Natural Product Isoliquiritigenin Activates GABAB Receptors to Decrease Voltage-Gate Ca2+ Channels and Glutamate Release in Rat Cerebrocortical Nerve Terminals.

Reduction in glutamate release is a key mechanism for neuroprotection and we investigated the effect of isoliquiritigenin (ISL), an active ingredient of Glycyrrhiza with neuroprotective activities, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). ISL produced a concentration-dependent inhibition of glutamate release and reduced the intraterminal [Ca2+] increase. The inhibition of glutamate release by ISL was prevented after removing extracellular Ca2+ or blocking P/Q-type Ca2+ channels. This inhibition was mediated through the γ-aminobutyric acid type B (GABAB) receptors because ISL was unable to inhibit glutamate release in the presence of baclofen (an GABAB agonist) or CGP3548 (an GABAB antagonist) and docking data revealed that ISL interacted with GABAB receptors. Furthermore, the ISL inhibition of glutamate release was abolished through the inhibition of Gi/o-mediated responses or Gßγ subunits, but not by 8-bromoadenosine 3',5'-cyclic monophosphate or adenylate cyclase inhibition. The ISL inhibition of glutamate release was also abolished through the inhibition of protein kinase C (PKC), and ISL decreased the phosphorylation of PKC. Thus, we inferred that ISL, through GABAB receptor activation and Gßγ-coupled inhibition of P/Q-type Ca2+ channels, suppressed the PKC phosphorylation to cause a decrease in evoked glutamate release at rat cerebrocortical nerve terminals.

Class II histone deacetylases require P/Q-type Ca2+ channels and CaMKII to maintain gamma oscillations in the pedunculopontine nucleus.

Epigenetic mechanisms (i.e., histone post-translational modification and DNA methylation) play a role in regulation of gene expression. The pedunculopontine nucleus (PPN), part of the reticular activating system, manifests intrinsic gamma oscillations generated by voltage-dependent, high threshold N- and P/Q-type Ca2+ channels. We studied whether PPN intrinsic gamma oscillations are affected by inhibition of histone deacetylation. We showed that, a) acute in vitro exposure to the histone deacetylation Class I and II inhibitor trichostatin A (TSA, 1 µM) eliminated oscillations in the gamma range, but not lower frequencies, b) pre-incubation with TSA (1 µM, 90-120 min) also decreased gamma oscillations, c) Ca2+ currents (ICa) were reduced by TSA, especially on cells with P/Q-type channels, d) a HDAC Class I inhibitor MS275 (500 nM), and a Class IIb inhibitor Tubastatin A (150-500 nM), failed to affect gamma oscillations, e) MC1568, a HDAC Class IIa inhibitor (1 µM), blocked gamma oscillations, and f) the effects of both TSA and MC1568 were blunted by blockade of CaMKII with KN-93 (1 µM). These results suggest a cell type specific effect on gamma oscillations when histone deacetylation is blocked, suggesting that gamma oscillations through P/Q-type channels modulated by CaMKII may be linked to processes related to gene transcription.

Upregulation of L-type calcium channels in colonic inhibitory motoneurons of P/Q-type calcium channel-deficient mice.

Enteric inhibitory motoneurons use nitric oxide and a purine neurotransmitter to relax gastrointestinal smooth muscle. Enteric P/Q-type Ca2+ channels contribute to excitatory neuromuscular transmission; their contribution to inhibitory transmission is less clear. We used the colon from tottering mice (tg/tg, loss of function mutation in the α1A pore-forming subunit of P/Q-type Ca2+ channels) to test the hypothesis that P/Q-type Ca2+ channels contribute to inhibitory neuromuscular transmission and colonic propulsive motility. Fecal pellet output in vivo and the colonic migrating motor complex (ex vivo) were measured. Neurogenic circular muscle relaxations and inhibitory junction potentials (IJPs) were also measured ex vivo. Colonic propulsive motility in vivo and ex vivo was impaired in tg/tg mice. IJPs were either unchanged or somewhat larger in tissues from tg/tg compared with wild-type (WT) mice. Nifedipine (L-type Ca2+ channel antagonist) inhibited IJPs by 35 and 14% in tissues from tg/tg and WT mice, respectively. The contribution of N- and R-type channels to neuromuscular transmission was larger in tissues from tg/tg compared with WT mice. The resting membrane potential of circular muscle cells was similar in tissues from tg/tg and WT mice. Neurogenic relaxations of circular muscle from tg/tg and WT mice were similar. These results demonstrate that a functional deficit in P/Q-type channels does not alter propulsive colonic motility. Myenteric neuron L-type Ca2+ channel function increases to compensate for loss of functional P/Q-type Ca2+ channels. This compensation maintains inhibitory neuromuscular transmission and normal colonic motility.

