Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue

Fiona E. Baird, Jorge J. Pinilla-Tenas, William L. J. Ogilvie, Vadival Ganapathy, Harinder S. Hundal, Peter M. Taylor

    Research output: Contribution to journalArticle

    26 Citations (Scopus)

    Abstract

    System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K-0.5(Na+)) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K-0.5(Na+) at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.

    Original languageEnglish
    Pages (from-to)369-375
    Number of pages7
    JournalBiochemical Journal
    Volume397
    Issue number2
    DOIs
    Publication statusPublished - 15 Jul 2006

    Fingerprint

    Allosteric Regulation
    Diethyl Pyrocarbonate
    Amino Acid Transport Systems
    Histidine
    Amino Acids
    Hydroxylamine
    Cell membranes
    Alanine
    Protein Isoforms
    Genes
    Cells
    Substrates
    Xenopus
    Oocytes
    Proteins
    Cell Membrane
    Phenotype
    Mutation

    Keywords

    • Allosteric regulation
    • Amino acid transport
    • Hydrogen ion
    • System A
    • System N

    Cite this

    Baird, Fiona E. ; Pinilla-Tenas, Jorge J. ; Ogilvie, William L. J. ; Ganapathy, Vadival ; Hundal, Harinder S. ; Taylor, Peter M. / Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue. In: Biochemical Journal. 2006 ; Vol. 397, No. 2. pp. 369-375.
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    abstract = "System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K-0.5(Na+)) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K-0.5(Na+) at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.",
    keywords = "Allosteric regulation, Amino acid transport, Hydrogen ion, System A, System N",
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    Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue. / Baird, Fiona E.; Pinilla-Tenas, Jorge J.; Ogilvie, William L. J.; Ganapathy, Vadival; Hundal, Harinder S.; Taylor, Peter M.

    In: Biochemical Journal, Vol. 397, No. 2, 15.07.2006, p. 369-375.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue

    AU - Baird, Fiona E.

    AU - Pinilla-Tenas, Jorge J.

    AU - Ogilvie, William L. J.

    AU - Ganapathy, Vadival

    AU - Hundal, Harinder S.

    AU - Taylor, Peter M.

    PY - 2006/7/15

    Y1 - 2006/7/15

    N2 - System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K-0.5(Na+)) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K-0.5(Na+) at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.

    AB - System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K-0.5(Na+)) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K-0.5(Na+) at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.

    KW - Allosteric regulation

    KW - Amino acid transport

    KW - Hydrogen ion

    KW - System A

    KW - System N

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    DO - 10.1042/BJ20060026

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    SP - 369

    EP - 375

    JO - Biochemical Journal

    JF - Biochemical Journal

    SN - 0264-6021

    IS - 2

    ER -