ABCMdb

ABCG2 p.Asn629Ala

None has been submitted yet.

Publications

PMID: 20739628 [PubMed] Ni Z et al: "Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport."

No. Sentence Comment

11 While T402A and T402R showed a significant global reduction in the efflux of mitoxantrone, Hoechst 33342, and BODIPY-prazosin, N629A exhibited significantly increased efflux activities for all of the substrates. Login to comment
15 Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Login to comment
68 The forward primers used for mutagenesis were as follows: N387A (5=- AAG CGT TCA TTC AAA GCC TTG CTG GGT AAT CCC-3=), Q398A (5=-CAG GCC TCT ATA GCT GCG ATC ATT GTC ACA GTC-3=), T402A (5=-GCT CAG ATC ATT GTC GCA GTC GTA CTG GGA CTG-3=), N629A (5=-CTG GGG CTT GTG GAA GGC TCA CGT GGC CTT GGC TTG-3=), T642A (5=-GAT TGT TAT TTT CCT CGC AAT TGC CTA CCT GAA ATT G-3=), and Y645F (5=-TTC CTC ACA ATT GCC TTC CTG AAA TTG TTA TTT C-3=). Login to comment
162 The levels of N387A, Q398A, T402A, N629A, T642A, and Y645F, determined by immunoblotting of whole cell lysates using beta-actin as an internal standard, were ϳ4.4-, 4.5-, 3.1-, 0.4-, 1.3-, and 1.5-fold that of wild-type BCRP (Fig. 2, A and B). Login to comment
192 In contrast, the efflux activities of N629A for all of the three substrates were increased up to fourfold. Login to comment
200 Representative areas of HEK-293 cells expressing wild-type BCRP and the mutants N387A, Q398A, T402A, N629A, T642A, and Y645F are shown. Login to comment
217 Thus N387A, Q398A, and T402A exhibited a significantly lower resistance to MX than wild-type BCRP, whereas N629A displayed an approximately threefold increase in resistance to MX. Login to comment
218 Similarly, resistance to SN-38 conferred by N629A was increased approximately threefold, and resistance to SN-38 conferred by other mutants was decreased by 60-90%. Login to comment
224 The Km values of N387A, T402A, T642A, and Y645F were comparable to that of wild-type protein; however, the Km values of Q398A and N629A were decreased by ϳ50-60%. Login to comment
225 This suggests that the ATP binding affinity to Q398A and N629A is increased. Login to comment
226 After normalization to the BCRP protein levels, the Vmax values of N387A, Q398A, and T402A were approximately one-half of that of wild-type BCRP, whereas the Vmax value of N629A was increased by ϳ90%. Login to comment
227 Importantly, the Vmax-to-Km ratio of N629A was increased approximately fourfold compared with that of wild-type protein, suggesting that this mutant possesses a more efficient intrinsic ATP hydrolysis capability than wild-type protein. Login to comment
231 FTC-inhibitable efflux activities of HEK-293 cells stably expressing wild-type and mutant BCRP Mitoxantrone BODIPY-Prazosin Hoechst 33342 ⌬F ⌬F= Ratio ⌬F ⌬F= Ratio ⌬F ⌬F= Ratio Wild-type BCRP 10.6 Ϯ 0.6 10.6 Ϯ 0.6 1.0 12.5 Ϯ 4.3 12.5 Ϯ 4.3 1.0 2150.0 Ϯ 25.5 2150.0 Ϯ 25.5 1.0 N387A 11.3 Ϯ 0.5 2.5 Ϯ 0.1* 0.2 54.5 Ϯ 5.8 12.3 Ϯ 1.3 1.0 5,024.7 Ϯ 570.5 1,131.7 Ϯ 128.5* 0.5 Q398A 28.1 Ϯ 5.2 6.3 Ϯ 1.2* 0.6 69.3 Ϯ 10.6 15.5 Ϯ 2.4 1.2 4,206.7 Ϯ 252.1 941.1 Ϯ 56.4* 0.4 T402A 12.3 Ϯ 2.4 4.0 Ϯ 0.8* 0.4 4.5 Ϯ 1.8 1.5 Ϯ 0.6* 0.1 999.3 Ϯ 352.9 322.4 Ϯ 113.8* 0.1 N629A 19.3 Ϯ 3.7 46.0 Ϯ 8.8* 4.3 12.8 Ϯ 1.5 30.5 Ϯ 3.6* 2.4 2,681.0 Ϯ 370.5 6,383.3 Ϯ 882.1* 3.0 T642A 17.0 Ϯ 0.3 12.9 Ϯ 0.2 1.2 15.4 Ϯ 1.3 11.8 Ϯ 1.0 0.9 1,860.7 Ϯ 462.3 1,420.4 Ϯ 352.9* 0.7 Y645F 16.8 Ϯ 2.3 11.6 Ϯ 1.6 1.1 15.1 Ϯ 4.6 10.4 Ϯ 3.2 0.8 1,358.7 Ϯ 35.5 937.0 Ϯ 24.5* 0.4 Values are means Ϯ SD of 3 independent experiments. Login to comment
