, the modulator nicardipine stimulated hydrolysis to an identical extent and at a similar potency in the G346C isoform compared that in the cysteine-less isoform. Login to comment
292 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:292:133
status: NEW
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This accounts for the unaffected nicardipine stimulation, reduced vinblastine stimulation, and yet unaffected vinblastine binding to G346C mutant. Login to comment
293 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 17696319:293:46
status: NEW
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ABCB1 p.Leu339Cys
X
ABCB1 p.Leu339Cys 17696319:293:115
status: NEW
view ABCB1 p.Leu339Cys details
ABCB1 p.Ser349Cys
X
ABCB1 p.Ser349Cys 17696319:293:56
status: NEW
view ABCB1 p.Ser349Cys details
A similar conclusion was demonstrated for the Q347C and S349C isoforms (Tables 3 and 4) and was also shown for the L339C isoform (29). Login to comment
301 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:301:78
status: NEW
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(b) Front view of the N-terminal half of the mutant P-gp model shows that the G346C side chain (CPK spacefill) is closely coordinated by one residue from TM6, A342 (red liquorice), and two residues from TM5, F303 (lower) and I306 (upper red liquorice). Login to comment
302 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:302:35
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:302:82
status: NEW
view ABCB1 p.Gly346Cys details
A342 and I306 sterically constrain G346C, while hydrogen bonds are formed between G346C and F303. Login to comment
307 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:307:40
status: NEW
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The fact that the primary effect of the G346C mutation is a reduction in basal ATP hydrolysis appears counter-intuitive since the mutated residue lies within the TMD, while ATP hydrolysis occurs at the NBDs. Login to comment
310 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:310:113
status: NEW
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As shown in Figure 8, ATP hydrolysis comprises multiple discrete steps and one, or more, will be affected by the G346C mutation. Login to comment
316 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:316:112
status: NEW
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The signature motif coordination of NBDs may be the rate-limiting step of the catalytic cycle, but how does the G346C mutation affect it, and is it required for basal ATP hydrolysis? Login to comment
322 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:322:4
status: NEW
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The G346C mutation prevents conformational changes within the TMD such that basal route B occurs at a slower rate or alternatively, prevents step (ii) from occurring, and the protein proceeds via sub-basal route A. Login to comment
323 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:323:17
status: NEW
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In addition, the G346C mutation prevents step (iia) (drug-stimulated ATP hydrolysis) from occurring for vinblastine but not nicardipine. Login to comment
325 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:325:130
status: NEW
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In order to provide a theoretical visualization (as opposed to a structure-based interpretation, which remains impossible) of the G346C mutation, we have constructed a P-gp homology model based upon the structure of Sav1866. Login to comment
326 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:326:353
status: NEW
view ABCB1 p.Gly346Cys details
The structure-based comparison of the X-ray crystallographic data for Sav1866 (33) with the 8 Å electron microscopy data for P-gp (30) demonstrates that several of the long R-helices in Sav1866 can be superimposed onto long rod-shaped EM densities observed by electron microscopy of 2D P-gp FIGURE 8: Catalytic cycle for P-gp, the influence of the G346C mutation. Login to comment
336 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:336:49
status: NEW
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Moreover, the regulation may be perturbed by the G346C mutation in TM6, suggesting a key role for this region of the protein for both the basal- and drug-stimulated activity. Login to comment
337 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 17696319:337:145
status: NEW
view ABCB1 p.Gly346Cys details
The rate-limiting step in ATP hydrolysis by P-gp (and other ABC transporters) is assumed to be the signature motif coordination of NBDs, and the G346C mutation in TM6 may retard the rate of this step. Login to comment