Membrane Topology of Human ASBT (SLC10A2) Determined by Dual Label Epitope Insertion Scanning Mutagenesis. New Evidence for Seven Transmembrane Domains

Abstract

The membrane topology of the human apical sodium-dependent bile acid transporter (hASBT) remains unresolved. Whereas N-glycosylation analysis favors a 7 transmembrane (TM) model, membrane insertion scanning supports a 9TM topology. In order to resolve this controversy, we used dual label epitope insertion to systematically examine the topological framework of hASBT. Two distinct epitopes, hemagglutinin (HA) and FLAG, were individually inserted by inverted PCR mutagenesis at strategic positions along the hASBT sequence. Cell surface biotinylation and immunoblotting with epitope-specific and anti-hASBT antibodies confirmed expression and trafficking of the mutants to the plasma membrane. Confocal microscopy confirmed membrane localization of epitope-tagged hASBT in saponin-treated (permeabilized) and non-permeabilized transfected COS-1 and MDCK cells. Tags at positions 116, 120, 186, 270 and 284 were accessible to the epitope antibodies in non-permeabilized cells, indicative of the extracellular localization of loops 1 (99-130), 2 (180-191), and 3 (253-287). The corresponding positions in the 9TM model were predicted to be intracellular or membrane bound. Epitope mutants at residues 56, 92, 156 and 221 were only detected after treatment with saponin, indicating the intracellular localizations of loops 1 (50-73), 2 (150-160), 3 (215-227) as predicted by a 7 TM model. Our results also confirm the exofacial and cytosolic localization of N-and C-terminal tails, respectively. With the exception of constructs inserted at position 120, epitope mutants displayed active, sodium-dependent taurocholate uptake. Consequently, our study strongly supports a 7 TM topology for hASBT and refutes the previously proposed 9 TM model. KeywordsSubstituted Cysteine Accessibility Method; bile acid; transporter; mutagenesis The apical localization of SLC10A2, the sodium-dependent bile acid transporter (1-3), on the distal ileum and cholangiocytes contributes to the enterohepatic circulation of bile acids (4) and cholesterol homeostasis (5-7). Although its crystal structure has not yet been elucidated, critical structural and functional determinants have been evaluated by biochemical methods (8), site-directed mutagenesis (9-12) and photo affinity labeling (13). Based on these data, structure-activity models for hASBT have been generated (14,15). Extending these studies to develop a three-dimensional representation of hASBT protein would require its essential domain structure and transmembrane (TM) topology (11).* To whom correspondence should be addressed: Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD 21201. Telephone: (410) Fax: (410) Hydropathy analysis proposes between seven to nine putative TM domains (TMDs) with an extracellular glycosylated N-terminus and a cytoplasmic C-terminus (16). However, there is controversy regarding the exact membrane topology of ASBT. Whereas in vitro translational studies based on membrane-insertion-scanning mutagenesis suggested the possibility of a 9TM hASBT t...

Conserved Aspartic Acid Residues Lining the Extracellular Loop I of Sodium-coupled Bile Acid Transporter ASBT Interact with Na+ and 7α-OH Moieties on the Ligand Cholestane Skeleton

“…Cysteine Scan of EL1-As predicted by our topology model (25,28), residues spanning Val-99 -Ser-126 form the EL1 protein region, which exhibits high sequence conservation among a range of evolutionarily diverse species (Fig. 1).…”

“…This uptake period ensures linear steady-state kinetics in conjunction with an optimal signal-to-noise ratio for subsequent [ 3 H]TCA analysis via liquid scintillation counting (22,23,28). Uptake was halted by a series of washes with ice-cold Dulbecco's phosphate-buffered saline, pH 7.4, containing 0.2% fatty acid free bovine serum albumin and 0.5 mM TCA.…”

Effects of Ala Substitution for Conserved Cys Residues in Mouse Ileal and Hepatic Na+-Dependent Bile Acid Transporters

“…A model with nine-TM segments has been proposed based on a prediction method using HMMTOP and in vitro translation analysis, 23) while scanning analyses of epitope and glycosylation site insertions suggested a seven-TM model. 22,26) Moreover, a novel seven-TM model, in which TM6 and TM7 of the nine-TM model are associated with the plasma membrane at the extracellular surface, is proposed for human SLC10A1. 25) Cys 51 in Slc10a2 is located just at the boundary between the first TM segment and the first intracellular loop in both the seven-TM and nine-TM models (Fig.…”

Apical Sodium Dependent Bile Acid Transporter (ASBT, SLC10A2): A Potential Prodrug Target

“…While precise information about the substrate binding domains of hASBT are not available yet, its structural and functional determinants have been studied using various biophysical methods [36][37][38][39][40] . These studies have indicated that hASBT exhibits a seven transmembrane (7 TM) topology similar to NTCP.…”