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

Abstract

The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) plays a critical role in the enterohepatic circulation of bile acids, as well as in cholesterol homeostasis. ASBT reclaims bile acids from the distal ileum via active sodium co-transport, in a multistep process, orchestrated by key residues in exofacial loop regions, as well as in membrane-spanning helices. Here, we unravel the functional contribution of highly conserved transmembrane helix 1 (TM1) on the hASBT transport cycle. Consecutive cysteine substitution of individual residues along the TM1 helix (Ile 29 -Gly 50 ), as well as exofacial Asn 27 and Asn 28 , resulted in functional impairment of ϳ70% of mutants, despite appreciable cell surface expression for all but G50C. Cell surface expression of G50C and G50A was rescued upon MG132 treatment as well as cyclosporine A, but not by FK506 or bile acids, suggesting that Gly 50 is involved in hASBT folding. TM1 accessibility to membrane-impermeant MTSET remains confined to the exofacial half of the helix along a single, discrete face. Substrate protection from MTSET labeling was temperature-dependent for L34C, T36C, and L38C, consistent with conformational changes playing a role in solvent accessibility for these mutants. Residue Leu 30 was shown to be critical for both bile acid and sodium affinity, while Asn 27 , Leu 38 , Thr 39 , and Met 46 participate in sodium co-transport. Combined, our data demonstrate that TM1 plays a pivotal role in ASBT function and stability, thereby providing further insight in its dynamic transport mechanism.Enterohepatic recirculation is a highly efficient mechanism for conserving the body's total bile acid pool. Whereas the majority of bile acids are reabsorbed passively throughout the small intestine, active reabsorption occurs in the distal ileum by the apical sodium-dependent bile acid transporter (ASBT, 2 SLC10A2). As a high-capacity, high-affinity co-transporter, ASBT effectively reclaims the vast majority of bile acids, such that less than 5% of the circulating bile acid pool is lost through fecal elimination (1). Defective ASBT transport is associated with various disease conditions (2-4). 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 (11-16). 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 facili...

Transmembrane Domain V Plays a Stabilizing Role in the Function of Human Bile Acid Transporter SLC10A2

“…Western blotting was also preformed as previously described. 17 Briefly, samples were loaded onto precast, 15 well, 12% Tris-HCl Polyacrylamide gel (BioRad 456–1046) along with 2.5 μL Li-cor Odyssey two-color protein molecular weight marker (928-40001). A custom antibody (1:1,000) was used to visualize the glycosylated (41 kDa) and unglycoslyated (38 kDa) hASBT protein.…”

Resveratrol promotes degradation of the human bile acid transporter ASBT (SLC10A2)

“…The procedure was followed as described previously [25]. Briefly, cells were washed with icecold DPBS twice and then incubated with EZ Link NHS-SS-biotin (1 mg/ml) for 30 min at 4 • C. The reaction was quenched with 1 M Tris/HCl (pH 7.4).…”

“…Medium was changed on alternate days after confluency. The uptake studies were carried out as described previously [25]. Briefly, cells were washed twice with DPBS (Dulbecco's PBS) and then incubated at 37 • C for 12 min (previous studies have shown that TCA uptake is linear up to 15 min and 10 min in COS-1 and Caco-2 cells respectively) [23,26] …”

Transmembrane Domain II of the Human Bile Acid Transporter SLC10A2 Coordinates Sodium Translocation

“…TCA Uptake Study-Uptake studies were carried out as previously described (11). Briefly, COS-1 cells were transiently transfected with pCMV5 plasmid containing C270A mutant, TM2 mutants, or WT_ASBT DNA.…”

“…For example, mutations on ϳ70% TM1 residues resulted in impaired function (11), with residue Leu 30 critical for both bile acid and sodium binding. The large hydrophilic region lining the cytosolic half of TM3 may be partially involved in substrate exit during bile acid translocation (12).…”