The relation between charge movement and transport‐associated currents in the rat GABA cotransporter rGAT1

Fesce, Riccardo; Giovannardi, Stefano; Binda, Francesca; Bossi, Elena; Peres, Antonio

doi:10.1113/jphysiol.2002.026823

Cited by 31 publications

(75 citation statements)

References 26 publications

(67 reference statements)

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“…1d, Table 1). Although it is commonly accepted that the slope of the I-Q relationship yields the transporter turnover rate (Loo et al, 1993;Mager et al, 1993;Fesce et al, 2002;Sacher et al, 2002;Karakossian et al, 2005), we provide evidence in this study that this slope underestimates the GAT1 turnover rate. Figure 2a shows GAT1 GABA-evoked (1 mM) responses at 21°C (left trace) and 31°C (right trace).…”

Section: Correlation Between the Gaba-evoked Current (contrasting

confidence: 56%

“…1a) and is directly proportional to (see Fig. 1d) (Mager et al, 1993;Fesce et al, 2002;Sacher et al, 2002;Karakossian et al, 2005).…”

Section: Freeze-fracture and Electron Microscopymentioning

confidence: 99%

“…An important factor that governs the ability of the GATs to shape fast synaptic events, such as those mediated by GABA A receptors, is the rate at which these proteins transport GABA across the plasma membrane per unit time (i.e., turnover rate). For the forward mode of transport (Na + /Cl − / GABA cotransport into the cell), estimates of the turnover rate range 1.5-25 s −1 at 21-23°C and voltages ranging from −50 to −120 mV (Radian, Bendahan & Kanner, 1986;Mager et al, 1993;Forlani et al, 2001b;Fesce et al, 2002;Sacher et al, 2002;Whitlow et al, 2003;Karakossian et al, 2005). For the reverse mode, the turnover rate has been estimated to range 3-60 s −1 at 33°C, and voltages ranging from −120 to +120 mV (Lu & Hilgemann, 1999b).…”

Section: Introductionmentioning

confidence: 99%

“…The turnover rate is estimated by obtaining the ratio of the maximum GABA-evoked inward current (I max ) and the maximum voltage-induced transporter charge movements (Q max ) (Loo et al, 1993;Mager et al, 1993;Lu & Hilgemann, 1999b;Forlani et al, 2001a,b;Fesce et al, 2002;Sacher et al, 2002;Karakossian et al, 2005). I max is necessarily obtained under saturating substrate concentrations and is defined as I max = N T meR T , where N T is the total number of functional transporters in the plasma membrane, m is the number of charges translocated across the plasma membrane per transport cycle, e is the elementary charge and R T is the unitary transporter turnover rate (cycles per second).…”

Section: Introductionmentioning

confidence: 99%

“…Because I max is a measure of the maximal rate of GABA translocation across the plasma membrane (Loo et al, 2000;Sacher et al, 2002;Whitlow et al, 2003;Karakossian et al, 2005) and Q max is proportional to the total number of transporters in the plasma membrane (Zampighi et al, 1995;Eskandari et al, 2000), the ratio of I max to Q max [(N T meR T )/(N T zδe)] has been used to estimate the GAT turnover rate (see Fig. 1d) (Mager et al, 1993;Forlani et al, 2001a,b;Fesce et al, 2002;Sacher et al, 2002;Karakossian et al, 2005) and other electrogenic Na + -coupled transporters (Loo et al, 1993;Wadiche et al, 1995;Mackenzie et al, 1996;Eskandari et al, 1997;Forster et al, 2002;Mim et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

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Turnover Rate of the γ-Aminobutyric Acid Transporter GAT1

et al. 2007

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We combined electrophysiological and freeze-fracture methods to estimate the unitary turnover rate of the γ-aminobutyric acid (GABA) transporter GAT1. Human GAT1 was expressed in Xenopus laevis oocytes, and individual cells were used to measure and correlate the macroscopic rate of GABA transport and the total number of transporters in the plasma membrane. The twoelectrode voltage-clamp method was used to measure the transporter-mediated macroscopic current evoked by GABA ( ), macroscopic charge movements (Q NaCl ) evoked by voltage pulses and whole-cell capacitance. The same cells were then examined by freeze-fracture and electron microscopy in order to estimate the total number of GAT1 copies in the plasma membrane. GAT1 expression in the plasma membrane led to the appearance of a distinct population of 9-nm freeze-fracture particles which represented GAT1 dimers. There was a direct correlation between Q NaCl and the total number of transporters in the plasma membrane. This relationship yielded an apparent valence of 8 ± 1 elementary charges per GAT1 particle. Assuming that the monomer is the functional unit, we obtained 4 ± 1 elementary charges per GAT1 monomer. This information and the relationship between and Q NaCl were used to estimate a GAT1 unitary turnover rate of 15 ± 2 s −1 (21°C, −50 mV). The temperature and voltage dependence of GAT1 were used to estimate the physiological turnover rate to be 79-93 s −1 (37°C, −50 to −90 mV).

