Molecular insights into the gating mechanisms of voltage-gated calcium channel CaV2.3.

High-voltage-activated R-type CaV2.3 channel plays pivotal roles in many physiological activities and is implicated in epilepsy, convulsions, and other neurodevelopmental impairments. Here, we determine the high-resolution cryo-electron microscopy (cryo-EM) structure of human CaV2.3 in complex with the α2δ1 and ß1 subunits. The VSDII is stabilized in the resting state. Electrophysiological experiments elucidate that the VSDII is not required for channel activation, whereas the other VSDs are essential for channel opening. The intracellular gate is blocked by the W-helix. A pre-W-helix adjacent to the W-helix can significantly regulate closed-state inactivation (CSI) by modulating the association and dissociation of the W-helix with the gate. Electrostatic interactions formed between the negatively charged domain on S6II, which is exclusively conserved in the CaV2 family, and nearby regions at the alpha-interacting domain (AID) and S4-S5II helix are identified. Further functional analyses indicate that these interactions are critical for the open-state inactivation (OSI) of CaV2 channels.

Non-Mendelian inheritance during inbreeding of Cav3.2 and Cav2.3 deficient mice.

The mating of 77 heterozygous pairs (Cav3.2[+|-] x Cav3.2[+|-]) revealed a significant deviation of genotype distribution from Mendelian inheritance in weaned pups. The mating of 14 pairs (Cav3.2[-|-] female x Cav3.2[+|-] male) and 8 pairs (Cav3.2[+|-] female x Cav3.2[-|-] male) confirmed the significant reduction of deficient homozygous Cav3.2[-|-] pups, leading to the conclusion that prenatal lethality may occur, when one or both alleles, encoding the Cav3.2T-type Ca2+ channel, are missing. Also, the mating of 63 heterozygous pairs (Cav2.3[+|-] x Cav2.3[+|-]) revealed a significant deviation of genotype distribution from Mendelian inheritance in weaned pups, but only for heterozygous male mice, leading to the conclusion that compensation may only occur for Cav2.3[-|-] male mice lacking both alleles of the R-type Ca2+ channel. During the mating of heterozygous parents, the number of female mice within the weaned population does not deviate from the expected Mendelian inheritance. During prenatal development, both, T- and R-type Ca2+ currents are higher expressed in some tissues than postnatally. It will be discussed that the function of voltage-gated Ca2+ channels during prenatal development must be investigated in more detail, not least to understand devastative diseases like developmental epileptic encephalopathies (DEE).

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.

Apamin Boosting of Synaptic Potentials in CaV2.3 R-Type Ca2+ Channel Null Mice.

SK2- and KV4.2-containing K+ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca2+ source that provides Ca2+-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca2+ entry through NMDARs, whereas KV4.2-containing channel availability is increased by Ca2+ entry through SNX-482 (SNX) sensitive CaV2.3 R-type Ca2+ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca2+ entry through R-type Ca2+ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca2+ channels and KV4.2-containing K+ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca2+ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from CaV2.3 null mice. The results show that in the absence of CaV2.3 channels, apamin application still boosted EPSPs. The boosting effect of CaV2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from CaV2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and KV4.2-containing channels to CaV2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.

Low concentrations of ethanol but not of dimethyl sulfoxide (DMSO) impair reciprocal retinal signal transduction.

BACKGROUND: The model of the isolated and superfused retina provides the opportunity to test drugs and toxins. Some chemicals have to be applied using low concentrations of organic solvents as carriers. Recently, E-/R-type (Cav2.3) and T-type (Cav3.2) voltage-gated Ca(2+) channels were identified as participating in reciprocal inhibitory retinal signaling. Their participation is apparent, when low concentrations of NiCl2 (15 µM) are applied during superfusion leading to an increase of the ERG b-wave amplitude, which is explained by a reduction of amacrine GABA-release onto bipolar neurons. During these investigations, differences were observed for the solvent carrier used. METHODS: Recording of the transretinal receptor potentials from the isolated bovine retina. RESULTS: The pretreatment of bovine retina with 0.01 % (v/v) dimethylsulfoxide did not impair the NiCl2-mediated increase of the b-wave amplitude, which was 1.31-fold ± 0.03 of initial value (n = 4). However, pretreatment of the retina with the same concentration of ethanol impaired reciprocal signaling (0.96-fold ± 0.05, n = 4). Further, the implicit time of the b-wave was increased, suggesting that ethanol itself but not DMSO may antagonize GABA-receptors. CONCLUSION: Ethanol itself but not DMSO may block GABA receptors and cause an amplitude increase by itself, so that reciprocal signaling is impaired.

