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Titolo:
GAT1 (GABA : Na+: Cl-) cotransport function - Kinetic studies in giant Xenopus oocyte membrane patches
Autore:
Lu, CC; Hilgemann, DW;
Indirizzi:
Univ Texas, SW Med Ctr, Dept Physiol, Dallas, TX 75235 USA Univ Texas Dallas TX USA 75235 ed Ctr, Dept Physiol, Dallas, TX 75235 USA
Titolo Testata:
JOURNAL OF GENERAL PHYSIOLOGY
fascicolo: 3, volume: 114, anno: 1999,
pagine: 445 - 457
SICI:
0022-1295(199909)114:3<445:G(:NCC>2.0.ZU;2-W
Fonte:
ISI
Lingua:
ENG
Soggetto:
GAMMA-AMINOBUTYRIC-ACID; NA,K PUMP; NA/K PUMP; ELECTROGENIC PROPERTIES; GLUCOSE COTRANSPORTER; VOLTAGE-DEPENDENCE; CHARGE MOVEMENTS; ION-BINDING; RAT-BRAIN; TRANSPORT;
Keywords:
charge movement; neurotransmitter transporter; NO-711; transport kinetics; voltage dependence;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
26
Recensione:
Indirizzi per estratti:
Indirizzo: Lu, CC Univ Texas, SW Med Ctr, Dept Physiol, 5323 Harry Hines Blvd, Dallas, TX 75325 USA Univ Texas 5323 Harry Hines Blvd Dallas TX USA 75325 , TX 75325 USA
Citazione:
C.C. Lu e D.W. Hilgemann, "GAT1 (GABA : Na+: Cl-) cotransport function - Kinetic studies in giant Xenopus oocyte membrane patches", J GEN PHYSL, 114(3), 1999, pp. 445-457

Abstract

TO explain cotransport function, the "alternating access" model requires that conformational changes of the empty transporter allow substrates to bind alternatively on opposite membrane sides. To test this principle for the GAT1 (GABA:Na+:Cl-) cotransporter, we have analyzed how its charge-moving partial reactions depend on substrates on both membrane sides in giant Xenopus oocyte membrane patches. (a) "Slow" charge movements, which require extracellular Na+ and probably reflect occlusion of Na+ by GAT1, were defined in three ways with similar results: by application of the high-affinity GAT1blocker (NO-711), by application of a high concentration (120 mM) of cytoplasmic Cl-, and by removal of extracellular Na+ via pipette perfusion. (b) Three results indicate that cytoplasmic Cl- and extracellular Na+ bind to the transporter in a mutually exclusive fashion: first, cytoplasmic Cl- (5-140 mM) shifts the voltage dependence of the slow charge movement to more negative potentials, specifically by slowing its "forward" rate (i.e., extracellular Na+ occlusion); second, rapid application of cytoplasmic Cl- induces an outward current transient that requires extracellular Na+, consistent with extracellular Na+ being forced out of its binding site; third, fast charge-moving reactions, which can be monitored as a capacitance, are "immobilized" both by cytoplasmic Cl- binding and by extracellular Na+ occlusion (i.e., by the slow charge movement). (c) In the absence of extracellular Na+, three fast (submillisecond) charge movements have been identified, but noslow components. The addition of cytoplasmic Cl- suppresses two components(tau < 1 ms and 13 mu s) and enables a faster component (tau < 1 mu s) (d)We failed to identify charge movements of fully loaded GAT1 transporters (i.e., with all substrates on both sides). (e) Under zero-trans conditions, inward (forward) GAT1 current shows pronounced pre-steady state transients,while outward (reverse) GAT1 current does not. (f) Turnover rates for reverse GAT1 transport (33 degrees C), calculated from the ratio of steady state current magnitude to total charge movement magnitude, can exceed 60 s(-1)at positive potentials.

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Documento generato il 17/01/21 alle ore 18:25:31