Structural insights into the allosteric effects of the antiepileptic drug topiramate on the CaV2.3 channel.

The R-type voltage-gated calcium channel CaV2.3 is predominantly located in the presynapse and is implicated in distinct types of epileptic seizures. It has consequently emerged as a molecular target in seizure treatment. Here, we determined the cryo-EM structure of the CaV2.3-α2δ1-ß1 complex in the topiramate-bound state at a 3.0 Å resolution. We provide a snapshot of the binding site of topiramate, a widely prescribed antiepileptic drug, on a voltage-gated ion channel. The binding site is located at an intracellular juxtamembrane hydrophilic cavity. Further structural analysis revealed that topiramate may allosterically facilitate channel inactivation. These findings provide fundamental insights into the mechanism underlying the inhibitory effect of topiramate on CaV and NaV channels, elucidating a previously unseen modulator binding site and thus pointing toward a route for the development of new drugs.

Association Study of CACNA1D, KCNJ11, KCNQ1, and CACNA1E Single-Nucleotide Polymorphisms with Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a complex chronic disease characterized by decreased insulin secretion and the development of insulin resistance. Previous genome-wide association studies demonstrated that single-nucleotide polymorphisms (SNPs) present in genes coding for ion channels involved in insulin secretion increase the risk of developing this disease. We determined the association of 16 SNPs found in CACNA1D, KCNQ1, KCNJ11, and CACNA1E genes and the increased probability of developing T2DM. In this work, we performed a case-control study in 301 Mexican adults, including 201 cases with diabetes and 100 controls without diabetes. Our findings indicate a moderate association between T2DM and the C allele, and the C/C genotype of rs312480 within CACNA1D. The CAG haplotype surprisingly showed a protective effect, whereas the CAC and CGG haplotypes have a strong association with T2DM. The C allele and C/C genotype of rs5219 were significantly associated with diabetes. Also, an association was observed between diabetes and the A allele and the A/A genotype of rs3753737 and rs175338 in CACNA1E. The TGG and CGA haplotypes were also found to be significantly associated. The findings of this study indicate that the SNPs examined could serve as a potential diagnostic tool and contribute to the susceptibility of the Mexican population to this disease.

L-cysteine modulates visceral nociception mediated by the CaV2.3 R-type calcium channels.

CaV2.3 channels are subthreshold voltage-gated calcium channels that play crucial roles in neurotransmitter release and regulation of membrane excitability, yet modulation of these channels with endogenous molecules and their role in pain processing is not well studied. Here, we hypothesized that an endogenous amino acid l-cysteine could be a modulator of these channels and may affect pain processing in mice. To test this hypothesis, we employed conventional patch-clamp technique in the whole-cell configuration using recombinant CaV2.3 subunit stably expressed in human embryonic kidney (HEK-293) cells. We found in our in vitro experiments that l-cysteine facilitated gating and increased the amplitudes of recombinant CaV2.3 currents likely by chelating trace metals that tonically inhibit the channel. In addition, we took advantage of mouse genetics in vivo using the acetic acid visceral pain model that was performed on wildtype and homozygous Cacna1e knockout male littermates. In ensuing in vivo experiments, we found that l-cysteine administered both subcutaneously and intraperitoneally evoked more prominent pain responses in the wildtype mice, while the effect was completely abolished in knockout mice. Conversely, intrathecal administration of l-cysteine lowered visceral pain response in the wildtype mice, and again the effect was completely abolished in the knockout mice. Our study strongly suggests that l-cysteine-mediated modulation of CaV2.3 channels plays an important role in visceral pain processing. Furthermore, our data are consistent with the contrasting roles of CaV2.3 channels in mediating visceral nociception in the peripheral and central pain pathways.

Direct inhibition of CaV2.3 by Gem is dynamin dependent and does not require a direct alfa/beta interaction.

The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel ß subunit (CaVß), and the binding between CaVα1/CaVß appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVß. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVß independent promotion of dynamin-dependent endocytosis.

De Novo Pathogenic Variants in CACNA1E Cause Developmental and Epileptic Encephalopathy with Contractures, Macrocephaly, and Dyskinesias.

