Using lithium to probe sequential cation interactions with GAT1.

Area of Science:

• Neuroscience
• Biochemistry
• Molecular Biology

Background:

• The sodium/chloride-dependent GABA transporter (GAT1) is crucial for regulating GABAergic neurotransmission.
• Lithium ions (Li(+)) are known to interact with GAT1, but their precise effects on transporter kinetics remain unclear.
• Understanding these interactions is vital for comprehending lithium's therapeutic effects and potential side effects.

Purpose of the Study:

• To investigate how Li(+) influences the kinetic parameters of GAT1-mediated GABA cotransport.
• To elucidate the mechanism by which Li(+) affects the binding and translocation steps of GAT1.
• To determine the impact of partial substitution of extracellular Na(+) with Li(+) on GAT1 function.

Main Methods:

• Expression of GAT1 in Xenopus oocytes.
• Two-electrode voltage clamp electrophysiology to assess currents and kinetics.
• (22)Na uptake assays to quantify ion transport.
• Kinetic analysis under varying Li(+) and Na(+) concentrations.

Main Results:

• Li(+) reduced the coupling ratio between Na(+) and net charge translocation during GABA cotransport.
• Li(+) increased the apparent Na(+) affinity for GAT1 without altering its voltage dependence.
• Li(+) modified the voltage dependence of presteady-state relaxations in the absence of GABA.

Conclusions:

• A novel ordered binding scheme for GAT1 cotransport is proposed, where Li(+) can occupy the first cation binding site (Na2).
• Li(+) binding at Na2 facilitates the subsequent binding of a second Na(+) ion (at Na1), enhancing transporter turnover.
• These findings provide a mechanistic basis for Li(+) modulation of GABAergic signaling.