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 -