Elucidating conformational changes in the gamma-aminobutyric acid transporter-1.
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.
Meinild
Anne-Kristine
AK
From the Departments of Biology, 2100 Copenhagen, Denmark. Electronic address: smeinild@bio.ku.dk.
Loo
Donald D F
DDF
Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095-1751.
Skovstrup
Soren
S
Medicinal Chemistry, 2100 Copenhagen, Denmark.
Gether
Ulrik
U
Neuroscience and Pharmacology, 2100 Copenhagen, Denmark.
MacAulay
Nanna
N
Cellular and Molecular Medicine, University of Copenhagen, 2100 Copenhagen, Denmark.
D000818:Animals; D002021:Buffers; D003553:Cystine / Q000737:chemistry; D005453:Fluorescence; D018682:GABA Agents / Q000494:pharmacology; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000502:physiology*; D018021:Lithium Chloride / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009557:Nipecotic Acids / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D012965:Sodium Chloride / Q000494:pharmacology*; D014982:Xenopus laevis
D002021:Buffers; D018682:GABA Agents; D050485:GABA Plasma Membrane Transport Proteins; D009557:Nipecotic Acids; D012965:Sodium Chloride; D003553:Cystine; C045598:N-(4,4-diphenyl-3-butenyl)nipecotic acid; D018021:Lithium Chloride