High Throughput Screening

Alberto di Silvio, JeanFrancois Rolland, Michela Stucchi, Silvio, Alberto di, Rolland, JeanFrancois, Stucchi, Michela

The FLIPR (Fluorescent Imaging Plate Reader) system has been extensively used in the early stages of drug discovery for the identification of small molecules as a starting point for drug development, and for the pharmacological characterization of compounds. The main application of the system has been the measurement of intracellular Ca(2+) signals using fluorescent calcium indicators.This chapter describes the application of a protocol for the study and characterization of state-dependent blockers of Voltage-Gated Calcium Channels (VGCC) on the FLIPR(TETRA).The cell line suitable for the application of the protocol, and described hereafter, co-expresses the human CaV1.2 channel and the human inward rectifier K(+) channel Kir2.3. The presence of Kir2.3 allows the modulation of the plasma membrane potential and consequently of the state of the CaV1.2 channel by changing the extracellular K(+) concentration. In this way, CaV1.2 activity can be measured at different membrane voltages, corresponding to either the resting or partial inactivated state, by loading the cells with a calcium probe in extracellular low or high potassium buffer.