Identification of state-dependent blockers for voltage-gated calcium channels using a FLIPR-based assay

Abstract

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 Ca2+ 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 FLIPRTETRA. 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.