238 Relative drug resistance of HEK-293 cells expressing wild-type and mutant BCRP MX SN-38 Dox Rho-123 IC50, nM RR (ratio) IC50, nM RR (ratio) IC50, nM RR IC50, nM RR pcDNA control 8.5 Ϯ 0.6 2.3 Ϯ 0.28 125.6 Ϯ 32.0 1,881 Ϯ 168.2 Wild-type BCRP 64.8 Ϯ 3.9 7.6 (1.0) 89.7 Ϯ 5.4 39.0 (1.0) 168.0 Ϯ 24.5 1.3 3,341 Ϯ 267.4 1.8 N387A 54.8 Ϯ 6.3 6.4 (0.2)* 44.8 Ϯ 6.3 19.5 (0.1)* 120.4 Ϯ 31.6 1.0 3,279 Ϯ 436.8 1.7 Q398A 49.9 Ϯ 4.6 5.9 (0.2)* 71.6 Ϯ 4.3 31.1 (0.2)* 173.4 Ϯ 36.5 1.4 2,534 Ϯ 376.5 1.3 T402A 43.3 Ϯ 7.6 5.1 (0.2)* 63.0 Ϯ 5.4 27.4 (0.2)* 129.4 Ϯ 31.4 1.0 2,140 Ϯ 210.7 1.1 N629A 76.5 Ϯ 12.3 9.0 (2.8)* 108.3 Ϯ 12.3 47.1 (2.9)* 164.6 Ϯ 14.8 1.3 3,051 Ϯ 286.5 1.6 T642A 50.0 Ϯ 9.5 5.9 (0.6) 49.9 Ϯ 9.9 21.7 (0.4)* 156.1 Ϯ 20.0 1.2 1,393 Ϯ 221.6 0.7 Y645F 42.8 Ϯ 6.8 5.0 (0.5)* 44.8 Ϯ 6.3 19.5 (0.3)* 132.4 Ϯ 18.4 1.1 1,846 Ϯ 206.2 1.0 The IC50 values shown are means Ϯ SD of 3 independent experiments. Login to comment
250 Only a slight decrease in 5D3-phycoerythrin fluorescence was observed for the vector control cells (Fig. 5); however, the 5D3-phycoerythrin fluorescence was differentially increased in a prazosin concentration-dependent manner for wild-type BCRP, N629A, T642A, and Y645F. Login to comment
254 In particular, significant differences in 5D3 binding were observed between wild-type BCRP and the mutants N387A, Q398A, T402A, and N629A at certain prazosin concentrations (Fig. 5). Login to comment
274 Kinetic parameters of ATP hydrolysis by wild-type and mutant BCRP Wild-type BCRP N387A Q398A T402A N629A T642A Y645F Vmax, nmol Pi ·min-1 ·mg protein-1 14.5 Ϯ 1.9 16.9 Ϯ 1.5 14.9 Ϯ 0.93 17.4 Ϯ 1.7 11.5 Ϯ 1.1 17.9 Ϯ 1.9 14.5 Ϯ 1.9 Vmax normalized to BCRP protein level, nmol Pi ·min-1 ·mg protein-1 14.5 Ϯ 1.9 7.0 Ϯ 0.63 6.5 Ϯ 0.41 8.3 Ϯ 0.81 28.0 Ϯ 2.7 16.3 Ϯ 1.7 12.0 Ϯ 1.6 Km for ATP, mM 0.89 Ϯ 0.40 0.90 Ϯ 0.27 0.48 Ϯ 0.12 1.1 Ϯ 0.35 0.40 Ϯ 0.15 0.93 Ϯ 0.34 0.89 Ϯ 0.43 Vmax/Km, nmol Pi ·min-1 ·mg protein-1 ·mM-1 16.3 7.8 13.5 7.5 70.0 17.5 13.5 Values are means Ϯ SD of 3 independent determinations. Login to comment
282 Significant differences (P Ͻ 0.05) in 5D3 binding analyzed by the Student`s t-test were observed between wild-type BCRP and the mutant N387A, Q398A, T402A, or N629A at certain prazosin concentrations and are marked (*) on the right of respective data points. Login to comment
332 Replacement of Asn629 with Ala significantly increased transport activity of BCRP for all of the substrates tested (Tables 1 and 2). Login to comment
336 Notably, N629A was associated with an ϳ60% decrease in Km and an approximately fourfold increase in Vmax/Km for ATP hydrolysis (Table 3). Login to comment
337 This suggests that ATP binding affinity and the efficiency of ATP hydrolysis are increased for N629A, which could be linked to the increased transport activity of the mutant. Login to comment
350 Prazosin differentially increased 5D3 binding to wild-type BCRP, N629A, T642A, and Y645F, whereas 5D3 binding to N387A and Q398A was slightly decreased (Fig. 5). Login to comment