Transmembrane Domain VII of the Human Apical Sodium-Dependent Bile Acid Transporter ASBT (SLC10A2) Lines the Substrate Translocation Pathway

Abstract

Recent evidence implicating transmembrane (TM) segment 7 of the apical sodium-dependent bile acid transporter (ASBT) in substrate interaction warranted examination of its aqueous accessibility. Therefore, cysteine substitution of 22 consecutive amino acids was performed against a methanethiosulfonate (MTS)-resistant background (C270A). Activity and susceptibility to polar MTS derivatives [(2-aminoethyl)-methanethiosulfonate (MTSEA), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and methanethiosulfonate ethylsulfonate (MTSES)] of mutants were evaluated in COS-1 cells. Thr289, Tyr293, Gln297, Ala301, Phe307, and Tyr308 represented loss-of-function mutants; furthermore, the measurable residual activities for T289C, Y293C, and A301C (Յ20% control) proved insensitive to MTS treatment. MTSES and MTSET inhibition was confined to residues lining the extracellular half of TM7; amino acids situated deeper within the membrane were unaffected. In contrast, the entire length of TM7 was susceptible to the relatively smaller MTSEA; moreover, MTSEA sensitivity was significantly amended by coapplication with substrates. This selective pattern of modification suggests that the highly conserved lower half of TM7 lies within a water-filled cavity easily accessible from the extracellular milieu, whereas residues approaching the cytosolic/membrane interface reside in pores for which accessibility is modulated by molecular volume. Functionally inactive and MTS-inaccessible residues (T289C, Y293C, Q297C, and A301C) within TM7 may play a structural role critical to transporter function; conversely, MTS-sensitive residues are spatially distinct and may demarcate a face of the TM involved in substrate translocation. In addition, computational analysis of solvent-accessible domains identified five key solvent pockets that predominantly line the hydrophilic face of TM7. Combined, our data suggest that TM7 plays a dominant role in the hASBT translocation process.

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

“…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.…”

“…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. Cells were lysed in 350 l of a 1 N NaOH, and aliquots were analyzed using an LS6500 liquid scintillation counter (Beckman Coulter, Inc., Fullerton, CA) and total protein quantification using the Bradford protein assay (Bio-Rad Protein Membrane Expression-Protein expression was determined as described previously (23). Briefly, transiently transfected COS-1 cells were lysed, separated on a 12.5% SDSpolyacrylamide gel, and transferred onto an immunoblot polyvinylidene difluoride membrane (Bio-Rad).…”

“…Blots were probed for positive and negative controls, the plasma membrane marker ␣-integrin (150 kDa), and the endoplasmic reticulum membrane protein calnexin (90 kDa) to assess the integrity of the biotinylation procedure (calnexin data not shown). Relative ASBT membrane expression was standardized to integrin expression and quantified via densitometry as previously described (23).…”

“…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.…”