title: A fluorescence-based high-throughput screening assay for the identification of T-type calcium channel blockers.
abstract: T-type voltage-gated Ca(2+) channels have been implicated in contributing to a broad variety of human disorders, including pain, epilepsy, sleep disturbances, cardiac arrhythmias, and certain types of cancer. However, potent and selective T-type Ca(2+) channel modulators are not yet available for clinical use. This may in part be due to their unique biophysical properties that have delayed the development of high-throughput screening (HTS) assays for identifying blockers. One notable challenge is that at the normal resting membrane potential (V(m)) of cell lines commonly utilized for drug screening purposes, T-type Ca(2+) channels are largely inactivated and thus cannot be supported by typical formats of functional HTS assays to both evoke and quantify the Ca(2+) channel signal. Here we describe a simple method that can successfully support a fluorescence-based functional assay for compounds that modulate T-type Ca(2+)channels. The assay functions by exploiting the pore-forming properties of gramicidin to control the cellular V(m) in advance of T-type Ca(2+) channel activation. Using selected ionic conditions in the presence of gramicidin, T-type Ca(2+) channels are converted from the unavailable, inactivated state to the available, resting state, where they can be subsequently activated by application of extracellular K(+). The fidelity of the assay has been pharmacologically characterized with sample T-type Ca(2+) channel blockers whose potency has been determined by conventional manual patch-clamp techniques. This method has the potential for applications in high-throughput fluorometric imaging plate reader (FLIPR(R), Molecular Devices, Sunnyvale, CA) formats with cell lines expressing either recombinant or endogenous T-type Ca(2+) channels.
authors.affiliation: Neuromed Pharmaceuticals Ltd., Vancouver, British Columbia, Canada.
authors.forename: Francesco
authors.initials: F
authors.lastname: Belardetti
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Tringham
authors.forename: Cyrus
authors.initials: C
authors.lastname: Eduljee
authors.forename: Xinpo
authors.initials: X
authors.lastname: Jiang
authors.forename: Haiheng
authors.initials: H
authors.lastname: Dong
authors.forename: Adam
authors.initials: A
authors.lastname: Hendricson
authors.forename: Yoko
authors.initials: Y
authors.lastname: Shimizu
authors.forename: Diana L
authors.initials: DL
authors.lastname: Janke
authors.forename: David
authors.initials: D
authors.lastname: Parker
authors.forename: Janette
authors.initials: J
authors.lastname: Mezeyova
authors.forename: Afsheen
authors.initials: A
authors.lastname: Khawaja
authors.forename: Hassan
authors.initials: H
authors.lastname: Pajouhesh
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fraser
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Arneric
authors.forename: Terrance P
authors.initials: TP
authors.lastname: Snutch
mesh_terms: D000465:Algorithms; D002121:Calcium Channel Blockers / Q000138:chemical synthesis / Q000494:pharmacology*; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism; D020908:Calcium Channels, R-Type / Q000187:drug effects / Q000378:metabolism; D020747:Calcium Channels, T-Type / Q000187:drug effects* / Q000378:metabolism; D027682:Cation Transport Proteins / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D004594:Electrophysiology; D006096:Gramicidin / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D019999:Pharmaceutical Solutions; D013050:Spectrometry, Fluorescence
chemical_list: C472073:CACNA1D protein, human; C423009:CACNA1E protein, human; C469757:CACNA1I protein, human; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D020908:Calcium Channels, R-Type; D020747:Calcium Channels, T-Type; D027682:Cation Transport Proteins; D018076:DNA, Complementary; D019999:Pharmaceutical Solutions; D006096:Gramicidin