R-Type Voltage-Gated Ca²âº Channels in Cardiac and Neuronal Rhythmogenesis.

During the past decades, an increasing number of ion channel and transporter types have been identified acting together to produce cardiac and neuronal pacemaker action potentials. The basis of pacemaker activity was understood in more detail by using single-microelectrode recordings on cells isolated from pacemaker regions. Meanwhile, this powerful technique was complemented by computer modeling and recombinant technologies, including gene inactivation of ion channels and transporters, which may be involved in the generation of the electrical activity of pacemaker cells. Several genes of the voltage-gated Ca(2+) channel (VGCC) family have been ablated, and their role in cardiac and neuronal pacemaking is compared in the present summary, focusing on the role of murine R-type voltage-gated Ca(2+) channels encoded by cacna1e and expressing the ion conducting subunit Cav2.3.

Cardiac functions of voltage-gated Ca(2+) channels: role of the pharmacoresistant type (E-/R-Type) in cardiac modulation and putative implication in sudden unexpected death in epilepsy (SUDEP).

Voltage-gated Ca(2+) channels (VGCCs) are ubiquitous in excitable cells. These channels play key roles in many physiological events like cardiac regulation/pacemaker activity due to intracellular Ca(2+) transients. In the myocardium, the Cav1 subfamily (L-type: Cav1.2 and Cav1.3) is the main contributor to excitation-contraction coupling and/or pacemaking, whereas the Cav3 subfamily (T-type: Cav3.1 and Cav3.2) is important in rhythmically firing of the cardiac nodal cells. No established cardiac function has been attributed to the Cav2 family (E-/R-type: Cav2.3) despite accumulating evidence of cardiac dysregulation observed upon deletion of the Cav2.3 gene, the only member of this family so far detected in cardiomyocytes. In this review, we summarize the pathophysiological changes observed after ablation of the E-/R-type VGCC and propose a cardiac mechanism of action for this channel. Also, considering the role played by this channel in epilepsy and its reported sensitivity to antiepileptic drugs, a putative involvement of this channel in the cardiac mechanism of sudden unexpected death in epilepsy is also discussed.

Lipid modulation of calcium flux through CaV2.3 regulates acrosome exocytosis and fertilization.

Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3's pore-forming α1E subunit showed altered Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1's lipid and sugar components and CaV2.3's α1E and α2δ subunits. Our results provide a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.

Different calcium sources control somatic versus dendritic SK channel activation during action potentials.

Small-conductance calcium-activated potassium (SK) channels play an important role in regulating neuronal excitability. While SK channels at the soma have long been known to contribute to the medium afterhyperpolarization (mAHP), recent evidence indicates they also regulate NMDA receptor activation in dendritic spines. Here we investigate the activation of SK channels in spines and dendrites of rat cortical pyramidal neurons during action potentials (APs), and compare this to SK channel activation at the soma. Using confocal calcium imaging, we demonstrate that the inhibition of SK channels with apamin results in a location-dependent increase in calcium influx into dendrites and spines during backpropagating APs (average increase, ~40%). This effect was occluded by block of R-type voltage-dependent calcium channels (VDCCs), but not by inhibition of N- or P/Q-type VDCCs, or block of calcium release from intracellular stores. During these experiments, we noticed that the calcium indicator (Oregon Green BAPTA-1) blocked the mAHP. Subsequent experiments using low concentrations of EGTA (1 mm) produced the same result, suggesting that somatic SK channels are not tightly colocalized with their calcium source. Consistent with this idea, all known subtypes of VDCCs except R-type were calcium sources for the apamin-sensitive mAHP at the soma. We conclude that SK channels in spines and dendrites of cortical pyramidal neurons regulate calcium influx during backpropagating APs in a distance-dependent manner, and are tightly coupled to R-type VDCCs. In contrast, SK channels activated by APs at the soma of these neurons are weakly coupled to a variety of VDCCs.

