Regulation Of Voltage-gated Calcium Channels Research Articles

Disrupted regulation of voltage-gated calcium channels by pathogenic calmodulin variants

Calmodulin (CaM) is an essential sensor of intracellular calcium in multiple cell types, including cardiac myocytes and neurons. In these cells, calcium ions enter the cell through voltage-gated calcium channels (VGCCs) and bind to CaM in one of its four EF-hand domains. These binding events trigger an array of important downstream cellular processes, including calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF) of multiple VGCC subtypes. In this way, CaM tunes VGCC activity and facilitates calcium signaling cascades.

AMPK mediates regulation of voltage-gated calcium channels by leptin in isolated neurons from arcuate nucleus.

Neuronal control of the energy homeostasis requires the arcuate nucleus of the hypothalamus. This structure integrates peripheral and central signals concerning the energy state of the body. It comprises two populations of neurons releasing anorexigenic and orexigenic peptides, among others. Both populations are regulated by leptin, an anorexigenic hormone, released by white adipose tissue. Voltage-gated calcium entry is critical to promote neurotransmitter and hormone release. It is already known that calcium channel current is inhibited by leptin in orexigenic neurons. However, fine-tuning details of calcium channel regulation in arcuate nucleus by leptin remain to be elucidated. This work aimed to investigate whether 5' adenosine monophosphate-activated protein kinase (AMPK) underlies the leptin-induced inhibition of calcium channels. By using patch-clamping methods, immunocytochemical, and biochemical reagents, we recorded calcium channel currents in orexigenic neuropeptide Y neurons of the arcuate nucleus of rats. Consistently, leptin inhibition of the calcium channel current was not only prevented by AMPK inhibition with Compound C but also hampered with 5-aminoimidazole-4-carboxamide ribonucleoside. Furthermore, leptin selectively inhibited L-type calcium channel current amplitude without major changes in voltage dependence or current kinetics. These results support for the first time the key role of AMPK in the maintenance and regulation of voltage-gated calcium channels. Together, they advance our understanding of the regulation of calcium channels in the central nervous system and emerging questions concerning food intake and energy balance.NEW & NOTEWORTHY Our results readily support the hypothesis that AMPK is responsible for the maintenance of the calcium current and mediates the fine-tuning modulation of the leptin response. The novelty of these results strengthens the critical role of AMPK in the general energy balance and homeostasis.

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.

Maintenance of CaV2.2 channel-current by PIP2 unveiled by neomycin in sympathetic neurons of the rat

Membrane lipids are key determinants in the regulation of voltage-gated ion channels. Phosphatidylinositol 4,5-bisphosphate (PIP2), a native membrane phospholipid, has been involved in the maintenance of the current amplitude and in the voltage-independent regulation of voltage-gated calcium channels (VGCC). However, the nature of the PIP2 regulation on VGCC has not been fully elucidated. This work aimed to investigate whether the interacting PIP2 electrostatic charges may account for maintaining the current amplitude of CaV2.2 channels. Furthermore, we tested whether charge shielding of PIP2 mimics the voltage-independent inhibition induced by M1 muscarinic acetylcholine receptor (M1R) activation. Therefore, neomycin, a polycation that has been shown to block electrostatic interactions of PIP2, was intracellularly dialyzed in superior cervical ganglion (SCG) neurons of the rat. Consistently, neomycin time-dependently diminished the calcium current amplitude letting the channel exhibit the hallmarks of the voltage-independent regulation. These results support that interacting PIP2 charges not only underly the maintenance of the channel-current but also that charge screening of PIP2 by itself unveils the voltage-independent features of CaV2.2 channels in SCG neurons.

Regulation of Dietary Amino Acids and Voltage-Gated Calcium Channels in Autism Spectrum Disorder.

Autism, or autism spectrum disorders (ASD), is one of the complex genetic diseases and its etiology is unknown for majority of the patients. It is characterized by deterioration in social interaction, communication, interests, imagination, and activities. As autism is a highly heterogeneous disorder, the symptoms can vary greatly in each affected individual. Oxidative stress implicates major pathogenesis of neurological disorders like ASD. Nutrients and dietary supplements play an important role in the health of an individual and there are several lines of evidence suggesting the role of dietary factors in the development or pathogenesis of ASD. The amino acids supplement has been found to reduce symptoms as they act as the precursors of neurotransmitters which in turn may extenuate mental disorders. The biosynthesis of amino acids in the brain is regulated by the concentration of amino acids in plasma. Amino acids are also considerable entities as they themselves, or peptides consisting of them, have profound antioxidant activities. Dietary constituents have an effect on the transport of amino acids across the blood-brain barrier (BBB) thus indirectly modulating the therapeutic value of amino acids. Among the other factors, voltage-gated calcium channels are directly linked to ASD as per results of genetic studies. Malfunctioning of these calcium channels causes ASD. The intricate biochemical and molecular machinery contributing to neurological disorders is still unknown. Here we discuss the preventive role of dietary amino acids against and regulation of voltage-gated calcium channels on ASD.

