A parasympathetic neurotransmitter induces myoepithelial cell differentiation during salivary gland development.

Myoepithelial cells (MECs) are responsible for receiving stimuli from the central nervous system and translating their responses into the form of secretion into glandular tissue, including salivary glands (SG), sweet glands, and mammary glands. SG MECs cause the secretion of serous saliva by contracting of acini/ductal cells with acetylcholine (Ach) from parasympathetic nerves via muscarinic receptors. To response the parasympathetic physiological stimulation, SG epithelial cell-derived MECs are supposed to be induced and placed adjacent to parasympathetic system nerve ends in SGs by forming a neuro-myoepithelial junction. For salivary secretion to function under parasympathetic control, therefore, specific regions of salivary gland epithelial cells must be mapped and the epithelium near the nerve must differentiate into MECs in order to form a nerve-myoepithelial junction during organogenesis. We hypothesized that the epithelium near the parasympathetic nerves is induced the differentiation into MECs by which the neurotransmitter acetylcholine via muscarinic receptors. qPCR and whole-mount immunohistochemical analysis in ex vivo organ culture system revealed that SG epithelial cells near a parasympathetic nerve were found to be induced to differentiate into MECs via the cholinergic receptor muscarinic 1 by carbachol (CCh), an acetylcholine agonist. In addition, CCh stimulated ERK and Akt signaling for the induction of MEC differentiation in rat submandibular gland epithelial cells. These findings indicate that muscarinic action is required for the induction of MECs and formation of a neuro-myoepithelial junction in developing SGs. This study proposes a novel concept for tissue architecture to form a neuro-myoepithelial junction during neurofunctional organogenesis including SGs.

Periodontal ligaments enhance neurite outgrowth in trigeminal ganglion neurons through Wnt5a production induced by mechanical stimulation.

The peripheral sensory nerve must be maintained to perceive environmental changes. Daily physiological mechanical stimulations, like gravity, floor reaction force, and occlusal force, influence the nerve homeostasis directly or indirectly. Although the direct axonal membrane stretch enhances axon outgrowth via mechanosensitive channel activation, the indirect mechanisms remain to be elucidated. In this study, we identified the indirect pathways where Wnt5a was a molecular cue released by mechanically stimulated rat periodontal ligament (rPDL) cells. qRT-PCR and ELISA showed that mechanically stimulated rPDL cells enhanced Wnt5a expression level and Wnt5a protein in a Ca2+-dependent manner. The inhibitors of PI3K (LY294002) and MEK1/2 (U0126) suppressed the Akt/PKB and ERK1/2 phosphorylation, respectively, in Western blotting analysis and consequently abolished the increase in Wnt5a expression. Similarly, PF573228, a focal adhesion kinase inhibitor, attenuated Akt- and ERK1/2-phosphorylation and Wnt5a expression. Importantly, the culture medium of stretched PDL cells enhanced neurite elongation, sprouting, and branching in trigeminal ganglion neurons that project to PDL. Moreover, treatment with an anti-Wnt5a antibody (to neutralize Wnt5a activity), AP7677a (anti-Ryk antibody, to block Ryk receptor activity), or strictinin (Ror1 inhibitor) suppressed the morphological changes. These findings reveal the indirect mechanisms that Wnt5a, released from the connective tissues in response to mechanical stimulation, enhances the outgrowth of the peripheral nerves. Our study suggests that the peripheral connective tissues regulate peripheral nerve homeostasis and that Wnt5a signaling could be targeted for the treatment of peripheral nerve disorders.

Mode-selective inhibitory effects of eugenol on the mouse TRPV1 channel.

The transient receptor potential vanilloid 1 (TRPV1) channel is a polymodal receptor in sensory nerves and involved in pain sensation. TRPV1 has at least three distinct activation modes that are selectively induced by different stimuli capsaicin, noxious heat, and protons. Although many mode-selective TRPV1 antagonists have been developed for their anticipated analgesic effects, there have been few successful reports because of adverse effects due to burn injuries and hyperthermia. Eugenol is a vanilloid that has been used as an analgesic in the dental treatment, and its TRPV1 activation ability has been reported. However, our knowledge about the underlying mechanisms of the antagonistic effects of eugenol on TRPV1 activation induced by three different modes is limited. Here, we show that eugenol dose-dependently inhibited the capsaicin-activated inward currents of mouse TRPV1 expressed in human embryonic kidney 293 (HEK293) cells. Under low pH conditions, low concentrations of eugenol only enhanced the proton-induced TRPV1 currents, whereas high eugenol concentrations initially potentiated but then immediately abrogated TRPV1 currents. Finally, eugenol had no modulatory effects on heat-activated TRPV1 in electrophysiological and Fura-2-based Ca2+ imaging experiments. Our results demonstrate that eugenol is a mode-selective antagonist of TRPV1 and can be evaluated as a lead compound of analgesics targeting TRPV1 without serious side effects.

