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.

The hASBT (human apical Na(+)-dependent bile acid transporter) constitutes a key target of anti-hypercholesterolaemic therapies and pro-drug approaches; physiologically, hASBT actively reclaims bile acids along the terminal ileum via Na(+) co-transport. Previously, TM (transmembrane segment) 7 was identified as part of the putative substrate permeation pathway using SCAM (substitute cysteine accessibility mutagenesis). In the present study, SCAM was extended through EL3 (extracellular loop 3; residues Arg(254)-Val(286)) that leads into TM7 from the exofacial matrix. Activity of most EL3 mutants was significantly hampered upon cysteine substitution, whereas ten (out of 31) were functionally inactive (<10% activity). Since only E282C lacked plasma membrane expression, EL3 amino acids predominantly fulfill critical functional roles during transport. Oppositely charged membrane-impermeant MTS (methanethiosulfonate) reagents {MTSET [(2-trimethylammonium) ethyl MTS] and MTSES [(2-sulfonatoethyl) MTS]} produced mostly similar inhibition profiles wherein only middle and descending loop segments (residues Thr(267)-Val(286)) displayed significant MTS sensitivity. The presence of bile acid substrate significantly reduced the rates of MTS modification for all MTS-sensitive mutants, suggesting a functional association between EL3 residues and bile acids. Activity assessments at equilibrative [Na(+)] revealed numerous Na(+)-sensitive residues, possibly performing auxiliary functions during transport such as transduction of protein conformational changes during translocation. Integration of these data suggests ligand interaction points along EL3 via electrostatic interactions with Arg(256), Glu(261) and probably Glu(282) and a potential cation-pi interaction with Phe(278). We conclude that EL3 amino acids are essential for hASBT activity, probably as primary substrate interaction points using long-range electrostatic attractive forces.

The objective was to investigate the interplay between transporter expression levels and substrate affinity in controlling the influence of aqueous boundary layer (ABL) resistance on transporter kinetics in an over-expression system. Taurocholate flux was measured across human apical sodium-dependent bile acid transporter (hASBT)-Madin-Darby canine kidney monolayers on different occasions and kinetic parameters estimated with and without considering ABL. In error-free simulation/ regression studies, flux values were generated across a range of J max , K t , and substrate concentrations. Similar evaluation was performed for transport inhibition studies. Additionally, simulation/regression studies were performed, incorporating 15% random error to estimate the probability of successfully estimating K t . Across different occasions, experimental J max and K t estimates for taurocholate were strongly associated (p Ͻ 0.001; r 2 ϭ 0.82) when ABL was not considered. Simulation/ regression results indicate that not considering ABL caused this association, such that K t estimates were highly positively biased at high hASBT expression. In reanalyzing taurocholate flux data using the ABL-present model, K t was relatively constant across occasions (ϳ5 M) and not associated with J max (p ϭ 0.24; r 2 ϭ 0.13). Simulations suggest that J max and K t collectively determined ABL influence, which is most prominent under conditions of low monolayer resistance. Additionally, not considering ABL lead to negatively biased K i estimates, especially at high J max . Error-inclusive simulation/regression studies indicated that the probability of successfully estimating K t depended on the contribution of ABL resistance to flux; when flux became increasingly ABL-limited, probability of success decreased. Results indicate that ABL resistance can bias K t and K i estimates from overexpression systems, where the extent of bias is determined by transporter expression level and substrate affinity.Transfected cell models overexpressing specific transporters are a powerful tool to characterize substrate requirements of the transporter, including its ability to translocate drugs and prodrugs (Herrera-Ruiz et al., 2003;Tolle-Sander et al., 2004;Balakrishnan et al., 2005;. Relative to native cells and in vivo systems, transfected cell models frequently have the advantage of characterizing a transporter without confounding variables, such as other simultaneously expressing transporters with overlapping substrate requirements. This benefit is achieved in part through high expression of the transporter of interest.We recently developed a stably transfected cell model for the human apical sodium-dependent bile acid transporter (hASBT), using MDCK cells (Balakrishnan et al., 2005) that possesses several favorable properties, including high hASBT expression. This hASBT-MDCK model was further developed to yield kinetic estimates of substrates and/or inhibitors (e.g., J max , K t , and K i ) that can be used for subsequent quantitative-structure acti...

Chlorpyrifos (CPF), an organophosphorus pesticide (OP), is one of the most widely used pesticides in the world. Subchronic exposures to CPF that do not cause cholinergic crisis are associated with problems in cognitive function (i.e., learning and memory deficits), but the biological mechanism(s) underlying this association remain speculative. To identify potential mechanisms of subchronic CPF neurotoxicity, adult male Long Evans (LE) rats were administered CPF at 3 or 10 mg/kg/d (s.c.) for 21 days. We quantified mRNA and non-coding RNA (ncRNA) expression profiles by RNA-seq, microarray analysis and small ncRNA sequencing technology in the CA1 region of the hippocampus. Hippocampal slice immunohistochemistry was used to determine CPF-induced changes in protein expression and localization patterns. Neither dose of CPF caused overt clinical signs of cholinergic toxicity, although after 21 days of exposure, cholinesterase activity was decreased to 58% or 13% of control levels in the hippocampus of rats in the 3 or 10 mg/kg/d groups, respectively. Differential gene expression in the CA1 region of the hippocampus was observed only in the 10 mg/kg/d dose group relative to controls. Of the 1382 differentially expressed genes identified by RNA-seq and microarray analysis, 67 were common to both approaches. Differential expression of six of these genes (Bdnf, Cort, Crhbp, Nptx2, Npy and Pnoc) was verified in an independent CPF exposure study; immunohistochemistry demonstrated that CRHBP and NPY were elevated in the CA1 region of the hippocampus at 10 mg/kg/d CPF. Gene ontology enrichment analysis suggested association of these genes with receptor-mediated cell survival signaling pathways. miR132/212 was also elevated in the CA1 hippocampal region, which may play a role in the disruption of neurotrophin-mediated cognitive processes after CPF administration. These findings identify potential mediators of CPF-induced neurobehavioral deficits following subchronic exposure to CPF at a level that inhibits hippocampal cholinesterase to less than 20% of control. An equally significant finding is that subchronic exposure to CPF at a level that produces more moderate inhibition of hippocampal cholinesterase (approximately 50% of control) does not produce a discernable change in gene expression.

Functional contributions of residues Val-99 -Ser-126 lining extracellular loop (EL) 1 of the apical sodium-dependent bile acid transporter were determined via cysteine-scanning mutagenesis, thiol modification, and in silico interpretation. Despite membrane expression for all but three constructs (S112C, Y117C, S126C), most EL1 mutants (64%) were inactivated by cysteine mutation, suggesting a functional role during sodium/bile acid co-transport. A negative charge at conserved residues Asp-120 and Asp-122 is required for transport function, whereas neutralization of charge at Asp-124 yields a functionally active transporter. D124A exerts low affinity for common bile acids except deoxycholic acid, which uniquely lacks a 7␣-hydroxyl (OH) group. Overall, we conclude that (i) Asp-122 functions as a Na ؉ sensor, binding one of two co-transported Na ؉ ions, (ii) Asp-124 interacts with 7␣-OH groups of bile acids, and (iii) apolar EL1 residues map to hydrophobic ligand pharmacophore features. Based on these data, we propose a comprehensive mechanistic model involving dynamic salt bridge pairs and hydrogen bonding involving multiple residues to describe sodium-dependent bile acid transporter-mediated bile acid and cation translocation.

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

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).

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...