Voltage-gated Ca(2+) channel mediated Ca(2+) influx in epileptogenesis.

Voltage- and ligand-gated ion channels are key elements in the etiopathogenesis of various forms of epilepsy. In this chapter, we present an overview of the functional implications of voltage-gated Ca(2+) channels in modulating internal Ca(2+) level fluctuations and generating ictiform/epileptiform cellular electrophysiological activity. A specific focus will be on the fascinating and evolving field of high-voltage activated (HVA) Non-L-type Ca(v)2.3 R-type channels and low-voltage activated (LVA) Ca(v)3.1-3.3 T-type Ca(2+) channels in the genesis of plateau potentials and excessive rebound bursting. Plateau potentials have been characterised in the hippocampus and were shown to be triggered by Ca(v)2.3 which subsequently activate CNG channels that mediate long-lasting plateaus. In the thalamocortical network, a complex ion channel armamentarium is involved in regulating a complex balance of burst and tonic mode activity. Recent findings point to an outstanding role of R- and T-type channels in both thalamocortical eurhythmia and pathophysiological -aberrations. Thus, pharmacological modulation of voltage-gated Ca(2+)-channels might prove more and more important in treatment of neurological and psychiatric disorder such as schizophrenia, mania, dementia and epilepsy.

Atropine-sensitive hippocampal θ oscillations are mediated by Cav2.3 R-type Ca²âº channels.

Hippocampal theta oscillations are key elements in numerous behavioral and cognitive processes. Based on the dualistic theory of theta oscillations, one can differentiate between atropine-sensitive and atropine-insensitive theta subtypes. Urethane-induced atropine-sensitive theta oscillations are driven by muscarinic signal transduction pathways through G protein q/11 alpha subunit (Gα(q/11)), phospholipase ß( ») (PLCß( »), inositol trisphosphate (InsP3), diacylglycerole (DAG), and protein kinase C (PKC). Recent findings illustrate that Ca(v)2.3 Ca²âº channels are important targets of muscarinic signaling in the hippocampus mediating plateau potential generation, epileptiform burst activity, and complex rhythm generation in the septohippocampal network. To investigate the physiological implications of Ca(v)2.3 Ca²âº channels in hippocampal theta oscillations we performed radiotelemetric intrahippocampal (cornu ammonis (CA1)) recordings in urethane (800 mg/kg, i.p.) and atropine (50 mg/kg, i.p.) treated Ca(v)2.3⁺/⁺ and Ca(v)2.3⁻/⁻ mice followed by wavelet analysis of EEG data. Our results demonstrate that Ca(v)2.3 ablation, unlike PLCß1 deletion, does not result in complete abolishment of urethane-induced theta oscillations and that both mean and total theta duration is not significantly inhibited by subsequent atropine treatment, indicating that Ca(v)2.3 Ca²âº channels are important mediators of atropine-sensitive theta. Although theta frequency remained unchanged between both genotypes, the temporal characteristics of theta distribution, that is, theta architecture were significantly affected by the loss of Ca(v)2.3 Ca²âº channels. Our data suggest, for the first time, that Ca(v)2.3 voltage-gated Ca²âº channels (VGCC) are an important factor in septohippocampal synchronization associated with theta oscillation.

Two separate Ni(2+) -sensitive voltage-gated Ca(2+) channels modulate transretinal signalling in the isolated murine retina.