Zebrafish calls for reinterpretation for the roles of P/Q calcium channels in neuromuscular transmission.

A long-held tenet of neuromuscular transmission is that calcium-dependent neurotransmitter release is mediated by N-type calcium channels in frog but P/Q-type channels in mammals. The N-type assignment in frog is based principally on pharmacological sensitivity to ω-conotoxin GVIA. Our studies show that zebrafish neuromuscular transmission is also sensitive to ω-conotoxin GVIA. However, positional cloning of a mutant line with compromised neuromuscular function identified a mutation in a P/Q- rather than N-type channel. Cloning and heterologous expression of this P/Q-type channel confirmed a block by ω-conotoxin GVIA raising the likelihood that all vertebrates, including frog, use the P/Q-type calcium channel for neuromuscular transmission. In addition, our P/Q defective mutant line offered a means of testing the ability of roscovitine, known to potentiate frog neuromuscular transmission, to mediate behavioral and functional rescue. Acute treatment led to rapid improvement of both, pointing to potential therapeutic benefit for myasthenic disorders involving calcium channel dysfunction.

Postnatal loss of P/Q-type channels confined to rhombic-lip-derived neurons alters synaptic transmission at the parallel fiber to purkinje cell synapse and replicates genomic Cacna1a mutation phenotype of ataxia and seizures in mice.

Ataxia, episodic dyskinesia, and thalamocortical seizures are associated with an inherited loss of P/Q-type voltage-gated Ca(2+) channel function. P/Q-type channels are widely expressed throughout the neuraxis, obscuring identification of the critical networks underlying these complex neurological disorders. We showed recently that the conditional postnatal loss of P/Q-type channels in cerebellar Purkinje cells (PCs) in mice (purky) leads to these aberrant phenotypes, suggesting that intrinsic alteration in PC output is a sufficient pathogenic factor for disease initiation. The question arises whether P/Q-type channel deletion confined to a single upstream cerebellar synapse might induce the pathophysiological abnormality of genomically inherited P/Q-type channel disorders. PCs integrate two excitatory inputs, climbing fibers from inferior olive and parallel fibers (PFs) from granule cells (GCs) that receive mossy fiber (MF) input derived from precerebellar nuclei. In this study, we introduce a new mouse model with a selective knock-out of P/Q-type channels in rhombic-lip-derived neurons including the PF and MF pathways (quirky). We found that in quirky mice, PF-PC synaptic transmission is reduced during low-frequency stimulation. Using focal light stimulation of GCs that express optogenetic light-sensitive channels, channelrhodopsin-2, we found that modulation of PC firing via GC input is reduced in quirky mice. Phenotypic analysis revealed that quirky mice display ataxia, dyskinesia, and absence epilepsy. These results suggest that developmental alteration of patterned input confined to only one of the main afferent cerebellar excitatory synaptic pathways has a significant role in generating the neurological phenotype associated with the global genomic loss of P/Q-type channel function.

Presence of metastasis-associated protein 1 in Sertoli cells is required for proper contact between Sertoli cells and adjacent germ cells.