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α1-subunit of the voltage-gated CaV2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

Disruption of GpI mGluR-Dependent Cav2.3 Translation in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is an inherited intellectual impairment that results from the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein that regulates mRNA translation at synapses. The absence of FMRP leads to neuronal and circuit-level hyperexcitability that is thought to arise from the aberrant expression and activity of voltage-gated ion channels, although the identification and characterization of these ion channels have been limited. Here, we show that FMRP binds the mRNA of the R-type voltage-gated calcium channel Cav2.3 in mouse brain synaptoneurosomes and represses Cav2.3 translation under basal conditions. Consequently, in hippocampal neurons from male and female FMRP KO mice, we find enhanced Cav2.3 protein expression by western blotting and abnormally large R currents in whole-cell voltage-clamp recordings. In agreement with previous studies showing that FMRP couples Group I metabotropic glutamate receptor (GpI mGluR) signaling to protein translation, we find that GpI mGluR stimulation results in increased Cav2.3 translation and R current in hippocampal neurons which is disrupted in FMRP KO mice. Thus, FMRP serves as a key translational regulator of Cav2.3 expression under basal conditions and in response to GpI mGluR stimulation. Loss of regulated Cav2.3 expression could underlie the neuronal hyperactivity and aberrant calcium spiking in FMRP KO mice and contribute to FXS, potentially serving as a novel target for future therapeutic strategies.SIGNIFICANCE STATEMENT Patients with fragile X syndrome (FXS) exhibit signs of neuronal and circuit hyperexcitability, including anxiety and hyperactive behavior, attention deficit disorder, and seizures. FXS is caused by the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein, and the neuronal hyperexcitability observed in the absence of FMRP likely results from its ability to regulate the expression and activity of voltage-gated ion channels. Here we find that FMRP serves as a key translational regulator of the voltage-gated calcium channel Cav2.3 under basal conditions and following activity. Cav2.3 impacts cellular excitability and calcium signaling, and the alterations in channel translation and expression observed in the absence of FMRP could contribute to the neuronal hyperactivity that underlies FXS.

Paradoxical Excitatory Impact of SK Channels on Dendritic Excitability.

Dendritic excitability regulates how neurons integrate synaptic inputs and thereby influences neuronal output. As active dendritic events are associated with significant calcium influx they are likely to be modulated by calcium-dependent processes, such as calcium-activated potassium channels. Here we investigate the impact of small conductance calcium-activated potassium channels (SK channels) on dendritic excitability in male and female rat cortical pyramidal neurons in vitro and in vivo Using local applications of the SK channel antagonist apamin in vitro, we show that blocking somatic SK channels enhances action potential output, whereas blocking dendritic SK channels paradoxically reduces the generation of dendritic calcium spikes and associated somatic burst firing. Opposite effects were observed using the SK channel enhancer NS309. The effect of apamin on dendritic SK channels was occluded when R-type calcium channels were blocked, indicating that the inhibitory impact of apamin on dendritic calcium spikes involved R-type calcium channels. Comparable effects were observed in vivo Intracellular application of apamin via the somatic whole-cell recording pipette reduced the medium afterhyperpolarization and increased action potential output during UP states. In contrast, extracellular application of apamin to the cortical surface to block dendritic SK channels shifted the distribution of action potentials within UP states from an initial burst to a more distributed firing pattern, while having no impact on overall action potential firing frequency or UP and DOWN states. These data indicate that somatic and dendritic SK channels have opposite effects on neuronal excitability, with dendritic SK channels counter-intuitively promoting rather than suppressing neuronal output.SIGNIFICANCE STATEMENT Neurons typically receive input from other neurons onto processes called dendrites, and use electrical events such as action potentials for signaling. As electrical events in neurons are usually associated with calcium influx they can be regulated by calcium-dependent processes. One such process is through the activation of calcium-dependent potassium channels, which usually act to reduce action potential signaling. Although this is the case for calcium-dependent potassium channels found at the cell body, we show here that calcium-dependent potassium channels in dendrites of cortical pyramidal neurons counter-intuitively promote rather than suppress action potential output.

Interaction of MDIMP with the Voltage-Gated Calcium Channels.

