ABCMdb

PMID: 19015228

Conseil G, Rothnie AJ, Deeley RG, Cole SP
Multiple roles of charged amino acids in cytoplasmic loop 7 for expression and function of the multidrug and organic anion transporter MRP1 (ABCC1).
Mol Pharmacol. 2009 Feb;75(2):397-406. Epub 2008 Nov 17., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC1 p.Glu1144Ala ABCC1 p.Arg1173Ala ABCC1 p.Asp1179Ala ABCC1 p.Asp1183Ala ABCC1 p.Glu1184Ala ABCC1 p.Lys1181Ala ABCC1 p.Arg1166Ala The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, 1169 AAQA). Login to comment 7 ABCC1 p.Asp1183Glu ABCC1 p.Arg1166Lys Same charge mutant D1183E was also not expressed, whereas expression and activity of R1166K were similar to wild-type MRP1. Login to comment 8 ABCC1 p.Glu1144Ala ABCC1 p.Asp1179Ala ABCC1 p.Lys1181Ala The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and 1169 AAQA were accompanied by changes in orthovanadate-induced trapping of [␣-32 P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. Login to comment 9 ABCC1 p.Glu1144Ala In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Login to comment 43 ABCC1 p.Lys1141Glu Substitution of Lys1141 with Glu also impairs expression of MRP1 at the plasma membrane as well as adversely affecting transport activity (Conseil et al., 2006). Login to comment 59 ABCC1 p.Glu1144Ala ABCC1 p.Arg1173Ala ABCC1 p.Asp1179Ala ABCC1 p.Asp1183Ala ABCC1 p.Glu1184Ala ABCC1 p.Lys1181Ala ABCC1 p.Arg1166Ala ABCC1 p.Asp1183Glu ABCC1 p.Arg1166Lys Mutations were first generated in the pGEM-3Z-XmaI/MRP1 plasmid according to the manufacturer`s instructions with the following mutagenic primers (Integrated DNA Technologies, Inc., Coralville, IA), which also introduced an additional restriction site (substituted nucleotides are underlined): E1144A (5Ј-G AAG CGC CTC GCG TCG GTC AGC-3Ј); R1166A (5Ј C AGC GTC ATT GCA GCA TTC GAG GAG CAG-3Ј); R1166K (5Ј C AGC GTC ATT AAG GCC TTC GAG G-3Ј); 1169 EEQE-1169 AAQA (5Ј-C ATT CGA GCC TTC GCG GCA CAG GCA CGC TTC ATC C-3Ј); R1173A (5Ј-C GAG GAG CAG GAG GCA TTC ATC CAC CAG AG-3Ј); D1179A (5Ј-C CAC CAG AGT GCC CTT AAG GTG GAC G-3Ј), K1181A (5Ј-G AGT GAC CTG GCA GTC GAC GAG AAC C-3Ј); D1183A (5Ј-CTG AAG GTG GCC GAG AAC CAG-3Ј); D1183E (5Ј- CTG AAG GTG GAA GAG AAC CAG-3Ј); and E1184A (5Ј-GT GAC CTG AAG GTA GAC GCG AAC CAG AAG GCC-3Ј). Login to comment 120 ABCC1 p.Asp1183Ala ABCC1 p.Arg1166Ala These experiments showed that two of the Ala-substituted mutants (R1166A and D1183A) were consistently expressed at very low levels (10-20% of wild-type MRP1) (Fig. 2A). Login to comment 124 ABCC1 p.Asp1183Ala ABCC1 p.Arg1166Ala Confocal microscopy of intact HEK cells using the MRP1-specific mAb QCRL-3 showed that despite their low expression levels, at least a portion of the R1166A and D1183A mutant proteins were correctly routed to the plasma mem- brane (not shown). Login to comment 126 ABCC1 p.Asp1183Ala ABCC1 p.Arg1166Ala However, even at the lower temperature, expression of the R1166A and D1183A mutants remained substantially below that of wild-type MRP1; furthermore, plasma membrane routing of the mutants as well as wild-type MRP1 was impaired (data not shown). Login to comment 128 ABCC1 p.Asp1183Glu ABCC1 p.Arg1166Lys To determine whether it was the charge or other property of the amino acid at positions 1166 and 1183 that was