Absence seizures are a common seizure type in children with genetic generalized epilepsy and are characterized by a temporary loss of awareness, arrest of physical activity, and accompanying spike-and-wave discharges on an electroencephalogram. They arise from abnormal, hypersynchronous neuronal firing in brain thalamocortical circuits. Currently available therapeutic agents are only partially effective and act on multiple molecular targets, including γ-aminobutyric acid (GABA) transaminase, sodium channels, and calcium (Ca(2+)) channels. We sought to develop high-affinity T-type specific Ca(2+) channel antagonists and to assess their efficacy against absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model. Using a rational drug design strategy that used knowledge from a previous N-type Ca(2+) channel pharmacophore and a high-throughput fluorometric Ca(2+) influx assay, we identified the T-type Ca(2+) channel blockers Z941 and Z944 as candidate agents and showed in thalamic slices that they attenuated burst firing of thalamic reticular nucleus neurons in GAERS. Upon administration to GAERS animals, Z941 and Z944 potently suppressed absence seizures by 85 to 90% via a mechanism distinct from the effects of ethosuximide and valproate, two first-line clinical drugs for absence seizures. The ability of the T-type Ca(2+) channel antagonists to inhibit absence seizures and to reduce the duration and cycle frequency of spike-and-wave discharges suggests that these agents have a unique mechanism of action on pathological thalamocortical oscillatory activity distinct from current drugs used in clinical practice.

Voltage-gated calcium channel (Ca v )2.2 (N-type calcium channels) are key components in nociceptive transmission pathways. Ziconotide, a state-independent peptide inhibitor of Ca v 2.2 channels, is efficacious in treating refractory pain but exhibits a narrow therapeutic window and must be administered intrathecally. We have discovered an N-triazole oxindole, (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1), as a small-molecule, state-dependent blocker of Ca v 2 channels, and we investigated the therapeutic advantages of this compound for analgesia. TROX-1 preferentially inhibited potassium-triggered calcium influx through recombinant Ca v 2.2 channels under depolarized conditions (IC 50 ϭ 0.27 M) compared with hyperpolarized conditions (IC 50 Ͼ 20 M). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited -conotoxin GVIA-sensitive calcium currents (Ca v 2.2 channel currents), with greater potency under depolarized conditions (IC 50 ϭ 0.4 M) than under hyperpolarized conditions (IC 50 ϭ 2.6 M), indicating state-dependent Ca v 2.2 channel block of native as well as recombinant channels. TROX-1 fully blocked calcium influx mediated by a mixture of Ca v 2 channels in calcium imaging experiments in rat DRG neurons, indicating additional block of all Ca v 2 family channels. TROX-1 reversed inflammatory-induced hyperalgesia with maximal effects equivalent to nonsteroidal anti-inflammatory drugs, and it reversed nerve injury-induced allodynia to the same extent as pregabalin and duloxetine. In contrast, no significant reversal of hyperalgesia was observed in Ca v 2.2 gene-deleted mice. Mild impairment of motor function in the Rotarod test and cardiovascular functions were observed at 20-to 40-fold higher plasma concentrations than required for analgesic activities. TROX-1 demonstrates that an orally available state-dependent Ca v 2 channel blocker may achieve a therapeutic window suitable for the treatment of chronic pain.Inflammatory diseases and neuropathic insults are frequently accompanied by severe debilitating pain, which can become chronic and unresponsive to conventional analgesic treatments. Intrathecal administration of conventional agents, including morphine, may be required in more severe C.A. and O.B.M. contributed equally to this work. Article, publication date, and citation information can be found at

Voltage-gated ion channels are implicated in pain sensation and transmission signaling mechanisms within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments have shown that specific channel isoforms, including Na(V)1.7 and Na(V)1.8 sodium channels and Ca(V)3.2 T-type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized a novel small organic compound (Z123212) that modulates both recombinant and native sodium and calcium channel currents by selectively stabilizing channels in their slow-inactivated state. Slow inactivation of voltage-gated channels can function as a brake during periods of neuronal hyperexcitability, and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal cord neurons in a state-dependent manner. In vivo experiments demonstrate that oral administration of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve ligation model of neuropathic pain and also produces acute antinociception in the hot-plate test. At therapeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse effects. Taken together, the state-dependent inhibition of sodium and calcium channels in both the peripheral and central pain signaling pathways may provide a synergistic mechanism toward the development of a novel class of pain therapeutics.

