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

Biochem Pharmacol. 2006 Sep 14;72(6):770-82. doi: 10.1016/j.bcp.2006.06.003. Epub 2006 Jul 17.


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.

MeSH terms

  • Calcium / metabolism*
  • Calcium Channel Blockers / pharmacology
  • Calcium Channels, N-Type / drug effects
  • Calcium Channels, N-Type / genetics
  • Calcium Channels, N-Type / metabolism*
  • Cells, Cultured
  • Electrophysiology
  • Humans
  • Signal Transduction / physiology*


  • Calcium Channel Blockers
  • Calcium Channels, N-Type
  • Calcium