L-type Ca2+ channel activation of STIM1-Orai1 signaling remodels the dendritic spine ER to maintain long-term structural plasticity.

Learning and memory require coordinated structural and functional plasticity at neuronal glutamatergic synapses located on dendritic spines. Here, we investigated how the endoplasmic reticulum (ER) controls postsynaptic Ca2+ signaling and long-term potentiation of dendritic spine size, i.e., sLTP that accompanies functional strengthening of glutamatergic synaptic transmission. In most ER-containing (ER+) spines, high-frequency optical glutamate uncaging (HFGU) induced long-lasting sLTP that was accompanied by a persistent increase in spine ER content downstream of a signaling cascade engaged by N-methyl-D-aspartate receptors (NMDARs), L-type Ca2+ channels (LTCCs), and Orai1 channels, the latter being activated by stromal interaction molecule 1 (STIM1) in response to ER Ca2+ release. In contrast, HFGU stimulation of ER-lacking (ER-) spines expressed only transient sLTP and exhibited weaker Ca2+ signals noticeably lacking Orai1 and ER contributions. Consistent with spine ER regulating structural metaplasticity, delivery of a second stimulus to ER- spines induced ER recruitment along with persistent sLTP, whereas ER+ spines showed no additional increases in size or ER content in response to sequential stimulation. Surprisingly, the physical interaction between STIM1 and Orai1 induced by ER Ca2+ release, but not the resulting Ca2+ entry through Orai1 channels, proved necessary for the persistent increases in both spine size and ER content required for expression of long-lasting late sLTP.

RIM and RIM-Binding Protein Localize Synaptic CaV2 Channels to Differentially Regulate Transmission in Neuronal Circuits.

At chemical synapses, voltage-gated Ca2+ channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca2+ microdomains they elicit must be located precisely to primed SVs to evoke rapid transmitter release. Localization is mediated by Rab3-interacting molecule (RIM) and RIM-binding proteins, which interact and bind to the C terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction of Caenorhabditis elegans hermaphrodites. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Although postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition (E-I) balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABAA or GABAB receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus, RIMB-1 function differentially affects excitation-inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission within circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 ß-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet it does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerge from VGCC regulation.

Anchored PKA synchronizes adrenergic phosphoregulation of cardiac Cav1.2 channels.

Adrenergic modulation of voltage gated Ca2+ currents is a context specific process. In the heart Cav1.2 channels initiate excitation-contraction coupling. This requires PKA phosphorylation of the small GTPase Rad (Ras associated with diabetes) and involves direct phosphorylation of the Cav1.2 α1 subunit at Ser1700. A contributing factor is the proximity of PKA to the channel through association with A-kinase anchoring proteins (AKAPs). Disruption of PKA anchoring by the disruptor peptide AKAP-IS prevents upregulation of Cav1.2 currents in tsA-201 cells. Biochemical analyses demonstrate that Rad does not function as an AKAP. Electrophysiological recording shows that channel mutants lacking phosphorylation sites (Cav1.2 STAA) lose responsivity to the second messenger cAMP. Measurements in cardiomyocytes isolated from Rad-/- mice show that adrenergic activation of Cav1.2 is attenuated but not completely abolished. Whole animal electrocardiography studies reveal that cardiac selective Rad KO mice exhibited higher baseline left ventricular ejection fraction, greater fractional shortening, and increased heart rate as compared to control animals. Yet, each parameter of cardiac function was slightly elevated when Rad-/- mice were treated with the adrenergic agonist isoproterenol. Thus, phosphorylation of Cav1.2 and dissociation of phospho-Rad from the channel are local cAMP responsive events that act in concert to enhance L-type calcium currents. This convergence of local PKA regulatory events at the cardiac L-type calcium channel may permit maximal ß-adrenergic influence on the fight-or-flight response.

Optogenetic stimulation reveals a latent tipping point in cortical networks during ictogenesis.

