BACKGROUND AND PURPOSE Voltage-gated calcium channels are involved in nociception in the CNS and in the periphery. N-type (Ca(v)2.2) and T-type (Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3) voltage-gated calcium channels are particularly important in studying and treating pain and epilepsy. EXPERIMENTAL APPROACH In this study, whole-cell patch clamp electrophysiology was used to assess the potency and mechanism of action of a novel ortho-phenoxylanilide derivative, MONIRO-1, against a panel of voltage-gated calcium channels including Ca(v)1.2, Ca(v)1.3, C(a)v2.1, Ca(v)2.2, Ca(v)2.3, Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3. KEY RESULTS MONIRO-1 was 5-to 20-fold more potent at inhibiting human T-type calcium channels, hCa(v)3.1, hCa(v)3.2 and hCa(v)3.3 (IC50: 3.3 +/- 0.3, 1.7 +/- 0.1 and 7.2 +/- 0.3 mu M, respectively) than N-type calcium channel, hCa(v)2.2 (IC50: 34.0 +/- 3.6 mu M). It interacted with L-type calcium channels Ca(v)1.2 and Ca(v)1.3 with significantly lower potency (IC50 > 100 mu M) and did not inhibit hCa(v)2.1 or hCa(v)2.3 channels at concentrations as high as 100 mu M. State-and use-dependent inhibition of hCav2.2 channels was observed, whereas stronger inhibition occurred at high stimulation frequencies for hCa(v)3.1 channels suggesting a different mode of action between these two channels. CONCLUSIONS AND IMPLICATIONS Selectivity, potency, reversibility and multi-modal effects distinguish MONIRO-1 from other low MW inhibitors acting on Ca-v channels involved in pain and/or epilepsy pathways. High-frequency firing increased the affinity for MONIRO-1 for both hCa(v)2.2 and hCa(v)3.1 channels. Such Ca-v channel modulators have potential clinical use in the treatment of epilepsies, neuropathic pain and other nociceptive pathophysiologies. LINKED ARTICLES This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary. wiley. com/doi/10.1111/bph. v175.12/issuetoc

a Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong NSW 2522, Australia. b Health Innovations Research Institute, RMIT University, Melbourne VIC 3083, Australia. c CSIRO Manufacturing, Bag 10, Clayton South VIC 3169, Australia. d School of Chemistry, Monash University, Clayton VIC 3800, Australia. e Institute for Molecular Bioscience, The University of Queensland, St Lucia QLD 4072, Australia. f School of Chemical and Physical Sciences, Flinders University, Adelaide SA 5042, Australia.

U50488H is a benzeneacetamide K-opioid receptor (K-OR) agonist analgesic, widely used for investigating the pharmacology of G protein-coupled K-ORs. However, U50488H is also known to directly block various voltage-gated ion channels in a G protein-independent manner. We investigated the direct actions of U50488H on various high voltage-activated (HVA) and low voltage-activated (LVA) neuronal Ca2+ channels heterologously expressed in human embryonic kidney (HEK293) cells. U50488H inhibited HVA rat Ca(v)1.3 (rCa(v)1.3), human Cav2.1 (hCav2.1), hCa(v)2.2, hCav2.3, and LVA hCa(v)3.1 and hCa(v)3.2 channels in a concentration-dependent manner, with similar potencies characterised with half-maximal inhibitory concentration (IC50) values of similar to 30 mu M. U50488H concentrations causing direct Ca-v inhibition are typically >100 times higher than those producing K-OR activation. Investigation of the mechanism of U50488H block of the Ca(v)2.2 channel revealed that U50488H interacted with all major kinetic states of the channel - resting, open, and inactivated. U50488H did not affect the voltage dependence of activation but shifted the steady-state inactivation curve by similar to 11 mV to more hyperpolarized potentials. U50488H also increased the rate of Ba2+ current inactivation during a step depolarization and significantly delayed recovery from slow inactivation, compared with control. Ca(v)2.2 current inhibition was frequency dependent during repetitive step depolarization at 1 Hz and 3 Hz, consistent with use dependent block. In summary, our results suggest that preferential interaction of U50488H with inactivated Ca(v)2.2 channels significantly contributes to reduced Ca(v)2.2 channel availability and slow recovery form inactivation. We conclude that U50488H non-selectively blocks heterologously expressed neuronal HVA and LVA Ca-v channels in the absence of kappa-ORs. This cross-reactivity also suggests potentially common U50488H binding motifs across Ca-v channel targets. (C) 2016 Elsevier Ltd. All rights reserved.