Alternative splicing in the C-terminal tail of Cav2.1 is essential for preventing a neurological disease in mice.

Alternative splicing (AS) that occurs at the final coding exon (exon 47) of the Cav2.1 voltage-gated calcium channel (VGCC) gene produces two major isoforms in the brain, MPI and MPc. These isoforms differ in their splice acceptor sites; human MPI is translated into a polyglutamine tract associated with spinocerebellar ataxia type 6 (SCA6), whereas MPc splices to an immediate stop codon, resulting in a shorter cytoplasmic tail. To gain insight into the functional role of the AS in vivo and whether modulating the splice patterns at this locus can be a potential therapeutic strategy for SCA6, here we created knockin mice that exclusively express MPc by inserting the splice-site mutation. The resultant Cacna1aCtmKO/CtmKO mice developed non-progressive neurological phenotypes, featuring early-onset ataxia and absence seizure without significant alterations in the basic properties of the channel. Interactions of Cav2.1 with Cavß4 and Rimbp2 were significantly reduced while those with GABAB2 were enhanced in the cerebellum of Cacna1aCtmKO/CtmKO mice. Treatment with the GABAB antagonist CGP35348 partially rescued the motor impairments seen in Cacna1aCtmKO/CtmKO mice. These results suggest that the carboxyl-terminal domain of Cav2.1 is not essential for maintaining the basic properties of the channel in the cerebellar Purkinje neurons but is involved in multiple interactions of Cav2.1 with other proteins, and plays an essential role in preventing a complex neurological disease.

Protective Effects of Duloxetine against Cerebral Ischemia-Reperfusion Injury via Transient Receptor Potential Melastatin 2 Inhibition.

Activation of transient receptor potential melastatin 2 (TRPM2), an oxidative stress-sensitive Ca2+-permeable channel, contributes to the aggravation of cerebral ischemia-reperfusion (CIR) injury. Recent studies indicated that treatment with the antidepressant duloxetine for 24 hours (long term) attenuates TRPM2 activation in response to oxidative stress in neuronal cells. To examine the direct effects of antidepressants on TRPM2 activation, we examined their short-term (0-30 minutes) treatment effects on H2O2-induced TRPM2 activation in TRPM2-expressing human embryonic kidney 293 cells using the Ca2+ indicator fura-2. Duloxetine exerted the strongest inhibitory effects on TRPM2 activation among the seven antidepressants tested. These inhibitory effects appeared to be due to the inhibition of H2O2-induced TRPM2 activation via an open-channel blocking-like mechanism, because duloxetine reduced the sustained phase but not the initial phase of increases in intracellular Ca2+ concentrations. In a whole-cell patch-clamp study, duloxetine reduced the TRPM2-mediated inward current during the channel opening state. We also examined the effects of duloxetine in a mouse model of CIR injury. The administration of duloxetine to wild-type mice attenuated CIR injury, similar to that in Trpm2 knockout (KO) mice. The administration of duloxetine did not reduce CIR injury further in Trpm2 KO mice, suggesting that it exerts neuroprotective effects against CIR injury by inhibiting TRPM2 activation. Regarding drug repositioning, duloxetine may be a useful drug in reperfusion therapy for ischemic stroke because it has already been used clinically in therapeutics for several disorders, including depression.

Inhibition of veratridine-induced delayed inactivation of the voltage-sensitive sodium channel by synthetic analogs of crambescin B.

