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Titolo:
RELAXATION KINETICS OF THE NA+ GLUCOSE COTRANSPORTER/
Autore:
LOO DDF; HAZAMA A; SUPPLISSON S; TURK E; WRIGHT EM;
Indirizzi:
UNIV CALIF LOS ANGELES,SCH MED,DEPT PHYSIOL LOS ANGELES CA 90024
Titolo Testata:
Proceedings of the National Academy of Sciences of the United Statesof America
fascicolo: 12, volume: 90, anno: 1993,
pagine: 5767 - 5771
SICI:
0027-8424(1993)90:12<5767:RKOTNG>2.0.ZU;2-D
Fonte:
ISI
Lingua:
ENG
Soggetto:
CHANNELS;
Keywords:
NA+; GLUCOSE SYMPORTER; PRE-STEADY-STATE CURRENT; CHARGE MOVEMENT;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
18
Recensione:
Indirizzi per estratti:
Citazione:
D.D.F. Loo et al., "RELAXATION KINETICS OF THE NA+ GLUCOSE COTRANSPORTER/", Proceedings of the National Academy of Sciences of the United Statesof America, 90(12), 1993, pp. 5767-5771

Abstract

An important class of integral membrane proteins, cotransporters, couple solute transport to electrochemical potential gradients; e.g., theNa+/glucose cotransporter uses the Na+ electrochemical potential gradient to accumulate sugar in cells. So far, kinetic analysis of cotransporters has mostly been limited to steady-state parameters. In this study, we have examined pre-steady-state kinetics of Na+/glucose cotransport. The cloned human transporter (hSGLT1) was expressed in Xenopus oocytes, and voltage-clamp techniques were used to monitor current transients after step changes in membrane potential. Transients exhibited a voltage-dependent time constant (tau) ranging between 2 and 10 ms. The charge movement Q was fitted to a Boltzmann relation with maximal charge Q(max) of almost-equal-to 20 nC, apparent valence z of 1, and potential V0.5 of -39 mV for 50% Q(max). Lowering external Na+ from 100 to 10 mM reduced Q(max) 40%, shifted V0.5 from -39 to -70 mV, had no effect on z, and reduced the voltage dependence of tau. Q(max) was independent of, but tau was dependent on, temperature (a 10-degrees-C increase increased tau by a factor of almost-equal-to 2.5 at -50 mV). Addition of sugar or phlorizin reduced Q(max). Analyses of hSGLT1 pre-steady-state kinetics indicate that charge transfer upon a step of membrane potential in the absence of sugar is due to two steps in the reaction cycle: Na+ binding/dissociation (30%) and reorientation of the protein in the membrane field (70%). The rate-limiting step appears to be Na+ binding/dissociation. Q(max) provides a measure of transporter density (almost-equal-to 10(4)/mum2). Charge transfer measurements give insight into the partial reactions of the Na+/glucose cotransporter, and, combined with genetic engineering of the protein, provide a powerfultool for studying transport mechanisms.

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Documento generato il 01/04/20 alle ore 11:39:18