Neuronal voltage-gated Cav2.3 channels are widely expressed in the central and peripheral nervous system where they contribute to neurotransmission and pain sensation. However, modulation of the Cav2.3 channel through G protein-coupled (GPC) μ- and δ-opioid receptors is poorly defined and has not previously been reported for κ-opioid receptors. We hypothesized that activation of human μ-, δ- or κ-opioid receptors modulates Cav2.3 channels via G protein signaling. Whole-cell Ba2+ currents were recorded in HEK293T cells co-expressing human Cav2.2 or Cav2.3 channels and μ-, δ- or κ-opioid receptors. Selective opioid receptor agonists and antagonists were used to study receptor modulation. The involvement of intracellular signaling pathways was investigated using specific inhibitors of GPC receptor-G protein coupling. Activation of μ-, δ- or κ-opioid receptors inhibited Cav2.3 and Cav2.2 channel current amplitude by ∼45% and ∼60%, respectively. Inhibition of Cav2.3 was not dependent on the type of subunit co-expressed. Inhibition of the Cav2.3 channel was primarily voltage independent, as depolarizing prepulses could not relieve the inhibited current. This was in marked contrast with the primarily voltage-dependent modulation of Cav2.2 channels that showed nearly complete recovery of the inhibited current with depolarizing prepulses. For all three types of opioid receptors, the pathway leading to Cav2.3 channel inhibition was sensitive to pertussis toxin and intracellular application of GDP-β-S. Similarly, the overexpression of a G protein subunit scavenger, myristoylated-phosducin, significantly reduced the magnitude of Cav2.3 channel inhibition. Here we demonstrate that Cav2.3 channels are efficiently inhibited by activation of μ-, δ- or κ-opioid receptors. Inhibition occurs via voltage-independent G protein signaling mechanisms. These results suggest opioid receptor controls specific members of the Cav2 channel family via differential signaling pathways. Neuronal Cav2.3 channels are therefore potential targets for opioid analgesics.

Nine different voltage-gated sodium channel isoforms are responsible for inducing and propagating action potentials in the mammalian nervous system. The Nav1.7 channel isoform plays an important role in conducting nociceptive signals. Specific mutations of this isoform may impair gating behavior of the channel resulting in several pain syndromes. In addition to channel mutations, similar or opposite changes in gating may be produced by spider and scorpion toxins binding to different parts of the voltage-gated sodium channel. In the present study, we analyzed the effects of the α-scorpion toxin OD1 and 2 synthetic toxin analogs on the gating properties of the Nav1.7 sodium channel. All toxins potently inhibited channel inactivation, however, both toxin analogs showed substantially increased potency by more than one order of magnitude when compared with that of wild-type OD1. The decay phase of the whole-cell Na(+) current was substantially slower in the presence of toxins than in their absence. Single-channel recordings in the presence of the toxins revealed that Na(+) current inactivation slowed due to prolonged flickering of the channel between open and closed states. Our findings support the voltage-sensor trapping model of α-scorpion toxin action, in which the toxin prevents a conformational change in the domain IV voltage sensor that normally leads to fast channel inactivation.

