Functional characterization of human GAT-1 using solid supported membrane electrophysiology and automated patch clamp

DRA lecture held by Senior Scientist Cecilia George and Application Scientist Rocco Zerlotti, Nanion Technologies, Germany

Solid supported membrane-based electrophysiology (SSME) is a method used to measure transport and electrogenic activity in transporters, pumps, and ion channels. In this technique, membrane vesicles or proteoliposomes are adsorbed onto an artificial membrane formed over a gold-coated sensor, creating a capacitive-coupled membrane system. A rapid solution exchange is facilitated by a fluidic system, and transport is initiated by substrate concentration gradients at zero membrane voltage. We investigated GAT-1, a secondary-active transporter that utilizes the Na⁺ gradient to drive the re-uptake of GABA from the synaptic cleft. Using SSME, GABA-induced currents were detected in vesicles overexpressing hGAT-1, with maximum amplitudes of 2-3 nA and a triphasic profile, indicating three distinct electrogenic events. Experiments with varying Na⁺ gradients revealed the disappearance of the middle component when the Na⁺ gradient was minimized. The transport component exhibited a K_M of 20 μM, and a Na⁺ stoichiometry of 2:1 was determined. Additionally, automated patch clamp recordings in living cells showed inward GABA-induced currents at a holding potential of -70 mV, with an I_max of approximately -150 pA and a K_M of 11-12 μM, consistent with published data.

The lecture is organised on behalf of the graduate programme in pharmaceutical sciences, Drug Research Academy, by Professor Petrine Wellendorph and Ass. Prof. Jonas S. Mortensen, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen.

The DRA lecture is free of charge and open for attendance by all interested parties. It is not necessary to pre-register.