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Titolo:
Engineered Zn2+ switches in the gamma-aminobutyric acid (GABA) transporter-1 - Differential effects on GABA uptake and currents
Autore:
MacAulay, N; Bendahan, A; Loland, CJ; Zeuthen, T; Kanner, BI; Gether, U;
Indirizzi:
Univ Copenhagen, Panum Inst, Dept Med Physiol 12 5 22, Div Cellular & Mel Physiol, DK-2200 Copenhagen N, Denmark Univ Copenhagen Copenhagen DenmarkN siol, DK-2200 Copenhagen N, Denmark Hebrew Univ Jerusalem, Hadassah Med Sch, Dept Biochem, IL-91120 Jerusalem,Israel Hebrew Univ Jerusalem Jerusalem Israel IL-91120 L-91120 Jerusalem,Israel
Titolo Testata:
JOURNAL OF BIOLOGICAL CHEMISTRY
fascicolo: 44, volume: 276, anno: 2001,
pagine: 40476 - 40485
SICI:
0021-9258(20011102)276:44<40476:EZSITG>2.0.ZU;2-1
Fonte:
ISI
Lingua:
ENG
Soggetto:
NA+CL COTRANSPORT FUNCTION; HUMAN DOPAMINE TRANSPORTER; OOCYTE MEMBRANE PATCHES; XENOPUS-OOCYTES; NEUROTRANSMITTER TRANSPORTERS; ELECTROGENIC PROPERTIES; GLUTAMATE TRANSPORTER; GLUCOSE COTRANSPORTER; CHLORIDE CHANNEL; RAT-BRAIN;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
43
Recensione:
Indirizzi per estratti:
Indirizzo: Gether, U Univ Copenhagen, Panum Inst, Dept Med Physiol 12 5 22, Div Cellular & Mel Physiol, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark Univ Copenhagen Blegdamsvej 3 Copenhagen Denmark N n N, Denmark
Citazione:
N. MacAulay et al., "Engineered Zn2+ switches in the gamma-aminobutyric acid (GABA) transporter-1 - Differential effects on GABA uptake and currents", J BIOL CHEM, 276(44), 2001, pp. 40476-40485

Abstract

Two high affinity Zn2+ binding sites were engineered in the otherwise Zn2+-insensitive rat gamma -aminobutyric acid (GABA) transporter-1 (rGAT-1) based on structural information derived from Zn2+ binding sites engineered previously in the homologous dopamine transporter. Introduction of a histidine(T349H) at the extracellular end of transmembrane segment (TM) 7 together with a histidine (E370H) or a cysteine (Q374C) at the extracellular end of TM 8 resulted in potent inhibition of [H-3]GABA uptake by Zn2+ (IC50 = 35 and 44 muM, respectively). Upon expression in Xenopus laevis oocytes it was similarly observed that Zn2+ was a potent inhibitor of the GABA-induced current (IC50 = 21 muM for T349H/E370H and 51 muM for T349H/Q374C), albeit maximum inhibition was only similar to 40% in T349H/E370H versus similar to 90% in T349H/Q374C. In the wild type, Zn2+ did not affect the Na+-dependent transient currents elicited by voltage jumps and thought to reflect capacitive charge movements associated with Na+ binding. However, in both mutants Zn2+ caused a reduction of the inward transient currents upon jumping to hyperpolarized potentials as reflected in rightward-shifted Q/V relationships. This suggests that Zn2+ is inhibiting transporter function by stabilizing the outward-facing Na+-bound state. Translocation of lithium by the transporter does not require GAGA binding and analysis of this uncoupled Li+ conductance revealed a potent inhibition by Zn2+ in T349H/E370H, whereas surprisingly the T349H/Q374C leak was unaffected. This differential effect supports that the leak conductance represents a unique operational mode of the transporter involving conformational changes different from those of the substrate translocation process. Altogether our results support both an evolutionary conserved structural organization of the TM 7/8 domain and a key role of this domain in GABA-dependent and -independent conformational changes ofthe transporter.

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Documento generato il 04/07/20 alle ore 21:30:08