Elucidating the mechanisms that modulate calcium channels via opioid receptor activation is fundamental to our understanding of both pain perception and how opioids modulate pain. Neuronal voltage-gated N-type calcium channels (Cav2.2) are inhibited by activation of G protein-coupled opioid receptors (ORs). However, inhibition of R-type (Cav2.3) channels by μ- or κ-ORs is poorly defined and has not been reported for δ-ORs. To investigate such interactions, we coexpressed human μ-, δ-, or κ-ORs with human Cav2.3 or Cav2.2 in human embryonic kidney 293 cells and measured depolarization-activated Ba(2+) currents (IBa). Selective agonists of μ-, δ-, and κ-ORs inhibited IBa through Cav2.3 channels by 35%. Cav2.2 channels were inhibited to a similar extent by κ-ORs, but more potently (60%) via μ- and δ-ORs. Antagonists of δ- and κ-ORs potentiated IBa amplitude mediated by Cav2.3 and Cav2.2 channels. Consistent with G protein βγ (Gβγ) interaction, modulation of Cav2.2 was primarily voltage-dependent and transiently relieved by depolarizing prepulses. In contrast, Cav2.3 modulation was voltage-independent and unaffected by depolarizing prepulses. However, Cav2.3 inhibition was sensitive to pertussis toxin and to intracellular application of guanosine 5'-[β-thio]diphosphate trilithium salt and guanosine 5'-[γ-thio]triphosphate tetralithium salt. Coexpression of Gβγ-specific scavengers-namely, the carboxyl terminus of the G protein-coupled receptor kinase 2 or membrane-targeted myristoylated-phosducin-attenuated or abolished Cav2.3 modulation. Our study reveals the diversity of OR-mediated signaling at Cav2 channels and identifies neuronal Cav2.3 channels as potential targets for opioid analgesics. Their novel modulation is dependent on pre-existing OR activity and mediated by membrane-delimited Gβγ subunits in a voltage-independent manner.

Scorpion α-toxins are invaluable pharmacological tools for studying voltage-gated sodium channels, but few structure-function studies have been undertaken due to their challenging synthesis. To address this deficiency, we report a chemical engineering strategy based upon native chemical ligation. The chemical synthesis of α-toxin OD1 was achieved by chemical ligation of three unprotected peptide segments. A high resolution X-ray structure (1.8 Å) of synthetic OD1 showed the typical βαββ α-toxin fold and revealed important conformational differences in the pharmacophore region when compared with other α-toxin structures. Pharmacological analysis of synthetic OD1 revealed potent α-toxin activity (inhibition of fast inactivation) at Nav1.7, as well as Nav1.4 and Nav1.6. In addition, OD1 also produced potent β-toxin activity at Nav1.4 and Nav1.6 (shift of channel activation in the hyperpolarizing direction), indicating that OD1 might interact at more than one site with Nav1.4 and Nav1.6. Investigation of nine OD1 mutants revealed that three residues in the reverse turn contributed significantly to selectivity, with the triple OD1 mutant (D9K, D10P, K11H) being 40-fold more selective for Nav1.7 over Nav1.6, while OD1 K11V was 5-fold more selective for Nav1.6 than Nav1.7. This switch in selectivity highlights the importance of the reverse turn for engineering α-toxins with altered selectivity at Nav subtypes.

Angiotensin II exerts glucose-dependent effects on K v currents in mouse pancreatic (cid:1) -cells via angiotensin II type 2 receptors. Am J Physiol Cell Physiol 298: C313–C323, 2010. doi:10.1152/ajpcell.00575.2008.—Hyper-glycemia-associated glucotoxicity induces (cid:1) -cell apoptosis but the underlying mechanisms are unknown. Interestingly, prolonged exposure to high glucose upregulates the expression and function of the renin-angiotensin system (RAS). We hypothesize that the voltage-gated outward potassium (K v ) current, which governs (cid:1) -cell membrane potential and insulin secretion, has a role in glucotoxicity. In this study, we investigated the effects of prolonged exposure to high glucose on mouse pancreatic (cid:1) -cells and concurrent effects on the RAS by examining changes in expression of angiotensin II (ANG II) receptors and changes in the expression and activity of K v channels. (cid:1) -Cells were incubated in high glucose medium for 1–7 days and then were examined with electrophysiological and molecular biology tech-niques. Prolonged exposure to high glucose produced a marked increase in (cid:1) -cell primary K v channel subunit, K v 2.1, expression and K v current amplitude. Enhanced expression of ANG II type 1 receptor (AT 1 R) was also observed under high glucose conditions, whereas blockade of AT 1 R by losartan did not alter K v channel expression. External application of ANG II reduced K v current amplitude under normal, but not high, glucose conditions. The effect of ANG II on K v channel gating was abolished by ANG II type 2 receptor (AT 2 R) antagonism. These data suggest that hyperglycemia alters (cid:1) -cell function through modification of the K v channel which may be associated with the RAS. Amersham) or peroxidase-labeled anti-mouse IgG secondary antibody (1:2,000, for 1 h Positive labeling was revealed using enhanced chemiluminescence plus Western blotting detection reagents and autoradiography film (Amersham). The chemiluminescence intensity of the bands was quantified using an image analyzer CA).

