Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
Structural probing of a microdomain in the dopamine transporter by engineering of artificial Zn2+ binding sites
Autore:
Norregaard, L; Visiers, I; Loland, CJ; Ballesteros, J; Weinstein, H; Gether, U;
Indirizzi:
Univ Copenhagen, Panum Inst, Dept Med Physiol, Dept Cellular & Mol Physiol, DK-2200 Copenhagen N, Denmark Univ Copenhagen Copenhagen Denmark N siol, DK-2200 Copenhagen N, Denmark NeuroSearch AS, Dept Neurochem, Ballerup, Denmark NeuroSearch AS Ballerup Denmark h AS, Dept Neurochem, Ballerup, Denmark Mt Sinai Sch Med, Dept Physiol & Biophys, New York, NY 10029 USA Mt Sinai Sch Med New York NY USA 10029 & Biophys, New York, NY 10029 USA
Titolo Testata:
BIOCHEMISTRY
fascicolo: 51, volume: 39, anno: 2000,
pagine: 15836 - 15846
SICI:
0006-2960(200012)39:51<15836:SPOAMI>2.0.ZU;2-S
Fonte:
ISI
Lingua:
ENG
Soggetto:
PROTEIN-COUPLED RECEPTORS; MEMBRANE-SPANNING SEGMENT; TACHYKININ NK-1 RECEPTOR; SEROTONIN TRANSPORTER; TRANSMEMBRANE DOMAIN; NEUROTRANSMITTER TRANSPORTERS; NOREPINEPHRINE TRANSPORTERS; HORMONE RECEPTOR; COCAINE BINDING; D2 RECEPTOR;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
51
Recensione:
Indirizzi per estratti:
Indirizzo: Gether, U Univ Copenhagen, Panum Inst, Dept Med Physiol, Dept Cellular & Mol Physiol, 12-5-22, DK-2200 Copenhagen N, Denmark Univ Copenhagen 12-5-22 Copenhagen Denmark N enhagen N, Denmark
Citazione:
L. Norregaard et al., "Structural probing of a microdomain in the dopamine transporter by engineering of artificial Zn2+ binding sites", BIOCHEM, 39(51), 2000, pp. 15836-15846

Abstract

Previously, we have identified three Zn2+ binding residues in an endogenous Zn2+ binding site in the human dopamine transporter (hDAT): (193)His in extracellular loop 2 (ECL 2), (375)His at the external end of transmembrane segment (TM) 7, and (396)Glu at the external end of TM 8. Here we have generated a series of artificial Zn2+ binding sites in a domain situated aroundthe external ends of TMs 7 and 8 by taking advantage of the well-defined structural constraints for binding of the zinc(II) ion. Initially, we found that the Zn2+-coordinating (193)His in ECL 2 could be substituted with a histidine inserted at the i - 4 position relative to (375)His in TM 7. In this mutant (H193K/M371H), Zn2+ potently inhibited [H-3]dopamine uptake with an IC50 value of 7 muM as compared to a value of 300 muM for the control (H193K). These data are consistent with the presence of an alpha -helical configuration of TM 7. This inference was further corroborated by the observation that no increase in the apparent Zn2+ affinity was observed following introduction of histidines at the i - 2, i - 3, and i - 5 positions. In contrast, introduction of histidines at positions i + 2, i + 3, and i + 4 all resulted in potent inhibition of [H-3]dopamine uptake by Zn2+ (IC50 = 3-32 muM). These observations are inconsistent with continuation of the helix beyond position 375 and indicate an approximate boundary between the end of thehelix and the succeeding loop. In summary, the data presented here providenew insight into the structure of a functionally important domain in the hDAT and illustrate how engineering of Zn2+ binding sites can be a useful approach for probing both secondary and tertiary structure relationships in membrane proteins of unknown structure.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 28/11/20 alle ore 04:27:45