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MA DPhil, Fellow of the HEA
Department of Biological and Medical Sciences
Faculty of Health and Life Sciences
Subject co-ordinator for BSc Biomedical Science and BSc Biological Sciences, and course co-ordinator for MSc Medical Genetics and Genomics.
The themes of my research
Health and environment
The Membrane Transport Group research involves investigating the structure-function relationship of membrane transport proteins, to try to elucidate how they bind and transport their substrates. In particular, we study (nutrient) transporters that are naturally present in the intestine with a view to designing small drug molecules that can be taken up through them. These transporters include the peptide transporter (PepT1), amino acid transporters (the PAT family), monocarboxylate transporters (MCTs) and organic anion transporters (OATPs). The techniques involved range from expression of wild-type and mutated transporter proteins in model systems, cell culture, organic chemistry (with Prof Pat Bailey, London South Bank University & Dr David Foley, University of Dundee) and protein crystallography and molecular modelling (with Dr Newstead and Prof Samson's groups, respectively, University of Oxford).
The organic anion transporting polypeptides (OATPs) encompass a family of membrane transport proteins responsible for the uptake of xenobiotic compounds. Human organic anion transporting polypeptide 1B1 (OATP1B1) mediates the uptake of clinically relevant compounds such as statins and chemotherapeutic agents into hepatocytes, playing an important role in drug delivery and detoxification. The OATPs have a putative 12-transmembrane domain topology and a highly conserved signature sequence (human OATP1B1: DSRWVGAWWLNFL), spanning the extracellular loop 3/TM6 boundary. The presence of three conserved tryptophan residues at the TM interface suggests a structural role for the sequence. This was investigated by site-directed mutagenesis of selected amino acids within the sequence D251E, W254F, W258/259F, and N261A. Transport was measured using the substrate estrone-3-sulfate and surface expression detected by luminometry and confocal microscopy, facilitated by an extracellular FLAG epitope. Uptake of estrone-3-sulfate and the surface expression of D251E, W254F, and W258/259F were both significantly reduced from the wild type OATP1B1-FLAG in transfected HEK293T cells. Confocal microscopy revealed that protein was produced but was retained intracellularly. The uptake and expression of N261A were not significantly different. The reduction in surface expression and intracellular protein retention indicates a structural and/or membrane localization role for these signature sequence residues in the human drug transporter OATP1B1.
In addition to being responsible for the majority of absorption of dietary nitrogen, the mammalian proton-coupled di- and tripeptide transporter PepT1 is also recognised as a major route of drug delivery for several important classes of compound, including lactam antibiotics and angiotensin-converting enzyme inhibitors. Thus there is considerable interest in the PepT1 protein and especially its substrate binding site. In the absence of a crystal structure, computer modelling has been used to try to understand the relationship between PepT1 3D structure and function. Two basic approaches have been taken: modelling the transporter protein, and modelling the substrate. For the former, computer modelling has evolved from early interpretations of the twelve transmembrane domain structure to more recent homology modelling based on recently crystallised bacterial members of the major facilitator superfamily (MFS). Substrate modelling has involved the proposal of a substrate binding template, to which all substrates must conform and from which the affinity of a substrate can be estimated relatively accurately, and identification of points of potential interaction of the substrate with the protein by developing a pharmacophore model of the substrates. Most recently, these two approaches have moved closer together, with the attempted docking of a substrate library onto a homology model of the human PepT1 protein. This article will review these two approaches in which computers have been applied to peptide transport and suggest how such computer modelling could affect drug design and delivery through PepT1.
The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [3H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 M. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 M). Other amino acids that inhibited [3H]proline were isoleucine (Ki 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (Ki >5mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (Ki 1.09 mM), whereas -aminobutyric acid (GABA), -alanine, L-Dopa, D-serine, and -aminolevulinic acid were without effect on [3H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid -œtransceptor- that plays a role in nutrient sensing.
A thiodipeptide carrier system is shown to be effective at enabling a range of covalently bound molecules, including benzyl, benzoyl and ibuprofen conjugates, to be transported via the intestinal peptide transporter PepT1, demonstrating its potential as a rational drug delivery target.
T1R taste receptors are present throughout the gastrointestinal tract. Glucose absorption comprises active absorption via SGLT1 and facilitated absorption via GLUT2 in the apical membrane. Trafficking of apical GLUT2 is rapidly up-regulated by glucose and artificial sweeteners, which act through T1R2 + T1R3/alpha-gustducin to activate PLC beta 2 and PKC beta II. We therefore investigated whether non-sugar nutrients are regulated by taste receptors using perfused rat jejunum in vivo. Under different conditions, we observed a Ca(2+)-dependent reciprocal relationship between the H(+)/oligopeptide transporter PepT1 and apical GLUT2, reflecting the fact that trafficking of PepT1 and GLUT2 to the apical membrane is inhibited and activated by PKC beta II, respectively. Addition of l-glutamate or sucralose to a perfusate containing low glucose (20 mm) each activated PKC beta II and decreased apical PepT1 levels and absorption of the hydrolysis-resistant dipeptide l-Phe(Psi S)-l-Ala (1 mm), while increasing apical GLUT2 and glucose absorption within minutes. Switching perfusion from mannitol to glucose (75 mm) exerted similar effects. l-Glutamate induced rapid GPCR internalization of T1R1, T1R3 and transducin, whereas sucralose internalized T1R2, T1R3 and alpha-gustducin. We conclude that l-glutamate acts via amino acid and glucose via sweet taste receptors to coordinate regulation of PepT1 and apical GLUT2 reciprocally through a common enterocytic pool of PKC beta II. These data suggest the existence of a wider Ca(2+) and taste receptor-coordinated transport network incorporating other nutrients and/or other stimuli capable of activating PKC beta II and additional transporters, such as the aspartate/glutamate transporter, EAAC1, whose level was doubled by l-glutamate. The network may control energy supply.
Chondrocytes, which control the turnover of cartilage, undergo predominantly glycolytic metabolism due to the avascular nature of the tissue. This will result in high levels of lactic acid production, and this lactic acid must leave the cells for their normal intracellular pH to be maintained. However to date the mechanism by which lactic acid is removed from the chondrocyteshas not been elucidated. In the present study lactic acid transport has been characterised using the intracellular pH-sensitive fluorimetric dye BCECF to measure intracellular pH (pH). Addition of extracellular lactic acid-induced an acidification which was sensitive to alpha-cyano-4-hydroxycinnamate (alpha-CHC) and phloretin indicating the involvement of isoform(s) of the monocarboxylate transporter (MCT) family. The results studies of transport kinetics were consistent with the MCT4 isoform (K-m 14.1 mM), common to other glycolytic cells. Western blotting confirmed that MCT4 was the predominantly expressed isoform, although both MCT1 and MCT4 transcripts were present when cells were assayed by RT-PCR. Through effects on pH(i), the activity of this transporter may therefore modify cartilage turnover. Copyright (C) 2002 S. Karger AG, Basel.
Member of The Physiological Society and the European Intestinal Transport Group
Fellow of the Higher Education Academy
Department of Biological and Medical Sciences - Faculty of Health and Life Sciences