Hyperglycemia-associated glucotoxicity induces beta-cell apoptosis but the underlying mechanisms are unknown. Interestingly, prolonged exposure to high glucose upregulates the expression and function of the renin-angiotensin system (RAS). We hypothesize that the voltage-gated outward potassium (K(v)) current, which governs beta-cell membrane potential and insulin secretion, has a role in glucotoxicity. In this study, we investigated the effects of prolonged exposure to high glucose on mouse pancreatic beta-cells and concurrent effects on the RAS by examining changes in expression of angiotensin II (ANG II) receptors and changes in the expression and activity of K(v) channels. beta-Cells were incubated in high glucose medium for 1-7 days and then were examined with electrophysiological and molecular biology techniques. Prolonged exposure to high glucose produced a marked increase in beta-cell primary K(v) channel subunit, K(v)2.1, expression and K(v) current amplitude. Enhanced expression of ANG II type 1 receptor (AT(1)R) was also observed under high glucose conditions, whereas blockade of AT(1)R by losartan did not alter K(v) channel expression. External application of ANG II reduced K(v) current amplitude under normal, but not high, glucose conditions. The effect of ANG II on K(v) channel gating was abolished by ANG II type 2 receptor (AT(2)R) antagonism. These data suggest that hyperglycemia alters beta-cell function through modification of the K(v) channel which may be associated with the RAS.

A number of ω-conotoxins are potent and selective antagonists of N-type voltage-gated calcium channels (VGCCs) and are potentially effective as analgesic agents. ω-Conotoxins CVID and CVIB, venom peptides from Conus catus, inhibit N-type and N/P/Q-type VGCCs, respectively, in rat dorsal root ganglion sensory neurons. In the present study, we tested the effects of five different ω-conotoxins, CVID, CVIB, MVIIA, MVIIC and GVIA, on excitatory synaptic transmission between primary afferents and dorsal horn superficial lamina neurons of rat spinal cord. The N-type VGCC antagonists CVID (200nM) and MVIIA (500nM) completely and irreversibly inhibited excitatory postsynaptic currents (EPSCs) in the dorsal horn superficial lamina. The N- and P/Q-type VGCC antagonist CVIB (200nM) reversibly reduced evoked EPSC amplitude an average of 34±8%, whereas MVIIC (200nM) had no effect on excitatory synaptic transmission. In neurons receiving polysynaptic input, CVIB reduced both the EPSC amplitude and the “success rate” calculated as the relative number of primary afferent stimulations that resulted in postsynaptic responses. These results indicate that (i) the analgesic action of ω-conotoxins that antagonise N-type VGCCs may be attributed to inhibition of neurotransmission between primary afferents and superficial dorsal horn neurons, (ii) nociceptive synaptic transmission between primary afferents and superficial lamina neurons is mediated predominantly by N-type VGCCs, and (iii) in contrast to the irreversible inhibition by CVID, MVIIA and GVIA, the inhibition of excitatory monosynaptic transmission by CVIB is reversible.

