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T-type Ca channels (IA fluorometric (fluorescence imaging plate reader [FLIPR]) assay was used to investigate modulation of human T-type IIn the FLIPR assay, all 11 phytocannabinoids tested modulated T-type IModulation of T-type calcium channels is a common property of phytocannabinoids, which all increase steady-state inactivation at physiological membrane potentials, with some also affecting channel activation. Thus, T-type I

IntroductionLow voltage-activated T-type calcium channels (T-type ICa), CaV3.1, CaV3.2, and CaV3.3 are opened by small depolarizations from the resting membrane potential in many cells and have been associated with neurological disorders including absence epilepsy and pain. Δ9-tetrahydrocannabinol (THC) is the principal psychoactive compound in Cannabis and also directly modulates T-type ICa, however, there is no information about functional activity of most phytocannabinoids on T-type calcium channels, including Δ9-tetrahydrocannabinol acid (THCA), the natural non-psychoactive precursor of THC. The aim of this work was to characterize THCA effects on T-type calcium channels.Materials and MethodsWe used HEK293 Flp-In-TREx cells stably expressing CaV3.1, 3.2 or 3.3. Whole-cell patch clamp recordings were made to investigate cannabinoid modulation of ICa.ResultsTHCA and THC inhibited the peak current amplitude CaV3.1 with a pEC50s of 6.0 ± 0.7 and 5.6 ± 0.4, respectively. 1μM THCA or THC produced a significant negative shift in half activation and inactivation of CaV3.1 and both drugs prolonged CaV3.1 deactivation kinetics. THCA (10 μM) inhibited CaV3.2 by 53% ± 4 and both THCA and THC produced a substantial negative shift in the voltage for half inactivation and modest negative shift in half activation of CaV3.2. THC prolonged the deactivation time of CaV3.2 while THCA did not. THCA inhibited the peak current of CaV3.3 by 43% ± 2 (10μM) but did not notably affect CaV3.3 channel activation or inactivation, however, THC caused significant hyperpolarizing shift in CaV3.3 steady state inactivation.DiscussionTHCA modulated T-type ICa currents in vitro, with significant modulation of kinetics and voltage dependence at low μM concentrations. This study suggests that THCA may have potential for therapeutic use in pain and epilepsy via T-type channel modulation without the unwanted psychoactive effects associated with THC.

Introduction: Low voltage-activated T-type calcium channels (T-type I(Ca)), Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3, are opened by small depolarizations from the resting membrane potential in many cells and have been associated with neurological disorders, including absence epilepsy and pain. Δ(9)-tetrahydrocannabinol (THC) is the principal psychoactive compound in Cannabis and also directly modulates T-type I(Ca); however, there is no information about functional activity of most phytocannabinoids on T-type calcium channels, including Δ(9)-tetrahydrocannabinolic acid (THCA), the natural nonpsychoactive precursor of THC. The aim of this work was to characterize THCA effects on T-type calcium channels. Materials and Methods: We used HEK293 Flp-In-TREx cells stably expressing Ca(V)3.1, 3.2, or 3.3. Whole-cell patch clamp recordings were made to investigate cannabinoid modulation of I(Ca). Results: THCA and THC inhibited the peak current amplitude Ca(V)3.1 with pEC(50)s of 6.0±0.7 and 5.6±0.4, respectively. THC (1 μM) or THC produced a significant negative shift in half activation and inactivation of Ca(V)3.1, and both drugs prolonged Ca(V)3.1 deactivation kinetics. THCA (10 μM) inhibited Ca(V)3.2 by 53%±4%, and both THCA and THC produced a substantial negative shift in the voltage for half inactivation and modest negative shift in half activation of Ca(V)3.2. THC prolonged the deactivation time of Ca(V)3.2, while THCA did not. THCA inhibited the peak current of Ca(V)3.3 by 43%±2% (10 μM) but did not notably affect Ca(V)3.3 channel activation or inactivation; however, THC caused significant hyperpolarizing shift in Ca(V)3.3 steady-state inactivation. Discussion: THCA modulated T-type I(Ca) currents in vitro, with significant modulation of kinetics and voltage dependence at low μM concentrations. This study suggests that THCA may have potential for therapeutic use in pain and epilepsy through T-type calcium channel modulation without the unwanted psychoactive effects associated with THC.