PURPOSE: Light-evoked responses from vertebrate retinas were recorded as an electroretinogram (ERG). The b-wave is the most prominent component of the ERG, and in the bovine retina its NiCl(2) -sensitive component was attributed to reciprocal signalling by pharmacoresistant R-type voltage-gated Ca(2+) channels, which similar to other voltage-dependent Ca(2+) channels trigger and control neurotransmitter release. The murine retina has the great advantage that the effect of gene inactivation for Ni(2+) -sensitive Ca(2+) channels can be analysed to prove or disprove that any of these Ca(2+) channels is involved in retinal signalling. METHODS: Superfused retinas from different murine genotypes lacking either one or both highly Ni(2+) -sensitive voltage-gated Ca(2+) channels were used to record their ex vivo ERGs. RESULTS: The isolated retinas from mice lacking Ca(v)2.3 R-type or Ca(v)3.2 T-type or both voltage-gated Ca(2+) channels were superfused with a NiCl(2) (15 µm) containing nutrient solution. The change in the b-wave amplitude and implicit time, caused by NiCl(2), was calculated as a difference spectrum and compared to data from control animals. From the results, it can be deduced that Ca(v)2.3 contributes rather to a later component in the b-wave response, while in the absence of Ca(v)3.2 the gain of Ni(2+) -mediated increase in the b-wave amplitude is significantly increased, probably due to a loss of reciprocal inhibition to photoreceptors. Thus, each of the Ni(2+)-sensitive Ca(2+) channels contributes to specific features of the b-wave response. CONCLUSION: Both high-affinity Ni(2+)-sensitive Ca(2+) channels contribute to transretinal signalling. Based on the results from the double knockout mice, additional targets for NiCl(2) must contribute to transretinal signalling, which will be most important for the structurally similar physiologically more important heavy metal cation Zn(2+).

Cav2.3 channels are critical for oscillatory burst discharges in the reticular thalamus and absence epilepsy.

Neurons of the reticular thalamus (RT) display oscillatory burst discharges that are believed to be critical for thalamocortical network oscillations related to absence epilepsy. Ca²+-dependent mechanisms underlie such oscillatory discharges. However, involvement of high-voltage activated (HVA) Ca²+ channels in this process has been discounted. We examined this issue closely using mice deficient for the HVA Ca(v)2.3 channels. In brain slices of Ca(v)2.3⁻/⁻, a hyperpolarizing current injection initiated a low-threshold burst of spikes in RT neurons; however, subsequent oscillatory burst discharges were severely suppressed, with a significantly reduced slow afterhyperpolarization (AHP). Consequently, the lack of Ca(v)2.3 resulted in a marked decrease in the sensitivity of the animal to γ-butyrolactone-induced absence epilepsy. Local blockade of Ca(v)2.3 channels in the RT mimicked the results of Ca(v)2.3⁻/⁻ mice. These results provide strong evidence that Ca(v)2.3 channels are critical for oscillatory burst discharges in RT neurons and for the expression of absence epilepsy.

R type anion channel: a multifunctional channel seeking its molecular identity.

Plant genomes code for channels involved in the transport of cations, anions and uncharged molecules through membranes. Although the molecular identity of channels for cations and uncharged molecules has progressed rapidly in the recent years, the molecular identity of anion channels has lagged behind. Electrophysiological studies have identified S-type (slow) and R-type (rapid) anion channels. In this brief review, we summarize the proposed functions of the R-type anion channels which, like the S-type, were first characterized by electrophysiology over 20 years ago, but unlike the S-type, have still yet to be cloned. We show that the R-type channel can play multiple roles.

A post-burst after depolarization is mediated by group i metabotropic glutamate receptor-dependent upregulation of Ca(v)2.3 R-type calcium channels in CA1 pyramidal neurons.