Brain-state transitions are readily apparent from changes in brain rhythms,1 but are difficult to predict, suggestive that the underlying cause is latent to passive recording methods. Among the most important transitions, clinically, are the starts of seizures. We here show that an 'active probing' approach may have several important benefits for epileptic management, including by helping predict these transitions. We used mice expressing the optogenetic actuator, channelrhodopsin, in pyramidal cells, allowing this population to be stimulated in isolation. Intermittent stimulation at frequencies as low as 0.033 Hz (period = 30 s) delayed the onset of seizure-like events in an acute brain slice model of ictogenesis, but the effect was lost if stimulation was delivered at even lower frequencies (1/min). Notably, active probing additionally provides advance indication of when seizure-like activity is imminent, revealed by monitoring the postsynaptic response to stimulation. The postsynaptic response, recorded extracellularly, showed an all-or-nothing change in both amplitude and duration, a few hundred seconds before seizure-like activity began-a sufficient length of time to provide a helpful warning of an impending seizure. The change in the postsynaptic response then persisted for the remainder of the recording, indicative of a state change from a pre-epileptic to a pro-epileptic network. This occurred in parallel with a large increase in the stimulation-triggered Ca2+ entry into pyramidal dendrites, and a step increase in the number of evoked postsynaptic action potentials, both consistent with a reduction in the threshold for dendritic action potentials. In 0 Mg2+ bathing media, the reduced threshold was not associated with changes in glutamatergic synaptic function, nor of GABAergic release from either parvalbumin or somatostatin interneurons, but simulations indicate that the step change in the optogenetic response can instead arise from incremental increases in intracellular [Cl-]. The change in the response to stimulation was replicated by artificially raising intracellular [Cl-], using the optogenetic chloride pump, halorhodopsin. By contrast, increases in extracellular [K+] cannot account for the firing patterns in the response to stimulation, although this, and other cellular changes, may contribute to ictal initiation in other circumstances. We describe how these various cellular changes form a synergistic network of positive feedback mechanisms, which may explain the precipitous nature of seizure onset. This model of seizure initiation draws together several major lines of epilepsy research as well as providing an important proof-of-principle regarding the utility of open-loop brain stimulation for clinical management of the condition.

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria.

CaV1 and CaV2 voltage-gated calcium channels evolved from an ancestral CaV1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian CaV1 channels are uniquely subject to pronounced, buffer-resistant Ca2+/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca2+ rises. Early diverging, nonbilaterian invertebrates also possess CaV1 and CaV2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both CaV1 and CaV2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of CaV1 and CaV2 channels. Here, we build upon previous characterization of Trichoplax CaV channels by determining the cellular expression and ion-conducting properties of the CaV1 channel orthologue, TCaV1. We show that TCaV1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage-activated Ca2+ currents with kinetics resembling those of rat CaV1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCaV1, but not TCaV2, exhibits buffer-resistant Ca2+/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca2+ signaling within various cellular contexts.

Fluorofenidone enhances cardiac contractility by stimulating CICR and CaV1.2.

Fluorofenidone (AKF-PD) is a novel pyridone derivative that inhibits fibrosis and inflammation in many tissues. Accordingly, it has been effective in disease models, such as liver failure, nephropathy, and pulmonary fibrosis. However, its potential role in cardiac physiology and pathology has yet to be elucidated. Thus, this paper investigated a possible functional impact of AKF-PD on adult rat cardiac myocytes. Cells were kept in culture for 1-2 days under either control conditions or the presence of AKF-PD (500 µM). They were next examined concerning cell contractility, intracellular Ca2+ homeostasis, and activity of voltage-gated Ca2+ channels. Remarkably, AKF-PD enhanced the percentage of cell shortening and rates of both contraction and relaxation by nearly 100%. A stimulus in Ca2+-induced Ca2+ release (CICR) most likely accounts for these effects because AKF-PD also increased the magnitude of electrically evoked Ca2+ transients. Of note, the compound did not alter the peak value of caffeine-elicited Ca2+ transients, indicating stimulation of CICR at constant sarcoplasmic reticulum Ca2+ load. Since CICR is triggered by the entry of Ca2+ through CaV1.2 (ICa), a possible effect on these Ca2+ channels was also investigated. AKF-PD increased the magnitude of both ICa and maximal macroscopic Ca2+ conductance (Gmax) by about 50%. However, no differences were found in either voltage dependence of inactivation or the amount of maximal immobilization-resistant charge movement (Qmax). Thus, the effect on ICa could be explained by a higher channel's open probability (Po) rather than a greater abundance of channel proteins. Additional data indicate that AKF-PD reduces the rate of Ca2+ extrusion in the presence of caffeine, suggesting inhibition of the Na/Ca exchanger. Overall, these results indicate that AKF-PD upregulates the Po of CaV1.2 and then sequentially enhances ICa, CICR, and contractility. Therefore, the novel compound is also a candidate to be tested in cardiac disease models.

