A fluorescence-based high-throughput screening assay for the identification of T-type calcium channel blockers.
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.
Belardetti
Francesco
F
Neuromed Pharmaceuticals Ltd., Vancouver, British Columbia, Canada.
Tringham
Elizabeth
E
Eduljee
Cyrus
C
Jiang
Xinpo
X
Dong
Haiheng
H
Hendricson
Adam
A
Shimizu
Yoko
Y
Janke
Diana L
DL
Parker
David
D
Mezeyova
Janette
J
Khawaja
Afsheen
A
Pajouhesh
Hassan
H
Fraser
Robert A
RA
Arneric
Stephen P
SP
Snutch
Terrance P
TP
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
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