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キーワード:
SURFE2R; SURFER; Transporter; Membrane transport; Solid-supported membrane; SSM; Transport assay; Electrophysiology; Bilayer; Pump; ATPase; Cell free
要旨:
Functional characterization of transport proteins using conventional electrophysiology can be challenging, especially for low turnover transporters or transporters from bacteria and intracellular compartments. Solid-supported membrane (SSM)-based electrophysiology is a sensitive and cell-free assay technique for the characterization of electrogenic membrane proteins. Purified proteins reconstituted into proteoliposomes or membrane vesicles from cell culture or native tissues are adsorbed to the sensor holding an SSM. A substrate or a ligand is applied via rapid solution exchange. The electrogenic transporter activity charges the sensor, which is recorded as a transient current. The high stability of the SSM allows cumulative measurements on the same sensor using different experimental conditions. This allows the determination of kinetic properties including EC50, IC50, Km, KD, and rate constants of electrogenic reactions. About 100 different transporters have been measured so far using this technique, among them symporters, exchangers, uniporters, ATP-, redox-, and light-driven ion pumps, as well as receptors and ion channels. Different instruments apply this technique: the laboratory setups use a closed flow-through arrangement, while the commercially available SURFE2R N1 resembles a pipetting robot. For drug screening purposes high-throughput systems, such as the SURFE2R 96SE enable the simultaneous measurement of up to 96 sensors.
