A fluorescence-based high-throughput screening assay for the identification of T-type calcium channel blockers.
Assay Drug Dev Technol
; 7(3): 266-80, 2009 Jun.
Article
em En
| MEDLINE
| ID: mdl-19530894
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Bloqueadores dos Canais de Cálcio
/
Canais de Cálcio Tipo T
Tipo de estudo:
Diagnostic_studies
/
Guideline
/
Screening_studies
Idioma:
En
Revista:
Assay Drug Dev Technol
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Canadá