OBJECTIVE: To investigate whether the normal expression of metastasis-associated protein 1 (MTA1) in Sertoli cells (SCs) is associated with adjacent germ cells (GCs) and to provide the functional relevance of MTA1 in this somatic cell. METHODS: The expression pattern of MTA1 in the SCs of impaired human spermatogenesis was determined using immunohistochemistry. The effect of the depletion of GCs on the expression of MTA1 in isolated SCs was evaluated using reverse transcriptase polymerase chain reaction in murine testes treated with busulphan. Finally, using multiple assays, the functional investigation of MTA1 by its specific knockdown was performed in SC-GC co-cultures. RESULTS: SCs were negatively immunolabeled in the tubules with impaired spermatogenesis. Depletion of murine GCs by treatment with busulphan resulted in a dramatic decrease of the MTA1 transcripts level in the isolated SCs on the 15th day of treatment and thereafter had totally abolished MTA1 expression by the 30th day of treatment, respectively. The addition of isolated round spermatids into SC culture could partially elevate MTA1 expression in the latter. Furthermore, MTA1 is crucial to maintain the GC nursery function and normal anchoring junction formation in SCs because ablation of MTA1 by siRNA induced extensive defects of genes related to SC homeostasis. CONCLUSION: We propose a novel role for SC-expressing MTA1, which is determined by the presence of surrounding GCs, in mediating the crosstalk between SCs and GCs by influencing a broad spectrum of gene changes.

Cerebellar ataxia by enhanced Ca(V)2.1 currents is alleviated by Ca2+-dependent K+-channel activators in Cacna1a(S218L) mutant mice.

Mutations in the CACNA1A gene are associated with neurological disorders, such as ataxia, hemiplegic migraine, and epilepsy. These mutations affect the pore-forming α(1A)-subunit of Ca(V)2.1 channels and thereby either decrease or increase neuronal Ca(2+) influx. A decreased Ca(V)2.1-mediated Ca(2+) influx has been shown to reduce the regularity of cerebellar Purkinje cell activity and to induce episodic cerebellar ataxia. However, little is known about how ataxia can be caused by CACNA1A mutations that increase the Ca(2+) influx, such as the S218L missense mutation. Here, we demonstrate that the S218L mutation causes a negative shift of voltage dependence of Ca(V)2.1 channels of mouse Purkinje cells and results in lowered thresholds for somatic action potentials and dendritic Ca(2+) spikes and in disrupted firing patterns. The hyperexcitability of Cacna1a(S218L) Purkinje cells was counteracted by application of the activators of Ca(2+)-dependent K(+) channels, 1-EBIO and chlorzoxazone (CHZ). Moreover, 1-EBIO also alleviated the irregularity of Purkinje cell firing both in vitro and in vivo, while CHZ improved the irregularity of Purkinje cell firing in vitro as well as the motor performance of Cacna1a(S218L) mutant mice. The current data suggest that abnormalities in Purkinje cell firing contributes to cerebellar ataxia induced by the S218L mutation and they advocate a general therapeutic approach in that targeting Ca(2+)-dependent K(+) channels may be beneficial for treating ataxia not only in patients suffering from a decreased Ca(2+) influx, but also in those suffering from an increased Ca(2+) influx in their Purkinje cells.

New ataxic tottering-6j mouse allele containing a Cacna1a gene mutation.

Voltage-gated Ca(2+) (Ca(v)) channels control neuronal functions including neurotransmitter release and gene expression. The Cacna1a gene encodes the α1 subunit of the pore-forming Ca(v)2.1 channel. Mice with mutations in this gene form useful tools for defining channel functions. The recessive ataxic tottering-6j strain that was generated in the Neuroscience Mutagenesis Facility at The Jackson Laboratory has a mutation in the Cacna1a gene. However, the effect of this mutation has not been investigated in detail. In this study, mutation analysis shows a base substitution (C-to-A) in the consensus splice acceptor sequence linked to exon 5, which results in the skipping of exon 5 and the splicing of exon 4 directly to exon 6. The effect of this mutation is expected to be severe as the expressed α1 subunit protein lacks a significant part of the S4-S5 linker, S5, and part of S5-S6 linker in domain I. Tottering-6j mice display motor dysfunctions in the footprint, rotating rod, and hind-limb extension tests. Although cytoarchitecture of the mutant brains appears normal, tyrosine hydroxylase was persistently expressed in cerebellar Purkinje cells in the adult mutant mice. These results indicate that tottering-6j is a useful model for functional studies of the Ca(v)2.1 channel.

