ABCMdb

ABCC3 p.Trp1242Cys

None has been submitted yet.

Publications

PMID: 12388190 [PubMed] Oleschuk CJ et al: "Substitution of Trp1242 of TM17 alters substrate specificity of human multidrug resistance protein 3."

No. Sentence Comment

59 Mutations for Trp1242 substitutions (underlined), silent DraI restriction sites (italicized), and their corresponding oligonucleotides were as follows: W1242A (5Ј-G CAG GTG ACA TTC GCT TTA AAC GCG ATG ATA CGA ATG ATG TCA G-3Ј), W1242C (5Ј-G CAG GTG ACA TTC GCT TTA AAC TGC ATG ATA CGA ATG ATG TCA G-3Ј), W1242F (5Ј-G CAG GTG Fig. 1. Login to comment
103 These included nonconservative substitutions with a nonaromatic nonpolar amino acid (Ala; W1242A-MRP3) and a nonaromatic polar amino acid (Cys; W1242C-MRP3), as well as conservative substitutions with aromatic polar (Tyr; W1242Y-MRP3) and nonpolar (Phe; W1242F-MRP3) amino acids. Login to comment
106 As shown in Fig. 2, wild-type MRP3 and the four mutants (W1242A-, W1242C-, W1242F-, and W1242Y-MRP3) were expressed at similar levels. Login to comment
108 Mean expression levels of the mutant MRP3 proteins relative to wild-type MRP3 were as follows: W1242A, 1.1 Ϯ 0.3; W1242C, 1.0 Ϯ 0.2; W1242Y, 1.0 Ϯ 0.1; W1242F, 1.0 Ϯ 0.3; and W1242P, 0.6 Ϯ 0.2 (4-6 independent transfections). Login to comment
110 Time courses of ATP-dependent [3 H]E217betaG uptake were determined for the W1242A-, W1242C-, W1242F-, W1242Y-, and W1242P-MRP3 mutants by using inside-out membrane vesicles prepared from transfected HEK293T cells (Fig. 3A). Login to comment
111 Unexpectedly, four of the five mutants (W1242A-, W1242C-, W1242F-, and W1242Y-MRP3) transported this glucuronide substrate at levels that were substantially higher than those of wild-type MRP3. Login to comment
113 At 3 min, [3 H]E217betaG uptake in membrane G282 CONSERVED TRP AND MRP3 (ABCC3) SUBSTRATE SPECIFICITY AJP-Gastrointest Liver Physiol • VOL 284 • FEBRUARY 2003 • www.ajpgi.org atUnivofNorthCarolina-AcqSrvcsonOctober,2012http://ajpgi.physiology.org/Downloadedfrom vesicles enriched for W1242A-, W1242C-, and W1242F-MRP3 was 2.5-to 3-fold higher than for wild-type MRP3, whereas [3 H]E217betaG uptake by the most conservatively substituted mutant, W1242Y-MRP3, was ϳ7-fold higher (Fig. 3B). Login to comment
121 At 5 min, levels of [3 H]LTC4 uptake by these three mutants were ϳ6270% those of wild-type MRP3, whereas uptake by the W1242C-MRP3 mutant was only 37% of wild-type levels (after subtraction of uptake by vector control membrane vesicles and normalization of mutant MRP3 protein levels to wild-type MRP3 protein levels) (Fig. 4B). Login to comment
128 At 10 min, ATP-dependent [3 H]MTX uptake levels by the W1242A, W1242C, W1242F, and W1242Y Fig. 3. Login to comment
130 A: time course of ATP-dependent [3 H]E217betaG uptake in membrane vesicles prepared from HEK293T cells transfected with empty vector (ᮀ), wild-type MRP3 (s), and mutant [W1242A (), W1242C (}), W1242F (F), W1242Y (Œ), and W1242P (ƒ)] cDNA expression vectors. Login to comment
135 Top: MRP3 expression in membrane vesicles prepared from human embryonic kidney (HEK) 293T cells transfected with empty vector [pcDNA3.1(ϩ)], wild-type (WT-MRP3), and mutant (W1242A, W1242C, W1242F, W1242Y, and W1242P) MRP3 cDNAs was determined by immunoblotting with MAb M3II-9, and relative levels of expression were estimated by densitometry. Login to comment
147 To determine if Trp1242 substitutions also affected MRP3-mediated transport of the latter substrate, [3 H]leucovorin uptake into membrane vesicles prepared from transfected cells expressing wild-type MRP3 and W1242A-, W1242C-, W1242F-, and W1242Y-MRP3 mutants was examined. Login to comment
151 Time course of [3 H]methotrexate (MTX) uptake and MTX- mediated inhibition of [3 H]E217betaG uptake by wild-type and Trp1242 mutant MRP3 proteins. A: ATP-dependent uptake of [3 H]MTX was measured in membrane vesicles prepared from HEK293T cells transfected with empty pcDNA3.1(ϩ) vector (ᮀ), wild-type (s), and mutant [W1242A (), W1242C (}), W1242F (F), and W1242Y (Œ)] MRP3 cDNA expression vectors. Membrane vesicles were incubated at 37°C with 1 ␮M [3 H]MTX and ATP or AMP in transport buffer. Login to comment
161 Taurocholate uptake by the W1242A-, W1242C-, W1242F-, and W1242Y-MRP3 mutants was then examined and, in all cases, was not significantly different from uptake by wild-type MRP3 (Fig. 7B). Login to comment
162 Consistent with this observation, [3 H]E217betaG uptake by W1242A-, W1242C-, W1242F-, and W1242Y-MRP3 could still be inhibited by taurocholic acid (40-60%) at concentrations of 50 and 100 ␮M (Fig. 7C). Login to comment
170 Cells were transfected with empty vector [pcDNA3.1(ϩ)], wild-type, and mutant (W1242A, W1242C, W1242F, and W1242Y) MRP3 cDNA expression. Login to comment
171 C: membrane vesicles from cells expressing wild-type and W1242A, W1242C, W1242F, and W1242Y mutant MRP3 were incubated at 37°C with 400 nM [3 H]E217betaG in transport buffer and other components for 3 min in the absence (open bars) or presence of taurocholic acid (50 ␮M, grey bar; 100 ␮M, solid bar). Login to comment
173 [3 H]leucovorin uptake by wild-type and Trp1242 mutant MRP3 proteins. A: uptake of [3 H]leucovorin was measured in membrane vesicles prepared from HEK293T cells transfected with empty vector [pcDNA3.1(ϩ)], wild-type, and Trp1242 mutant (W1242A, W1242C, W1242F, and W1242Y) MRP3 cDNA expression vectors. Membrane vesicles were incubated at 37°C with 250 ␮M [3 H]leucovorin and ATP or AMP in transport buffer for 20 min. Login to comment
196 Membrane vesicles from cells expressing wild-type MRP3 (WT-MRP3) and W1242A, W1242C, W1242F, and W1242Y mutant MRP3 were incubated at 37°C with 400 nM [3 H]E217betaG in transport buffer and other components for 3 min in the absence (open bars) or presence of glycocholate (50 ␮M, grey bar; 100 ␮M, solid bar). Login to comment
199 Transport activities of variously substituted MRP3-Trp1242 mutants Substrate Transport Activity W1242A W1242C W1242F W1242Y W1242P E217betaG 1 1 1 11 222 LTC4 2 22 2 2 222 MTX 222 222 222 222 n.d. Login to comment