Cav3.2 calcium channels are important mediators of nociceptive signaling in the primary afferent pain pathway, and their expression is increased in various rodent models of chronic pain. Previous work from our laboratory has shown that this is in part mediated by an aberrant expression of deubiquitinase USP5, which associates with these channels and increases their stability. Here, we report on a novel bioactive rhodanine compound (II-1), which was identified in compound library screens. II-1 inhibits biochemical interactions between USP5 and the Cav3.2 domain III-IV linker in a dose-dependent manner, without affecting the enzymatic activity of USP5. Molecular docking analysis reveals two potential binding pockets at the USP5-Cav3.2 interface that are distinct from the binding site of the deubiquitinase inhibitor WP1130 (a.k.a. degrasyn). With an understanding of the ability of some rhodanines to produce false positives in high-throughput screening, we have conducted several orthogonal assays to confirm the validity of this hit, including in vivo experiments. Intrathecal delivery of II-1 inhibited both phases of formalin-induced nocifensive behaviors in mice, as well as abolished thermal hyperalgesia induced by the delivery of complete Freund's adjuvant (CFA) to the hind paw. The latter effects were abolished in Cav3.2 null mice, thus confirming that Cav3.2 is required for the action of II-1. II-1 also mediated a robust inhibition of mechanical allodynia induced by injury to the sciatic nerve. Altogether, our data uncover a novel class of analgesics─well suited to rapid structure-activity relationship studies─that target the Cav3.2/USP5 interface.

Synthetic cannabinoid receptor agonists (SCRAs) are new psychoactive substances associated with acute intoxication and even death. However, the molecular mechanisms through which SCRAs may exert their toxic effects remain unclear—including the potential differential activation of G protein subtypes by cannabinoid receptor type 1 (CB1), a major target of SCRA. We measured CB1‐mediated activation of Gαs and Gαi/o proteins by SCRAs by examining stimulation (pertussis toxin, PTX treated) as well as inhibition (non‐PTX treated) of forskolin (FSK)‐induced cyclic adenosine monophosphate (cAMP) accumulation in human embryonic kidney (HEK) cells stably expressing CB1. Real‐time measurements of stimulation and inhibition of cAMP levels were made using a BRET biosensor. We found that the maximum concentration of SCRAs tested (10 µmol L−1), increased cAMP levels 12%‐45% above that produced by FSK alone, while the phytocannabinoid THC did not significantly alter cAMP levels in PTX‐treated HEK‐CB1 cells. All SCRAs had greater potency to inhibit FSK‐induced cAMP levels than to stimulate cAMP levels. The rank order of potencies for SCRA stimulation of cAMP (Gαs) was PB‐22 > 5F‐MDMB‐PICA > JWH‐018 ≈ AB‐FUBINACA > XLR‐11. By contrast, the potency of SCRAs for inhibition of cAMP (Gαi/o) was 5F‐MDMB‐PICA > AB‐FUBINACA > PB‐22 > JWH‐018 > XLR‐11. The different rank order of potency and EMax of the SCRAs to stimulate Gαs‐like signaling compared to Gαi/o signaling suggests differences in G protein preference between SCRAs. Understanding the apparent differences among these drugs may contribute to unravelling their complex effects in humans.

Background and purpose 5-HT1B receptors are widely expressed GPCRs and a target of triptans, the most commonly prescribed anti-migraine drugs. There is very limited information about the acute, agonist-induced regulation of 5-HT1B receptor signalling and so we sought to characterize this in a neuron-like system. Experimental approach Epitope-tagged human 5-HT1B receptors were expressed in mouse AtT20 cells. 5-HT1B receptor signalling was assessed using whole-cell patch-clamp recordings of endogenous G protein-gated inwardly rectified potassium (GIRK) channels, and receptor localization measured using immunofluorescence. Key results 5-HT (EC50 65 nM) and sumatriptan (EC50 165 nM) activated GIRK channels in AtT20 cells expressing 5-HT1B receptors. Continuous application of both 5-HT (EC50 120 nM) and sumatriptan (EC50 280 nM) produced profound desensitization of 5-HT1B receptor signalling within a few minutes. Complete recovery from desensitization was observed after 10 min. Both 5-HT and sumatriptan induced significant heterologous desensitization of SRIF (somatostatin)-activated GIRK currents, with the 5-HT-induced heterologous desensitization being blocked by the protein kinase inhibitor staurosporine. Both agonists induced modest 5-HT1B receptor internalization, with a time course much slower than receptor desensitization. Conclusions and implications In AtT-20 cells, 5-HT1B receptors undergo rapid and reversible desensitization at concentrations of agonist similar to those required to activate the receptor. Desensitization is incomplete, and the continued signalling of the receptor in the presence of the agonist may lead to cellular adaptations. Finally, 5-HT1B receptor activation causes significant heterologous desensitization, which may lead to a reduced effectiveness of unrelated drugs in vivo.

Synthetic cannabinoid receptor agonists (SCRAs) are new psychoactive substances associated with acute intoxication and even death. However, the molecular mechanisms through which SCRAs may exert their toxic effects remain unclear - including the potential differential activation of G protein subtypes by CB1, a major target of SCRA. We measured CB1-mediated activation of Gαs and Gαi/o proteins by SCRAs by examining stimulation (PTX-treated) as well as inhibition (non-PTX treated) of forskolin-induced cAMP accumulation in HEK cells stably expressing CB1. Real-time measurements of stimulation and inhibition of cAMP levels were made using a BRET biosensor. We found that the maximum concentration of SCRAs tested (10 μM), increased cAMP levels 12 to 45% above that produced by forskolin alone, while the phytocannabinoid THC did not significantly alter cAMP levels in PTX-treated HEK-CB1 cells. All SCRAs had greater potency to inhibit of forskolin-induced cAMP levels than to stimulate cAMP levels. The rank order of potencies for SCRA stimulation of cAMP (Gαs) was PB-22 > 5F-MDMB-PICA > JWH-018 > AB-FUBINACA > XLR-11. By contrast, the potency of SCRAs for inhibition of cAMP (Gαi/o) was 5F-MDMB-PICA > AB-FUBINACA > PB-22 > JWH-018 > XLR-11. The different rank order of potency of the SCRAs to stimulate Gαs-like signalling compared to Gαi/o signalling suggests differences in G protein preference between SCRAs. Understanding the apparent differences among these drugs may contribute to unravelling their complex effects in humans.