Nanoscale organization of CaV2.1 splice isoforms at presynaptic terminals: implications for synaptic vesicle release and synaptic facilitation.

The distance between CaV2.1 voltage-gated Ca2+ channels and the Ca2+ sensor responsible for vesicle release at presynaptic terminals is critical for determining synaptic strength. Yet, the molecular mechanisms responsible for a loose coupling configuration of CaV2.1 in certain synapses or developmental periods and a tight one in others remain unknown. Here, we examine the nanoscale organization of two CaV2.1 splice isoforms (CaV2.1[EFa] and CaV2.1[EFb]) at presynaptic terminals by superresolution structured illumination microscopy. We find that CaV2.1[EFa] is more tightly co-localized with presynaptic markers than CaV2.1[EFb], suggesting that alternative splicing plays a crucial role in the synaptic organization of CaV2.1 channels.

Ambroxol-Enhanced Frequency and Amplitude of Beating Cilia Controlled by a Voltage-Gated Ca2+ Channel, Cav1.2, via pHi Increase and [Cl-]i Decrease in the Lung Airway Epithelial Cells of Mice.

Ambroxol (ABX), a frequently prescribed secretolytic agent which enhances the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30%, activates a voltage-dependent Ca2+ channel (CaV1.2) and a small transient Ca2+ release in the ciliated lung airway epithelial cells (c-LAECs) of mice. The activation of CaV1.2 alone enhanced the CBF and CBA by 20%, mediated by a pHi increasei and a [Cl-]i decrease in the c-LAECs. The increase in pHi, which was induced by the activation of the Na+-HCO3- cotransporter (NBC), enhanced the CBF (by 30%) and CBA (by 15-20%), and a decrease in [Cl-]i, which was induced by the Cl- release via anoctamine 1 (ANO1), enhanced the CBA (by 10-15%). While a Ca2+-free solution or nifedipine (an inhibitor of CaV1.2) inhibited 70% of the CBF and CBA enhancement using ABX, CaV1.2 enhanced most of the CBF and CBA increases using ABX. The activation of the CaV1.2 existing in the cilia stimulates the NBC to increase pHi and ANO1 to decrease the [Cl-]i in the c-LAECs. In conclusion, the pHi increase and the [Cl-]i decrease enhanced the CBF and CBA in the ABX-stimulated c-LAECs.

Activation of Endothelial Large Conductance Potassium Channels Protects against TNF-α-Induced Inflammation.

Elevated TNF-α levels in serum and broncho-alveolar lavage fluid of acute lung injury patients correlate with mortality rates. We hypothesized that pharmacological plasma membrane potential (Em) hyperpolarization protects against TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells through inhibition of inflammatory Ca2+-dependent MAPK pathways. Since the role of Ca2+ influx in TNF-α-mediated inflammation remains poorly understood, we explored the role of L-type voltage-gated Ca2+ (CaV) channels in TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells. The CaV channel blocker, Nifedipine, decreased both CCL-2 and IL-6 secretion, suggesting that a fraction of CaV channels is open at the significantly depolarized resting Em of human microvascular pulmonary endothelial cells (-6 ± 1.9 mV), as shown by whole-cell patch-clamp measurements. To further explore the role of CaV channels in cytokine secretion, we demonstrated that the beneficial effects of Nifedipine could also be achieved by Em hyperpolarization via the pharmacological activation of large conductance K+ (BK) channels with NS1619, which elicited a similar decrease in CCL-2 but not IL-6 secretion. Using functional gene enrichment analysis tools, we predicted and validated that known Ca2+-dependent kinases, JNK-1/2 and p38, are the most likely pathways to mediate the decrease in CCL-2 secretion.

