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Abstract Phα1β is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1β to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1β (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1β antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

At early life, the adrenal chromaffin cells respond with a catecholamine surge under hypoxic conditions. This response depends on Ca2+ entry through voltage-activated calcium channels (VACCs). We have investigated here three unresolved questions that concern this response in rat embryo chromaffin cells (ECCs): 1) the relative contribution of L (α1D, Cav1.3), N (α1B, Cav2.2), and PQ (α1A, Cav2.1) to the whole cell Ca2+ current (ICa); 2) the relative contribution of L and N/PQ channels to the cytosolic Ca2+ elevations triggered by hypoxia (Δ[Ca2+]c); and 3) the role of L and non-L high-VACCs in the regulation of the catecholamine surge occurring during prolonged (1 min) hypoxia exposure of ECCs. Nimodipine halved peak ICa and blocked 60% the total Ca2+ entry during a 50-ms depolarizing pulse to 0 mV (QCa). Combined ω-agatoxin IVA plus ω-conotoxin GVIA (Aga/GVIA) blocked 30% of both ICa peak and QCa. This relative proportion of L- and non-L VACCs was corroborated by Western blot that indicated 55, 23, and 25% relative expression of L, N, and PQ VACCs. Exposure of ECCs to hypoxia elicited a mild but sustained Δ[Ca2+]c; the area of Δ[Ca2+]c was blocked 50% by nifedipine and 10% by Aga/GVIA. Exposure of ECCs to 1-min hypoxia elicited an initial transient burst of amperometric secretory spikes followed by scattered spikes along the time of cell exposure to hypoxia. This bulk response was blocked 85% by nimodipine and 35% by Aga/GVIA. Histograms on secretory spike frequency vs. time indicated a faster initial inactivation when Ca2+ entry took place through N/PQ channels; more sustained secretion but at a lower rate was associated to Ca2+ entry through L channels. The results suggest that the HIS response may initially be controlled by L and P/Q channels, but later on, N/PQ channels inactivate and the delayed HIS response is maintained at lower rate by slow-inactivating L channels. [ABSTRACT FROM AUTHOR]

Abstract: Distinctly different effects of two closely related contryphans have been demonstrated on voltage-activated Ca2+ channels. The peptides Lo959 and Am975 were isolated from Conus loroisii, a vermivorous marine snail and Conus amadis, a molluscivore, respectively. The sequences of Lo959 and Am975 were deduced by mass spectrometric sequencing (MALDI-MS/MS) and confirmed by chemical synthesis. The sequences of Lo959, GCP D WDPWC-NH 2 and Am975, GCO D WDPWC-NH 2 (O: 4-trans-hydroxyproline: Hyp), differ only at residue 3; Pro in Lo959, Hyp in Am975, which is identical to contryphan-P, previously isolated from Conus purpurascens, a piscivore; while Lo959 is a novel peptide. Both Lo959 and Am975 undergo slow conformational interconversion under reverse-phase chromatographic conditions, a characteristic feature of all contryphans reported thus far. Electrophysiological studies performed using dorsal root ganglion neurons reveal that both peptides target high voltage-activated Ca2+ channels. While Lo959 increases the Ca2+ current, Am975 causes inhibition. The results establish that subtle sequence effects, which accompany post-translational modifications in Conus peptides, can have dramatic effects on target ion channels. [Copyright &y& Elsevier]

