Inhibition of human N- and T-type calcium channels by an ortho-phenoxyanilide derivative, MONIRO-1.

BACKGROUND AND PURPOSE: Voltage-gated calcium channels are involved in nociception in the CNS and in the periphery. N-type (Cav 2.2) and T-type (Cav 3.1, Cav 3.2 and Cav 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 Cav 1.2, Cav 1.3, Cav 2.1, Cav 2.2, Cav 2.3, Cav 3.1, Cav 3.2 and Cav 3.3. KEY RESULTS: MONIRO-1 was 5- to 20-fold more potent at inhibiting human T-type calcium channels, hCav 3.1, hCav 3.2 and hCav 3.3 (IC50 : 3.3 ± 0.3, 1.7 ± 0.1 and 7.2 ± 0.3 µM, respectively) than N-type calcium channel, hCav 2.2 (IC50 : 34.0 ± 3.6 µM). It interacted with L-type calcium channels Cav 1.2 and Cav 1.3 with significantly lower potency (IC50  > 100 µM) and did not inhibit hCav 2.1 or hCav 2.3 channels at concentrations as high as 100 µM. State- and use-dependent inhibition of hCav 2.2 channels was observed, whereas stronger inhibition occurred at high stimulation frequencies for hCav 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 Cav channels involved in pain and/or epilepsy pathways. High-frequency firing increased the affinity for MONIRO-1 for both hCav 2.2 and hCav 3.1 channels. Such Cav 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.

Mechanism of direct Cav2.2 channel block by the κ-opioid receptor agonist U50488H.

U50488H is a benzeneacetamide κ-opioid receptor (κ-OR) agonist analgesic, widely used for investigating the pharmacology of G protein-coupled κ-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 Ca(2+) channels heterologously expressed in human embryonic kidney (HEK293) cells. U50488H inhibited HVA rat Cav1.3 (rCav1.3), human Cav2.1 (hCav2.1), hCav2.2, hCav2.3, and LVA hCav3.1 and hCav3.2 channels in a concentration-dependent manner, with similar potencies characterised with half-maximal inhibitory concentration (IC50) values of ∼30 µM. U50488H concentrations causing direct Cav inhibition are typically >100 times higher than those producing κ-OR activation. Investigation of the mechanism of U50488H block of the Cav2.2 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 ∼11 mV to more hyperpolarized potentials. U50488H also increased the rate of Ba(2+) current inactivation during a step depolarization and significantly delayed recovery from slow inactivation, compared with control. Cav2.2 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 Cav2.2 channels significantly contributes to reduced Cav2.2 channel availability and slow recovery form inactivation. We conclude that U50488H non-selectively blocks heterologously expressed neuronal HVA and LVA Cav channels in the absence of κ-ORs. This cross-reactivity also suggests potentially common U50488H binding motifs across Cav channel targets.

Voltage-Gated R-Type Calcium Channel Inhibition via Human µ-, δ-, and κ-opioid Receptors Is Voltage-Independently Mediated by Gßγ Protein Subunits.

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 ßγ (Gßγ) 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'-[ß-thio]diphosphate trilithium salt and guanosine 5'-[γ-thio]triphosphate tetralithium salt. Coexpression of Gßγ-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 Gßγ subunits in a voltage-independent manner.

Modulation of human Nav1.7 channel gating by synthetic α-scorpion toxin OD1 and its analogs.

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.

Chemical engineering and structural and pharmacological characterization of the α-scorpion toxin OD1.

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 ßαßß 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 ß-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 Kv currents in mouse pancreatic beta-cells via angiotensin II type 2 receptors.

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.

omega-Conotoxin inhibition of excitatory synaptic transmission evoked by dorsal root stimulation in rat superficial dorsal horn.

A number of omega-conotoxins are potent and selective antagonists of N-type voltage-gated calcium channels (VGCCs) and are potentially effective as analgesic agents. omega-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 omega-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 omega-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-conotoxin CVIB differentially inhibits native and recombinant N- and P/Q-type calcium channels.

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.

Intravenous anaesthetics inhibit nicotinic acetylcholine receptor-mediated currents and Ca2+ transients in rat intracardiac ganglion neurons.

The effects of intravenous (i.v.) anaesthetics on nicotinic acetylcholine receptor (nAChR)-induced transients in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and membrane currents were investigated in neonatal rat intracardiac neurons. In fura-2-loaded neurons, nAChR activation evoked a transient increase in [Ca(2+)](I), which was inhibited reversibly and selectively by clinically relevant concentrations of thiopental. The half-maximal concentration for thiopental inhibition of nAChR-induced [Ca(2+)](i) transients was 28 microM, close to the estimated clinical EC(50) (clinically relevant (half-maximal) effective concentration) of thiopental. In fura-2-loaded neurons, voltage clamped at -60 mV to eliminate any contribution of voltage-gated Ca(2+) channels, thiopental (25 microM) simultaneously inhibited nAChR-induced increases in [Ca(2+)](i) and peak current amplitudes. Thiopental inhibited nAChR-induced peak current amplitudes in dialysed whole-cell recordings by approximately 40% at -120, -80 and -40 mV holding potential, indicating that the inhibition is voltage independent. The barbiturate, pentobarbital and the dissociative anaesthetic, ketamine, used at clinical EC(50) were also shown to inhibit nAChR-induced increases in [Ca(2+)](i) by approximately 40%. Thiopental (25 muM) did not inhibit caffeine-, muscarine- or ATP-evoked increases in [Ca(2+)](i), indicating that inhibition of Ca(2+) release from internal stores via either ryanodine receptor or inositol-1,4,5-trisphosphate receptor channels is unlikely. Depolarization-activated Ca(2+) channel currents were unaffected in the presence of thiopental (25 microM), pentobarbital (50 microM) and ketamine (10 microM). In conclusion, i.v. anaesthetics inhibit nAChR-induced currents and [Ca(2+)](i) transients in intracardiac neurons by binding to nAChRs and thereby may contribute to changes in heart rate and cardiac output under clinical conditions.

Allosteric alpha 1-adrenoreceptor antagonism by the conopeptide rho-TIA.

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.