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Titolo:
Defining proximity relationships in the tertiary structure of the dopaminetransporter - Identification of a conserved glutamic acid as a third coordinate in the endogenous Zn2+-binding site
Autore:
Loland, CJ; Norregaard, L; Gether, U;
Indirizzi:
Univ Copenhagen, Panum Inst, Dept Med Physiol 12522, Div Cellular& Mol Physiol, DK-2200 Copenhagen N, Denmark Univ Copenhagen Copenhagen Denmark N siol, DK-2200 Copenhagen N, Denmark
Titolo Testata:
JOURNAL OF BIOLOGICAL CHEMISTRY
fascicolo: 52, volume: 274, anno: 1999,
pagine: 36928 - 36934
SICI:
0021-9258(199912)274:52<36928:DPRITT>2.0.ZU;2-8
Fonte:
ISI
Lingua:
ENG
Soggetto:
TACHYKININ NK-1 RECEPTOR; METAL-BINDING SITES; SEROTONIN TRANSPORTER; NEUROTRANSMITTER TRANSPORTERS; NOREPINEPHRINE TRANSPORTERS; TRANSMEMBRANE DOMAIN; COCAINE BINDING; AMINO-ACID; ZINC IONS; RECOGNITION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
49
Recensione:
Indirizzi per estratti:
Indirizzo: Gether, U Univ Copenhagen, Panum Inst, Dept Med Physiol 12522, Div Cellular& Mol Physiol, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark Univ Copenhagen Blegdamsvej 3 Copenhagen Denmark N n N, Denmark
Citazione:
C.J. Loland et al., "Defining proximity relationships in the tertiary structure of the dopaminetransporter - Identification of a conserved glutamic acid as a third coordinate in the endogenous Zn2+-binding site", J BIOL CHEM, 274(52), 1999, pp. 36928-36934

Abstract

Recently, we have described a distance constraint in the unknown tertiary structure of the human dopamine transporter (hDAT) by identification of twohistidines, His(193) in the second extracellular loop and His(375) at the top of transmembrane (TM) 7, that form two coordinates in an endogenous, high affinity Zn2+-binding site. To achieve further insight into the tertiaryorganization of hDAT, we set out to identify additional residues involved in Zn2+ binding and subsequently to engineer artificial Zn2+-binding sites. Ten aspartic acids and glutamic acids, predicted to be on the extracellular side, were mutated to asparagine and glutamine, respectively. Mutation ofGlu(396) (E396Q) at the top of TM 8 increased the IC50 value for Zn2+ inhibition of [H-3]dopamine uptake from 1.1 to 530 mu M and eliminated Zn2+ induced potentiation of [H-3]WIN 35,428 binding. These data suggest that Glu(396) is involved in Zn2+ binding to hDAT, Importantly, Zn2+ sensitivity was preserved following substitution of Glu(396) with histidine, indicating that the effect of mutating Glu(396) is not an indirect effect because of the removal of a negatively charged residue. The common participation of Glu(396), His(193), and His(375) in binding the small Zn2+ ion implies their proximity in the unknown tertiary structure of hDAT, The close association between TM. 7 and 8 was further established by engineering of a Zn2+-binding site between His(375) and a cysteine inserted in position 400 in TM 8, Summarized, our data define an important set of proximity relationships in hDAT that should prove an important template for further exploring the molecular architecture of Na+/Cl--dependent neurotransmitter transporters.

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Documento generato il 28/11/20 alle ore 03:08:22