The First Cytoplasmic Loop in the Core Structure of the ABCC1 (Multidrug Resistance Protein 1; MRP1) Transporter Contains Multiple Amino Acids Essential for Its Expression.

Gwenaëlle Conseil,Susan P C Cole

doi:10.3390/ijms22189710

Gwenaëlle Conseil, Susan P C Cole

Open Access

https://doi.org/10.3390/ijms22189710

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Abstract

ABCC1 (human multidrug resistance protein 1 (hMRP1)) is an ATP-binding cassette transporter which effluxes xeno- and endobiotic organic anions and confers multidrug resistance through active drug efflux. The 17 transmembrane α-helices of hMRP1 are distributed among three membrane spanning domains (MSD0, 1, 2) with MSD1,2 each followed by a nucleotide binding domain to form the 4-domain core structure. Eight conserved residues in the first cytoplasmic loop (CL4) of MSD1 in the descending α-helix (Gly392, Tyr404, Arg405), the perpendicular coupling helix (Asn412, Arg415, Lys416), and the ascending α-helix (Glu422, Phe434) were targeted for mutagenesis. Mutants with both alanine and same charge substitutions of the coupling helix residues were expressed in HEK cells at wild-type hMRP1 levels and their transport activity was only moderately compromised. In contrast, mutants of the flanking amino acids (G392I, Y404A, R405A/K, E422A/D, and F434Y) were very poorly expressed although Y404F, E422D, and F434A were readily expressed and transport competent. Modeling analyses indicated that Glu422 and Arg615 could form an ion pair that might stabilize transporter expression. However, this was not supported by exchange mutations E422R/R615E which failed to improve hMRP1 levels. Additional structures accompanied by rigorous biochemical validations are needed to better understand the bonding interactions crucial for stable hMRP1 expression.

Highlights

Cellular efflux of many physiological organic anions and pharmacological metabolites is mediated by one or more of the 12 members of the mammalian ATP-binding cassette (ABC) (ABCC) family of membrane proteins [1,2]
Our finding that CL4-Lys406 was crucial for human MRP1/ABCC1 (hMRP1) expression was somewhat unexpected because we previously showed that non-conservative substitutions of only one (Asp430 ) of five (i.e., Arg394, Lys396, Arg433, and Asp436 ) charged residues in CL4 had any adverse effect on hMRP1 levels [22,23]
To determine which CL4 residues to target for substitution, multiple sequences of hMRP1 amino acids 390-439 with its human homologs ABCC2-ABCC7 (MRP2-5, ABCC6, CFTR; P33527, Q92887, O15438, O15439, O15440, O95255, P13569) were retrieved from

Summary

Introduction

Cellular efflux of many physiological organic anions and pharmacological metabolites is mediated by one or more of the 12 members of the mammalian ATP-binding cassette (ABC) (subfamily C) (ABCC) family of membrane proteins [1,2]. HMRP1 has been shown to efflux a diverse array of conjugated xenobiotic and physiologic organic anions [4,5]. Foremost among the latter are the proinflammatory cysteinyl leukotriene C4 (LTC4 ) [7] and the conjugated estrogens, estradiol glucuronide (E2 17βG) and estrone sulfate (E1 3SO4 ) [8,9]. The Walker A and B motifs of NBD1 and the ‘C’

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