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Abstract: The N-type voltage-gated calcium channel (Cav2.2) 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 Cav2.2 pharmacology in two stable Cav2.2 cell lines: α1B, α2δ, β3-HEK-293 and α1B, β3-HEK-293. Cav2.2 channels were opened by addition of KCl and Ca2+ mobilization was measured by Fluo-4 fluorescence on a fluorescence imaging plate reader (FLIPR96). Cav2.2 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 IC50 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 Cav2.2 blockers were characterized including cadmium, flunarizine, fluspirilene, and mibefradil. IC50 values correlated with literature EP-derived values. Novel Cav2.2 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 Cav2.2 were identified in the phenoxyphenyl pyridine, phenoxyphenyl pyrazole, and other classes. The highest potency at Cav2.2 tricyclic antidepressant identified was desipramine. [Copyright &y& Elsevier]