ABCMdb

ABCC1 p.Arg1202Asp

Predicted by SNAP2:	A: D (85%), C: D (85%), D: D (95%), E: D (91%), F: D (91%), G: D (85%), H: D (66%), I: D (80%), K: D (85%), L: D (85%), M: D (85%), N: D (71%), P: D (95%), Q: D (85%), S: D (85%), T: D (80%), V: D (85%), W: D (85%), Y: D (85%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Mutational analysis of ionizable residues proximal... J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18. Situ D, Haimeur A, Conseil G, Sparks KE, Zhang D, Deeley RG, Cole SP
Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter.
J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18., 2004-09-10 [PMID:15208328]

Abstract [show]
The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4). In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding.

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No. Sentence Comment
108 In contrast, expression levels of the R1202D and E1204K mutants were substantially reduced (by Ͼ75%). Login to comment
109 A Northern blot analysis performed on total RNA isolated from the transfected cells indicated that the R1202D and E1204K mRNA levels were comparable with wild-type MRP1 mRNA levels, thus excluding the possibility that the low R1202D and E1204K protein levels could be caused by differences in transfection efficiency (results not shown). Login to comment
113 Membrane vesicles prepared from HEK293T cells transfected with empty vector [pcDNA3.1] and vector containing the wild-type (WT-MRP1) and mutant (R1197E, R1202D, E1204K, and R1249D) cDNAs were immunoblotted with mAb QCRL-1. Shown is a representative immunoblot of membrane vesicles (1 and 2 ␮g of protein) from a single transfection. Login to comment
141 Transport Activities of TM16/17 Arg1197 and Arg1249 Mutants-Unlike the R1202D and E1204K mutants, oppositely charged substitutions of Arg1197 (R1197E) and Arg1249 (R1249D) did not adversely affect expression of MRP1 (Fig. 3A). Login to comment
189 This idea is supported by the observation that membrane expression of the R1202D and E1204K mutants in mammalian cells can be substantially increased when transfected cells are grown at lower temperatures (30 °C) where the stringency of the proofreading machinery for monitoring protein folding is diminished.2 Because neutral (Leu, Gly) substitutions of TM16 Arg1202 and Glu1204 did not affect MRP1 expression in any significant way, it may be concluded that it is the opposite charge of the side chains of the substituting amino acids in the nonexpress- 2 A. Haimeur, R. G. Deeley, and S. P. C. Cole, unpublished observations. Login to comment
199 ing R1202D and E1204K mutants that is key to MRP1 protein destabilization. Login to comment
200 In this regard, it is worth noting that replacing Arg1202 with an Asp (or Glu1204 with a Lys) results in a potential net gain of two charges in the cytoplasmic half of TM16 as well as placing two same-charge ionizable residues in relatively close proximity to one another which could well perturb the ␣-helical geometry of TM16 and contribute to misfolding or aberrant TM helix-packing. Login to comment