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.
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