Amino acid-derived isoindolines are synthetic compounds that were created with the idea of investigating their biological actions. The amino acid moiety was included on the grounds that it may help to avoid toxic effects. Recently, the isoindoline MDIMP was shown to inhibit both cardiac excitation-contraction coupling and voltage-dependent calcium channels. Here, we revealed that MDIMP binds preferentially to low-voltage-activated (LVA) channels. Using a holding potential of -90 mV, the following IC50 values were found (in micromolars): >1000 (CaV2.3), 957 (CaV1.3), 656 (CaV1.2), 219 (CaV3.2), and 132 (CaV3.1). Moreover, the isoindoline also promoted both accelerated inactivation kinetics of high-voltage-activated Ca2+ channels and a modest upregulation of CaV1.3 and CaV2.3. Additional data indicate that although MDIMP binds to the closed state of the channels, it has more preference for the inactivated one. Concerning CaV3.1, the compound did not alter the shape of the instantaneous current-voltage curve, and substituting one or two residues in the selectivity filter drastically increased the IC50 value, suggesting that MDIMP binds to the extracellular side of the pore. However, an outward current failed in removing the inhibition, which implies an alternative mechanism may be involved. The enantiomer (R)-MDIMP [methyl (R)-2-(1,3-dihydroisoindol-2-yl)-4-methylpentanoate], on the other hand, was synthesized and evaluated, but it did not improve the affinity to LVA channels. Implications of these findings are discussed in terms of the possible underlying mechanisms and pharmacological relevance. SIGNIFICANCE STATEMENT: We have studied the regulation of voltage-gated calcium channels by MDIMP, which disrupts excitation-contraction coupling in cardiac myocytes. The latter effect is more potent in atrial than ventricular myocytes, and this could be explained by our results showing that MDIMP preferentially blocks low-voltage-activated channels. Our data also provide mechanistic insights about the blockade and suggest that MDIMP is a promising member of the family of Ca2+ channel blockers, with possible application to the inhibition of subthreshold membrane depolarizations.

Cav2.3 R-type calcium channels: from its discovery to pathogenic de novo CACNA1E variants: a historical perspective.

So-called pharmacoresistant (R-type) voltage-gated Ca2+ channels are structurally only partially characterized. Most of them are encoded by the CACNA1E gene and are expressed as different Cav2.3 splice variants (variant Cav2.3a to Cav2.3e or f) as the ion conducting subunit. So far, no inherited disease is known for the CACNA1E gene but recently spontaneous mutations leading to early death were identified, which will be brought into focus. In addition, a short historical overview may highlight the development to understand that upregulation during aging, easier activation by spontaneous mutations or lack of bioavailable inorganic cations (Zn2+ and Cu2+) may lead to similar pathologies caused by cellular overexcitation.

Experimentally Induced Convulsive Seizures Are Modulated in Part by Zinc Ions through the Pharmacoresistant Cav2.3 Calcium Channel.

BACKGROUND/AIMS: Still in 1999 the first hints were published for the pharmacoresistant Cav2.3 calcium channel to be involved in the generation of epileptic seizures, as transcripts of alpha1E (Cav2.3) and alpha1G (Cav3.1) are changed in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Consecutively, the seizure susceptibility of mice lacking Cav2.3 was analyzed in great detail by using 4-aminopyridine, pentylene-tetrazol, N-methyl-D-aspartate and kainic acid to induce experimentally convulsive seizures. Further, γ-hydroxybutyrolactone was used for the induction of non-convulsive absence seizures. For all substances tested, Cav2.3-competent mice differed from their knockout counterparts in the sense that for convulsive seizures the deletion of the pharmacoresistant channel was beneficial for the outcome during experimentally induced seizures [1]. The antiepileptic drug lamotrigine reduces seizure activity in Cav2.3-competent but increases it in Cav2.3-deficient mice. In vivo, Cav2.3 must be under tight control by endogenous trace metal cations (Zn2+ and Cu2+). The dyshomeostasis of either of them, especially of Cu2+, may alter the regulation of Cav2.3 severely and its activity for Ca2+ conductance, and thus may change hippocampal and neocortical signaling to hypo- or hyperexcitation. METHODS: To investigate by telemetric EEG recordings the mechanism of generating hyperexcitation by kainate, mice were tested for their sensitivity of changes in neuronal (intracerebroventricular) concentrations of the trace metal cation Zn2+. As the blood-brain barrier limits the distribution of bioavailable Zn2+ or Cu2+ into the brain, we administered micromolar Zn2+ ions intracerebroventricularly in the presence of 1 mM histidine as carrier and compared the effects on behavior and EEG activity in both genotypes. RESULTS: Kainate seizures are more severe in Cav2.3-competent mice than in KO mice and histidine lessens seizure severity in competent but not in Cav2.3-deficient mice. Surprisingly, Zn2+ plus histidine resembles the kainate only control with more seizure severity in Cav2.3-competent than in deficient mice. CONCLUSION: Cav2.3 represents one important Zn2+-sensitive target, which is useful for modulating convulsive seizures.