Omega-conotoxins are routinely used as selective inhibitors of different classes of voltage-gated calcium channels (VGCCs) in excitable cells. In the present study, we examined the potent N-type VGCC antagonist omega-conotoxin CVID and non-selective N- and P/Q-type antagonist CVIB for their ability to block native VGCCs in rat dorsal root ganglion (DRG) neurons and recombinant VGCCs expressed in Xenopus oocytes. Omega-conotoxins CVID and CVIB inhibited depolarization-activated whole-cell VGCC currents in DRG neurons with pIC50 values of 8.12 +/- 0.05 and 7.64 +/- 0.08, respectively. Inhibition of Ba2+ currents in DRG neurons by CVID (approximately 66% of total) appeared to be irreversible for > 30 min washout, whereas Ba2+ currents exhibited rapid recovery from block by CVIB (> or = 80% within 3 min). The recoverable component of the Ba2+ current inhibited by CVIB was mediated by the N-type VGCC, whereas the irreversibly blocked current (approximately 22% of total) was attributable to P/Q-type VGCCs. Omega-conotoxin CVIB reversibly inhibited Ba2+ currents mediated by N- (Ca(V)2.2) and P/Q- (Ca(V)2.1), but not R- (Ca(V)2.3) type VGCCs expressed in Xenopus oocytes. The alpha2delta1 auxiliary subunit co-expressed with Ca(V)2.2 and Ca(V)2.1 reduced the sensitivity of VGCCs to CVIB but had no effect on reversibility of block. Determination of the NMR structure of CVIB identified structural differences to CVID that may underlie differences in selectivity of these closely related conotoxins. Omega-conotoxins CVIB and CVID may be useful as antagonists of N- and P/Q-type VGCCs, particularly in sensory neurons involved in processing primary nociceptive information.

A peptide contained in the venom of the predatory marine snail Conus tulipa, rho-TIA, has previously been shown to possess alpha1-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, rho-TIA inhibited alpha1-adrenoreceptor-mediated increases in cytosolic Ca2+ concentration that were triggered by norepinephrine, but did not affect presynaptic alpha2-adrenoreceptor-mediated responses. In radioligand binding assays using [125I]HEAT, rho-TIA displayed slightly greater potency at the alpha 1B than at the alpha 1A or alpha 1D subtypes. Moreover, although it did not affect the rate of association for [3H]prazosin binding to the alpha 1B-adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by rho-TIA. N-terminally truncated analogs of rho-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of rho-TIA (Arg4). An alanine walk of rho-TIA confirmed the importance of Arg4 for activity and revealed a number of other residues clustered around Arg4 that contribute to the potency of rho-TIA. The unique allosteric antagonism of rho-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive alpha1-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.

Fast inactivation of Na channels in neonatal cardiac cells was removed by the action of proteolytic enzymes trypsin or papain. Two stages were apparent in the time course of this process. During the first one, both number of channel reopenings and the mean open time increased markedly even though fast inactivation remained complete. The second stage was manifested by the disappearance of all signs of fast inactivation without further noticeable changes in channel mean open time. At the same time the nonrandom clustering of blank response (response without channel openings) trials became prominent. The data obtained support the interpretation of two separate fast inactivation states in cardiac Na channels as suggested in our previous papers (Zilberter et al. (1989) in Neuromuscular Junction (Sellin, L.C., Libelius, R. and Thesleff, S., eds.), pp. 43-50, Elsevier, Amsterdam, and Zilberter et al. (1991) J. Mol. Cell. Cardiol. 23, (Suppl.) 61-72).

“Fast chemical stimulation” was shown to induce potentiation of glutamate-activated currents in neurons isolated from rat hippocampus. A fast application system allowed solution changes up to a rate of 20 Hz. In Mg2+-free solution, the response to glutamate application immediately after repetitive stimulation with glutamate plus glycine was increased by 25%–88%, returning to control levels over 10–15 min. Enhancement of glutamate-induced currents was also seen after stimulation with solutions containing aspartate or NMDA plus glycine. Aspartate-induced currents were not potentiated. These and other observations demonstrate that in a purely “postsynaptic” system, short-term potentiation can be induced and is mediated via NMDA receptors whereas the potentiated current is carried via non-NMDA glutamate receptor channels.