Background and purpose Consumption of Synthetic Cannabinoid Receptor agonists (SCRAs) is associated with severe adverse reactions including seizures, arrhythmias and death, but the molecular mechanisms surrounding SCRA toxicity are not yet established. These disease-like symptoms are also synonymous with altered T-type calcium channel activity which controls rhythmicity in the heart and brain. This study examined whether SCRAs alter T-type activity and whether this represents a possible mechanism of toxicity. Experimental approach Fluorescence-based and electrophysiology assays were used to screen 16 structurally related synthetic cannabinoids for their ability to inhibit human T-type calcium channels expressed in HEK293 cells. The most potent compounds were then further examined using patch clamp electrophysiology. Key results MDMB-CHMICA and AMB-CHMINACA potently blocked Cav3.2 with IC50 values of 1.5 and 0.74 μM respectively. Current inhibition increased from 47 to 80% and 45–87% respectively when the channel was in slow-inactivated state. Both SCRAs had little effect on steady state inactivation, however MDMB-CHMICA significantly shifted the half activation potential by -7mV. Neither drug produced frequency dependent block, in contrast to the phytocannabinoid Δ9-THC. Conclusions and implications SCRAs are potent agonists of CB1 receptors and can be extremely toxic, but observed toxicity also resembles symptoms associated with altered Cav3.2 activity. Many SCRAs tested were potent modulators of Cav3.2, raising the possibility that SC toxicity may be due in part to Cav3.2 modulation. This potent T-type channel modulation suggests the possibility of SCRAs as a new drug class with potential to treat diseases associated with altered T-type channel activity. This article is part of the special issue on ‘Cannabinoids’.

N-Methyl-D-aspartate receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are two major types of ionotropic glutamate receptors involved in synaptic transmission. However, excessive activity of these receptors can be cytotoxic and thus their function must be precisely controlled. We have previously reported that NMDA receptor activity is dysregulated following genetic knockout of cellular prion protein (PrPC), and that PrPC regulation of NMDA receptors is copper-dependent. Here, we employed electrophysiological methods to study NMDAR and AMPAR currents of cultured hippocampal neurons from PrPC overexpresser mice. We show that NMDA receptor current amplitude and kinetics are differentially modulated by overexpression of human or mouse PrPC. By contrast, AMPA receptor activity was unaffected. Nonetheless, AMPA receptor activity was modulated by copper ions in a manner similar to what we previously reported for NMDA receptors. Taken together, our findings reveal that AMPA and NMDA receptors are differentially regulated by PrPC, but share common modulation by copper ions.

International audience; Spider venoms are rich sources of peptidic ion channel modulators with important therapeutical potential. We screened a panel of 60 spider venoms to find modulators of ion channels involved in pain transmission. We isolated, synthesized and pharmacologically characterized Cd1a, a novel peptide from the venom of the spider Ceratogyrus darlingi. Cd1a reversibly paralysed sheep blowflies (PD50 of 1318 pmol/g) and inhibited human Cav2.2 (IC50 2.6 μM) but not Cav1.3 or Cav3.1 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp electrophysiological assays Cd1a inhibited rat Cav2.2 with similar potency (IC50 3 μM) without influencing the voltage dependence of Cav2.2 activation gating, suggesting that Cd1a doesn't act on Cav2.2 as a classical gating modifier toxin. The Cd1a binding site on Cav2.2 did not overlap with that of the pore blocker ω-conotoxin GVIA, but its activity at Cav2.2-mutant indicated that Cd1a shares some molecular determinants with GVIA and MVIIA, localized near the pore region. Cd1a also inhibited human Nav1.1-1.2 and Nav1.7-1.8 (IC50 0.1-6.9 μM) but not Nav1.3-1.6 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp assays, Cd1a strongly inhibited human Nav1.7 (IC50 16 nM) and produced a 29 mV depolarising shift in Nav1.7 voltage dependence of activation. Cd1a (400 pmol) fully reversed Nav1.7-evoked pain behaviours in mice without producing side effects. In conclusion, Cd1a inhibited two anti-nociceptive targets, appearing to interfere with Cav2.2 inactivation gating, associated with the Cav2.2 α-subunit pore, while altering the activation gating of Nav1.7. Cd1a was inactive at some of the Nav and Cav channels expressed in skeletal and cardiac muscles and nodes of Ranvier, apparently contributing to the lack of side effects at efficacious doses, and suggesting potential as a lead for development of peripheral pain treatments.