Roles and Regulation of Voltage-gated Calcium Channels in Arrhythmias.

Calcium flowing through voltage-dependent calcium channels into cardiomyocytes mediates excitation–contraction coupling, controls action-potential duration and automaticity in nodal cells, and regulates gene expression. Proper surface targeting and basal and hormonal regulation of calcium channels are vital for normal cardiac physiology. In this review, we discuss the roles of voltage-gated calcium channels in the heart and the mechanisms by which these channels are regulated by physiological signaling pathways in health and disease.

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

AimVitamin 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. MethodsHuman 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. ResultsVdr 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. ConclusionThese 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.

Novel approaches to examine the regulation of voltage-gated calcium channels in the heart.

The cardiac L-type Ca(2+) channel plays a key role in cardiac excitation-contraction coupling, action potential duration, and gene expression. Abnormalities in CaV1.2 function, including increased long-opening-mode gating and blunted adrenergic responsiveness, are associated with heart failure and hypertrophy. The increased activation of CaV1.2, in turn, triggers Ca(2+) -responsive signaling pathways, which contribute to the pathogenesis of heart failure and hypertrophy. CaV1.2 in heart is associated with large supramolecular complexes that impact on channel trafficking, localization, turnover, and function. Much of the prevailing dogma relating to mechanisms underlying CaV1.2 trafficking and modulation is derived from studies using recombinant channels reconstituted in heterologous expression systems. However, recent results using knock-in mice indicate that several long-standing "facts" about CaV1.2 regulation derived from heterologous expression studies are not replicated in native heart, emphasizing the critical need for mechanistic studies in the context of actual cardiomyocytes. In this review, we discuss the use of the use of knockin and knockout mice as well as new tools, including doxycycline-induced expression of informative α1C mutants within cardiomyocytes to probe adrenergic regulation of CaV1.2.

Mechanisms of Auxiliary α2δ-1 Subunit Regulation of Voltage-Gated Calcium Channels

The voltage-gated calcium (CaV) channel α2δ-1 subunit is implicated in a variety of cardiovascular and neurological diseases and is the molecular target of gabapentin, a drug used for the treatment of epilepsy and neuropathic pain. The precise mechanisms by which α2δ-1 dysfunction causes disease and how its targeting by gabapentin is therapeutic are unclear, in part due to incomplete understanding of how α2δ-1 binds to and regulates CaV channels. Here, we investigated mechanisms underlying α2δ-1 regulation of neuronal CaV2.2 (α1B+β2a) channels reconstituted in HEK293 cells. Using whole-cell electrophysiology, α2δ-1 caused a dramatic four to five-fold increase in whole-cell current amplitude compared to channels reconstituted with α1B+β2a alone. The increase in current was wholly accounted for by an increase in channel surface density as reported by a quantum dot labeling technique. An optical pulse-chase assay showed that the increased surface density was achieved solely through reducing the rate of CaV2.2 endocytosis rather than by enhancing the rate of forward trafficking. To map the site of interaction between α2δ-1 and α1B, we inserted a small epitope tag into judiciously selected extracellular regions of the α1B subunit. We determined accessibility of the tag to quantum dot and modulation of whole-cell currents in the presence or absence of α2δ-1. From the pattern of this dual readout, we inferred an α2δ-1 binding site in the α1B domain III extracellular S5-S6 loop. Alanine scanning mutagenesis revealed discrete residues in this loop that was necessary for α2δ-1 regulation of α1B. These results provide novel insights into mechanisms underlying α2δ-1 regulation of CaV channels, focus the putative modes of gabapentin action, and offer a new target site for potential development of future therapeutic molecules.

RGK regulation of voltage-gated calcium channels.

Voltage-gated calcium channels (VGCCs) play critical roles in cardiac and skeletal muscle contractions, hormone and neurotransmitter release, as well as slower processes such as cell proliferation, differentiation, migration and death. Mutations in VGCCs lead to numerous cardiac, muscle and neurological disease, and their physiological function is tightly regulated by kinases, phosphatases, G-proteins, calmodulin and many other proteins. Fifteen years ago, RGK proteins were discovered as the most potent endogenous regulators of VGCCs. They are a family of monomeric GTPases (Rad, Rem, Rem2, and Gem/Kir), in the superfamily of Ras GTPases, and they have two known functions: regulation of cytoskeletal dynamics including dendritic arborization and inhibition of VGCCs. Here we review the mechanisms and molecular determinants of RGK-mediated VGCC inhibition, the physiological impact of this inhibition, and recent evidence linking the two known RGK functions.

