Conotoxin contulakin-G engages a neurotensin receptor 2/R-type calcium channel (Cav2.3) pathway to mediate spinal antinociception.

ABSTRACT: Intrathecal application of contulakin-G (CGX), a conotoxin peptide and a neurotensin analogue, has been demonstrated to be safe and potentially analgesic in humans. However, the mechanism of action for CGX analgesia is unknown. We hypothesized that spinal application of CGX produces antinociception through activation of the presynaptic neurotensin receptor (NTSR)2. In this study, we assessed the mechanisms of CGX antinociception in rodent models of inflammatory and neuropathic pain. Intrathecal administration of CGX, dose dependently, inhibited thermal and mechanical hypersensitivities in rodents of both sexes. Pharmacological and clustered regularly interspaced short palindromic repeats/Cas9 editing of NTSR2 reversed CGX-induced antinociception without affecting morphine analgesia. Electrophysiological and gene editing approaches demonstrated that CGX inhibition was dependent on the R-type voltage-gated calcium channel (Cav2.3) in sensory neurons. Anatomical studies demonstrated coexpression of NTSR2 and Cav2.3 in dorsal root ganglion neurons. Finally, synaptic fractionation and slice electrophysiology recordings confirmed a predominantly presynaptic effect. Together, these data reveal a nonopioid pathway engaged by a human-tested drug to produce antinociception.

R-type voltage-gated Ca2+ channels mediate A-type K+ current regulation of synaptic input in hippocampal dendrites.

The subthreshold voltage-gated transient K+ current (IA) carried by pore-forming Kv4.2 subunits regulates the propagation of synaptic input, dendritic excitability, and synaptic plasticity in CA1 pyramidal neuron dendrites of the hippocampus. We report that the Ca2+ channel subunit Cav2.3 regulates IA in this cell type. We initially identified Cav2.3 as a Kv4.2-interacting protein in a proteomic screen and we confirmed Cav2.3-Kv4.2 complex association using multiple techniques. Functionally, Cav2.3 Ca2+-entry increases Kv4.2-mediated whole-cell current due to an increase in Kv4.2 surface expression. Using pharmacology and Cav2.3 knockout mice, we show that Cav2.3 regulates the dendritic gradient of IA. Furthermore, the loss of Cav2.3 function leads to the enhancement of AMPA receptor-mediated synaptic currents and NMDA receptor-mediated spine Ca2+ influx. These results propose that Cav2.3 and Kv4.2 are integral constituents of an ion channel complex that affects synaptic function in the hippocampus.

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.

GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals.

The synaptic connection from medial habenula (MHb) to interpeduncular nucleus (IPN) is critical for emotion-related behaviors and uniquely expresses R-type Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates or inhibits transmitter release from MHb terminals depending on the IPN subnucleus, but the role of KCTDs is unknown. We therefore examined the localization and function of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3 currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b, and Cav2.3 co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3 with KCTDs therefore scales synaptic strength independent of GBR activation.

Cav2.3 channel function and Zn2+-induced modulation: potential mechanisms and (patho)physiological relevance.

Voltage-gated calcium channels (VGCCs) are critical for Ca2+ influx into all types of excitable cells, but their exact function is still poorly understood. Recent reconstruction of homology models for all human VGCCs at atomic resolution provides the opportunity for a structure-based discussion of VGCC function and novel insights into the mechanisms underlying Ca2+ selective flux through these channels. In the present review, we use these data as a basis to examine the structure, function, and Zn2+-induced modulation of Cav2.3 VGCCs, which mediate native R-type currents and belong to the most enigmatic members of the family. Their unique sensitivity to Zn2+ and the existence of multiple mechanisms of Zn2+ action strongly argue for a role of these channels in the modulatory action of endogenous loosely bound Zn2+, pools of which have been detected in a number of neuronal, endocrine, and reproductive tissues. Following a description of the different mechanisms by which Zn2+ has been shown or is thought to alter the function of these channels, we discuss their potential (patho)physiological relevance, taking into account what is known about the magnitude and function of extracellular Zn2+ signals in different tissues. While still far from complete, the picture that emerges is one where Cav2.3 channel expression parallels the occurrence of loosely bound Zn2+ pools in different tissues and where these channels may serve to translate physiological Zn2+ signals into changes of electrical activity and/or intracellular Ca2+ levels.

An ancestral MAGUK protein supports the modulation of mammalian voltage-gated Ca2+ channels through a conserved CaVß-like interface.