Synaptic Contributions to Cochlear Outer Hair Cell Ca2+ Dynamics.

For normal cochlear function, outer hair cells (OHCs) require a precise control of intracellular Ca2+ levels. In the absence of regulatory elements such as proteinaceous buffers or extrusion pumps, OHCs degenerate, leading to profound hearing impairment. Influx of Ca2+ occurs both at the stereocilia tips and the basolateral membrane. In this latter compartment, two different origins for Ca2+ influx have been poorly explored: voltage-gated L-type Ca2+ channels (VGCCs) at synapses with Type II afferent neurons, and α9α10 cholinergic nicotinic receptors at synapses with medio-olivochlear complex (MOC) neurons. Using functional imaging in mouse OHCs, we dissected Ca2+ influx individually through each of these sources, either by applying step depolarizations to activate VGCC, or stimulating MOC axons. Ca2+ ions originated in MOC synapses, but not by VGCC activation, was confined by Ca2+-ATPases most likely present in nearby synaptic cisterns. Although Ca2+ currents in OHCs are small, VGCC Ca2+ signals were comparable in size to those elicited by α9α10 receptors, and were potentiated by ryanodine receptors (RyRs). In contrast, no evidence of potentiation by RyRs was found for MOC Ca2+ signals over a wide range of presynaptic stimulation strengths. Our study shows that despite the fact that these two Ca2+ entry sites are closely positioned, they differ in their regulation by intracellular cisterns and/or organelles, suggesting the existence of well-tuned mechanisms to separate the two different OHC synaptic functions.SIGNIFICANCE STATEMENT Outer hair cells (OHCs) are sensory cells in the inner ear operating under very special constraints. Acoustic stimulation leads to fast changes both in membrane potential and in the intracellular concentration of metabolites such as Ca2+ Tight mechanisms for Ca2+ control in OHCs have been reported. Interestingly, Ca2+ is crucial for two important synaptic processes: inhibition by efferent cholinergic neurons, and glutamate release onto Type II afferent fibers. In the current study we functionally imaged Ca2+ at these two different synapses, showing close positioning within the basolateral compartment of OHCs. In addition, we show differential regulation of these two Ca2+ sources by synaptic cisterns and/or organelles, which could result crucial for functional segregation during normal hearing.

Deletion of Voltage-Gated Calcium Channels in Astrocytes during Demyelination Reduces Brain Inflammation and Promotes Myelin Regeneration in Mice.

To determine whether Cav1.2 voltage-gated Ca2+ channels contribute to astrocyte activation, we generated an inducible conditional knock-out mouse in which the Cav1.2 α subunit was deleted in GFAP-positive astrocytes. This astrocytic Cav1.2 knock-out mouse was tested in the cuprizone model of myelin injury and repair which causes astrocyte and microglia activation in the absence of a lymphocytic response. Deletion of Cav1.2 channels in GFAP-positive astrocytes during cuprizone-induced demyelination leads to a significant reduction in the degree of astrocyte and microglia activation and proliferation in mice of either sex. Concomitantly, the production of proinflammatory factors such as TNFα, IL1ß and TGFß1 was significantly decreased in the corpus callosum and cortex of Cav1.2 knock-out mice through demyelination. Furthermore, this mild inflammatory environment promotes oligodendrocyte progenitor cells maturation and myelin regeneration across the remyelination phase of the cuprizone model. Similar results were found in animals treated with nimodipine, a Cav1.2 Ca2+ channel inhibitor with high affinity to the CNS. Mice of either sex injected with nimodipine during the demyelination stage of the cuprizone treatment displayed a reduced number of reactive astrocytes and showed a faster and more efficient brain remyelination. Together, these results indicate that Cav1.2 Ca2+ channels play a crucial role in the induction and proliferation of reactive astrocytes during demyelination; and that attenuation of astrocytic voltage-gated Ca2+ influx may be an effective therapy to reduce brain inflammation and promote myelin recovery in demyelinating diseases.SIGNIFICANCE STATEMENT Reducing voltage-gated Ca2+ influx in astrocytes during brain demyelination significantly attenuates brain inflammation and astrocyte reactivity. Furthermore, these changes promote myelin restoration and oligodendrocyte maturation throughout remyelination.