Low-voltage-activated (T-type) calcium channels are important regulators of the transmission of nociceptive information in the primary afferent pathway and finding ligands that modulate these channels is a key focus of the drug discovery field. Recently, we characterized a set of novel compounds with mixed cannabinoid receptor/T-type channel blocking activity and examined their analgesic effects in animal models of pain. Here, we have built on these previous findings and synthesized a new series of small organic compounds. We then screened them using whole-cell voltage clamp techniques to identify the most potent T-type calcium channel inhibitors. The two most potent blockers (compounds 9 and 10) were then characterized using radioligand binding assays to determine their affinity for CB1 and CB2 receptors. The structure-activity relationship and optimization studies have led to the discovery of a new T-type calcium channel blocker, compound 9. Compound 9 was efficacious in mediating analgesia in mouse models of acute inflammatory pain and in reducing tactile allodynia in the partial nerve ligation model. This compound was shown to be ineffective in Cav3.2 T-type calcium channel null mice at therapeutically relevant concentrations, and it caused no significant motor deficits in open field tests. Taken together, our data reveal a novel class of compounds whose physiological and therapeutic actions are mediated through block of Cav3.2 calcium channels.

International audience; T-type calcium channels are essential contributors to the transmission of nociceptive signals in the primary afferent pain pathway. Here, we show that T-type calcium channels are ubiquitinated by WWP1, a plasma-membrane-associated ubiquitin ligase that binds to the intracellular domain III-IV linker region of the Cav3.2 T-type channel and modifies specific lysine residues in this region. A proteomic screen identified the deubiquitinating enzyme USP5 as a Cav3.2 III-IV linker interacting partner. Knockdown of USP5 via shRNA increases Cav3.2 ubiquitination, decreases Cav3.2 protein levels, and reduces Cav3.2 whole-cell currents. In vivo knockdown of USP5 or uncoupling USP5 from native Cav3.2 channels via intrathecal delivery of Tat peptides mediates analgesia in both inflammatory and neuropathic mouse models of mechanical hypersensitivity. Altogether, our experiments reveal a cell signaling pathway that regulates T-type channel activity and their role in nociceptive signaling.

1,4-Dihydropyridines (DHPs) are an important class of L-type calcium channel blockers that are used to treat conditions such as hypertension and angina. Their primary target in the cardiovascular system is the Cav1.2 L-type calcium channel isoform, however, a number of DHPs also block low-voltage-activated T-type calcium channels. Here, we describe the synthesis of a series of novel DHP derivatives that have a condensed 1,4-DHP ring system (hexahydroquinoline) and report on their abilities to block both L- and T-type calcium channels. Within this series of compounds, modification of a key ester moiety not only regulates the blocking affinity for both L- and T-type channels, but also allows for the development of DHPs with 30-fold selectivity for T-type channels over the L-type. Our data suggest that a condensed dihydropyridine-based scaffold may serve as a pharmacophore for a new class of T-type selective inhibitors.

Voltage-gated ion channels are implicated in pain sensation and transmission signaling mechanisms within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments have shown that specific channel isoforms, including Na(V)1.7 and Na(V)1.8 sodium channels and Ca(V)3.2 T-type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized a novel small organic compound (Z123212) that modulates both recombinant and native sodium and calcium channel currents by selectively stabilizing channels in their slow-inactivated state. Slow inactivation of voltage-gated channels can function as a brake during periods of neuronal hyperexcitability, and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal cord neurons in a state-dependent manner. In vivo experiments demonstrate that oral administration of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve ligation model of neuropathic pain and also produces acute antinociception in the hot-plate test. At therapeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse effects. Taken together, the state-dependent inhibition of sodium and calcium channels in both the peripheral and central pain signaling pathways may provide a synergistic mechanism toward the development of a novel class of pain therapeutics.

Piperine, a natural compound isolated from the fruits of Piper, is known to modulate several neurotransmitter systems such as serotonin, norepinephrine, and GABA, all of which have been linked to the development of convulsions. Fruits of Piper species have been suggested as means for managing seizure disorders. The present study was designed to elucidate the anticonvulsant effect of piperine and its mechanisms of action using in-silico, in-vivo and in-vitro techniques.PASS software was used to determine its possible activity and mechanisms. Furthermore the latency for development of convulsions and mortality rate was recorded in different experimental mouse models of epilepsy (pentylenetetrazole, maximal electroshock, NMDA, picrotoxin, bicuculline, BAYK-8644, strychnine-induced convulsions) after administration of various doses of piperine (5, 10 and 20 mg/kg, i.p.). Finally, the effect of piperine on Na(+) and Ca(2+) channels were evaluated using the whole cell patch clamp techniqueOur results revealed that piperine decreased mortality in the MES-induced seizure model. Moreover, piperine (10 mg/kg) delayed the onset of tonic clonic convulsions in the pentylenetetrazole test and reduced associated mortality. Furthermore, an anticonvulsant dose of piperine also delayed the onset of tonic clonic seizures in strychnine, picrotoxin and BAY K-8644. Complete protection against mortality was observed in BAYK-8644 induced convulsions. Finally, whole cell patch clamp analysis suggested an inhibitory effect of piperine on Na(+) channels. Together, our data suggest Na(+) channel antagonist activity as a contributor to the complex anticonvulsant mechanisms of piperine.

