title: A high-throughput assay for evaluating state dependence and subtype selectivity of Cav2 calcium channel inhibitors.
abstract: Cav2.2 channels play a critical role in pain signaling by controlling synaptic transmission between dorsal root ganglion neurons and dorsal horn neurons. The Cav2.2-selective peptide blocker ziconotide (Prialt, Elan Pharmaceuticals, Dublin, Ireland) has proven efficacious in pain relief, but has a poor therapeutic index and requires intrathecal administration. This has provided impetus for finding an orally active, state-dependent Cav2.2 inhibitor with an improved safety profile. Members of the Cav2 subfamily of calcium channels are the main contributors to central and peripheral synaptic transmission, but the pharmacological effects of blocking each subtype is not yet defined. Here we describe a high-throughput fluorescent assay using a fluorometric imaging plate reader (FLIPR [Molecular Devices, Sunnyvale, CA]) designed to quickly evaluate the state dependence and selectivity of inhibitors across the Cav2 subfamily. Stable cell lines expressing functional Cav2 channels (Ca(V)alpha, beta(3), and alpha(2)delta subunits) were co-transfected with an inward rectifier (Kir2.3) so that membrane potential, and therefore channel state, could be controlled by external potassium concentration. Following cell incubation in drug with varying concentrations of potassium, a high potassium trigger was added to elicit calcium influx through available, unblocked channels. State-dependent inhibitors that preferentially bind to channels in the open or inactivated state can be identified by their increased potency at higher potassium concentrations, where cells are depolarized and channels are biased towards these states. Although the Cav2 channel subtypes differ in their voltage dependence of inactivation, by adjusting pre-trigger potassium concentrations, the degree of steady-state inactivation can be more closely matched across Cav2 subtypes to assess molecular selectivity.
authors.affiliation: Merck Research Laboratories, Rahway, NJ 07065, USA.
authors.forename: Ge
authors.initials: G
authors.lastname: Dai
authors.forename: Rodolfo J
authors.initials: RJ
authors.lastname: Haedo
authors.forename: Vivien A
authors.initials: VA
authors.lastname: Warren
authors.forename: Kevin S
authors.initials: KS
authors.lastname: Ratliff
authors.forename: Randal M
authors.initials: RM
authors.lastname: Bugianesi
authors.forename: Alison
authors.initials: A
authors.lastname: Rush
authors.forename: Mark E
authors.initials: ME
authors.lastname: Williams
authors.forename: James
authors.initials: J
authors.lastname: Herrington
authors.forename: McHardy M
authors.initials: MM
authors.lastname: Smith
authors.forename: Owen B
authors.initials: OB
authors.lastname: McManus
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Swensen
mesh_terms: D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D051243:Caveolin 2 / Q000187:drug effects* / Q000502:physiology*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004594:Electrophysiology; D006801:Humans; D007150:Immunohistochemistry; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000494:pharmacology; D024661:Potassium Channels, Inwardly Rectifying / Q000187:drug effects / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C495544:CAV2 protein, human; D002121:Calcium Channel Blockers; D051243:Caveolin 2; C099489:KCNJ4 protein, human; D024661:Potassium Channels, Inwardly Rectifying; D011188:Potassium; D002118:Calcium