Presynaptic CaV2.1 calcium channels carrying familial hemiplegic migraine mutation R192Q allow faster recovery from synaptic depression in mouse calyx of Held.

Ca(V)2.1 Ca(2+) channels have a dominant and specific role in initiating fast synaptic transmission at central excitatory synapses, through a close association between release sites and calcium sensors. Familial hemiplegic migraine type 1 (FHM-1) is an autosomal-dominant subtype of migraine with aura, caused by missense mutations in the CACNA1A gene that encodes the α(1A) pore-forming subunit of Ca(V)2.1 channel. We used knock-in (KI) transgenic mice harboring the FHM-1 mutation R192Q to study the consequences of this mutation in neurotransmission at the giant synapse of the auditory system formed by the presynaptic calyx of Held terminal and the postsynaptic neurons of the medial nucleus of the trapezoid body (MNTB). Although synaptic transmission seems unaffected by low-frequency stimulation in physiological Ca(2+) concentration, we observed that with low Ca(2+) concentrations (<1 mM) excitatory postsynaptic currents (EPSCs) showed increased amplitudes in R192Q KI mice compared with wild type (WT), meaning significant differences in the nonlinear calcium dependence of nerve-evoked transmitter release. In addition, when EPSCs were evoked by broadened presynaptic action potentials (achieved by inhibition of K(+) channels) via Ca(v)2.1-triggered exocytosis, R192Q KI mice exhibited further enhancement of EPSC amplitude and charge compared with WT mice. Repetitive stimulation of afferent axons to the MNTB at different frequencies caused short-term depression of EPSCs that recovered significantly faster in R192Q KI mice than in WT mice. Faster recovery in R192Q KI mice was prevented by the calcium chelator EGTA-AM, pointing to enlarged residual calcium as a key factor in accelerating the replenishment of synaptic vesicles.

Active zone protein Bassoon co-localizes with presynaptic calcium channel, modifies channel function, and recovers from aging related loss by exercise.

The P/Q-type voltage-dependent calcium channels (VDCCs) are essential for synaptic transmission at adult mammalian neuromuscular junctions (NMJs); however, the subsynaptic location of VDCCs relative to active zones in rodent NMJs, and the functional modification of VDCCs by the interaction with active zone protein Bassoon remain unknown. Here, we show that P/Q-type VDCCs distribute in a punctate pattern within the NMJ presynaptic terminals and align in three dimensions with Bassoon. This distribution pattern of P/Q-type VDCCs and Bassoon in NMJs is consistent with our previous study demonstrating the binding of VDCCs and Bassoon. In addition, we now show that the interaction between P/Q-type VDCCs and Bassoon significantly suppressed the inactivation property of P/Q-type VDCCs, suggesting that the Ca(2+) influx may be augmented by Bassoon for efficient synaptic transmission at NMJs. However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon. Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months. The training increased Bassoon immunoreactivity in NMJs without affecting synapse size. These results demonstrated that the P/Q-type VDCCs preferentially accumulate at NMJ active zones and play essential role in synaptic transmission in conjunction with the active zone protein Bassoon. This molecular mechanism becomes impaired by aging, which suggests altered synaptic function in aged NMJs. However, Bassoon level in aged NMJs can be improved by muscle exercise.

Ca2+ channel currents in dorsal root ganglion neurons of P/Q-type voltage-gated Ca2+ channel mutant mouse, rolling mouse Nagoya.