F0F1 ATP synthase regulates extracellular calcium influx in human neutrophils by interacting with Cav2.3 and modulates neutrophil accumulation in the lipopolysaccharide-challenged lung.

BACKGROUND: Neutrophils form the first line of innate host defense against invading microorganisms. We previously showed that F0F1 ATP synthase (F-ATPase), which is widely known as mitochondrial respiratory chain complex V, is expressed in the plasma membrane of human neutrophils and is involved in regulating cell migration. Whether F-ATPase performs cellular functions through other pathways remains unknown. METHODS: Blue native polyacrylamide gel electrophoresis followed by nano-ESI-LC MS/MS identification and bioinformatic analysis were used to identify protein complexes containing F-ATPase. Then, the identified protein complexes containing F-ATPase were verified by immunoblotting, immunofluorescence colocalization, immunoprecipitation, real-time RT-PCR and agarose gel electrophoresis. Immunoblotting, flow cytometry and a LPS-induced mouse lung injury model were used to assess the effects of the F-ATPase-containing protein complex in vitro and in vivo. RESULTS: We found that the voltage-gated calcium channel (VGCC) α2δ-1 subunit is a binding partner of cell surface F-ATPase in human neutrophils. Further investigation found that the physical connection between the two proteins may exist between the F1 part (α and ß subunits) of F-ATPase and the α2 part of VGCC α2δ-1. Real-time RT-PCR and PCR analyses showed that Cav2.3 (R-type) is the primary type of VGCC expressed in human neutrophils. Research on the F-ATPase/Cav2.3 functional complex indicated that it can regulate extracellular Ca2+ influx, thereby modulating ERK1/2 phosphorylation and reactive oxygen species production, which are typical features of neutrophil activation. In addition, the inhibition of F-ATPase can reduce neutrophil accumulation in the lungs of mice that were intratracheally instilled with lipopolysaccharide, suggesting that the inhibition of F-ATPase may prevent neutrophilic inflammation-induced tissue damage. CONCLUSIONS: In this study, we identified a mechanism by which neutrophil activity is modulated, with simultaneous regulation of neutrophil-mediated pulmonary damage. These results show that surface F-ATPase of neutrophils is a potential innate immune therapeutic target.

Submicromolar copper (II) ions stimulate transretinal signaling in the isolated retina from wild type but not from Cav2.3-deficient mice.

BACKGROUND: So far, only indirect evidence exists for the pharmacoresistant R-type voltage-gated Ca2+ channel (VGCC) to be involved in transretinal signaling by triggering GABA-release onto ON-bipolar neurons. This release of inhibitory neurotransmitters was deduced from the sensitivity of the b-wave to stimulation by Ni2+, Zn2+ and Cu2+. To further confirm the interpretation of these findings, we compared the effects of Cu2+ application and chelation (using kainic acid, KA) on the neural retina from wildtype and Cav2.3-deficient mice. Furthermore, the immediately effect of KA on the ERG b-wave modulation was assessed. METHODS: Transretinal signaling was recorded as an ERG from the superfused murine retina isolated from wildtype and Cav2.3-deficient mice. RESULTS: In mice, the stimulating effect of 100 nM CuCl2 is absent in the retinae from Cav2.3-deficient mice, but prominent in Cav2.3-competent mice. Application of up to 3 mM tricine does not affect the murine b-wave in both genotypes, most likely because of chelating amino acids present in the murine nutrient solution. Application of 27 µM KA significantly increased the b-wave amplitude in wild type and Cav2.3 (-|-) mice. This effect can most likely be explained by the stimulation of endogenous KA-receptors described in horizontal, OFF-bipolar, amacrine or ganglion cells, which could not be fully blocked in the present study. CONCLUSION: Cu2+-dependent modulation of transretinal signaling only occurs in the murine retina from Cav2.3 competent mice, supporting the ideas derived from previous work in the bovine retina that R-type Ca2+ channels are involved in shaping transretinal responses during light perception.