Nitric oxide-cyclic guanosine monophosphate (NO-cGMP)-mediated relaxation of cavernosal smooth muscle during erection is accompanied by a decrease in intracellular calcium concentrations ([Ca2+]i). However, it is not known whether and how an increase in [Ca2+]i is responsible for (i) initiating smooth muscle contraction/detumescence following relaxation; and (ii) maintaining the penis in a flaccid state under nonstimulating conditions. To elucidate (i) the mechanism(s) of [Ca2+]i homeostasis regulation in human cavernosal smooth muscle cells (HCSMC); and (ii) how NO-cGMP interacts with such [Ca2+]i homeostasis. We evaluated the expression and function of both T-type and L-type Ca2+ channels in HCSMC by employing selective probes/inhibitors using various cellular and molecular techniques (e.g., reverse transcriptase and real-time polymerase chain reaction, cell proliferation assay, fura-2 Ca2+ fluorescence spectroscopy, enzyme-linked immuno-absorbent assay (ELISA)). We have demonstrated for the first time significant interactions of NO-cGMP with the T-type (α1G) Ca2+ channel in HCSMC. Our results suggest that in addition to NO-induced rapid and transient decrease in [Ca2+]i that results in smooth muscle relaxation, NO-cGMP also enhanced mRNA expression of the T-type (α1G) Ca2+ channel resulting in delayed elevation of [Ca2+]i. This could be abolished by a selective T-channel blocker, NNC 55-0396. Another unique finding of this study is that dose-dependent HCSMC proliferation in vitro by NO is associated with the activation of the T-type (α1G) Ca2+ channel that regulates [Ca2+]i homeostasis in these cells. Human cavernosal cells express T-type (α1G) Ca2+ channels that are involved in maintaining [Ca2+]i homeostasis and regulation of NO-cGMP-induced smooth muscle relaxation–contraction responsible for penile erection, flaccidity, and tonicity. Targeting these Ca2+ channels may (i) associate various comorbidities with the onset of erectile dysfunction; (ii) provide a biochemical basis for differences between therapeutic profiles of various phosphodiesterase type 5 inhibitors, especially in nonresponders to current therapy; and (iii) provide biochemical basis in understanding mechanism(s) of drug tolerance. Zeng X, Keyser B, Li M, and Sikka SC. T-type (α1G) low voltage-activated calcium channel interactions with nitric oxide-cyclic guanosine monophosphate pathway and regulation of calcium homeostasis in human cavernosal cells. J Sex Med 2005;2:620–633. [ABSTRACT FROM AUTHOR]

Phα1β is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1β to treat chronic pain reverted opioid tolerance with a safer profile than ω -conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1β (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1β antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Introduction: Altered homeostasis of cell calcium movement is a central stage in multiple diseases of CNS. This explains the great therapeutic interest in blockers for the various subtypes of voltage-activated calcium channels (VACCs) expressed in neurons. Mitigation of Ca(2+) entry excess elicited by those blockers may restore the altered synaptic transmission, synaptic plasticity and gene expression to normal parameters, ending the enhanced neuronal vulnerability., Areas Covered: This review summarize 23 patents on ligands for L-, N- or T-type channels, claimed to have potential therapeutic interest in epilepsy, pain, migraine and neurodegenerative diseases., Expert Opinion: Collections of compounds are generally screened in cell lines expressing a given subtype of VACCs. IC50 to block such channels are often, but not always, provided. In few instances, compounds exhibiting the highest potency in in vitro experiments are also tested in animal models of pain, behavior, epilepsy or Alzheimer's disease. Attempts to develop selectivity for a given VACC subtype with non-peptidic organic ligands have so far failed. Due to their wide tissue expression, such selectivity is crucial for minimizing possible side effects. However, the few data reported by patents does not allow prediction of selectivity of the new compounds in many cases.

R-type Ca(v)2.3 high voltage-activated Ca(2+) channels in peripheral sensory neurons contribute to pain transmission. Recently we have demonstrated that, among the six Ca(v)2.3 isoforms (Ca(v)2.3a~Ca(v)2.3e), the Ca(v)2.3e isoform is primarily expressed in trigeminal ganglion (TG) nociceptive neurons. In the present study, we further investigated expression patterns of Ca(v)2.3 isoforms in the dorsal root ganglion (DRG) neurons. As in TG neurons, whole tissue RT-PCR analyses revealed the presence of two isoforms, Ca(v)2.3a and Ca(v)2.3e, in DRG neurons. Single-cell RT-PCR detected the expression of Ca(v)2.3e mRNA in 20% (n=14/70) of DRG neurons, relative to Ca(v)2.3a expression in 2.8% (n=2/70) of DRG neurons. Ca(v)2.3e mRNA was mainly detected in small-sized neurons (n=12/14), but in only a few medium-sized neurons (n=2/14) and not in large-sized neurons, indicating the prominence of Ca(v)2.3e in nociceptive DRG neurons. Moreover, Ca(v)2.3e was preferentially expressed in tyrosine-kinase A (trkA)-positive, isolectin B4 (IB4)-negative and transient receptor potential vanilloid 1 (TRPV1)-positive neurons. These results suggest that Ca(v)2.3e may be the main R-type Ca(2+) channel isoform in nociceptive DRG neurons and thereby a potential target for pain treatment, not only in the trigeminal system but also in the spinal system.