Abstract
Within the continuous quest for the discovery of pharmacologically interesting compounds, the development
of new and superior drug screening assays is desired. In recent years, the use of label-free techniques
has paved the way for an alternative high-throughput screening method. An example is the Epic® opticalbased
biosensor that relies on dynamic mass redistribution (DMR) for detection. So far, DMR assays have
been mostly used to study G protein-coupled receptor (GPCR) pharmacology. Here, we demonstrate the
utility of this assay for investigating ligand-gated ion channel receptors. Using the immortalized IMR-32
neuroblastoma cell line, which expresses relatively high levels of several endogenous GABAA receptor subunits,
we show that GABA produces concentration-dependent cellular responses that can be measured
and quantified in real-time. With the aid of the GABAA receptor-specific agonist muscimol and the selective
antagonists gabazine and bicuculline, we confirm that the data corresponds to that of a GABAA receptor.
Based on quantitative real-time PCR measurements, the subunits α3, α5, β3 and θ are the most likely candidates
for integration into functional receptors. Our demonstration that label-free methods such as the Epic
technology can be used to characterize endogenous GABAA receptors in the IMR-32 cell line is exemplary
for the superfamily of ligand-gated ion channel receptors, and holds interesting perspectives in relation to
identifying novel mechanisms of action.
of new and superior drug screening assays is desired. In recent years, the use of label-free techniques
has paved the way for an alternative high-throughput screening method. An example is the Epic® opticalbased
biosensor that relies on dynamic mass redistribution (DMR) for detection. So far, DMR assays have
been mostly used to study G protein-coupled receptor (GPCR) pharmacology. Here, we demonstrate the
utility of this assay for investigating ligand-gated ion channel receptors. Using the immortalized IMR-32
neuroblastoma cell line, which expresses relatively high levels of several endogenous GABAA receptor subunits,
we show that GABA produces concentration-dependent cellular responses that can be measured
and quantified in real-time. With the aid of the GABAA receptor-specific agonist muscimol and the selective
antagonists gabazine and bicuculline, we confirm that the data corresponds to that of a GABAA receptor.
Based on quantitative real-time PCR measurements, the subunits α3, α5, β3 and θ are the most likely candidates
for integration into functional receptors. Our demonstration that label-free methods such as the Epic
technology can be used to characterize endogenous GABAA receptors in the IMR-32 cell line is exemplary
for the superfamily of ligand-gated ion channel receptors, and holds interesting perspectives in relation to
identifying novel mechanisms of action.
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | MedChemComm |
| Vol/bind | 7 |
| Sider (fra-til) | 426-432 |
| Antal sider | 7 |
| ISSN | 2040-2503 |
| DOI | |
| Status | Udgivet - 1 mar. 2016 |