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Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Published on: July 28, 2014
Pharmacological characterization of recombinant N-type calcium channel (Cav2.2) mediated calcium mobilization using
Elfrida R Benjamin1, Farhana Pruthi, Shakira Olanrewaju
1Purdue Pharma Discovery Research, 6 Cedarbrook Drive, Cranbury, NJ 08512, USA. ebenjamin@amicustherapeutics.com
Abstract:
The N-type voltage-gated calcium channel (Ca(v)2.2) functions in neurons to regulate neurotransmitter release. It comprises a clinically relevant target for chronic pain. We have validated a calcium mobilization approach to assessing Ca(v)2.2 pharmacology in two stable Ca(v)2.2 cell lines: alpha1(B), alpha2delta, beta(3)-HEK-293 and alpha1(B), beta(3)-HEK-293. Ca(v)2.2 channels were opened by addition of KCl and Ca(2+) mobilization was measured by Fluo-4 fluorescence on a fluorescence imaging plate reader (FLIPR(96)). Ca(v)2.2 expression and biophysics were confirmed by patch-clamp electrophysiology (EP). Both cell lines responded to KCl with adequate signal-to-background. Signals from both cell lines were inhibited by omega-conotoxin (ctx)-MVIIa and omega-conotoxin (ctx)-GVIa with IC(50) values of 1.8 and 1nM, respectively, for the three-subunit stable, and 0.9 and 0.6nM, respectively, for the two-subunit stable. Other known Ca(v)2.2 blockers were characterized including cadmium, flunarizine, fluspirilene, and mibefradil. IC(50) values correlated with literature EP-derived values. Novel Ca(v)2.2 pharmacology was identified in classes of compounds with other primary pharmacological activities, including Na(+) channel inhibitors and antidepressants. Novel Na(+) channel compounds with high potency at Ca(v)2.2 were identified in the phenoxyphenyl pyridine, phenoxyphenyl pyrazole, and other classes. The highest potency at Ca(v)2.2 tricyclic antidepressant identified was desipramine.
Insights
A novel calcium mobilization assay effectively characterizes N-type voltage-gated calcium channel (Ca(v)2.2) pharmacology. This method identified new blockers, including antidepressants, for potential chronic pain therapies.
Area of Science:
- Neuroscience
- Pharmacology
- Molecular Biology
Background:
- N-type voltage-gated calcium channels (Ca(v)2.2) are crucial for neurotransmitter release in neurons.
- Ca(v)2.2 channels represent a significant therapeutic target for managing chronic pain.
- Assessing Ca(v)2.2 pharmacology is vital for drug discovery and development.
Purpose of the Study:
- To validate a calcium mobilization assay for evaluating Ca(v)2.2 channel pharmacology.
- To characterize the pharmacological profiles of known and novel Ca(v)2.2 modulators.
- To identify new classes of compounds targeting Ca(v)2.2 channels.
Main Methods:
- Utilized two stable HEK-293 cell lines expressing different Ca(v)2.2 subunit compositions.
- Measured Ca(2+) mobilization using Fluo-4 fluorescence and a fluorescence imaging plate reader (FLIPR(96)) upon KCl stimulation.
- Confirmed Ca(v)2.2 expression and biophysical properties via patch-clamp electrophysiology (EP).
Main Results:
- Both cell lines demonstrated robust responses to KCl stimulation with good signal-to-background ratios.
- The assay successfully reproduced known Ca(v)2.2 blocker potencies, including omega-conotoxins (ctx)-MVIIa and (ctx)-GVIa, with low nanomolar IC(50) values.
- Identified novel Ca(v)2.2 activity in Na(+) channel inhibitors and antidepressants, with desipramine showing high potency.
Conclusions:
- The calcium mobilization assay is a validated and effective method for high-throughput screening of Ca(v)2.2 modulators.
- This approach facilitates the discovery of novel pharmacological agents targeting Ca(v)2.2 channels.
- Findings suggest potential therapeutic applications for identified compounds in chronic pain management.
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