Conserved biophysical features of the CaV2 presynaptic Ca2+ channel homologue from the early-diverging animal Trichoplax adhaerens.

The dominant role of CaV2 voltage-gated calcium channels for driving neurotransmitter release is broadly conserved. Given the overlapping functional properties of CaV2 and CaV1 channels, and less so CaV3 channels, it is unclear why there have not been major shifts toward dependence on other CaV channels for synaptic transmission. Here, we provide a structural and functional profile of the CaV2 channel cloned from the early-diverging animal Trichoplax adhaerens, which lacks a nervous system but possesses single gene homologues for CaV1-CaV3 channels. Remarkably, the highly divergent channel possesses similar features as human CaV2.1 and other CaV2 channels, including high voltage-activated currents that are larger in external Ba2+ than in Ca2+; voltage-dependent kinetics of activation, inactivation, and deactivation; and bimodal recovery from inactivation. Altogether, the functional profile of Trichoplax CaV2 suggests that the core features of presynaptic CaV2 channels were established early during animal evolution, after CaV1 and CaV2 channels emerged via proposed gene duplication from an ancestral CaV1/2 type channel. The Trichoplax channel was relatively insensitive to mammalian CaV2 channel blockers ω-agatoxin-IVA and ω-conotoxin-GVIA and to metal cation blockers Cd2+ and Ni2+ Also absent was the capacity for voltage-dependent G-protein inhibition by co-expressed Trichoplax Gßγ subunits, which nevertheless inhibited the human CaV2.1 channel, suggesting that this modulatory capacity evolved via changes in channel sequence/structure, and not G proteins. Last, the Trichoplax channel was immunolocalized in cells that express an endomorphin-like peptide implicated in cell signaling and locomotive behavior and other likely secretory cells, suggesting contributions to regulated exocytosis.

Interactions with PDZ proteins diversify voltage-gated calcium channel signaling.

Voltage-gated Ca2+ (CaV ) channels are crucial for neuronal excitability and synaptic transmission upon depolarization. Their properties in vivo are modulated by their interaction with a variety of scaffolding proteins. Such interactions can influence the function and localization of CaV channels, as well as their coupling to intracellular second messengers and regulatory pathways, thus amplifying their signaling potential. Among these scaffolding proteins, a subset of PDZ (postsynaptic density-95, Drosophila discs-large, and zona occludens)-domain containing proteins play diverse roles in modulating CaV channel properties. At the presynaptic terminal, PDZ proteins enrich CaV channels in the active zone, enabling neurotransmitter release by maintaining a tight and vital link between channels and vesicles. In the postsynaptic density, these interactions are essential in regulating dendritic spine morphology and postsynaptic signaling cascades. In this review, we highlight the studies that demonstrate dynamic regulations of neuronal CaV channels by PDZ proteins. We discuss the role of PDZ proteins in controlling channel activity, regulating channel cell surface density, and influencing channel-mediated downstream signaling events. We highlight the importance of PDZ protein regulations of CaV channels and evaluate the link between this regulatory effect and human disease.

De novo CACNA1D Ca2+ channelopathies: clinical phenotypes and molecular mechanism.