Modulation of Cav2.3 channels by unconjugated bilirubin (UCB) - Candidate mechanism for UCB-induced neuromodulation and neurotoxicity.

Elevated levels of unbound unconjugated bilirubin (UCB) can lead to bilirubin encephalopathy and kernicterus. In spite of a large number of studies demonstrating UCB-induced changes in central neurotransmission, it is still unclear whether these effects involve alterations in the function of specific ion channels. To assess how different UCB concentrations and UCB:albumin (U/A) molar ratios affect neuronal R-type voltage-gated Ca2+ channels, we evaluated their effects on whole-cell currents through recombinant Cav2.3 + ß3 channel complexes and ex-vivo electroretinograms (ERGs) from wildtype and Cav2.3-deficient mice. Our findings show that modestly elevated levels of unbound UCB (U/A = 0.5) produce subtle but significant changes in the voltage-dependence of activation and prepulse inactivation, resulting in a stimulation of currents activated by weak depolarization and inhibition at potentials on the plateau of the activation curve. Saturation of the albumin binding capacity (U/A = 1) produced additional suppression that became significant when albumin was omitted completely and might involve a complete loss of channel function. Acutely administered UCB (U/A = 0.5) has recently been shown to affect transsynaptic signaling in the isolated vertebrate retina. The present report reveals that sustained exposure of the murine retina to UCB significantly suppresses also late responses of the inner retina (b-wave) from wildtype compared to Cav2.3-deficient mice. In addition, recovery during washout was significantly more complete and faster in retinae lacking Cav2.3 channels. Together, these findings show that UCB affects cloned and native Cav2.3 channels at clinically relevant U/A molar ratios and indicate that supersaturation of albumin is not required for modulation but associated with a loss of channel functional that could contribute to chronic neuronal dysfunction.

Reciprocal modulation of Cav 2.3 voltage-gated calcium channels by copper(II) ions and kainic acid.

Kainic acid (KA) is a potent agonist at non-N-methyl-D-aspartate (non-NMDA) ionotropic glutamate receptors and commonly used to induce seizures and excitotoxicity in animal models of human temporal lobe epilepsy. Among other factors, Cav 2.3 voltage-gated calcium channels have been implicated in the pathogenesis of KA-induced seizures. At physiologically relevant concentrations, endogenous trace metal ions (Cu2+ , Zn2+ ) occupy an allosteric binding site on the domain I gating module of these channels and interfere with voltage-dependent gating. Using whole-cell patch-clamp recordings in human embryonic kidney (HEK-293) cells stably transfected with human Cav 2.3d and ß3 -subunits, we identified a novel, glutamate receptor-independent mechanism by which KA can potently sensitize these channels. Our findings demonstrate that KA releases these channels from the tonic inhibition exerted by low nanomolar concentrations of Cu2+ and produces a hyperpolarizing shift in channel voltage-dependence by about 10 mV, thereby reconciling the effects of Cu2+ chelation with tricine. When tricine was used as a surrogate to study the receptor-independent action of KA in electroretinographic recordings from the isolated bovine retina, it selectively suppressed a late b-wave component, which we have previously shown to be enhanced by genetic or pharmacological ablation of Cav 2.3 channels. Although the pathophysiological relevance remains to be firmly established, we speculate that reversal of Cu2+ -induced allosteric suppression, presumably via formation of stable kainate-Cu2+ complexes, could contribute to the receptor-mediated excitatory effects of KA. In addition, we discuss experimental implications for the use of KA in vitro, with particular emphasis on the seemingly high incidence of trace metal contamination in common physiological solutions.

