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: α1 B , α2δ, β 3 -HEK-293 and α1 B , β 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 ω-conotoxin (ctx)-MVIIa and ω-conotoxin (ctx)-GVIa with IC 50 values of 1.8 and 1 nM, respectively, for the three-subunit stable, and 0.9 and 0.6 nM, 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.