Eukaryote voltage-gated Ca2+ channels of the CaV2 channel family are hetero-oligomers formed by the pore-forming CaVα1 protein assembled with auxiliary CaVα2δ and CaVß subunits. CaVß subunits are formed by a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain connected through a HOOK domain. The GK domain binds a conserved cytoplasmic region of the pore-forming CaVα1 subunit referred as the "AID". Herein we explored the phylogenetic and functional relationship between CaV channel subunits in distant eukaryotic organisms by investigating the function of a MAGUK protein (XM_004990081) cloned from the choanoflagellate Salpingoeca rosetta (Sro). This MAGUK protein (Sroß) features SH3 and GK structural domains with a 25% primary sequence identity to mammalian CaVß. Recombinant expression of its cDNA with mammalian high-voltage activated Ca2+ channel CaV2.3 in mammalian HEK cells produced robust voltage-gated inward Ca2+ currents with typical activation and inactivation properties. Like CaVß, Sroß prevents fast degradation of total CaV2.3 proteins in cycloheximide assays. The three-dimensional homology model predicts an interaction between the GK domain of Sroß and the AID motif of the pore-forming CaVα1 protein. Substitution of AID residues Trp (W386A) and Tyr (Y383A) significantly impaired co-immunoprecipitation of CaV2.3 with Sroß and functional upregulation of CaV2.3 currents. Likewise, a 6-residue deletion within the GK domain of Sroß, similar to the locus found in mammalian CaVß, significantly reduced peak current density. Altogether our data demonstrate that an ancestor MAGUK protein reconstitutes the biophysical and molecular features responsible for channel upregulation by mammalian CaVß through a minimally conserved molecular interface.

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.

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.

Cav2.3 channels contribute to dopaminergic neuron loss in a model of Parkinson's disease.

Degeneration of dopaminergic neurons in the substantia nigra causes the motor symptoms of Parkinson's disease. The mechanisms underlying this age-dependent and region-selective neurodegeneration remain unclear. Here we identify Cav2.3 channels as regulators of nigral neuronal viability. Cav2.3 transcripts were more abundant than other voltage-gated Ca2+ channels in mouse nigral neurons and upregulated during aging. Plasmalemmal Cav2.3 protein was higher than in dopaminergic neurons of the ventral tegmental area, which do not degenerate in Parkinson's disease. Cav2.3 knockout reduced activity-associated nigral somatic Ca2+ signals and Ca2+-dependent after-hyperpolarizations, and afforded full protection from degeneration in vivo in a neurotoxin Parkinson's mouse model. Cav2.3 deficiency upregulated transcripts for NCS-1, a Ca2+-binding protein implicated in neuroprotection. Conversely, NCS-1 knockout exacerbated nigral neurodegeneration and downregulated Cav2.3. Moreover, NCS-1 levels were reduced in a human iPSC-model of familial Parkinson's. Thus, Cav2.3 and NCS-1 may constitute potential therapeutic targets for combatting Ca2+-dependent neurodegeneration in Parkinson's disease.

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.

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.

1,25-Dihydroxyvitamin D3 enhances glucose-stimulated insulin secretion in mouse and human islets: a role for transcriptional regulation of voltage-gated calcium channels by the vitamin D receptor.

AIM: Vitamin D deficiency in rodents negatively affects glucose-stimulated insulin secretion (GSIS) and human epidemiological studies connect poor vitamin D status with type 2 diabetes. Previous studies performed primarily in rat islets have shown that vitamin D can enhance GSIS. However the molecular pathways linking vitamin D and insulin secretion are currently unknown. Therefore, experiments were undertaken to elucidate the transcriptional role(s) of the vitamin D receptor (VDR) in islet function. METHODS: Human and mouse islets were cultured with vehicle or 1,25-dihydroxyvitamin-D3 (1,25D3) and then subjected to GSIS assays. Insulin expression, insulin content, glucose uptake and glucose-stimulated calcium influx were tested. Microarray analysis was performed. In silico analysis was used to identify VDR response elements (VDRE) within target genes and their activity was tested using reporter assays. RESULTS: Vdr mRNA is abundant in islets and Vdr expression is glucose-responsive. Preincubation of mouse and human islets with 1,25D3 enhances GSIS and increases glucose-stimulated calcium influx. Microarray analysis identified the R-type voltage-gated calcium channel (VGCC) gene, Cacna1e, which is highly upregulated by 1,25D3 in human and mouse islets and contains a conserved VDRE in intron 7. Results from GSIS assays suggest that 1,25D3 might upregulate a variant of R-type VGCC that is resistant to chemical inhibition. CONCLUSION: These results suggest that the role of 1,25D3 in regulating calcium influx acts through the R-Type VGCC during GSIS, thereby modulating the capacity of beta cells to secrete insulin.

