Identification of Functionally Relevant Residues of the Rat Ileal Apical Sodium-dependent Bile Acid Cotransporter

Abstract

The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na ؉ ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na ؉ ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na ؉ .

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.…”

“…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. (i) The functional importance of charged protein residues during Na ϩ and bile acid binding events, as demonstrated by our group (21,25) and others (26), suggests functional involvement of Asp-120, Asp-122, and Asp-124, which are clustered along the distal half of EL1 during Na ϩ binding events. Moreover, numerous polar residues, also capable of long range electrostatic interactions with charged substrates, are present throughout the EL1 segment.…”

“…Moreover, numerous polar residues, also capable of long range electrostatic interactions with charged substrates, are present throughout the EL1 segment. Prior studies using ASBT orthologs (26) and paralogs (27) concluded that the EL1-localized Asp-122 (Asp-115 in NTCP) may form a sodium sensor, binding one of the two co-transported Na ϩ ions during the ASBT transport cycle. (ii) Both EL1 and EL3 protein segments have been speculated to act as re-entrant loop segments by Zahner et al (27), who postulated the formation of a P-loop structure by EL1/3 to explain transport mechanisms of the rat Ntcp and recently purported for ASBT (16).…”

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

“…The multidrug transporter ABCB1, for example, is functional after all the Cys residues are replaced with Ala; thus, Cys-less ABCB1 can be used as a scaffold in Cys-insertion scanning analysis, 27,28) but some Cys residues are essential for the function and correct intracellular localization of both ileal and hepatic SBATs. 1,9,10,12,13,29,30) In addition to considerable similarity between SLC10A1 and SLC10A2 in amino acid sequences (alignment of known mammalian SBATs results in 30% identity and 65% similarity), they share structural similarities, particularly in their hydrophobicity profiles, indicating that their membrane topology is similar. Nevertheless, the distribution of functional regions within the polypeptide chain has been suggested to be different between SLC10A1 and SLC10A2.…”

“…[1][2][3][4][5][6][7] These transporters and their homologous transporters do not share structural similarities with other Na þ -dependent secondary transporters. Even though analysis of naturally occurring and artificially induced mutations has identified some residues critical for the function of these transporters, [8][9][10][11][12][13][14][15][16][17][18][19][20][21] the strict localization of functionally important regions, such as substrate binding sites, has not been decided yet, and the secondary structure of SLC10A1 and SLC10A2 is still controversial. Cys insertion scanning and chemical modification using thiol modifiers are useful methods to determine functionally important regions and the topology of membrane transporters.…”