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Biochemistry; Issue 75; Biophysics; Molecular Biology; Cellular Biology; Physiology; Proteins; Membrane Lipids; Membrane Transport; Proteins; Kinetics; Electrophysiology; solid supported membrane; SSM; membrane transporter; lactose permease; lacy; capacitive coupling; solution exchange; model membrane; membrane protein; transporter; kinetics; transport mechanism
Abstract:
The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire. After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage. The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis. This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.