Proteomic analysis of olfactory bulb suggests CACNA1E as a promoter of CREB signaling in microbiota-induced depression.

Major depressive disorders impact approximately 17% of the population worldwide, whose high morbidity and considerable adversity have resulted in enormous social and economic burden. In addition, clinically depressed patients often show reduced volume of olfactory bulb (OB) and decreased olfactory sensitivity. Although mounting evidence conveyed that the gut microbiota may implicate the pathophysiology of major depressive disorder (MDD) via the microbe-gut-brain axis, knowledge about its distinctive molecular mechanism is rudimentary. Herein, iTRAQ coupled with LC-MS/MS was applied to compare the OB proteome between "pathological microbiota" and "healthy microbiota" germ-free mice. A set of 367 proteins were differentially identified in the OB, including 119 up-regulated and 248 down-regulated proteins compared with the levels in controls. A combined analysis with significantly changed OB proteins from CUMS depression model supported the role of CREB signaling, whose dysregulation is likely to disrupt the axonogenesis of OB under microbiota condition. With that, the down-regulated CACNA1E and its downstream proteins (CALM/ CaMKII/ CREB/ BDNF) in CREB pathway were validated by Western blot. Meanwhile, the canonical pathways involved Nuclear Receptor Signaling highlighted the fecal microbiota transplantation (FMT) model, which would be a new breakthrough for depressive research. These findings enrich the previous research achievements about the gut microbiota in psychiatric disorders, providing a creative insight into the intricate mechanisms of OB dysfunction in depression. SIGNIFICANCE: Emerging evidence has shown that gut microbiota can greatly influence brain functions and even behaviors. As one of the post-developmental neurogenesis areas for the adult brain, the OB is becoming increasingly important in the study of the pathogenesis of depression. Using an iTRAQ-based proteomics, we identified 367 altered proteins in the OB of fecal microbiota transplanted mouse, which provide a novel insight for further research of the "microbiota-gut-brain axis". In addition, combined analyses with the CUMS depression model and the validation of key proteins by Western blot may assist in the investigation of OB dysfunction in mental sickness.

A Dendritic Substrate for the Cholinergic Control of Neocortical Output Neurons.

The ascending cholinergic system dynamically regulates sensory perception and cognitive function, but it remains unclear how this modulation is executed in neocortical circuits. Here, we demonstrate that the cholinergic system controls the integrative operations of neocortical principal neurons by modulating dendritic excitability. Direct dendritic recordings revealed that the optogenetic-evoked release of acetylcholine (ACh) transformed the pattern of dendritic integration in layer 5B pyramidal neurons, leading to the generation of dendritic plateau potentials which powerfully drove repetitive action potential output. In contrast, the synaptic release of ACh did not positively modulate axo-somatic excitability. Mechanistically, the transformation of dendritic integration was mediated by the muscarinic ACh receptor-dependent enhancement of dendritic R-type calcium channel activity, a compartment-dependent modulation which decisively controlled the associative computations executed by layer 5B pyramidal neurons. Our findings therefore reveal a biophysical mechanism by which the cholinergic system controls dendritic computations causally linked to perceptual detection.

Calcium Channel CaV2.3 Subunits Regulate Hepatic Glucose Production by Modulating Leptin-Induced Excitation of Arcuate Pro-opiomelanocortin Neurons.

Leptin acts on hypothalamic pro-opiomelanocortin (POMC) neurons to regulate glucose homeostasis, but the precise mechanisms remain unclear. Here, we demonstrate that leptin-induced depolarization of POMC neurons is associated with the augmentation of a voltage-gated calcium (CaV) conductance with the properties of the "R-type" channel. Knockdown of the pore-forming subunit of the R-type (CaV2.3 or Cacna1e) conductance in hypothalamic POMC neurons prevented sustained leptin-induced depolarization. In vivo POMC-specific Cacna1e knockdown increased hepatic glucose production and insulin resistance, while body weight, feeding, or leptin-induced suppression of food intake were not changed. These findings link Cacna1e function to leptin-mediated POMC neuron excitability and glucose homeostasis and may provide a target for the treatment of diabetes.