Voltage-activated calcium channels are important facilitators of nociceptive transmission in the primary afferent pathway. Consequently, molecules that block these channels are of potential use as pain therapeutics. Our group has recently reported on the identification of a novel class of dihydropyridines (DHPs) that included compounds with preferential selectivity for T-type over L-type channels. Among those compounds, M4 was found to be an equipotent inhibitor of both Cav1.2 L- and Cav3.2 T-type calcium channels. Here, we have further characterized the effects of this compound on other types of calcium channels and examined its analgesic effect when delivered either spinally (i.t.) or systemically (i.p.) to mice. Both delivery routes resulted in antinociception in a model of acute pain. Furthermore, M4 was able to reverse mechanical hyperalgesia produced by nerve injury when delivered intrathecally. M4 retained partial activity when delivered to Cav3.2 null mice, indicating that this compound acts on multiple targets. Additional whole-cell patch clamp experiments in transfected tsA-201 cells revealed that M4 also effectively blocks Cav3.3 (T-type) and Cav2.2 (N-type) currents. Altogether, our data indicate that broad-spectrum inhibition of multiple calcium channel subtypes can lead to potent analgesia in rodents.

We examined ion channels derived from a chloroform extract of isolated, dehydrated rat liver mitochondria. The extraction method was previously used to isolate a channel-forming complex containing poly-3-hydroxybutyrate and calcium polyphosphate from Escherichia coli. This complex is also present in eukaryotic membranes, and is located primarily in mitochondria. Reconstituted channels showed multiple subconductance levels and were voltage-dependent, showing an increased probability of higher conductance states at voltages near zero. In symmetric 150 mM KCl, the maximal conductance of the channel ranged from 350 pS to 750 pS. For voltages >+/-60 mV, conductance fluctuated in the range of approximately 50- approximately 200 pS. In the presence of a 1:3 gradient of KCl, at pH = 7.4, selectivity periodically switched between different states ranging from weakly anion-selective (V(rev) approximately -15 mV) to ideally cation-selective (V(rev) approximately +29 mV), without a significant change in its conductance. Overall, the diverse, but highly reproducible, channel activity most closely resembled the behavior of the permeability transition pore channel seen in patch-clamp experiments on native mitoplasts. We suggest that the isolated complex may represent the ion-conducting module from the permeability transition pore.

We applied reverse transcription-PCR to examine the gene expression of cyclic GMP (cGMP)-dependent protein kinase in the rat brain. A PCR product with the size predicted from the type II cGMP-dependent protein kinase (cGK II) cDNA was detected in various regions of the brain, with highest expression in the thalamus. The amplified product of this cDNA was subcloned, sequenced, and consequently shown to be cGK II. Northern analysis confirmed that this kinase was highly expressed in the thalamus. In situ hybridization with riboprobes derived from this cDNA indicated that cGK II mRNA was highly expressed in the outer layers of the cortex, the septum, amygdala, and olfactory bulb with highest levels in the thalamus. High amounts of cGK II mRNA were also found in specific brainstem loci, including the medial habenula, the subthalamic nucleus, the locus ceruleus, the pontine nucleus, the inferior olivary nuclei, and the nucleus of the solitary tract. Only low levels of cGK II mRNA were detected in the striatum, cerebellum, and hippocampus. These data suggest that the effects of guanylyl cyclase activators, such as nitric oxide and the atriopeptides, in various regions of the CNS may be mediated through cGK II.

We have recently identified a class of dihydropyridine (DHP) analogues with 30-fold selectivity for T-type over L-type calcium channels that could be attributed to a modification of a key ester moiety. Based on these results, we examined a second series of compounds with similar attributes to determine if they had enhanced affinity for T-type channels. Whole-cell patch clamp experiments in transfected tsA-201 cells were used to screen these DHP derivatives for high affinity and selectivity for Cav3.2 over Cav1.2 L-type channels. The effects of the two lead compounds, termed N10 and N12, on Cav3.2 channel activity and gating were characterized in detail. When delivered intrathecally or intraperitoneally, these compounds mediated analgesia in a mouse model of acute inflammatory pain. The best compound from the initial screening, N12, was also able to reverse mechanical hyperalgesia produced by nerve injury. The compounds were ineffective in Cav3.2 null mice. Altogether, our data reveal a novel class of T-type channel blocking DHPs for potential pain therapies.