α-Conotoxin Vc1.1 inhibits human dorsal root ganglion neuroexcitability and mouse colonic nociception via GABAB receptors.

OBJECTIVE: α-Conotoxin Vc1.1 is a small disulfide-bonded peptide from the venom of the marine cone snail Conus victoriae. Vc1.1 has antinociceptive actions in animal models of neuropathic pain, but its applicability to inhibiting human dorsal root ganglion (DRG) neuroexcitability and reducing chronic visceral pain (CVP) is unknown. DESIGN: We determined the inhibitory actions of Vc1.1 on human DRG neurons and on mouse colonic sensory afferents in healthy and chronic visceral hypersensitivity (CVH) states. In mice, visceral nociception was assessed by neuronal activation within the spinal cord in response to noxious colorectal distension (CRD). Quantitative-reverse-transcription-PCR, single-cell-reverse-transcription-PCR and immunohistochemistry determined γ-aminobutyric acid receptor B (GABABR) and voltage-gated calcium channel (CaV2.2, CaV2.3) expression in human and mouse DRG neurons. RESULTS: Vc1.1 reduced the excitability of human DRG neurons, whereas a synthetic Vc1.1 analogue that is inactive at GABABR did not. Human DRG neurons expressed GABABR and its downstream effector channels CaV2.2 and CaV2.3. Mouse colonic DRG neurons exhibited high GABABR, CaV2.2 and CaV2.3 expression, with upregulation of the CaV2.2 exon-37a variant during CVH. Vc1.1 inhibited mouse colonic afferents ex vivo and nociceptive signalling of noxious CRD into the spinal cord in vivo, with greatest efficacy observed during CVH. A selective GABABR antagonist prevented Vc1.1-induced inhibition, whereas blocking both CaV2.2 and CaV2.3 caused inhibition comparable with Vc1.1 alone. CONCLUSIONS: Vc1.1-mediated activation of GABABR is a novel mechanism for reducing the excitability of human DRG neurons. Vc1.1-induced activation of GABABR on the peripheral endings of colonic afferents reduces nociceptive signalling. The enhanced antinociceptive actions of Vc1.1 during CVH suggest it is a novel candidate for the treatment for CVP.

Cav2.3 (R-Type) Calcium Channels are Critical for Mediating Anticonvulsive and Neuroprotective Properties of Lamotrigine In Vivo.

BACKGROUND/AIMS: Lamotrigine (LTG) is a popular modern antiepileptic drug (AED), however, its mechanism of action has yet to be fully understood, as it is known to modulate many members of several ion channel families. In heterologous systems, LTG inhibits Cav2.3 (R-type) calcium currents, which contribute to kainic-acid- (KA) induced epilepsy in vivo. To gain insight into the role of R-type currents in LTG drug action in vivo, we compared the effects of LTG to topiramate and lacosamide in Cav2.3-deficient mice and controls on KA-induced seizures. METHODS: Behavioral seizure rating and quantitative electrocorticography were performed after injection of 20 mg/kg [and 30 mg/kg] KA. One hour before KA injection, mice were pretreated with either 30 mg/kg LTG, 50 mg/kg topiramate (TPM) or 30 mg/kg lacosamide (LSM). RESULTS: Ablation of Cav2.3 reduced total seizure scores by 28.6% (p=0.0012) and pretreatment with LTG reduced seizure activity of control mice by 23.2% (p=0.02). In Cav2.3-deficient mice LTG pretreatment increased seizure activity by 22.1% (p=0.018) and increased the percentage of degenerated CA1 pyramidal neurons (p=0.02). All three tested AEDs reduced seizure activity in control mice, however only the non-calcium channel modulating AED, LSM had an anticonvulsive effect in Cav2.3-deficient mice. Furthermore LTG altered electrocorticographic parameters differently in the two genotypes, decreasing relative power of ictal spikes in control mice compared to Cav2.3-defcient mice. CONCLUSION: These findings give first in vivo evidence for an essential role for Cav2.3 in LTG pharmacology and shed light on a paradoxical effect of LTG in their absence. Furthermore, LTG appears to promote ictal activity in Cav2.3-deficient mice resulting in increased neurotoxicity in the CA1 region. This paradoxical mechanism, possibly reflecting rebound hyperexcitation of pyramidal CA1 neurons after increased inhibition, may be key in understanding LTG-induced seizure aggravation, observed in clinical practice.

