Identification of State-Dependent Blockers for Voltage-Gated Calcium Channels Using a FLIPR-Based Assay.
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.
Screening Technologies Unit, AXXAM SpA, via Meucci 3, 20091, Bresso (Milan), Italy.
Alberto
A
di Silvio
Screening Technologies Unit, AXXAM SpA, via Meucci 3, 20091, Bresso (Milan), Italy.
JeanFrancois
J
Rolland
Screening Technologies Unit, AXXAM SpA, via Meucci 3, 20091, Bresso (Milan), Italy. michela.stucchi.ms@axxam.com.
Michela
M
Stucchi
D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D061848:Optical Imaging / Q000379:methods*; D024661:Potassium Channels, Inwardly Rectifying / Q000037:antagonists & inhibitors / Q000378:metabolism*
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; C099489:KCNJ4 protein, human; C419385:L-type calcium channel alpha(1C); D024661:Potassium Channels, Inwardly Rectifying; D002118:Calcium
Calcium fluorescent dye; FLIPR; State-dependent blockers; Voltage-gated calcium channel