In vitro and in vivo phosphorylation of the Cav2.3 voltage-gated R-type calcium channel.

During the recording of whole cell currents from stably transfected HEK-293 cells, the decline of currents carried by the recombinant human Cav2.3+ß3 channel subunits is related to adenosine triphosphate (ATP) depletion after rupture of the cells. It reduces the number of functional channels and leads to a progressive shift of voltage-dependent gating to more negative potentials (Neumaier F., et al., 2018). Both effects can be counteracted by hydrolysable ATP, whose protective action is almost completely prevented by inhibition of serine/threonine but not tyrosine or lipid kinases. These findings indicate that ATP promotes phosphorylation of either the channel or an associated protein, whereas dephosphorylation during cell dialysis results in run-down. Protein phosphorylation is required for Cav2.3 channel function and could directly influence the normal features of current carried by these channels. Therefore, results from in vitro and in vivo phosphorylation of Cav2.3 are summarized to come closer to a functional analysis of structural variations in Cav2.3 splice variants.

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.

Protein phosphorylation maintains the normal function of cloned human Cav2.3 channels.

R-type currents mediated by native and recombinant Cav2.3 voltage-gated Ca2+ channels (VGCCs) exhibit facilitation (run-up) and subsequent decline (run-down) in whole-cell patch-clamp recordings. A better understanding of the two processes could provide insight into constitutive modulation of the channels in intact cells, but low expression levels and the need for pharmacological isolation have prevented investigations in native systems. Here, to circumvent these limitations, we use conventional and perforated-patch-clamp recordings in a recombinant expression system, which allows us to study the effects of cell dialysis in a reproducible manner. We show that the decline of currents carried by human Cav2.3+ß3 channel subunits during run-down is related to adenosine triphosphate (ATP) depletion, which reduces the number of functional channels and leads to a progressive shift of voltage-dependent gating to more negative potentials. Both effects can be counteracted by hydrolysable ATP, whose protective action is almost completely prevented by inhibition of serine/threonine but not tyrosine or lipid kinases. Protein kinase inhibition also mimics the effects of run-down in intact cells, reduces the peak current density, and hyperpolarizes the voltage dependence of gating. Together, our findings indicate that ATP promotes phosphorylation of either the channel or an associated protein, whereas dephosphorylation during cell dialysis results in run-down. These data also distinguish the effects of ATP on Cav2.3 channels from those on other VGCCs because neither direct nucleotide binding nor PIP2 synthesis is required for protection from run-down. We conclude that protein phosphorylation is required for Cav2.3 channel function and could directly influence the normal features of current carried by these channels. Curiously, some of our findings also point to a role for leupeptin-sensitive proteases in run-up and possibly ATP protection from run-down. As such, the present study provides a reliable baseline for further studies on Cav2.3 channel regulation by protein kinases, phosphatases, and possibly proteases.

Unconjugated bilirubin modulates neuronal signaling only in wild-type mice, but not after ablation of the R-type/Cav 2.3 voltage-gated calcium channel.

INTRODUCTION: The relationship between blood metabolites and hemoglobin degradation products (BMHDPs) formed in the cerebrospinal fluid and the development of vasospasm and delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) has been the focus of several previous studies, but their molecular and cellular targets remain to be elucidated. METHODS: Because BMHDP-induced changes in Cav 2.3 channel function are thought to contribute to DCI after aSAH, we studied their modulation by unconjugated bilirubin (UCB) in an organotypical neuronal network from wild-type (WT) and Cav 2.3-deficient animals (KO). Murine retinae were isolated from WT and KO and superfused with nutrient solution. Electroretinograms were recorded before, during, and after superfusion with UCB. Transretinal signaling was analyzed as b-wave, implicit time, and area under the curve (AUC). RESULTS: Superfusion of UCB significantly attenuated the b-wave amplitude in the isolated retina from wild-type mice by 14.9% (P < 0.05), followed by gradual partial recovery (P = 0.09). Correspondingly, AUC decreased significantly with superfusion of UCB (P < 0.05). During washout, the b-wave amplitude returned to baseline (P = 0.2839). The effects of UCB were absent in Cav 2.3-deficient mice, lacking the expression of Cav 2.3 as proofed on the biochemical level. CONCLUSIONS: Ex vivo neuronal recording in the murine retina is able to detect transient impairment of transretinal signaling by UCB in WT, but not in KO. This new model may be useful to further clarify the role of calcium channels in neuronal signal alteration in the presence of BHMDPs.