critical for efficient plasma membrane expression of MRP1, same-charge mutants of Arg1166 (R1166K) and Asp1183 (D1183E) were created and again expressed in HEK cells. Login to comment 129 ABCC1 p.Asp1183Glu ABCC1 p.Arg1166Lys At 37°C, the MRP1 expression levels in whole-cell lysates were increased to 50% of wild-type MRP1 levels for the D1183E mutant, and for the R1166K mutant, expression was comparable with that of wild-type MRP1 (Fig. 2B). Login to comment 130 ABCC1 p.Asp1183Glu ABCC1 p.Arg1166Lys Transport Activity of R1166K and D1183E Mutant MRP1 Proteins. Login to comment 131 ABCC1 p.Asp1183Glu ABCC1 p.Arg1166Lys To characterize the transport properties of the same-charge R1166K and D1183E mutants, membrane vesicles were prepared from transfected HEK cells. Login to comment 133 ABCC1 p.Arg1166Lys However, for the R1166K mutant, levels of LTC4 and E217betaG transport (Fig. 3, B and C) were comparable with that of wild-type MRP1 after correcting for relative expression levels of MRP1 in the vesicles. Login to comment 134 ABCC1 p.Asp1183Glu Transport activity of the D1183E mutant was not assessed because its very low expression levels relative to wild-type MRP1 made measurements unreliable (Fig. 3A). Login to comment 138 ABCC1 p.Asp1183Ala ABCC1 p.Arg1166Ala A, immunoblot of whole cell lysates (10 ␮g of protein) prepared from HEK 293T cells transfected with wild-type (WT-MRP1), and R1166A and D1183A mutant MRP1 expression vectors. Login to comment 140 ABCC1 p.Asp1183Ala ABCC1 p.Arg1166Ala MRP1 levels in whole-cell lysates were detected with mAb QCRL-1, and the relative protein expression levels are shown under the blot and were estimated by densitometry as described under Materials and Methods. B, immunoblots of whole-cell lysates (10 ␮g of protein) prepared from HEK 293T cells transfected with R1166A/K and D1183A/E mutant and wild-type (WT) MRP1 cDNA expression vectors. MRP1 proteins were detected with mAb QCRL-1 and lysates of untransfected cells were included as a negative control as above. Login to comment 144 ABCC1 p.Asp1183Glu ABCC1 p.Arg1166Lys A, representative immunoblot of membrane vesicles (1 ␮g of protein per lane) prepared from HEK 293T cells transfected with wild-type (WT), R1166K, and D1183E cDNA expression vectors. MRP1 proteins were detected with mAb QCRL-1 and membrane vesicles from untransfected cells were included as a negative control. Login to comment 146 ABCC1 p.Arg1166Lys B and C, ATP-dependent uptake of [3 H]LTC4 (B) and [3 H]E217betaG (C) was measured in membrane vesicles prepared from HEK 293T cells transfected with wild-type MRP1 (WT-MRP1) and R1166K mutant MRP1 cDNA expression vectors. Login to comment 154 ABCC1 p.Glu1144Ala Thus, E217betaG, E13SO4, and MTX transport by E1144A was moderately reduced (by ϳ50%), whereas uptake of GSH and LTC4 was comparable with that of wild-type MRP1. Login to comment 155 ABCC1 p.Asp1179Ala The D1179A mutant exhibited a selective but modest reduction (35% decrease) in LTC4 and E13SO4 transport, whereas the 1169 AAQA triple mutant showed a more substantial decrease (55%) in E13SO4 transport. Login to comment 156 ABCC1 p.Lys1181Ala On the other hand, the K1181A mutant exhibited a global reduction (ϳ50%) in transport of all tested organic anion substrates. Login to comment 158 ABCC1 