Eukaryotic voltage-sensitive sodium and calcium channels have a major structural subunit that consists of four linked, homologous domains, which contain six putative transmembrane (TM) segments for an overall total of 24. In the putative pore-lining S5-S6 linker, a glutamate residue appears in the position homologous to that acknowledged as the selectivity filter in eukaryotic sodium and calcium channels. Members of the sodium channel family typically have a selectivity ring consisting of the aspartate, glutamate, lysine, and alanine residues contributed from domains I through IV, respectively. The possibility of bacterial sodium channels being involved in rapid flagellar movement has been raised by Clapham and collaborators. Voltage-gated ion channels (VICs) are a subset of the larger P-loop ion channel family. Most voltage-activated channels exhibit two competing responses to membrane depolarization, which initiates both an activation process that results in channel opening and an inactivation process that ultimately results in channel closing. Analyses of the crystal structures of two 2-TM bacterial potassium channels led to the conclusion that the structure of KcsA represented a closed state and that of the related calcium-gated channel, MthK, crystallized in the presence of bound calcium, represented an open state. The 6-TM channels such as Shaker offer easier genetic manipulation, including the possibility of changing four residues in a functional channel for the price of a single mutation. A strong argument has been made that the bacterial sodium channels represent, or at least are closely related to, progenitors of eukaryotic channels.

Background Low-voltage-activated (T-type) calcium channels play a crucial role in a number of physiological processes, including neuronal and cardiac pacemaker activity and nociception. Therefore, finding specific modulators and/or blockers of T-type channels has become an important field of drug discovery. One characteristic of T-type calcium channels is that they share several structural similarities with voltage-gated sodium channels (VGSCs). We therefore hypothesized that binding sites for certain sodium channel blocking peptide toxins may be present in T-type calcium channels. Findings The sodium channel blocker ProTx I tonically blocked native and transiently expressed T-type channels in the sub- to low micro molar range with at least a ten-fold selectivity for the T-type calcium channel hCav3.1 over hCav3.3, and more than one hundred fold selectivity over hCav3.2. Using chimeras of hCav3.1 and hCav3.3, we determined that the domain IV region of hCav3.1 is a major determinant of toxin affinity, with a minor contribution from domain II. Further analysis revealed several residues in a highly conserved region between T-type and sodium channels that may correspond to toxin binding sites. Mutagenesis of several of these residues on an individual basis, however, did not alter the blocking effects of the toxin. ProTx II on the other hand preferentially blocked hCav3.2 and significantly shifted the steady state inactivation of this channel. Conclusions ProTx I blocks hCav3.1 both selectively and with high affinity. Domain IV appears to play a major role in this selectivity with some contribution from domain II. Given the structural similarities between sodium and T-type calcium channels and the apparent conservation in toxin binding sites, these data could provide insights into the development and synthesis of novel T-type channel antagonists.

Chlorophyll (Chl) retention by mature seed of canola as the result of an early frost or other environmental factors (the “green seed problem”) causes serious economic losses. The relationship of seed degreening to rate of moisture loss by seed and silique and the role of ABA in this process were investigated as a function of developmental age. During the normal predesiccation stage (28–45 days after pollination), seed of Brassica napus (cv. Westar) loses Chl rapidly but seed moisture slowly. After a mild freezing stress, there is a rapid loss of moisture from silique walls, followed by accelerated loss of seed moisture. Chl degradation ceases at 35–45% seed moisture. ABA levels in silique walls of frozen plants (determined by enzyme-linked immunosorbant assay) increased after freezing, apparently in response to moisture loss. In contrast, ABA levels in the seed increased dramatically 1 day after freezing, then decreased to control levels. The influence of the rate of seed moisture loss on Chl degradation was investigated by fast and slow drying of isolated seed under controlled humidity conditions. Seed dried rapidly at 22% RH retained most of its Chl, whereas seed dried slowly at 86% RH lost Chl as fast or faster than seed on control (unfrozen) plants. In all treatments, Chl loss stopped at about 40% seed moisture.

Voltage-gated sodium (Na(v)) and calcium (Ca(v)) channels play important roles in physiological processes, including neuronal and cardiac pacemaker activity, vascular smooth muscle contraction, and nociception. They are thought to share a common ancestry, and, in particular, T-type calcium (T-type) channels share structural similarities with Na(v) channels, both with regard to membrane topology and with regard to gating kinetics, including rapid inactivation. We thus reasoned that certain drugs acting on Na(v) channels may also modulate the activities of T-type channels. Here we show that the specific Na(v)1.8 blocker 5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide (A803467) tonically blocks T-type channels in the low micromolar range. Similarly to Na(v)1.8, this compound causes a significant hyperpolarizing shift in the voltage dependence of inactivation and seems to promote a slow inactivation-like phenotype. We further hypothesized that the structural similarity between T-type and Na(v) channels may extend to structurally similar drug-binding sites. Sequence alignment revealed several highly conserved regions between T-type and Na(v) channels that corresponded to drug-binding sites known to alter voltage-dependent gating kinetics. Mutation of amino acid residues in this regions within human Ca(v)3.2 T-type channels altered A803467 blocking affinity severalfold, suggesting that these sites may be exploited for the design of mixed T-type and Na(v) channel blockers that could potentially act synergistically to normalize aberrant neuronal activity.

