Investigation of GABA transport and the GABA/Na+ relationship in human GAT1 using solid-supported membrane-based electrophysiology

The neurotransmitter γ-aminobutyric acid (GABA) plays a pivotal role as the primary inhibitory neurotransmitter in the brain, and its regulation primarily relies on the activity of the GABA transporter hGAT1 within the central nervous system (CNS). hGAT1 is classified as a secondary-active transport protein, utilizing the sodium ion (Na+) gradient to power the transport of GABA from the synaptic cleft back into the presynaptic neuron. Given its association with numerous neurological disorders, dysregulation in GABA transport underscores the significance of hGAT1 as a therapeutic target. Solid-supported membrane-based electrophysiology (SSME) stands as a valuable technique enabling the measurement of electrogenic events in transporters, pumps, and channels. In this method, the sample, comprising native membrane vesicles or proteoliposomes, adheres to an artificial bilayer situated atop a gold-coated sensor, generating a capacitive-coupled system. Transport is initiated by concentration gradients of substrates as the driving force, while the membrane voltage is zero. Using SSME, we observed GABA-induced currents in CHO membrane vesicles that overexpressed hGAT1, with a maximum amplitude ranging from 3 to 5 nA. These currents exhibited a triphasic pattern, prompting the identification of three distinct electrogenic events. We successfully pinpointed the transport component, revealing a KM value within the range of 15 to 20 µM. Additionally, we investigated the cooperativity between Na+ and GABA, confirming a Na+:GABA stoichiometry of 2:1.