p.Arg1173Ala ABCC1 p.Glu1184Ala For this reason, the R1173A and E1184A mutants were not investigated further. Login to comment 160 ABCC1 p.Glu1144Ala ABCC1 p.Lys1181Ala Because the single E1144A and K1181A mutants and the 1169 AAQA triple mutant exhibited the most substantial changes in transport activity, their ATP binding and hydrolysis properties were examined. Login to comment 164 ABCC1 p.Lys1181Ala Thus, trapping of 8-azido-[␣-32 P]ADP by the K1181A mutant was 40% lower than for wild-type MRP1. Login to comment 165 ABCC1 p.Glu1144Ala ABCC1 p.Glu1144Ala In contrast, increased dinucleotide trapping (1.5to 1.7-fold) was observed for the E1144A and 1169 AAQA mutants (after corrections were made to take into account differences in MRP1 expression) (Fig. 5B). Login to comment 167 ABCC1 p.Glu1144Ala To determine whether the increased photolabeling under trapping conditions was due to a change in the affinity for azidoADP, additional trapping experiments were performed on the E1144A and 1169 AAQA mutants. Login to comment 172 ABCC1 p.Glu1144Ala Effect of E217betaG on 8-Azido-[␣-32 P]ATP Photolabeling and Vanadate-Induced 8-Azido-[␣-32 P]ADP Trapping by E1144A Mutant MRP1. Login to comment 173 ABCC1 p.Glu1144Ala Because E217betaG transport by the E1144A mutant was substantially reduced, the effect of this organic anion substrate on the interactions of the mutant protein with ATP was examined. Login to comment 174 ABCC1 p.Glu1144Ala As shown in Fig. 6A, neither 25 nor 50 ␮M E217betaG caused any detectable change in 8-azido-[␣-32 P]ATP photolabeling of either the E1144A mutant or wild-type MRP1 at 4°C. Login to comment 176 ABCC1 p.Glu1144Ala In contrast, E217betaG had no effect on vanadate-induced trapping of azidoADP by the E1144A mutant (Fig. 6B). Login to comment 177 ABCC1 p.Lys1181Ala [3 H]LTC4 Photolabeling of K1181A Mutant MRP1 Protein. Login to comment 178 ABCC1 p.Lys1181Ala Because the K1181A mutant showed the most significant decrease (45%) in LTC4 transport activity of the seven single Ala mutants, it was of interest to determine whether binding of this substrate was affected by this mutation. Login to comment 179 ABCC1 p.Lys1181Ala However, as shown in Fig. 7, no differences in [3 H]LTC4 photolabeling of the K1181A mutant and wild-type MRP1 in their nucleotide-free states were detected after taking in account relative levels of MRP1 expression (Fig. 4). Login to comment 180 ABCC1 p.Lys1181Ala Because vanadate-induced trapping of azidoADP by the K1181A mutant was significantly reduced compared with wild-type MRP1 (Fig. 5, B and C), [3 H]LTC4 photolabeling experiments were also carried out using MRP1-enriched membranes in the ATP-bound and "ADP-trapped" (low affinity) states. Login to comment 181 ABCC1 p.Lys1181Ala Under both conditions, low levels of [3 H]LTC4 photolabeling were observed for the K1181A mutant (Fig. 7). Login to comment 185 ABCC1 p.Glu1144Ala ABCC1 p.Arg1173Ala ABCC1 p.Asp1179Ala ABCC1 p.Glu1184Ala ABCC1 p.Lys1181Ala Shown is an immunoblot of membrane vesicles (1 ␮g of protein) prepared from HEK 293T cells transfected with cDNA expression vectors encoding wild-type, E1144A, 1169 AAQA, R1173A, D1179A, K1181A, and E1184A MRP1 mutants. Login to comment 191 ABCC1 p.Arg1173Ala ABCC1 p.Glu1184Ala Of the seven single-Ala-substituted