The role of the P/Q-type voltage-gated Ca(2+) channels (VGCCs) in release of neurotransmitters involved in nociception is not fully understood. Rolling mouse Nagoya (tg(rol)), a P/Q-type channel mutant mouse, expresses P/Q-type VGCC whose activation curve has a higher half activation potential and a smaller slope factor than the wild type channel. We previously reported that tg(rol) mice showed hypoalgesic responses to noxious stimuli. In this study, we examined the VGCC current in dorsal root ganglion (DRG) neurons by the whole-cell patch-clamp method. Both ω-agatoxin IVA (0.1 µM) and ω-conotoxin GVIA (1 µM) inhibited the VGCC current by about 40-50% in both the homozygous tg(rol) (tg(rol)/tg(rol)) and wild type (+/+) mice. The voltage-activation relationships of the total VGCC current and the ω-agatoxin IVA-sensitive component in the tg(rol)/tg(rol) mice shifted positively compared to the +/+ mice, whereas that sensitive to the ω-conotoxin GVIA was not different between the two genotypes. The time constant of activation of the VGCC current at -20 mV was longer in the tg(rol)/tg(rol) mice than in the +/+ mice. These changes in the properties of the VGCC in the tg(rol)/tg(rol) mouse may reduce the amount of the released neurotransmitters and account for the hypoalgesic responses.

Activity-dependent maturation of climbing fiber to Purkinje cell synapses during postnatal cerebellar development.

Cerebellar Purkinje cells (PCs) of newborn rodents are innervated by multiple climbing fibers (CFs). During the first postnatal week, single CFs are strengthened relative to other CFs on the somata of individual PCs. Then, the strengthened CFs undergo translocation to PC dendrites after P9. Elimination of the weaker CFs occurs in two distinct steps, namely the early phase from P7 to around P12 and the late phase from about P12 to around P17. Our previous study demonstrates that CF synapse elimination is severely impaired in null mutant mice lacking Ca(v)2.1, a pore-forming component of P/Q-type voltage-dependent Ca(2+) channel (VDCC). To examine the contribution of postsynaptic P/Q-type VDCC to postnatal rearrangement of CFs, we generated mice with PC-selective deletion of Ca(v)2.1 (PC-Ca(v)2.1 KO). We made whole-cell recordings from PCs in cerebellar slices and examined CF-mediated excitatory postsynaptic currents. We found that PC-Ca(v)2.1 KO PCs had severe defects in selective strengthening of single CFs during the first postnatal week and subsequent CF synapse elimination from P7. Moreover, our morphological analysis revealed that multiple CFs abnormally underwent translocation to PC dendrites in PC-Ca(v)2.1 KO mice. These results indicate that Ca(2+) influx through P/Q-type VDCC into PCs is crucial for selective strengthening of single CFs, early phase elimination and selective translocation of single strengthened CFs to PC dendrites.

ELKS1 and Ca(2+) channel subunit ß4 interact and colocalize at cerebellar synapses.

The cytoskeletal matrix of the active zone and synaptic voltage-dependent calcium channels (VDCCs) are both necessary components for the organization and regulation of synaptic vesicle release. In this study, we report a novel interaction between the cytoskeletal matrix of the active zone protein, ELKS1b, and the VDCC subunit, ß4, in the molecular layer of the cerebellum. We found that the two proteins coimmunoprecipitated using antibodies against each protein. Using fluorescent immunohistochemistry, we observed colocalization between ELKS1b and VDCC ß4 in the molecular layer of the cerebellum, suggesting that these proteins are both present in molecular layer synapses. Analysis of a P/Q-type VDCC knockout mouse (Cacna1a(-/-)) revealed that the localization of the VDCC ß4 subunit to the molecular layer was disrupted, although ELKS1b protein localization was not affected. These results demonstrate that these two proteins interact in vitro and colocalize in the cerebellum, and suggest that their interaction may play a role at the molecular layer synapses of the cerebellum.

Pharmacological validation of candidate causal sleep genes identified in an N2 cross.

