Abstract
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Journal of Biological Chemistry |
| Vol/bind | 284 |
| Udgave nummer | 24 |
| Sider (fra-til) | 16226-35 |
| Antal sider | 9 |
| ISSN | 0021-9258 |
| DOI | |
| Status | Udgivet - 2009 |
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Elucidating Conformational Changes in the {gamma}-Aminobutyric Acid Transporter-1. / Meinild, Anne-Kristine; Loo, Donald D F; Hansen, Søren Skovstrup; Gether, Ulrik; Macaulay, Nanna.
I: Journal of Biological Chemistry, Bind 284, Nr. 24, 2009, s. 16226-35.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review
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TY - JOUR
T1 - Elucidating Conformational Changes in the {gamma}-Aminobutyric Acid Transporter-1
AU - Meinild, Anne-Kristine
AU - Loo, Donald D F
AU - Hansen, Søren Skovstrup
AU - Gether, Ulrik
AU - Macaulay, Nanna
PY - 2009
Y1 - 2009
N2 - The GABA transporter-1 (GAT-1) has three current-generating modes: GABA-coupled current, Li(+)-induced leak current, and Na(+)-dependent transient currents. We earlier hypothesized that Li(+) is able to substitute for the first Na(+) in the transport cycle and thereby induce a distinct conformation in GAT-1 and that the onset of the Li(+)-induced leak current at membrane potentials more negative than -50 mV was due to a voltage-dependent conformational change of the Li(+)-bound transporter. In this study, we set out to verify this hypothesis and seek insight into the structural dynamics underlying the leak current, as well as the sodium-dependent transient currents, by applying voltage clamp fluorometry to tetramethylrhodamine 6-maleimide-labeled GAT-1 expressed in Xenopus laevis oocytes. MTSET accessibility studies demonstrated the presence of two distinct conformations of GAT-1 in the presence of Na(+) or Li(+). The voltage-dependent fluorescence intensity changes obtained in Li(+) buffer correlated with the Li(+)-induced leak currents, i.e. both were highly voltage-dependent and only present at hyperpolarized potentials (<-50 mV). The transient currents correlated directly with the voltage-dependent fluorescence data obtained in sodium buffer and the associated conformational changes were distinct from those associated with the Li(+)-induced leak current. The inhibitor potency of SKF89976A of the Li(+)- versus Na(+)-bound transporter confirmed the cationic dependence of the conformational occupancy. Our observations suggest that the microdomain situated at the external end of transmembrane I is involved in different conformational changes taking place either during the binding and release of sodium or during the initiation of the Li(+)-induced leak current.
AB - The GABA transporter-1 (GAT-1) has three current-generating modes: GABA-coupled current, Li(+)-induced leak current, and Na(+)-dependent transient currents. We earlier hypothesized that Li(+) is able to substitute for the first Na(+) in the transport cycle and thereby induce a distinct conformation in GAT-1 and that the onset of the Li(+)-induced leak current at membrane potentials more negative than -50 mV was due to a voltage-dependent conformational change of the Li(+)-bound transporter. In this study, we set out to verify this hypothesis and seek insight into the structural dynamics underlying the leak current, as well as the sodium-dependent transient currents, by applying voltage clamp fluorometry to tetramethylrhodamine 6-maleimide-labeled GAT-1 expressed in Xenopus laevis oocytes. MTSET accessibility studies demonstrated the presence of two distinct conformations of GAT-1 in the presence of Na(+) or Li(+). The voltage-dependent fluorescence intensity changes obtained in Li(+) buffer correlated with the Li(+)-induced leak currents, i.e. both were highly voltage-dependent and only present at hyperpolarized potentials (<-50 mV). The transient currents correlated directly with the voltage-dependent fluorescence data obtained in sodium buffer and the associated conformational changes were distinct from those associated with the Li(+)-induced leak current. The inhibitor potency of SKF89976A of the Li(+)- versus Na(+)-bound transporter confirmed the cationic dependence of the conformational occupancy. Our observations suggest that the microdomain situated at the external end of transmembrane I is involved in different conformational changes taking place either during the binding and release of sodium or during the initiation of the Li(+)-induced leak current.
U2 - 10.1074/jbc.M109.003137
DO - 10.1074/jbc.M109.003137
M3 - Journal article
C2 - 19363027
VL - 284
SP - 16226
EP - 16235
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 24
ER -