Redox Regulation of Neuronal Voltage-Gated Calcium Channels

Voltage-gated calcium channels are ubiquitously expressed in neurons and are key regulators of cellular excitability and synaptic transmitter release. There is accumulating evidence that multiple subtypes of voltage-gated calcium channels may be regulated by oxidation and reduction. However, the redox mechanisms involved in the regulation of channel function are not well understood. Several studies have established that both T-type and high-voltage-activated subtypes of voltage-gated calcium channel can be redox-regulated. This article reviews different mechanisms that can be involved in redox regulation of calcium channel function and their implication in neuronal function, particularly in pain pathways and thalamic oscillation. A current critical issue in the field is to decipher precise mechanisms of calcium channel modulation via redox reactions. In this review we discuss covalent post-translational modification via oxidation of cysteine molecules and chelation of trace metals, and reactions involving nitric oxide-related molecules and free radicals. Improved understanding of the roles of redox-based reactions in regulation of voltage-gated calcium channels may lead to improved understanding of novel redox mechanisms in physiological and pathological processes. Identification of redox mechanisms and sites on voltage-gated calcium channel may allow development of novel and specific ion channel therapies for unmet medical needs. Thus, it may be possible to regulate the redox state of these channels in treatment of pathological process such as epilepsy and neuropathic pain.

Mapping CRMP3 domains involved in dendrite morphogenesis and voltage-gated calcium channel regulation

Although hippocampal neurons are well-distinguished by the morphological characteristics of their dendrites and their structural plasticity, the mechanisms involved in regulating their neurite initiation, dendrite growth, network formation and remodeling are still largely unknown, in part because the key molecules involved remain elusive. Identifying new dendrite-active cues could uncover unknown molecular mechanisms that would add significant understanding to the field and possibly lead to the development of novel neuroprotective therapy because these neurons are impaired in many neuropsychiatric disorders. In our previous studies, we deleted the gene encoding CRMP3 in mice and identified the protein as a new endogenous signaling molecule that shapes diverse features of the hippocampal pyramidal dendrites without affecting axon morphology. We also found that CRMP3 protects dendrites against dystrophy induced by prion peptide PrP(106-126). Here, we report that CRMP3 has a profound influence on neurite initiation and dendrite growth of hippocampal neurons in vitro. Our deletional mapping revealed that the C-terminus of CRMP3 probably harbors its dendritogenic capacity and supports an active transport mechanism. By contrast, overexpression of the C-terminal truncated CRMP3 phenocopied the effect of CRMP3 gene deletion with inhibition of neurite initiation or decrease in dendrite complexity, depending on the stage of cell development. In addition, this mutant inhibited the activity of CRMP3, in a similar manner to siRNA. Voltage-gated calcium channel inhibitors prevented CRMP3-induced dendritic growth and somatic Ca(2+) influx in CRMP3-overexpressing neurons was augmented largely via L-type channels. These results support a link between CRMP3-mediated Ca(2+) influx and CRMP3-mediated dendritic growth in hippocampal neurons.

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Calcium binding protein-mediated regulation of voltage-gated calcium channels linked to human diseases

Calcium ion entry through voltage-gated calcium channels is essential for cellular signalling in a wide variety of cells and multiple physiological processes. Perturbations of voltage-gated calcium channel function can lead to pathophysiological consequences. Calcium binding proteins serve as calcium sensors and regulate the calcium channel properties via feedback mechanisms. This review highlights the current evidences of calcium binding protein-mediated channel regulation in human diseases.

Neuropsycological gender differences in healthy individuals and in pediatric neurodevelopmental disorders. A role for SNAP-25

Synaptosomal-associated protein of 25 KD (SNAP-25) is a protein that participates in synaptic vesicle exocytosis through the formation of a SNARE complex; SNAP-25 also plays a pivotal role in modulating calcium homeostasis through negative regulation of voltage-gated calcium channels. SNAP-25 has been involved in different neuropsychiatric disorders, including attention deficit hyperactivity disorder. There are well known physiological gender differences in many neuropsychological skills, and there are even more striking gender differences in patients with attention deficit hyperactivity disorder and autism spectrum disorders. We hypothesize that these differences are the result of a mechanism involving SNAP-25 polymorphisms and its differential expression in specific brain areas.