Detailed kinetic studies of the binding of the calcium channel antagonist (+)-[3H]PN200-110 to membrane preparations from rabbit skeletal muscle have demonstrated that, in addition to the high-affinity sites (Kd = 0.30 +/- 0.05 nM) that are readily measured in equilibrium and kinetic experiments, there are also dihydropyridine binding sites with much lower affinities. These sites were detected by the ability of micromolar concentrations of several dihydropyridines to accelerate the rate of dissociation of (+)-[3H]-PN200-110 from its high-affinity sites. The observed increase in rate was dependent on the concentration of competing ligand, and half-maximal effects occurred at approximately 10 microM for the agonist (+/-)-Bay K8644 and for the antagonists nifedipine, (+/-)-nitrendipine, and (+)-PN200-110. The low-affinity sites appear to be stereospecific since (-)-PN200-110 (1-200 microM) did not affect the dissociation rate. The possible involvement of guanine nucleotide binding proteins in dihydropyridine binding has been investigated by studying the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) on binding parameters. At a concentration of 10 microM, neither GTP gamma S nor GDP beta S significantly affected the binding of dihydropyridines to their high-affinity sites. GTP gamma S did, however, increase the ability of (+/-)-Bay K8644, but not of (+/-)-nitrendipine, to accelerate the rate of dissociation of tightly bound (+)-[3H]PN200-110. GDP beta S did not affect the dose dependence of either the agonist or the antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: The relationship between genetic variation in the T-type calcium channel gene CACNA1H and childhood absence epilepsy is well established. The purpose of this study was to investigate the range of epilepsy syndromes for which CACNA1H variants may contribute to the genetic susceptibility architecture and determine the electrophysiological effects of these variants in relation to proposed mechanisms underlying seizures. Methods: Exons 3 to 35 of CACNA1H were screened for variants in 240 epilepsy patients (167 unrelated) and 95 control subjects by single-stranded conformation analysis followed by direct sequencing. Cascade testing of families was done by sequencing or single-stranded conformation analysis. Selected variants were introduced into the CACNA1H protein by sitedirected mutagenesis. Constructs were transiently transfected into human embryo kidney cells, and electrophysiological data were acquired. Results: More than 100 variants were detected, including 19 novel variants leading to amino acid changes in subjects with phenotypes including childhood absence, juvenile absence, juvenile myoclonic and myoclonic astatic epilepsies, as well as febrile seizures and temporal lobe epilepsy. Electrophysiological analysis of 11 variants showed that 9 altered channel properties, generally in ways that would be predicted to increase calcium current. Interpretation: Variants in CACNA1H that alter channel properties are present in patients with various generalized epilepsy syndromes. We propose that these variants contribute to an individual’s susceptibility to epilepsy but are not sufficient to cause epilepsy on their own. The genetic architecture is dominated by rare functional variants; therefore, CACNA1H would not be easily identified as a susceptibility gene by a genome-wide case–control study seeking a statistical association. Ann Neurol 2007;62:560 –568

Summary: Purpose: Childhood absence epilepsy (CAE) is an idiopathic form of seizure disorder that is believed to have a genetic basis. Methods: We examined the biophysical consequences of seven mutations in the Cav3.2 T-type calcium channel gene linked to CAE. Results: Of the channel variants examined, one of the mutants, a replacement of glycine 848 in the domain II-S2 region with serine, resulted in significant slowing of the time courses of both activation and inactivation across a wide range of membrane potentials. These changes are consistent with increased channel activity in response to prolonged membrane depolarizations. Conclusions: Taken together, these findings suggest that such little changes in channel gating may contribute to the etiology of CAE.

Evidence that a proton (H+)-activated conductance mechanism may be involved in nociception was first reported back in 1981 when Krishtal and Pidoplichko discovered that lowering external pH induced...

Potassium channels that are inhibited by internal ATP (K ATP channels) provide a critical link between metabolism and cellular excitability. Protein kinase C (PKC) acts on K ATP channels to regulate diverse cellular processes, including cardioprotection by ischemic preconditioning and pancreatic insulin secretion. PKC action decreases the Hill coefficient of ATP binding to cardiac K ATP channels, thereby increasing their open probability at physiological ATP concentrations. We show that PKC similarly regulates recombinant channels from both the pancreas and heart. Surprisingly, PKC acts via phosphorylation of a specific, conserved threonine residue (T180) in the pore-forming subunit (Kir6.2). Additional PKC consensus sites exist on both Kir and the larger sulfonylurea receptor (SUR) subunits. Nonetheless, T180 controls changes in open probability induced by direct PKC action either in the absence of, or in complex with, the accessory SUR1 (pancreatic) or SUR2A (cardiac) subunits. The high degree of conservation of this site among different K ATP channel isoforms suggests that this pathway may have wide significance for the physiological regulation of K ATP channels in various tissues and organelles.