Despite the substantial impact of sleep disturbances on human health and the many years of study dedicated to understanding sleep pathologies, the underlying genetic mechanisms that govern sleep and wake largely remain unknown. Recently, the authors completed large-scale genetic and gene expression analyses in a segregating inbred mouse cross and identified candidate causal genes that regulate the mammalian sleep-wake cycle, across multiple traits including total sleep time, amounts of rapid eye movement (REM), non-REM, sleep bout duration, and sleep fragmentation. Here the authors describe a novel approach toward validating candidate causal genes, while also identifying potential targets for sleep-related indications. Select small-molecule antagonists and agonists were used to interrogate candidate causal gene function in rodent sleep polysomnography assays to determine impact on overall sleep architecture and to evaluate alignment with associated sleep-wake traits. Significant effects on sleep architecture were observed in validation studies using compounds targeting the muscarinic acetylcholine receptor M3 subunit (Chrm3) (wake promotion), nicotinic acetylcholine receptor alpha4 subunit (Chrna4) (wake promotion), dopamine receptor D5 subunit (Drd5) (sleep induction), serotonin 1D receptor (Htr1d) (altered REM fragmentation), glucagon-like peptide-1 receptor (Glp1r) (light sleep promotion and reduction of deep sleep), and calcium channel, voltage-dependent, T type, alpha 1I subunit (Cacna1i) (increased bout duration of slow wave sleep). Taken together, these results show the complexity of genetic components that regulate sleep-wake traits and highlight the importance of evaluating this complex behavior at a systems level. Pharmacological validation of genetically identified putative targets provides a rapid alternative to generating knock out or transgenic animal models, and may ultimately lead towards new therapeutic opportunities.

Immunohistochemical characterization of calcitonin gene-related peptide in the trigeminal system of the familial hemiplegic migraine 1 knock-in mouse.

INTRODUCTION: Familial hemiplegic migraine type 1 (FHM-1) is caused by mutations in the CACNA1A gene, with the R192Q mutation being the most common. Elevated calcitonin gene-related peptide (CGRP) levels in acute migraine and clinical trials using CGRP receptor antagonists suggest CGRP-related mechanisms are important in migraine. METHODS: Wild-type and R192Q knock-in mice were anaesthetized and perfused. Using immunohistochemical staining, the expression of CGRP in the trigeminocervical complex (TCC) and in the trigeminal and dorsal root ganglia was characterized. RESULTS: There was a 38% reduction in the percentage of CGRP-immunoreactive cells in the trigeminal ganglia (p < 0.001) of R192Q knock-in mice compared to wild-type animals. The size distribution profile of CGRP-immunoreactive cells within the trigeminal ganglia demonstrated no significant difference in cell diameter between the two groups (p ≥ 0.56). CGRP expression was also reduced in thoracic ganglia of R192Q knock-in mice (21% vs. 27% in wild-type group; p < 0.05), but not in other ganglia. In addition, decreased CGRP immunoreactivity was observed in the superficial laminae of the TCC in R192Q knock-in mice, when compared to the control group (p < 0.005). CONCLUSION: The data demonstrates that the FHM-1 CACNA1A mutation alters CGRP expression in the trigeminal ganglion and TCC. This suggests further study of these animals is warranted to characterize better the role of these mutations in the neurobiology of migraine.

Functional importance of L- and P/Q-type voltage-gated calcium channels in human renal vasculature.

Calcium channel blockers are widely used for treatment of hypertension, because they decrease peripheral vascular resistance through inhibition of voltage-gated calcium channels. Animal studies of renal vasculature have shown expression of several types of calcium channels that are involved in kidney function. It was hypothesized that human renal vascular excitation-contraction coupling involves different subtypes of channels. In human renal artery and dissected intrarenal blood vessels from nephrectomies, PCR analysis showed expression of L-type (Ca(v) 1.2), P/Q-type (Ca(v) 2.1), and T-type subtype (Ca(v) 3.1 and Ca(v) 3.2) voltage-gated calcium channels (Ca(v)s), and quantitative PCR showed highest expression of L-type channels in renal arteries and variable expression between patients of subtypes of calcium channels in intrarenal vessels. Immunohistochemical labeling of kidney sections revealed signals for Ca(v) 2.1 and Ca(v) 3.1 associated with smooth muscle cells of preglomerular and postglomerular vessels. In human intrarenal arteries, depolarization with potassium induced a contraction inhibited by the L-type antagonist nifedipine, EC(50) 1.2×10(-8) mol/L. The T-type antagonist mibefradil inhibited the potassium-induced constriction with large variations between patients. Interestingly, the P/Q-type antagonist, ω-agatoxin IVA, inhibited significantly the contraction with 24% at 10(-9) mol/L. In conclusion L-, P/Q, and T-type channels are expressed in human renal blood vessels, and L- and P/Q-type channels are of functional importance for the depolarization-induced vasoconstriction. The contribution of P/Q-type channels to contraction in the human vasculature is a novel mechanism for the regulation of renal blood flow and suggests that clinical treatment with calcium blockers might affect vascular reactivity also through P/Q-type channel inhibition.