Crambescin B carboxylic acid, a synthetic analog of crambescin B, was recently found to inhibit the voltage-sensitive sodium channels (VSSC) in a cell-based assay using neuroblastoma Neuro 2A cells. In the present study, whole-cell patch-clamp recordings were conducted with three heterologously expressed VSSC subtypes, Nav1.2, Nav1.6 and Nav1.7, in a human embryonic kidney cell line HEK293T to further characterize the inhibition of VSSC by crambescin B carboxylic acid. Contrary to the previous observation, crambescin B carboxylic acid did not inhibit peak current evoked by depolarization from the holding potential of -100mV to the test potential of -10mV in the absence or presence of veratridine (VTD). In the presence of VTD, however, crambescin B carboxylic acid diminished VTD-induced sustained and tail currents through the three VSSC subtypes in a dose-dependent manner, whereas TTX inhibited both the peak current and the VTD-induced sustained and tail currents through all subtypes of VSSC tested. We thus concluded that crambescin B carboxylic acid does not block VSSC in a similar manner to TTX but modulate the action of VTD, thereby causing an apparent block of VSSC in the cell-based assay.

Compromised maturation of GABAergic inhibition underlies abnormal network activity in the hippocampus of epileptic Ca2+ channel mutant mice, tottering.

Cholinergically induced network activity is a useful analogue of theta rhythms involved in memory processing or epileptiform activity in the hippocampus, providing a powerful tool to elucidate the mechanisms of synchrony in neuronal networks. In absence epilepsy, although its association with cognitive impairments has been reported, the mechanisms underlying hippocampal synchrony remain poorly investigated. Here we simultaneously recorded electrical activities from 64 sites in hippocampal slices of CaV2.1 Ca(2+) channel mutant tottering (tg) mice, a well-established mouse model of spontaneous absence epilepsy, to analyze the spatiotemporal pattern of cholinergically induced hippocampal network activity. The cholinergic agonist carbachol induced oscillatory discharges originating from the CA3 region. In tg/tg mice, this hippocampal network activity was characterized by enhanced occupancy of discharges of relatively high frequency (6-10 Hz) compared to the wild type. Pharmacological analyses of slices, patch clamp electrophysiological characterization of isolated neurons, and altered patterns of hippocampal GABAA receptor subunit and Cl(-) transporter messenger RNA (mRNA) transcript levels revealed that this abnormality is attributable to a developmental retardation of GABAergic inhibition caused by immature intracellular Cl(-) regulation. These results suggest that the inherited CaV2.1 Ca(2+) channel mutation leads to developmental abnormalities in Cl(-) transporter expression and GABAA receptor compositions in hippocampal neurons and that compromised maturation of GABAergic inhibition contributes to the abnormal synchrony in the hippocampus of tg absence epileptic mice.

Development of Purkinje cell degeneration in a knockin mouse model reveals lysosomal involvement in the pathogenesis of SCA6.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease caused by the expansion of a polyglutamine tract in the Ca(v)2.1 voltage-gated calcium channel. To elucidate how the expanded polyglutamine tract in this plasma membrane protein causes the disease, we created a unique knockin mouse model that modestly overexpressed the mutant transcripts under the control of an endogenous promoter (MPI-118Q). MPI-118Q mice faithfully recapitulated many features of SCA6, including selective Purkinje cell degeneration. Surprisingly, analysis of inclusion formation in the mutant Purkinje cells indicated the lysosomal localization of accumulated mutant Ca(v)2.1 channels in the absence of autophagic response. The lack of cathepsin B, a major lysosomal cysteine proteinase, exacerbated the loss of Purkinje cells and was accompanied by an acceleration of inclusion formation in this model. Thus, the pathogenic mechanism of SCA6 involves the endolysosomal degradation pathway, and unique pathological features of this model further illustrate the pivotal role of protein context in the pathogenesis of polyglutamine diseases.

Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound.

Canonical transient receptor potential (TRPC) channels control influxes of Ca(2+) and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca(2+) influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced Ca(2+) response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca(2+)-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.

Identification of ultra-rare disruptive variants in voltage-gated calcium channel-encoding genes in Japanese samples of schizophrenia and autism spectrum disorder.