Activation of group I metabotropic glutamate receptors (subtypes mGluR1 and mGluR5) regulates neural activity in a variety of ways. In CA1 pyramidal neurons, activation of group I mGluRs eliminates the post-burst afterhyperpolarization (AHP) and produces an afterdepolarization (ADP) in its place. Here we show that upregulation of Ca(v)2.3 R-type calcium channels is responsible for a component of the ADP lasting several hundred milliseconds. This medium-duration ADP is rapidly and reversibly induced by activation of mGluR5 and requires activation of phospholipase C (PLC) and release of calcium from internal stores. Effects of mGluR activation on subthreshold membrane potential changes are negligible but are large following action potential firing. Furthermore, the medium ADP exhibits a biphasic activity dependence consisting of short-term facilitation and longer-term inhibition. These findings suggest that mGluRs may dramatically alter the firing of CA1 pyramidal neurons via a complex, activity-dependent modulation of Ca(v)2.3 R-type channels that are activated during spiking at physiologically relevant rates and patterns.

Calcium channel subtypes for cholinergic and nonadrenergic noncholinergic neurotransmission in isolated guinea pig trachea.

The Ca(2+) channel subtypes in the neurotransmission of isolated guinea pig trachea were elucidated by monitoring the effects of specific Ca(2+) channel blockers on cholinergic contractions and nonadrenergic noncholinergic (NANC) relaxation elicited by electrical field stimulation (EFS). In isolated guinea pig trachea, cholinergic contractile responses to low- and high-frequency EFS were inhibited by the selective N-type calcium channel blocker, ω-conotoxin MVIIA. ω-Agatoxin IVA (a selective P-type blocker), ω-conotoxin MVIIC (a nonselective N-, Q-, and P-type blocker), and nifedipine (a selective L-type blocker) were ineffective, whereas Ni(2+) (a T- and R-type blocker) facilitated cholinergic contractions and produced a late contracture when its concentration exceeded 30 µM. The more the concentration of Ni(2+) increased, the greater the number of incidences and the late contracture areas which occurred. Late contracture did not seem to be due to the effects of acetylcholine, tachykinins, or other polypeptides, but disappeared in the absence of indomethacin. The NANC relaxant responses elicited by the low- and high-frequency EFS were inhibited by ω-conotoxin MVIIA or Ni(2+), but unaffected by ω-Agatoxin IVA, ω-conotoxin MVIIC, and nifedipine. In the absence of indomethacin, Ni(2+) did not alter the ω-conotoxin MVIIA (100 nM)-resistant component of cholinergic contraction, but significantly further inhibited that of NANC relaxation. These results suggest that in isolated guinea pig trachea, cholinergic contraction is regulated by N-type calcium channels which may mask T- and R-type calcium channels and may be co-modulated by both, while NANC relaxation is mainly and independently controlled by N-, T-, and R-type calcium channels.

The N terminus of a schistosome beta subunit regulates inactivation and current density of a Cav2 channel.

The ß subunit of high voltage-activated Ca(2+) (Ca(v)) channels targets the pore-forming α(1) subunit to the plasma membrane and tunes the biophysical phenotype of the Ca(v) channel complex. We used a combination of molecular biology and whole-cell patch clamp to investigate the functional role of a long N-terminal polyacidic motif (NPAM) in a Ca(v)ß subunit of the human parasite Schistosoma mansoni (ß(Sm)), a motif that does not occur in other known Ca(v)ß subunits. When expressed in human embryonic kidney cells stably expressing Ca(v)2.3, ß(Sm) accelerates Ca(2+)/calmodulin-independent inactivation of Ca(v)2.3. Deleting the first 44 amino acids of ß(Sm), a region that includes NPAM, significantly slows the predominant time constant of inactivation (τ(fast)) under conditions that prevent Ca(2+)/CaM-dependent inactivation (ß(Sm): τ(fast) = 66 ms; ß(SmΔ2-44): τ(fast) = 111 ms, p < 0.01). Interestingly, deleting the amino acids that are N-terminal to NPAM (2-24 or 2-17) results in faster inactivation than with an intact N terminus (τ(fast) = 42 ms with ß(SmΔ2-17); τ(fast) = 40 ms with ß(SmΔ2-24), p < 0.01). This suggests that NPAM is the structural determinant for accelerating Ca(2+)/calmodulin-independent inactivation. We also created three chimeric subunits that contain the first 44 amino acids of ß(Sm) attached to mammalian ß(1b), ß(2a), and ß(3) subunits. For any given mammalian ß subunit, inactivation was faster if it contained the N terminus of ß(Sm) than if it did not. Co-expression of the mammalian α(2)δ-1 subunit resulted in doubling of the inactivation rate, but the effects of NPAM persisted. Thus, it appears that the schistosome Ca(v) channel complex has acquired a new function that likely contributes to reducing the amount of Ca(2+) that enters the cells in vivo. This feature is of potential interest as a target for new antihelminthics.