S17.3 Voltage-gated calcium channel regulation by arachidonic acid: A fatty acid tail

S17.3 Voltage-gated calcium channel regulation by arachidonic acid: A fatty acid tail

Sigma-1 Receptor Regulation of Voltage-Gated Calcium Channels Involves a Direct Interaction

The sigma-1 receptor belongs to a recently discovered family of transmembrane proteins expressed in the central nervous system, including the eye, and mediates the regulation of ion channels. The exact function of sigma receptors remains to be elucidated. The purpose of this study was to investigate the effect of sigma-1 receptor ligands on calcium homeostasis in a retinal ganglion cell line (RGC)-5 and rat primary RGCs. Calcium imaging was used to assess the effect of sigma-1 receptor agonist (+)-N-allylnormetazocine ((+)-SKF10047) on potassium chloride (KCl)-induced calcium influx in RGC-5. The whole-cell patch clamp technique was used to analyze the effect of (+)-SKF10047 on calcium currents in primary RGCs. Coimmunoprecipitation assessed the interaction between the sigma-1 receptor and the L-type voltage-gated calcium channel. The sigma-1 receptor agonist (+)-SKF10047 inhibited potassium chloride (KCl)-induced calcium influx. The sigma-1 receptor antagonist, BD1047, reversed the inhibitory effect of (+)-SKF10047. Whole-cell patch clamp recordings of rat cultured primary RGCs demonstrated that (+)-SKF10047 inhibited calcium currents. Coimmunoprecipitation studies demonstrated an association between L-type calcium channels and the sigma-1 receptors. These results suggest that sigma-1 receptor activation can regulate calcium homeostasis and signaling in RGCs, likely by directly influencing the activity of L-type voltage-gated calcium channels. Regulation of calcium influx in RGCs by sigma-1 receptor ligands may represent in part the neuroprotective effect of sigma-1 receptors.

Role of PIP2 in regulating versus modulating Ca2+ channel activity

Role of PIP₂ in regulating <i>versus</i> modulating Ca²⁺ channel activity

PKCε upregulates voltage‐dependent calcium channels in cultured astrocytes

Astrocytes express voltage-gated calcium channels (VGCCs) that are upregulated in the context of the reactive astrogliosis occurring in several CNS pathologies. Moreover, the ability of selective calcium channel blockers to inhibit reactive astrogliosis has been revealed in a variety of experimental models. However, the functions and regulation of VGCC in astrocytes are still poorly understood. Interestingly, protein kinase C epsilon (PKCepsilon), one of the known regulators of VGCC in several cell types, induces in astrocytes a stellated morphology similar to that associated to gliosis. Thereby, here we explored the possible regulation of VGCC by adenovirally expressed PKCepsilon in astrocytes. We found that PKCepsilon potently increases the mRNA levels of two different calcium channel alpha(1) subunits, Ca(V)1.2 (L-type channel) and Ca(V)2.1 (P/Q-type channel). The mRNA upregulation was followed by a robust increase in the corresponding peptides. Moreover, the new calcium channels formed as a consequence of PKCepsilon activation are functional, since overexpression of constitutively-active PKCepsilon increased significantly the calcium current density in astrocytes. PKCepsilon raised currents carried by both L- and P/Q-type channels. However, the effect on the P/Q-type channel was more prominent since an increase of the relative contribution of this channel to the whole cell calcium current was observed. Finally, we found that PKCepsilon-induced stellation was significantly reduced by the specific L-type channel blocker nifedipine, indicating that calcium influx through VGCC mediates the change in astrocyte morphology induced by PKCepsilon. Therefore, here we describe a novel regulatory pathway involving VGCC that participates in PKCepsilon-dependent astrocyte activation.

A single amino acid mutation attenuates rundown of voltage-gated calcium channels

The activity of voltage-gated calcium channels (VGCCs) decreases with time in whole-cell and inside-out patch-clamp recordings. In this study we found that substituting a single amino acid (I1520) at the intracellular end of IIIS6 in the α 1 subunit of P/Q-type Ca 2+ channels with histidine or aspartate greatly attenuated channel rundown in inside-out patch-clamp recordings. The homologous mutations also slowed rundown of N- and L-type Ca 2+ channels, albeit to a lesser degree. In P/Q-type channels, the attenuation of rundown is accompanied by an increased apparent affinity for phosphatidylinositol-4,5-bisphosphate, which has been shown to be critical for maintaining Ca 2+ channel activity [L. Wu, C.S. Bauer, X.-G. Zhen, C. Xie, J. Yang, Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2. Nature 419 (2002) 947–952]. Furthermore, the histidine mutation significantly stabilized the open state, making the channels easier to open, slower to close, harder to inactivate and faster to recover from inactivation. Our finding that mutation of a single amino acid can greatly attenuate rundown provides an easy and efficient way to slow the rundown of VGCCs, facilitating functional studies that require direct access to the cytoplasmic side of the channel.