Pharmacological characterization of recombinant N-type calcium channel (Cav2.2) mediated calcium mobilization using FLIPR

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: alpha 1(B), alpha 2 delta, beta(3)-HEK-293 and alpha 1(B), beta(3)-HEK-293. Ca(v)2.2 channels were opened by addition of KCl and Ca2+, mobilization was measured by Fluo-4 fluorescence on a fluorescence imaging plate reader (FLIPR96). Ca(v)2.2 expression and biophysics were confirmed by patch-clamp electrophysiology (EP). Both cell lines responded to KC1 with adequate signal-to-background. Signals from both cell lines were inhibited by omega-conotoxin (ctx)-MVIIa and omega-conotoxin (ctx)-GVIa with IC50 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. IC50 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. (c) 2006 Elsevier Inc. All rights reserved.