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Measuring transporter currents

Solid supported membrane (SSM)-based electrophysiology differs from conventional electrophysiology such as patch-clamp, since no living cells are required, but rather diverse native or artificial membrane vesicles. The samples used range from reconstituted protein in proteoliposomes to membrane preparations from organelles or plasma membrane. For the sample preparation bacterial cells, eukaryotic cell culture or native tissue can be used. The membrane sample is added to an SSM in advance of the experiment. This leads to the stable adsorption of the membranes to the SSM and the formation of a capacitively coupled compound membrane. The SSM itself consists of a lipid monolayer on top of a thiolated gold coated sensor chip. One important advantage compared to patch-clamp is the large sensor size of up to 3 mm. This allows the measurement of about 109 transporters at the same time and yields a significant improvement of signal to noise ratio. Therefore low-turnover targets become accessible to electrophysiological characterization.

Difference to conventional electrophysiology

An important difference between SSM-based electrophysiology and conventional electrophysiology is the principle of measurement. In patch-clamp electrophysiology, stationary currents can be obtained due to the fact that the voltage and, therefore, the driving force is clamped during the measurement. In SSM-based electrophysiology a substrate gradient established by a fast solution exchange is the main driving force. The transport of charged substrates or ions into the liposomes or vesicles generates a membrane potential. This potential can be detected via capacitive coupling between the membrane and the SSM on the gold layer of the sensor. In short: The change in membrane potential due to electrogenic transport is measured. At some point the membrane potential equals the chemical driving force and the transport process comes to a halt. This is why any current measured with SSM-based electrophysiology is transient. The peak current amplitude reflects the transporter activity under steady-state conditions. Since the current decay is fast, one measurement takes only one second. Due to the high stability of the SSM, multiple measurements can be performed using the same sensor and different buffer conditions to determine kinetic parameters such as EC50, IC50 or even rate constants.

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