The Cytosolic Half of Helix III Forms the Substrate Exit Route during Permeation Events of the Sodium/Bile Acid Cotransporter ASBT

Abstract

Site-directed alkylation of consecutively introduced cysteines was employed to probe the solventaccessible profile of highly conserved transmembrane helix 3 (TM3), spanning residues V127-T149 of the apical sodium-dependent bile acid transporter (ASBT), a key membrane protein involved in cholesterol homeostasis. Sequence alignment of SLC10 family members has previously identified a signature motif (ALGMMPL) localized to TM3 of ASBT with as yet undetermined function. Cysteine mutagenesis of this motif resulted in severe decreases in uptake activity only for mutants M141C and P142C. Additional conservative and nonconservative replacement of P142 suggests its structural and functional importance during the ASBT transport cycle. Significant decreases in transport activity were also observed for three cysteine mutants clustered along the exofacial half of the helix (M129C, T130C, S133C) and five mutants consecutively lining the cytosolic half of TM3 (L145C-T149C). Measurable surface expression was detected for all TM3 mutants. Using physicochemically different alkylating reagents, sites predominantly lining the cytosolic half of the TM3 helix were found to be solvent accessible (i.e. S128C, L143C-T149C). Analysis of substrate kinetics for select TM3 mutants demonstrates significant loss of taurocholic acid affinity for mutants S128C and L145C-T149C. Overall, we conclude: (i) the functional and structural importance of P142 during the transport cycle; and (ii) presence of a large hydrophilic cleft region lining the cytosolic half of TM3 that may form portions of the substrate exit route during permeation. Our studies provide unique insight into molecular mechanisms guiding the ASBT transport cycle with respect to substrate binding and translocation events. Efficient recirculation of the body's bile acid pool proceeds via multiple active and passive transport routes lining the enterohepatic pathway. Along the distal ileum, the apical sodium/ bile acid co-transporter ASBT (SLC10A2) constitutes the chief mechanism for reclaiming bile acids secreted into the gut in response to food intake (1). Functioning as a sodium symporter, the relatively small ASBT protein (348 amino acids, 41 kDa) transduces the free energy stored in electrochemical ion gradients into solute concentration gradients, resulting in the translocation of one bile acid molecule per (approximately) two sodium ions per transport cycle (2). As bile acids are the catabolic product of cholesterol metabolism, ASBT also plays a physiologically critical role in cholesterol homeostasis (3). Pharmaceutically, the intimate link between cholesterol and ASBT can be exploited to generate therapeutics aimed at hypercholesterolemia indications (4-10) and prodrug approaches to increase oral bioavailability (11,12).Previous work from our laboratory has confirmed a 7-transmembrane (7TM) spanning topology for ASBT (13,14) from which an in silico homology model was generated (14).

Transmembrane Helix 1 Contributes to Substrate Translocation and Protein Stability of Bile Acid Transporter SLC10A2

“…Further, ASBT constitutes a pharmacologic target for improving oral drug bioavailability (5-7) as well as hypocholesterolemic agents, because cholesterol metabolism is induced upon bile acid depletion (8,9). To elucidate the structure-function relationship of ASBT, our laboratory has previously employed cysteine scanning mutagenesis and site-directed alkylation techniques (10,11) to determine structural requirements for substrates and their turnover (11)(12)(13)(14)(15)(16). We demonstrate that residues lining TM6 (12) and TM7 (15) participate in substrate recognition and protein entry from the exofacial matrix, while the cytosolic half of TM3 mediates substrate release into the cytosolic milieu (14), putatively in conjunction with TM4 (18).…”

“…To elucidate the structure-function relationship of ASBT, our laboratory has previously employed cysteine scanning mutagenesis and site-directed alkylation techniques (10,11) to determine structural requirements for substrates and their turnover (11)(12)(13)(14)(15)(16). We demonstrate that residues lining TM6 (12) and TM7 (15) participate in substrate recognition and protein entry from the exofacial matrix, while the cytosolic half of TM3 mediates substrate release into the cytosolic milieu (14), putatively in conjunction with TM4 (18). Moreover, the extracellular loop (EL) 1 (13) and EL3 (16) regions mediate initial bile acid and sodium recognition and binding and may facilitate movement of ligands in solvent-accessible pockets situated deeper into the protein core, for ultimate translocation along membrane-spanning domains.…”

“…Mutants were verified by sequencing and transiently transfected in COS-1 cells as described (14). Protein expression at the cell surface was determined by biotinylation with the membrane impermeable EZ Link NHS-SS-Biotin (Pierce) followed by Western blot as previously described (14) and detailed in the figure legends. An Odyssey imaging system (Licor, NE) was used to visualize protein bands.…”

“…Rates of uptake were calculated as pmols of [ 3 H]TCA internalized per min per mg of protein using the Bradford assay for total protein quantification. The influence of sodium on transport function was determined by [ 3 H]TCA uptake under equilibrating (12 mM) versus physiological (137 mM) sodium concentrations, as described previously (14). Choline chloride (ChCl) is used as a replacement for sodium and added to the 12 mM sodium solution to maintain osmolarity.…”

“…MTSET Inhibition and Substrate Protection Studies-Transiently transfected COS-1 cells were pretreated with 1 mM MTSET for 10 min at room temperature, and evaluated for [ 3 H]TCA uptake, as previously described (13,14). Control cells (without MTSET) were treated identically and run in parallel.…”