Several large-scale whole-exome sequencing studies in patients with schizophrenia (SCZ) and autism spectrum disorder (ASD) have identified rare variants with modest or strong effect size as genetic risk factors. Dysregulation of cellular calcium homeostasis might be involved in SCZ/ASD pathogenesis, and genes encoding L-type voltage-gated calcium channel (VGCC) subunits Cav1.1 (CACNA1S), Cav1.2 (CACNA1C), Cav1.3 (CACNA1D), and T-type VGCC subunit Cav3.3 (CACNA1I) recently were identified as risk loci for psychiatric disorders. We performed a screening study, using the Ion Torrent Personal Genome Machine (PGM), of exon regions of these four candidate genes (CACNA1C, CACNA1D, CACNA1S, CACNA1I) in 370 Japanese patients with SCZ and 192 with ASD. Variant filtering was applied to identify biologically relevant mutations that were not registered in the dbSNP database or that have a minor allele frequency of less than 1% in East-Asian samples from databases; and are potentially disruptive, including nonsense, frameshift, canonical splicing site single nucleotide variants (SNVs), and non-synonymous SNVs predicted as damaging by five different in silico analyses. Each of these filtered mutations were confirmed by Sanger sequencing. If parental samples were available, segregation analysis was employed for measuring the inheritance pattern. Using our filter, we discovered one nonsense SNV (p.C1451* in CACNA1D), one de novo SNV (p.A36V in CACNA1C), one rare short deletion (p.E1675del in CACNA1D), and 14 NSstrict SNVs (non-synonymous SNV predicted as damaging by all of five in silico analyses). Neither p.A36V in CACNA1C nor p.C1451* in CACNA1D were found in 1871 SCZ cases, 380 ASD cases, or 1916 healthy controls in the independent sample set, suggesting that these SNVs might be ultra-rare SNVs in the Japanese population. The neuronal splicing isoform of Cav1.2 with the p.A36V mutation, discovered in the present study, showed reduced Ca2+-dependent inhibition, resulting in excessive Ca2+ entry through the mutant channel. These results suggested that this de novo SNV in CACNA1C might predispose to SCZ by affecting Ca2+ homeostasis. Thus, our analysis successfully identified several ultra-rare and potentially disruptive gene variants, lending partial support to the hypothesis that VGCC-encoding genes may contribute to the risk of SCZ/ASD.

A missense mutation of the gene encoding voltage-dependent sodium channel (Nav1.1) confers susceptibility to febrile seizures in rats.

Although febrile seizures (FSs) are the most common convulsive syndrome in infants and childhood, the etiology of FSs has remained unclarified. Several missense mutations of the Na(v)1.1 channel (SCN1A), which alter channel properties, have been reported in a familial syndrome of GEFS+ (generalized epilepsy with febrile seizures plus). Here, we generated Scn1a-targeted rats carrying a missense mutation (N1417H) in the third pore region of the sodium channel by gene-driven ENU (N-ethyl-N-nitrosourea) mutagenesis. Despite their normal appearance under ordinary circumstances, Scn1a mutant rats exhibited remarkably high susceptibility to hyperthermia-induced seizures, which involve generalized clonic and/or tonic-clonic convulsions with paroxysmal epileptiform discharges. Whole-cell patch-clamp recordings from HEK cells expressing N1417H mutant channels and from hippocampal GABAergic interneurons of N1417H mutant rats revealed a significant shift of the inactivation curve in the hyperpolarizing direction. In addition, clamp recordings clearly showed the reduction in action potential amplitude in the hippocampal interneurons of these rats. These findings suggest that a missense mutation (N1417H) of the Na(v)1.1 channel confers susceptibility to FS and the impaired biophysical properties of inhibitory GABAergic neurons underlie one of the mechanisms of FS.

Rab3-interacting molecule gamma isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.

Assembly of voltage-dependent Ca(2+) channels (VDCCs) with their associated proteins regulates the coupling of VDCCs with upstream and downstream cellular events. Among the four isoforms of the Rab3-interacting molecule (RIM1 to -4), we have previously reported that VDCC beta-subunits physically interact with the long alpha isoform of the presynaptic active zone scaffolding protein RIM1 (RIM1alpha) via its C terminus containing the C(2)B domain. This interaction cooperates with RIM1alpha-Rab3 interaction to support neurotransmitter exocytosis by anchoring vesicles in the vicinity of VDCCs and by maintaining depolarization-triggered Ca(2+) influx as a result of marked inhibition of voltage-dependent inactivation of VDCCs. However, physiological functions have not yet been elucidated for RIM3 and RIM4, which exist only as short gamma isoforms (gamma-RIMs), carrying the C-terminal C(2)B domain common to RIMs but not the Rab3-binding region and other structural motifs present in the alpha-RIMs, including RIM1alpha. Here, we demonstrate that gamma-RIMs also exert prominent suppression of VDCC inactivation via direct binding to beta-subunits. In the pheochromocytoma PC12 cells, this common functional feature allows native RIMs to enhance acetylcholine secretion, whereas gamma-RIMs are uniquely different from alpha-RIMs in blocking localization of neurotransmitter-containing vesicles near the plasma membrane. Gamma-RIMs as well as alpha-RIMs show wide distribution in central neurons, but knockdown of gamma-RIMs attenuated glutamate release to a lesser extent than that of alpha-RIMs in cultured cerebellar neurons. The results suggest that sustained Ca(2+) influx through suppression of VDCC inactivation by RIMs is a ubiquitous property of neurons, whereas the extent of vesicle anchoring to VDCCs at the plasma membrane may depend on the competition of alpha-RIMs with gamma-RIMs for VDCC beta-subunits.

RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC beta-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The beta construct beta-AID dominant negative, which disrupts the RIM1-beta association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with beta in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.

Capsaicin and Proton Differently Modulate Activation Kinetics of Mouse Transient Receptor Potential Vanilloid-1 Channel Induced by Depolarization.

The transient receptor potential vanilloid type 1 (TRPV1) channel is a non-selective cation channel expressed with transient receptor potential ankyrin type 1 (TRPA1) in small and medial size neurons of the dorsal root ganglions and trigeminal ganglions. TRPV1 is activated by capsaicin, thermal stimuli higher than 43°C, mechanical stress, and protons (H+). Although the TRPV1 channel does not have positively charged residues at regular intervals on its transmembrane segments, alterations in membrane potential also affect the state of TRPV1 channel. In the presence of capsaicin, voltage-dependent probability of opening of the TRPV1 channel and its kinetics have been examined, but the characteristics in the low pH remain unclear. To understand the voltage-dependency of the TRPV1 channel activation, we recorded capsaicin- and proton-induced mouse TRPV1 channel currents in a heterologous expression system. Outward current evoked by depolarizing square pulses in the presence of capsaicin or protons was fitted to a two-exponential function with a time-independent component. The voltage-dependent changes in amplitude of the three components displayed shallow curves and the changes in their ratio to the total current display similar tendencies in the presence of capsaicin and under the low pH. However, the fast and slow time constants in the presence of capsaicin were respectively 5- and 8-fold lower than those obtained under low pH conditions. These results suggest that the TRPV1 channel slowly drives the feed-forward cycle of pain sensation, and capsaicin and protons differently modulate the voltage-dependent TRPV1 channel gating.

A pathogenic C terminus-truncated polycystin-2 mutant enhances receptor-activated Ca2+ entry via association with TRPC3 and TRPC7.

Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca(2+) release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca(2+) influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca(2+) influx that may lead to dysregulated cell growth in ADPKD.

Transient receptor potential 1 regulates capacitative Ca(2+) entry and Ca(2+) release from endoplasmic reticulum in B lymphocytes.

Capacitative Ca(2+) entry (CCE) activated by release/depletion of Ca(2+) from internal stores represents a major Ca(2+) influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca(2+) release-activated Ca(2+) currents and inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor-mediated Ca(2+) oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP(3) receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca(2+) signaling in B lymphocytes.

Knockdown of Cav2.1 calcium channels is sufficient to induce neurological disorders observed in natural occurring Cacna1a mutants in mice.

The CACNA1A gene encodes the poreforming, voltage-sensitive subunit of the voltage-dependent Ca(v)2.1 calcium channel. Mutations in this gene have been linked to several human disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. In mice, mutations of the homolog Cacna1a cause recessively inherited phenotypes in tottering, rolling Nagoya, rocker, and leaner mice. Here we describe two knockdown mice with 28.4+/-3.4% and 13.8+/-3.3% of the wild-type Ca(v)2.1 quantity. 28.4+/-3.4% level mutants displayed ataxia, absence-like seizures and progressive cerebellar atrophy, although they had a normal life span. Mutants with 13.8+/-3.3% level exhibited ataxia severer than the 28.4+/-3.4% level mutants, absence-like seizures and additionally paroxysmal dyskinesia, and died premature around 3 weeks of age. These results indicate that knock down of Ca(v)2.1 quantity to 13.8+/-3.3% of the wild-type level are sufficient to induce the all neurological disorders observed in natural occurring Cacna1a mutants. These knockdown animals with Ca(v)2.1 calcium channels intact can contribute to functional studies of the molecule in the disease.

