title: Pharmacological characterization of recombinant N-type calcium channel (Cav2.2) mediated calcium mobilization using FLIPR.
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.
authors.affiliation: Purdue Pharma Discovery Research, 6 Cedarbrook Drive, Cranbury, NJ 08512, USA. ebenjamin@amicustherapeutics.com
authors.forename: Elfrida R
authors.initials: ER
authors.lastname: Benjamin
authors.forename: Farhana
authors.initials: F
authors.lastname: Pruthi
authors.forename: Shakira
authors.initials: S
authors.lastname: Olanrewaju
authors.forename: Shen
authors.initials: S
authors.lastname: Shan
authors.forename: Denise
authors.initials: D
authors.lastname: Hanway
authors.forename: Xuesong
authors.initials: X
authors.lastname: Liu
authors.forename: Rok
authors.initials: R
authors.lastname: Cerne
authors.forename: Daniel
authors.initials: D
authors.lastname: Lavery
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Valenzano
authors.forename: Richard M
authors.initials: RM
authors.lastname: Woodward
authors.forename: Victor I
authors.initials: VI
authors.lastname: Ilyin
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004594:Electrophysiology; D006801:Humans; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D002118:Calcium