Conformational Flexibility of Helix VI Is Essential for Substrate Permeation of the Human Apical Sodium-Dependent Bile Acid Transporter

Abstract

The present study characterizes the methanethiosulfonate (MTS) inhibition profiles of 26 consecutive cysteine-substituted mutants comprising transmembrane (TM) helix 6 of the human apical Na ϩ

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

“…Note that substrate binding and translocation does not require fulfillment of all five features simultaneously. As mapped to the protein, our studies have putatively identified residues satisfying the first two features (21,(23)(24)(25); studies with ASBT orthologs and paralogs corroborate these assignments (26,27,37). However, there has been minimal progress toward identification of apolar residues involved in hydrophobic substrate-protein interactions.…”

“…Using this approach, we have thus far identified putative portions of the substrate permeation path, including specific protein regions likely interacting or binding substrates of ASBT (21)(22)(23)(24). Here, we focused on the highly conserved, large extracellular loop 1 (EL1) spanning 27 amino acids (Val-99 -Ser-126) based on the following rationale.…”

Mutational Analysis of Uncharged Polar Residues and Proline in the Distal One-Third (Thr130–Pro142) of the Highly Conserved Region of Mouse Slc10a2

“…18) A difference in residues at position 294 and 295 in mammalian SLC10A2/Slc10a2 has been reported to determine sensitivity to the inhibitor, ((3R,5R)-3-butyl-3-ethyl-5-phenyl-2,3,4,5-tetrahydro-1,4-benzothiazepine 1,1-dioxide (known as 2164U90). 29) [30][31][32] In spite of such extensive research, it is still unclear how SLC10A2 interacts and transports its substrates, and most of the residues proposed to interact with bile acids have not been mapped in the highly conserved region (except for Asp 122 and Asp 124 ). The regular function of SLC10A2 is important for reabsorption of bile acids in the ileum, and surgical elimination of this function increased colonic tumorigenesis in the rat fed deoxycholic acid.…”

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.…”