Arachidonic acid can function as a signaling modulator by activating the TRPM5 cation channel in taste receptor cells.

Vertebrate sensory cells such as vomeronasal neurons and Drosophila photoreceptor cells use TRP channels to respond to exogenous stimuli. In mammalian taste cells, bitter and sweet substances as well as some amino acids are received by G protein-coupled receptors (T2Rs or T1Rs). As a result of activation of G protein and phospholipase Cbeta2, the TRPM5 channel is activated. Intracellular Ca(2+) is known to be a TRPM5 activator, but the participation of lipid activators remains unreported. To clarify the effect of arachidonic acid on TRPM5 in taste cells, we investigated the expression profile of a series of enzymes involved in controlling the intracellular free arachidonic acid level, with the result that in a subset of taste bud cells, monoglyceride lipase (MGL) and cyclooxygenase-2 (COX-2) are expressed as well as the previously reported group IIA phospholipase A(2) (PLA(2)-IIA). Double-labeling analysis revealed that MGL, COX-2 and PLA(2)-IIA are co-expressed in some cells that express TRPM5. We then investigated whether arachidonic acid activates TRPM5 via a heterologous expression system in HEK293 cells, and found that its activation occurred at 10 microM arachidonic acid. These results strongly suggest the possibility that arachidonic acid acts as a modulator of TRPM5 in taste signaling pathways.

Solution structure of agelenin, an insecticidal peptide isolated from the spider Agelena opulenta, and its structural similarities to insect-specific calcium channel inhibitors.

Agelenin, isolated from the Agelenidae spider Agelena opulenta, is a peptide composed of 35 amino acids. We determined the three-dimensional structure of agelenin using two-dimensional NMR spectroscopy. The structure is composed of a short antiparallel beta-sheet and four beta-turns, which are stabilized by three disulfide bonds. Agelenin has characteristic residues, Phe9, Ser28 and Arg33, which are arranged similarly to the pharmacophore of the insect channel inhibitor, omega-atracotoxin-Hv1a. These observations suggest that agelenin and omega-atracotoxin-Hv1a bind to insect calcium channels in a similar manner. We also suggest that another mode of action may operate in the channel inhibition by omega-agatoxin-IVA and omega-atracotoxin-Hv2a.

Pharmacological properties of SAK3, a novel T-type voltage-gated Ca2+ channel enhancer.

T-type voltage-gated Ca2+ channels (T-VGCCs) function in the pathophysiology of epilepsy, pain and sleep. However, their role in cognitive function remains unclear. We previously reported that the cognitive enhancer ST101, which stimulates T-VGCCs in rat cortical slices, was a potential Alzheimer's disease therapeutic. Here, we introduce a more potent T-VGCC enhancer, SAK3 (ethyl 8'-methyl-2',4-dioxo-2-(piperidin-1-yl)-2'H-spiro[cyclopentane-1,3'-imidazo [1,2-a]pyridin]-2-ene-3-carboxylate), and characterize its pharmacological properties in brain. Based on whole cell patch-clamp analysis, SAK3 (0.01-10 nM) significantly enhanced Cav3.1 currents in neuro2A cells ectopically expressing Cav3.1. SAK3 (0.1-10 nM nM) also enhanced Cav3.3 but not Cav3.2 currents in the transfected cells. Notably, Cav3.1 and Cav3.3 T-VGCCs were localized in cholinergic neurve systems in hippocampus and in the medial septum. Indeed, acute oral administration of SAK3 (0.5 mg/kg, p.o.), but not ST101 (0.5 mg/kg, p.o.) significantly enhanced acetylcholine (ACh) release in the hippocampal CA1 region of naïve mice. Moreover, acute SAK3 (0.5 mg/kg, p.o.) administration significantly enhanced hippocampal ACh levels in olfactory-bulbectomized (OBX) mice, rescuing impaired memory-related behaviors. Treatment of OBX mice with the T-VGCC-specific blocker NNC 55-0396 (12.5 mg/kg, i.p.) antagonized both enhanced ACh release and memory improvements elicited by SAK3 administration. We also observed that SAK3-induced ACh releases were significantly blocked in the hippocampus from Cav3.1 knockout (KO) mice. These findings suggest overall that T-VGCCs play a key role in cognition by enhancing hippocampal ACh release and that the cognitive enhancer SAK3 could be a candidate therapeutic in Alzheimer's disease.