Rocco Zerlotti- Functional characterization of human GAT1 through SSM-based electrophysiology

The γ-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the brain, and its homeostasis is mainly regulated by the GABA transporter hGAT1 in the central nervous system (CNS). GAT1 is a secondary-active transport protein that exploits the Na+ gradient to energize the uphill re-uptake of GABA from the synaptic cleft into the presynaptic neuron. Since dysregulations in GABA transport are linked to many neurological disorders, hGAT1 is an important target for their medical treatment.
The solid supported membrane-based electrophysiology (SSME) is a technique that allows the measurement of electrogenic events in transporters, pumps, and channels. The sample (i.e., native membrane vesicles or proteoliposomes) is adsorbed on an artificial bilayer on top of a gold-coated sensor and a capacitive-coupled system is generated. The transport is triggered by substrates concentration gradients as driving force, while membrane voltage is zero.
Through SSME we detected GABA-induced currents on CHO membrane vesicles overexpressing hGAT1, with a maximum amplitude of 3-5 nA, that showed a triphasic behavior leading us to identify three different electrogenic events. The transport component has been identified, showing a KM of 15-20 μM. We could also assess the stoichiometry of GABA versus Na+, which was found to be in agreement with the currently most accepted one of GABA:Na+=1:2.
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