Nitric oxide (NO) acts via soluble guanylyl cyclase to increase cyclic GMP (cGMP), which can regulate various targets including protein kinases. Western blotting showed that type II cGMP-dependent protein kinase (cGK II) is widely expressed in various brain regions, especially in the thalamus. In thalamic extracts, the phosphorylation of several proteins, including cGK II, was increased by exogenous NO or cGMP. In vivo pretreatment with a NO synthase inhibitor reduced the phosphorylation of cGK II, and this could be reversed by exogenous NO or cGMP. Conversely, brainstem electrical stimulation, which enhances thalamic NO release, caused a NO synthase-dependent increase in the phosphorylation of thalamic cGK II. These results indicate that endogenous NO regulates cGMP-dependent protein phosphorylation in the thalamus. The activation of cGKII by NO may play a role in thalamic mechanisms underlying arousal.

The fluorescence changes accompanying the binding of the fluorescent calcium channel antagonist, felodipine, to transverse tubule membranes from rabbit skeletal muscle have been used to characterize low-affinity binding sites for 1,4-dihydropyridine derivatives in these preparations. In competition experiments, felodipine inhibited the high-affinity binding of (+)-[3H]PN200-110 to transverse tubule membranes with an apparent Ki of 5 +/- 2 nM. Binding of felodipine to additional low-affinity sites resulted in a large, saturable (Kd = 6 +/- 2 microM) increase in its fluorescence which could be excited either directly (380 nm) or indirectly via energy transfer from membrane protein (290 nm). The observed fluorescence enhancement was competitively inhibited by other 1,4-dihydropyridines with inhibition constants of 3-21 microM but was unaffected by the structurally unrelated calcium channel antagonists, diltiazem and verapamil, or by Ca2+, Cd2+, and La3+. Both high- and low-affinity binding sites appear to be localized in the transverse tubular system, since the magnitude of the observed fluorescence enhancement was higher in these membranes than in microsomal preparations and was directly proportional to the density of high-affinity sites for (+)-[3H]PN200-110. Furthermore, both high- and low-affinity sites appear to be conformationally coupled since, over the same concentration range that the fluorescence changes were observed, felodipine accelerated the rate of dissociation of [3H]PN200-110 previously bound to its high-affinity sites. Similar behavior has previously been reported for other 1,4-dihydropyridines [Dunn, S. M. J., & Bladen, C. (1991) Biochemistry 30, 5716-5721].(ABSTRACT TRUNCATED AT 250 WORDS)

The immunosuppressant drug [3H]cyclosporin A binds specifically and with high affinity to rat brain membrane preparations. The highest density of binding sites was observed in the hippocampus, cerebellum, cortex and basal ganglia. A similar distribution pattern was seen using a quantitative autoradiographic analysis. This distribution agrees with the localizations of cyclophilin and calcineurin reported in immunohistochemical and in situ hybridization studies. Thus inhibition of calcineurin activity following cyclosporin A binding to cyclophilin may occur in neurones, as it does in T-cells. These results suggest that the neurological side-effects of cyclosporin A may be mediated through its interaction with these proteins in neurones.

The expression of the olfactory cyclic nucleotide-gated channel (CNG1) was studied in the rat brain. Using RT-PCR, levels of CNG1 mRNA were determined relative to the expression of a constantly expressed gene, alpha-tubulin. RT-PCR showed that CNG1 mRNA was detectable in the pituitary gland, the olfactory bulb, and the cerebellum of adult and 5-day-old rats. A 3.4 kb mRNA was detected in the olfactory bulb by Northern blotting. In situ hybridization analysis showed that CNG1 mRNA expression is present in the olfactory bulb and in the Purkinje cells of the cerebellum. RT-PCR studies on Purkinje cell-enriched cultures obtained from the cerebellum of 16-day-old embryos (E16) confirmed the expression of CNG1 mRNA in these neurones. Our results show that CNG1 is not restricted to the olfactory epithelium but is also present in specific regions of the brain. These results suggest that cyclic nucleotides may act in the regions that possess CNG1 gene expression to affect the electrical activity of certain neurones directly.

Low-voltage-activated T-type calcium channels play important roles in neuronal physiology where they control cellular excitability and synaptic transmission. Alteration in T-type channel expression has been linked to various pathophysiological conditions such as pain arising from diabetic neuropathy. In the present study, we looked at the role of asparagine (N)-linked glycosylation on human Cav3.2 T-type channel expression and function. Manipulation of N-glycans on cells expressing a recombinant Cav3.2 channel revealed that N-linked glycosylation is critical for proper functional expression of the channel. Using site-directed mutagenesis to disrupt the canonical N-linked glycosylation sites of Cav3.2 channel, we show that glycosylation at asparagine N192 is critical for channel expression at the surface, whereas glycosylation at asparagine N1466 controls channel activity. Moreover, we demonstrate that N-linked glycosylation of Cav3.2 not only controls surface expression and activity of the channel but also underlies glucose-dependent potentiation of T-type Ca(2+) current. Our data suggest that N-linked glycosylation of T-type channels may play an important role in aberrant upregulation of T-type channel activity in response to glucose elevations.