ABCB1 p.Ala348Cys
| Predicted by SNAP2: | C: N (66%), D: D (80%), E: D (75%), F: D (75%), G: N (61%), H: D (71%), I: D (59%), K: D (75%), L: D (66%), M: D (59%), N: D (71%), P: D (80%), Q: D (71%), R: D (71%), S: N (53%), T: N (78%), V: D (59%), W: D (80%), Y: D (75%), |
| Predicted by PROVEAN: | C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: N, T: N, V: D, W: D, Y: D, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[hide] Residue G346 in transmembrane segment six is invol... Biochemistry. 2007 Sep 4;46(35):9899-910. Epub 2007 Aug 14. 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 [PMID:17696319]
Abstract [show]
Multidrug transporters such as P-glycoprotein require considerable inter-domain communication to couple energy utilization with substrate translocation. Elucidation of the regions or residues involved in these communication pathways is a key step in the eventual molecular description of multidrug transport. We used cysteine-scanning mutagenesis to probe the functional involvement of residues along the cytoplasmic half of transmembrane segment 6 (TM6) and its extension toward the nucleotide binding domain. The mutation of one residue (G346C) in this segment adversely affected drug transport in cells. Further investigation using purified protein revealed that the underlying biochemical effect was a reduction in basal ATP hydrolysis. This G346C mutation also affected the stimulation of ATPase activity in a drug dependent manner but had no effect on drug binding, ATP binding, or ADP release. Homology modeling of P-glycoprotein indicated that the G346C mutation caused a steric interaction between TM5 and TM6, thereby precluding a helical movement required to support ATP hydrolysis.
Comments [show]
None has been submitted yet.
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.
X
ABCB1 p.Ala348Cys 17696319:66:368
status: NEW77 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.
X
ABCB1 p.Ala348Cys 17696319:77:23
status: NEW187 The Vmax in the presence of nicardipine was also reduced for Q347C but increased for the A348C and A354C isoforms.
X
ABCB1 p.Ala348Cys 17696319:187:89
status: NEW188 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.
X
ABCB1 p.Ala348Cys 17696319:188:0
status: NEW218 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.
X
ABCB1 p.Ala348Cys 17696319:218:135
status: NEW229 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.
X
ABCB1 p.Ala348Cys 17696319:229:588
status: NEW231 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.
X
ABCB1 p.Ala348Cys 17696319:231:383
status: NEW234 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).
X
ABCB1 p.Ala348Cys 17696319:234:126
status: NEW238 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.
X
ABCB1 p.Ala348Cys 17696319:238:411
status: NEW