Sexual divergence in microtubule function: the novel intranasal microtubule targeting SKIP normalizes axonal transport and enhances memory.

Activity-dependent neuroprotective protein (ADNP), essential for brain formation, is a frequent autism spectrum disorder (ASD)-mutated gene. ADNP associates with microtubule end-binding proteins (EBs) through its SxIP motif, to regulate dendritic spine formation and brain plasticity. Here, we reveal SKIP, a novel four-amino-acid peptide representing an EB-binding site, as a replacement therapy in an outbred Adnp-deficient mouse model. We discovered, for the first time, axonal transport deficits in Adnp(+/-) mice (measured by manganese-enhanced magnetic resonance imaging), with significant male-female differences. RNA sequencing evaluations showed major age, sex and genotype differences. Function enrichment and focus on major gene expression changes further implicated channel/transporter function and the cytoskeleton. In particular, a significant maturation change (1 month-five months) was observed in beta1 tubulin (Tubb1) mRNA, only in Adnp(+/+) males, and sex-dependent increase in calcium channel mRNA (Cacna1e) in Adnp(+/+) males compared with females. At the protein level, the Adnp(+/-) mice exhibited impaired hippocampal expression of the calcium channel (voltage-dependent calcium channel, Cacnb1) as well as other key ASD-linked genes including the serotonin transporter (Slc6a4), and the autophagy regulator, BECN1 (Beclin1), in a sex-dependent manner. Intranasal SKIP treatment normalized social memory in 8- to 9-month-old Adnp(+/-)-treated mice to placebo-control levels, while protecting axonal transport and ameliorating changes in ASD-like gene expression. The control, all d-amino analog D-SKIP, did not mimic SKIP activity. SKIP presents a novel prototype for potential ASD drug development, a prevalent unmet medical need.

R-Type Ca2+ channels couple to inhibitory neurotransmission to the longitudinal muscle in the guinea-pig ileum.

NEW FINDINGS: What is the central question of this study? Subtypes of enteric neurons are coded by the neurotransmitters they synthesize, but it is not known whether enteric neuron subtypes might also be coded by other proteins, including calcium channel subtypes controlling neurotransmitter release. What is the main finding and its importance? Our data indicate that guinea-pig ileum myenteric neuron subtypes may be coded by calcium channel subtypes. We found that R-type calcium channels are expressed by inhibitory but not excitatory longitudinal muscle motoneurons. R-Type calcium channels are also not expressed by circular muscle inhibitory motoneurons. Calcium channel subtype-selective antagonists could be used to target subtypes of neurons to treat gastrointestinal motility disorders. There is evidence that R-type Ca2+ channels contribute to synaptic transmission in the myenteric plexus. It is unknown whether R-type Ca2+ channels contribute to neuromuscular transmission. We measured the effects of the nitric oxide synthase inhibitor nitro-l-arginine (NLA), Ca2+ channel blockers and apamin (SK channel blocker) on neurogenic relaxations and contractions of the guinea-pig ileum longitudinal muscle-myenteric plexus (LMMP) in vitro. We used intracellular recordings to measure inhibitory junction potentials. Immunohistochemical techniques localized R-type Ca2+ channel protein in the LMMP and circular muscle. Cadmium chloride (pan-Ca2+ channel blocker) blocked and NLA and NiCl2 (R-type Ca2+ channel blocker) reduced neurogenic relaxations in a non-additive manner. Nickel chloride did not alter neurogenic cholinergic contractions, but it potentiated neurogenic non-cholinergic contractions. Relaxations were inhibited by apamin, NiCl2 and NLA and were blocked by combined application of these drugs. Relaxations were reduced by NiCl2 or ω-conotoxin (N-type Ca2+ channel blocker) and were blocked by combined application of these drugs. Longitudinal muscle inhibitory junction potentials were inhibited by NiCl2 but not MRS 2179 (P2Y1 receptor antagonist). Circular muscle inhibitory junction potentials were blocked by apamin, MRS 2179, ω-conotoxin and CdCl2 but not NiCl2 . We conclude that neuronal R-type Ca2+ channels contribute to inhibitory neurotransmission to longitudinal muscle but less so or not all in the circular muscle of the guinea-pig ileum.