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Section: Correlation Between the Gaba-evoked Current (contrasting

confidence: 56%

“…1a) and is directly proportional to (see Fig. 1d) (Mager et al, 1993;Fesce et al, 2002;Sacher et al, 2002;Karakossian et al, 2005).…”

Section: Freeze-fracture and Electron Microscopymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Turnover Rate of the γ-Aminobutyric Acid Transporter GAT1

et al. 2007

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Revised Ion/Substrate Coupling Stoichiometry of GABA Transporters

Eskandari

Willford

Anderson

2017

Advances in Neurobiology

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The purpose of this review is to highlight recent evidence in support of a 3 Na: 1 Cl: 1 GABA coupling stoichiometry for plasma membrane GABA transporters (SLC6A1 , SLC6A11 , SLC6A12 , SLC6A13 ) and how the revised stoichiometry impacts our understanding of the contribution of GABA transporters to GABA homeostasis in synaptic and extrasynaptic regions in the brain under physiological and pathophysiological states. Recently, our laboratory probed the GABA transporter stoichiometry by analyzing the results of six independent measurements, which included the shifts in the thermodynamic transporter reversal potential caused by changes in the extracellular Na, Cl, and GABA concentrations, as well as the ratio of charge flux to substrate flux for Na, Cl, and GABA under voltage-clamp conditions. The shifts in the transporter reversal potential for a tenfold change in the external concentration of Na, Cl, and GABA were 84 ± 4, 30 ± 1, and 29 ± 1 mV, respectively. Charge flux to substrate flux ratios were 0.7 ± 0.1 charges/Na, 2.0 ± 0.2 charges/Cl, and 2.1 ± 0.1 charges/GABA. We then compared these experimental results with the predictions of 150 different transporter stoichiometry models, which included 1-5 Na, 0-5 Cl, and 1-5 GABA per transport cycle. Only the 3 Na: 1 Cl: 1 GABA stoichiometry model correctly predicts the results of all six experimental measurements. Using the revised 3 Na: 1 Cl: 1 GABA stoichiometry, we propose that the GABA transporters mediate GABA uptake under most physiological conditions. Transporter-mediated GABA release likely takes place under pathophysiological or extreme physiological conditions.

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Functionally independent subunits in the oligomeric structure of the GABA cotransporter rGAT1

Soragna

Bossi

Giovannardi

et al. 2005

Cell. Mol. Life Sci.

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We have combined structural and functional approaches to investigate the role of oligomerization in the operation of the GABA transporter rGAT1. Xenopus laevis oocytes were induced to express, either separately or simultaneously, the wild-type form of rGAT1 and a mutated (Y140W) form, unable to translocate GABA and to generate transport currents, although its intramembrane charge movement properties are only slightly affected. These characteristics, together with the insensitivity of Y140W to the blocking action of SKF89976A, were used to study the possible functional interaction of the two forms in an heteromeric structure. The electrophysiological data from oocytes coexpressing wild-type and Y140W rGAT1 were consistent with a completely independent activity of the two forms. Oligomerization was also studied by fluorescence resonance energy transfer (FRET) in tsA201 cells expressing the transporters fused with cyan and yellow fluorescent proteins (ECFP and EYFP). All combinations tested (WT-ECFP/WTEYFP, Y140W-ECFP/Y140W-EYFP and WT-ECFP/ Y140W-EYFP) were able to give rise to FRET, confirming the formation of homo- as well as heterooligomers. We conclude that, although rGAT1 undergoes structural oligomerization, each monomer operates independently.

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The relation between charge movement and transport‐associated currents in the rat GABA cotransporter rGAT1

Cited by 31 publications

References 26 publications

Turnover Rate of the γ-Aminobutyric Acid Transporter GAT1

Turnover Rate of the γ-Aminobutyric Acid Transporter GAT1

Revised Ion/Substrate Coupling Stoichiometry of GABA Transporters

Functionally independent subunits in the oligomeric structure of the GABA cotransporter rGAT1

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