Alternative splicing generates a smaller assortment of CaV2.1 transcripts in cerebellar Purkinje cells than in the cerebellum. Physiol Genomics 24: 86 -96, 2006. First published November 8, 2005 doi:10.1152/physiolgenomics.00149.2005.-P/Qtype calcium channels control many calcium-driven functions in the brain. The CACNA1A gene encoding the pore-forming CaV2.1 (␣1A) subunit of P/Q-type channels undergoes alternative splicing at multiple loci. This results in channel variants with different phenotypes. However, the combinatorial patterns of alternative splice events at two or more loci, and hence the diversity of CaV2.1 transcripts, are incompletely defined for specific brain regions and types of brain neurons. Using RT-PCR and splice variant-specific primers, we have identified multiple CaV2.1 transcript variants defined by different pairs of splice events in the cerebellum of adult rat. We have uncovered new splice variations between exons 28 and 34 (some of which predict a premature stop codon) and a new variation in exon 47 (which predicts a novel extended COOH-terminus). Single cell RT-PCR reveals that each individual cerebellar Purkinje neuron also expresses multiple alternative CaV2.1 transcripts, but the assortment is smaller than in the cerebellum. Two of these variants encode different extended COOH-termini which are not the same as those previously reported in Purkinje cells of the mouse. Our patch-clamp recordings show that calcium channel currents in the soma and dendrites of Purkinje cells are largely inhibited by a concentration of -agatoxin IVA selective for P-type over Q-type channels, suggesting that the different transcripts may form phenotypic variants of P-type calcium channels in Purkinje cells. These results expand the known diversity of Ca V2.1 transcripts in cerebellar Purkinje cells, and propose the selective expression of distinct assortments of Ca V2.1 transcripts in different brain neurons and species. splice variants; P type; calcium channels; Purkinje neurons 0 VOLTAGE-GATED calcium (Ca 2ϩ ) channels regulate numerous cellular functions, from neuronal electrical activity to intracellular signaling pathways. Diversity of Ca 2ϩ channels in the brain is apparent from the many roles that Ca 2ϩ channels play in different cell types and subcellular compartments (3). The diversity reflects, in part, the existence of nine classes of the pore-forming Ca V (␣1) subunit in the brain and alternative pre-mRNA splicing of the gene encoding each class of Ca V subunit (12, 17). P/Q-type Ca 2ϩ channels control many functions throughout the brain and contain a Ca V 2.1 subunit (also known as ␣ 1A ). However, information about splicing of the CACNA1A gene encoding Ca V 2.1 in different brain regions and neurons is incomplete (2,5,16,30,33,35,37). Augmentation of what is currently known about Ca V 2.1 splicing in the cerebellum and cerebellar Purkinje cells is of particular interest, because P-type currents in Purkinje cells are the prototypical P-type currents against which putative P-type and Q-...

To determine if the properties of Ca2+ channels in cerebellar Purkinje cells change during postnatal development, we recorded Ca 2+ channel currents from Purkinje cells in cerebellar slices of mature (postnatal days (P) 40-50) and immature (P13-20) rats. We found that at P40-50, the somatic Ca 2+ channel current was inhibited by ω-agatoxin IVA at concentrations selective for P-type Ca 2+ channels (∼85%; IC 50 , <1 nM) and by the dihydropyridine ( Voltage-gated Ca 2+ channels regulate electrical activity and many intracellular processes (Catterall et al. 2005). The classification of Ca 2+ channels as T-, N-, L-, P-, Q-or R-type is based on the electrophysiological and pharmacological properties of Ca 2+ channel currents in different cell types (Catterall et al. 2005). A finer subdivision of this classification follows from the identification of 10 classes of the pore-forming Ca V (α1) Ca 2+ channel subunit and alternative pre-mRNA splicing of the gene encoding each Ca V class, as well as different classes and splice variants of the accessory α 2 δ, β and γ subunits (Catterall et al. 2005).Currents mediated by native L-type channels or by recombinant channels consisting of one of the four classes of the Ca V 1 family (Ca V 1.1, Ca V 1.2, Ca V 1.3 and Ca V