CL7 mutants generated, only two (R1173A and E1184A) displayed properties that were comparable with those of wild-type MRP1, demonstrating the extreme sensitivity of this cytoplasmic loop to mutation. Login to comment 192 ABCC1 p.Glu1144Ala ABCC1 p.Asp1179Ala ABCC1 p.Asp1183Ala ABCC1 p.Lys1181Ala ABCC1 p.Arg1166Ala The remaining five single mutants were either poorly expressed (R1166A, D1183A) (Fig. 2) or exhibited significantly reduced transport levels of one or more organic anions (E1144A, D1179A, K1181A) (Table 1). Login to comment 194 ABCC1 p.Asp1183Ala ABCC1 p.Arg1166Ala The poor expression of the R1166A and D1183A mutants indicates that Arg1166 and Asp1183 contribute to the stability of MRP1, probably by influencing the proper folding of the transporter during its biosynthesis. Login to comment 199 ABCC1 p.Asp1183Ala ABCC1 p.Arg1166Ala Elucidation of the post-translational events leading to poor expression of R1166A and D1183A and other low or nonexpressing MRP1 mutants is currently under investigation. Login to comment 201 ABCC1 p.Asp1183Ala ABCC1 p.Asp1183Glu Our observation that the same-charge D1183E mutant was as poorly expressed at the plasma membrane as the neutrally substituted D1183A is somewhat unexpected, because previously identified expression impaired mutants involving charged residues have typically not exhibited this property (Haimeur et al., 2004; Situ et al., 2004). Login to comment 208 ABCC1 p.Asp1183Glu ABCC1 p.Arg1166Lys In contrast to D1183E, the same charge mutant of Arg1166 , R1166K, was expressed in membrane vesicles at levels that approached 50% those of wild-type MRP1, and the relative levels of LTC4 and E217betaG transport activity of this mutant were comparable with those of wild-type MRP1 after normalization for differences in protein expression levels (Fig. 3). Login to comment 212 ABCC1 p.Glu1144Ala ABCC1 p.Arg1173Ala ABCC1 p.Asp1179Ala ABCC1 p.Glu1184Ala ABCC1 p.Lys1181Ala Percentage Wild-Type MRP1 Uptake Activity E1144A 1169 AAQA R1173A D1179A K1181A E1184A % LTC4 90 Ϯ 5 70 Ϯ 5 120 Ϯ 5 65 Ϯ 5 55 Ϯ 5 85 Ϯ 5 E217bG 55 Ϯ 5 80 Ϯ 0 130 Ϯ 10 90 Ϯ 5 65 Ϯ 5 95 Ϯ 15 E13SO4 45 Ϯ 5 45 Ϯ 5 125 Ϯ 0 65 Ϯ 0 50 Ϯ 5 90 Ϯ 10 MTX 55 Ϯ 5 105 Ϯ 5 N.D. N.D. 45 Ϯ 5 N.D. GSH 80 Ϯ 5 95 Ϯ 10 N.D. N.D. 30 Ϯ 5 N.D. N.D., not determined. Login to comment 219 ABCC1 p.Glu1144Ala ABCC1 p.Lys1181Ala 8-Azido-[32 P]ATP photolabeling and vanadate-dependent 8-azido- [32 P]ADP trapping of CL7 mutants E1144A, 1169 AAQA and K1181A of MRP1. Login to comment 230 ABCC1 p.Glu1144Ala Effect of E217betaG on 8-azido-[32 P]ATP photolabeling and vanadate-dependent azido-[32 P]ADP trapping by MRP1 mutant E1144A. Login to comment 232 ABCC1 p.Glu1144Ala The relative levels of labeling were estimated by densitometric analysis of the films, and for wild-type and E1144A mutant MRP1 are expressed relative to labeling in the absence of substrate. Login to comment 233 ABCC1 p.Glu1144Ala In the lower part of B, the relative levels of vanadate-induced azidoADP trapping by wild-type (F) and E1144A (E) MRP1 have also been plotted against concentration of E217betaG. Login to comment 235 ABCC1 p.Lys1181Ala [3 H]LTC4 photolabeling of wild-type and K1181A mutant MRP1 in the presence of ATP and trapped ADP. Login to comment 236 ABCC1 p.Lys1181Ala Wild-type (WT-MRP1) and K1181A mutant membrane vesicles (50 ␮g of protein) were incubated in transport buffer containing 5 mM MgCl2 for 20 min in the absence (-) or presence (ϩ) of ATP (1 mM) and sodium orthovanadate (Vi) (1 mM), alone or in combination, and then incubated with [3 H]LTC4 (200 nM, 120 nCi) for a further 30 min followed by UV cross-linking, SDS-PAGE, and fluorography. Login to comment 238 ABCC1 p.Arg1166Ala ABCC7 p.Arg1066Cys Fanen et al. (1997) further found that transfected cells expressing CFTR-R1066C did not respond to cAMP stimulation, and, similar to what we observed with MRP1-R1166A, the mutant CFTR protein was poorly expressed. Login to comment 239 ABCC7 p.Arg1066Cys Moreover, unlike other CFTR-processing mutations, the defect exhibited by CFTR-R1066C could not be corrected in vitro by reduced temperature or treatment with sodium butyrate. Login to comment 240 ABCC1 p.Arg1166Ala Likewise, we found that expression of MRP1-R1166A could not be improved under comparable conditions. Login to comment 244 ABCC1 p.Glu1144Ala ABCC1 p.Asp1179Ala ABCC1 p.Asp1183Ala ABCC1 p.Lys1181Ala ABCC1 p.Arg1166Ala Unlike the R1166A and D1183A mutants, the second group of functionally altered single Ala-substituted mutants (E1144A, D1179A, K1181A) and the 1169 AAQA triple mutant were all expressed at levels comparable with or greater than that of wild-type MRP1 (Fig. 4). Login to comment 245 ABCC1 p.Asp1179Ala However, none of these residues is absolutely critical for MRP1 function because their replacement with Ala caused only moderate (and in the case of D1179A very moderate) and substrate-selective changes in transport activity (Table 1). Login to comment 255 ABCC1 p.Glu1144Ala ABCC1 p.Lys1181Ala The altered transport activity and nucleotide interactions of the E1144A, K1181A, and 1169 AAQA mutants may thus result from perturbation of this signaling (Table 1, Fig. 5). Login to comment 256 ABCC1 p.Glu1144Ala ABCC1 p.Glu1144Ala The observation that E217betaG failed to inhibit vanadate-induced trapping of azidoADP by the E1144A mutant, as it does with wild-type MRP1 (Fig. 6), suggests that the signaling that occurs after E217betaG interacts with its binding site in the MSDs of E1144A has become at least partially uncoupled from the hydrolysis of ATP (or release of ADP) at NBD2/ NBS2. Login to comment 264 ABCC1 p.Glu1157Leu ABCC1 p.Gly1161Leu Ren et al. (2006) have recently confirmed the importance of CL7 in MRP1 function by substituting the highly conserved Glu1157 and Gly1161 with Leu and Pro, respectively. Login to comment 265 ABCC1 p.Gly1161Pro ABCC1 p.Glu1157Leu They found that the double-mutant E1157L/G1161P no longer transported LTC4 and could not be labeled with the photoaffinity ligand azidoAgosterol A. Login to comment 267 ABCC1 p.Gly1161Pro ABCC1 p.Glu1157Leu Thus, for reasons that are presently unclear, the interactions of the double E1157L/G1161P mutant with nucleotide differ substantially from those of the CL7 mutants we have described here and elsewhere (Conseil et al., 2006). Login to comment 268 ABCC1 p.Glu507Leu ABCC1 p.Gly511Pro Ren et al. (2006) also created the analogous double mutant (E507L/G511P) in CL5, the cytoplasmic loop that connects TM10 to TM11, and is predicted to be in contact with NBD2 (DeGorter et al., 2008). Login to comment