Inactivation kinetics of voltage-gated calcium channels in glutamatergic neurons are influenced by SNAP-25.

SNAP-25 forms part of the SNARE core complex that mediates membrane fusion. Biochemical and electrophysiological evidence supports an accessory role for SNAP-25 in interacting with voltage-gated calcium channels (VGCCs) to modulate channel activity. We recently reported that endogenous SNAP-25 negatively regulates VGCC activity in glutamatergic neurons from rat hippocampal cultures by shifting the voltage-dependence of inactivation of the predominant P/Q-type channel current in these cells. In the present study, we extend these findings by investigating the effect that manipulating endogenous SNAP-25 expression has on the inactivation kinetics of VGCC current in both glutamatergic and GABAergic cells recorded from 9-13 DIV cultures. Silencing SNAP-25 in glutamatergic neurons significantly slowed the inactivation rate of P/Q-type VGCC current whereas alterations in SNAP-25 expression did not alter inactivation rates in GABAergic neurons. These results indicate that endogenous SNAP-25 plays an important role in P/Q-type channel regulation in glutamatergic neurons.

Dramatically different levels of Cacna1a gene expression between pre-weaning wild type and leaner mice.

Loss of function mutations of the CACNA1A gene, coding for the α1A subunit of P/Q type voltage-gated calcium channel (Ca(V)2.1), are responsible for Episodic Ataxia type 2 (EA2), an autosomal dominant disorder. A dominant negative effect of the EA2 mutated protein, rather than a haploinsufficiency mechanism, has been hypothesised both for protein-truncating and missense mutations. We analysed the cacna1a mRNA expression in leaner mice carrying a cacna1a mutation leading to a premature stop codon. The results showed a very low mutant mRNA expression compared to the wild type allele. Although the mutant mRNA slightly increases with age, its low level is likely due to degradation by nonsense mediated decay, a quality control mechanism that selectively degrades mRNA harbouring premature stop codons. These data have implications for EA2 in humans, suggesting a haploinsufficiency mechanism at least for some of the CACNA1A mutations leading to a premature stop codon.

Delayed postnatal loss of P/Q-type calcium channels recapitulates the absence epilepsy, dyskinesia, and ataxia phenotypes of genomic Cacna1a mutations.

Inherited loss of P/Q-type calcium channel function causes human absence epilepsy, episodic dyskinesia, and ataxia, but the molecular "birthdate" of the neurological syndrome and its dependence on prenatal pathophysiology is unknown. Since these channels mediate transmitter release at synapses throughout the brain and are expressed early in embryonic development, delineating the critical circuitry and onset underlying each of the emergent phenotypes requires targeted control of gene expression. To visualize P/Q-type Ca(2+) channels and dissect their role in neuronal networks at distinct developmental stages, we created a novel conditional Cacna1a knock-in mouse by inserting the floxed green fluorescent protein derivative Citrine into the first exon of Cacna1a and then crossed it with a postnatally expressing PCP2-Cre line for delayed Purkinje cell (PC) gene deletion within the cerebellum and sparsely in forebrain (purky). PCs in purky mice lacked P/Q-type calcium channel protein and currents within the first month after birth, displayed altered spontaneous firing, and showed impaired neurotransmission. Unexpectedly, adult purky mice exhibited the full spectrum of neurological deficits seen in mice with genomic Cacna1a ablation. Our results show that the ataxia, dyskinesia, and absence epilepsy caused by inherited disorders of the P/Q-type channel arise from signaling defects beginning in late infancy, revealing an early window of opportunity for therapeutic intervention.