Possible Involvement of the CACNA1E Gene in Migraine: A Search for Single Nucleotide Polymorphism in Different Clinical Phenotypes.

OBJECTIVE: To search for differences in prevalence of a CACNA1E variant between migraine without aura, various phenotypes of migraine with aura, and healthy controls. BACKGROUND: Familial hemiplegic migraine type 1 (FHM1) is associated with mutations in the CACNA1A gene coding for the alpha 1A (Cav 2.1) pore-forming subunit of P/Q voltage-dependent Ca2+ channels. These mutations are not found in the common forms of migraine with or without aura. The alpha 1E subunit (Cav 2.3) is the counterpart of Cav 2.1 in R-type Ca2+ channels, has different functional properties, and is encoded by the CACNA1E gene. METHODS: First, we performed a total exon sequencing of the CACNA1E gene in three probands selected because they had no abnormalities in the three FHM genes. In a patient suffering from basilar-type migraine, we identified a single nucleotide polymorphism (SNP) in exon 20 of the CACNA1E gene (Asp859Glu - rs35737760; Minor Allele Frequency 0.2241) hitherto not studied in migraine. In a second step, we determined its occurrence in four groups by direct sequencing on blood genomic DNA: migraine patients without aura (N = 24), with typical aura (N = 55), complex neurological auras (N = 19; hemiplegic aura: N = 15; brain stem aura: N = 4), and healthy controls (N = 102). RESULTS: The Asp859Glu - rs35737760 SNP of the CACNA1E gene was present in 12.7% of control subjects and in 20.4% of the total migraine group. In the migraine group it was significantly over-represented in patients with complex neurological auras (42.1%), OR 4.98 (95% CI: 1.69-14.67, uncorrected P = .005, Bonferroni P = .030, 2-tailed Fisher's exact test). There was no significant difference between migraine with typical aura (10.9%) and controls. CONCLUSIONS: We identified a polymorphism in exon 20 of the CACNA1E gene (Asp859Glu - rs35737760) that is more prevalent in hemiplegic and brain stem aura migraine. This missense variant causes a change from aspartate to glutamate at position 859 of the Cav 2.3 protein and might modulate the function of R-type Ca2+ channels. It could thus be relevant for migraine with complex neurological aura, although this remains to be proven.

Effect of electroacupuncture on gene expression in calcium signaling pathway in hippocampal cells in mice with cerebral ischemia reperfusion.

OBJECTIVE: To observe the regulation of electroacupuncture on gene expression at calcium signaling pathways in mice with cerebral ischemia reperfusion. METHODS: Sixty male, inbred Kunming mice were randomly assigned to three groups: repeated cerebral ischemia reperfusion group (RG, n = 24), sham-operated group (SG, n = 12), and electroacupuncture group (EG, n = 24). Mice in RG and EG groups were modeled by repeated cerebral ischemia reperfusion surgery, and EG mice were treated with electroacupuncture for 30 min after recovery from anesthesia. Changes in gene expression profile of mice hippocampi were analyzed by global expression profile microarray. Genes that were up-regulated or down-regulated greater than 1.5 folds were considered to be biologically meaningful. Real-time quantitative polymerase chain reaction (q-PCR) method was used to verify the expression of selected genes based on the algorithm [2^ (ΔΔCt)]. RESULTS: Compared with SG mice, 242 genes showed different in expressions in RG mice: 107 down-regulated and 135 up-regulated. Compared with RG mice, 609 genes showed a difference of expression in EG mice: 315 down-regulated and 375 up-regulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated two pathways: calcium signaling and long-term potentiation in which 11 differentially expressed genes selected. Six of the 11 genes in the calcium signaling pathway were verified after real-time q-PCR testing. CONCLUSION: Electroacupuncture treatment of cerebral ischemia reperfusion appears to regulate Atp2a2, Cacna1e, Camk2a, Gnas, Grm1, Rapgef3 genes in the calcium signaling pathway.