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

Purdue Pharma Discovery Research, 6 Cedarbrook Drive, Cranbury, NJ 08512, USA.
Biochemical Pharmacology (Impact Factor: 5.01). 10/2006; 72(6):770-82. DOI: 10.1016/j.bcp.2006.06.003
Source: PubMed


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.

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Available from: Victor Ilyin, Jun 18, 2015
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    • "(Fig. 3D, Table 4). These results are consistent with previous studies on MVIIA [22]–[24], [26] and CVID [22]–[24] inhibition of N-type responses in native and recombinant systems, when Cav2.2 was co-expressed with β and α2δ subunits [22]–[24]. In contrast, GVIA potency at Cav2.2 expressed in SH-SY5Y cells (IC50 of 0.15 µM; pIC50 6.8±0.072) was consistently lower than previously described for heterologous expressed rat [24], [26] and human [25] α1B co-expressed with α2δ1 and β3, but similar to data obtained using native expression systems such as dissociated rat DRG cells [30] and chicken synaptosomes [31]. "
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    ABSTRACT: SH-SY5Y human neuroblastoma cells provide a useful in vitro model to study the mechanisms underlying neurotransmission and nociception. These cells are derived from human sympathetic neuronal tissue and thus, express a number of the Cav channel subtypes essential for regulation of important physiological functions, such as heart contraction and nociception, including the clinically validated pain target Cav2.2. We have detected mRNA transcripts for a range of endogenous expressed subtypes Cav1.3, Cav2.2 (including two Cav1.3, and three Cav2.2 splice variant isoforms) and Cav3.1 in SH-SY5Y cells; as well as Cav auxiliary subunits α2δ1-3, β1, β3, β4, γ1, γ4-5, and γ7. Both high- and low-voltage activated Cav channels generated calcium signals in SH-SY5Y cells. Pharmacological characterisation using ω-conotoxins CVID and MVIIA revealed significantly (∼ 10-fold) higher affinity at human versus rat Cav2.2, while GVIA, which interacts with Cav2.2 through a distinct pharmacophore had similar affinity for both species. CVID, GVIA and MVIIA affinity was higher for SH-SY5Y membranes vs whole cells in the binding assays and functional assays, suggesting auxiliary subunits expressed endogenously in native systems can strongly influence Cav2.2 channels pharmacology. These results may have implications for strategies used to identify therapeutic leads at Cav2.2 channels.
    PLoS ONE 03/2013; 8(3):e59293. DOI:10.1371/journal.pone.0059293 · 3.23 Impact Factor
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    • "Antidepressants tend to accumulate in tissues because of their lipophilic nature [34], so in the central nervous system they may reach the effective range for inhibition of P2X4 receptors observed in this experiment. Antidepressants modulate many kinds of ion channels at a wide range of concentrations (0.1 to 1000 μM) in vitro [20,35,36], but only the effects observed near the serum concentration are considered to have an influence in vivo. Sometimes, the analgesic effect of antidepressants is explained by their inhibitory effects on voltage-dependent sodium channels and calcium channels, that are observed at relatively low concentrations (0.1 to 10 μM) in vitro [19]. "
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    ABSTRACT: Neuropathic pain is characterized by pain hypersensitivity to innocuous stimuli (tactile allodynia) that is nearly always resistant to known treatments such as non-steroidal anti-inflammatory drugs or even opioids. It has been reported that some antidepressants are effective for treating neuropathic pain. However, the underlying molecular mechanisms are not well understood. We have recently demonstrated that blocking P2X4 receptors in the spinal cord reverses tactile allodynia after peripheral nerve injury in rats, implying that P2X4 receptors are a key molecule in neuropathic pain. We investigated a possible role of antidepressants as inhibitors of P2X4 receptors and analysed their analgesic mechanism using an animal model of neuropathic pain. Antidepressants strongly inhibited ATP-mediated Ca2+ responses in P2X4 receptor-expressing 1321N1 cells, which are known to have no endogenous ATP receptors. Paroxetine exhibited the most powerful inhibition of calcium influx via rat and human P2X4 receptors, with IC50 values of 2.45 microM and 1.87 microM, respectively. Intrathecal administration of paroxetine produced a striking antiallodynic effect in an animal model of neuropathic pain. Co-administration of WAY100635, ketanserin or ondansetron with paroxetine induced no significant change in the antiallodynic effect of paroxetine. Furthermore, the antiallodynic effect of paroxetine was observed even in rats that had received intrathecal pretreatment with 5,7-dihydroxytryptamine, which dramatically depletes spinal 5-hydroxytryptamine. These results suggest that paroxetine acts as a potent analgesic in the spinal cord via a mechanism independent of its inhibitory effect on serotonin transporters. Powerful inhibition on P2X4 receptors may underlie the analgesic effect of paroxetine, and it is possible that some antidepressants clinically used in patients with neuropathic pain show antiallodynic effects, at least in part via their inhibitory effects on P2X4 receptors.
    Molecular Pain 05/2009; 5(1):20. DOI:10.1186/1744-8069-5-20 · 3.65 Impact Factor
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    • "Several studies document TRPM8 modulation by a variety of stimulus demonstrating an important flexibility in the temperature response curve of TRPM8 channels which can vary by more than 15°C [24]. Examples of TRPM8 modulators are: phosphoinositides [25], PIP2 [26]–[28], phosphorylation [28], [29], inorganic polyphosphate [30] channel density, intracellular Ca2+ levels [27], the variable expression ratio of K+/TRPM8 channels [31], [32] and lipid rafts [33]. "
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    ABSTRACT: The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca(2+) dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca(2+) ([Ca(2+)]i). Ca(2+) triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 microM) and 80% by BCTC (1.6 microM). Activation of TRPM8 either by temperature or menthol induced [Ca(2+)]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 microM) and BCTC (1.6 microM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis.
    PLoS ONE 02/2009; 4(6):e6095. DOI:10.1371/journal.pone.0006095 · 3.23 Impact Factor
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