Identification of subunits of voltage-gated calcium channels and actions of pregabalin on intrinsic primary afferent neurons in the guinea-pig ileum.

BACKGROUND: The intrinsic primary afferent neurons (IPANs) in the intestine are the first neurons of intrinsic reflexes. Action potential currents of IPANs flow partly through calcium channels, which could feasibly be targeted by pregabalin. The aim was to determine whether pregabalin-sensitive α2δ1 subunits associate with calcium channels of IPANs and whether α2δ1 subunit ligands influence IPAN neuronal properties. METHODS: We used intracellular electrophysiological recording and in situ hybridisation to investigate calcium channel subunit expression in guinea-pig enteric neurons. KEY RESULTS: The α subunits of N (α1B) and R (α1E) type calcium channels, and the auxiliary α2δ1 subunit, were expressed by IPANs. This is the first discovery of the α2δ1 subunit in enteric neurons; we therefore investigated its functional role, by determining effects of the α2δ1 subunit ligand, pregabalin, that inhibits currents carried by channels incorporating this subunit. Pregabalin (10 µmol L(-1)) reduced the action potential duration. The effect was not increased with increase in concentration to 100 µmol L(-1). If N channels were first blocked by ω-conotoxin GVIA (0.5 µmol L(-1)), pregabalin had no effect on the residual inward calcium current. Reduction of the calcium current by pregabalin substantially inhibited the after-hyperpolarising potential (AHP) and increased neuron excitability. CONCLUSION & INFERENCES: Intrinsic primary afferent neurons express functional N (α1B) channel-forming subunits that are associated with α2δ1 modulatory subunits and are inhibited by pregabalin, plus functional R (α1E) channels that are not sensitive to binding of pregabalin to α2δ subunits. The positive effects of pregabalin in irritable bowel syndrome (IBS) patients might be partly mediated by its effect on enteric neurons.

Analgesic effects of a substituted N-triazole oxindole (TROX-1), a state-dependent, voltage-gated calcium channel 2 blocker.

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) = 0.27 microM) compared with hyperpolarized conditions (IC(50) > 20 microM). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited omega-conotoxin GVIA-sensitive calcium currents (Ca(v)2.2 channel currents), with greater potency under depolarized conditions (IC(50) = 0.4 microM) than under hyperpolarized conditions (IC(50) = 2.6 microM), 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.

Functional interplay between NMDA receptors, SK channels and voltage-gated Ca2+ channels regulates synaptic excitability in the medial prefrontal cortex.

Synaptic activity in the medial prefrontal cortex (mPFC) is fundamental for higher cognitive functions such as working memory. The present study shows that small conductance (SK) calcium-activated potassium channels attenuate excitatory synaptic transmission at layer 2/3 and layer 5 inputs to layer 5 pyramidal neurons in the mPFC. SK channels are located postsynaptically at synapses where they are activated during synaptic transmission by calcium influx through NMDA receptors, L-type calcium channels, R-type calcium channels and by calcium release from IP(3)-sensitive stores. Removal of the SK channel-mediated shunt of synaptic transmission reveals significant NMDA receptor-mediated activation during basal synaptic transmission, which is greater at layer 5 inputs (approximately 30%) than at layer 2/3 inputs (approximately 20%). These findings show that interactions between NMDA receptors, SK channels and voltage-gated calcium channels play a critical role in regulating excitatory synaptic transmission in layer 5 pyramidal neurons in the mPFC.