The identification of rare disease-causing variants in humans by large-scale next-generation sequencing (NGS) studies has also provided us with new insights into the pathophysiological role of de novo missense variants in the CACNA1D gene that encodes the pore-forming α1-subunit of voltage-gated Cav1.3 L-type Ca2+ channels. These CACNA1D variants have been identified somatically in aldosterone-producing adenomas as well as germline in patients with neurodevelopmental and in some cases endocrine symptoms. In vitro studies in heterologous expression systems have revealed typical gating changes that indicate enhanced Ca2+ influx through Cav1.3 channels as the underlying disease-causing mechanism. Here we summarize the clinical findings of 12 well-characterized individuals with a total of 9 high-risk pathogenic CACNA1D variants. Moreover, we propose how information from somatic mutations in aldosterone-producing adenomas could be used to predict the potential pathogenicity of novel germline variants. Since these pathogenic de novo variants can cause a channel-gain-of function, we also discuss the use of L-type Ca2+ channel blockers as a potential therapeutic option.

Ion channels and myogenic activity in retinal arterioles.

Retinal pressure autoregulation is an important mechanism that protects the retina by stabilizing retinal blood flow during changes in arterial or intraocular pressure. Similar to other vascular beds, retinal pressure autoregulation is thought to be mediated largely through the myogenic response of small arteries and arterioles which constrict when transmural pressure increases or dilate when it decreases. Over recent years, we and others have investigated the signaling pathways underlying the myogenic response in retinal arterioles, with particular emphasis on the involvement of different ion channels expressed in the smooth muscle layer of these vessels. Here, we review and extend previous work on the expression and spatial distribution of the plasma membrane and sarcoplasmic reticulum ion channels present in retinal vascular smooth muscle cells (VSMCs) and discuss their contribution to pressure-induced myogenic tone in retinal arterioles. This includes new data demonstrating that several key players and modulators of the myogenic response show distinctively heterogeneous expression along the length of the retinal arteriolar network, suggesting differences in myogenic signaling between larger and smaller pre-capillary arterioles. Our immunohistochemical investigations have also highlighted the presence of actin-containing microstructures called myobridges that connect the retinal VSMCs to one another. Although further work is still needed, studies to date investigating myogenic mechanisms in the retina have contributed to a better understanding of how blood flow is regulated in this tissue. They also provide a basis to direct future research into retinal diseases where blood flow changes contribute to the pathology.

The voltage-gated T-type Ca2+ channel is key to the sperm motility of Atlantic salmon (Salmo salar).

Ca2+ is a key element in the sperm activation process of Salmo salar. However, the molecular mechanisms by which this ion enters the sperm cell have been poorly studied. In this study, we examined, for the first time, the role of the voltage-gated T-type Ca2+ channel in the activation of sperm motility of Salmo salar. Using an in vitro inhibition assay, a significant decrease in total and progressive motility (P < 0.0001) was observed in Salmo salar sperm when they were treated with NNC-55-0396, a highly selective blocker. The in silico analysis showed that this blocker is docked with a strong affinity for the pore of the voltage-gated T-type calcium channel suggesting the blocking of Ca2+ ions. The results show that the T-type voltage-gated Ca2+ channel is key to sperm motility in Salmo salar.

Redox regulation of the yeast voltage-gated Ca2+ channel homolog Cch1p by glutathionylation of specific cysteine residues.

Cch1p, the yeast homolog of the pore-forming subunit α1 of the mammalian voltage-gated Ca2+ channel (VGCC), is located on the plasma membrane and mediates the redox-dependent influx of Ca2+ Cch1p is known to undergo both rapid activation (after oxidative stress and or a change to high pH) and slow activation (after ER stress and mating pheromone activation), but the mechanism of activation is not known. We demonstrate here that both the fast activation (exposure to pH 8-8.5 or treatment with H2O2) and the slow activation (treatment with tunicamycin or α-factor) are mediated through a common redox-dependent mechanism. Furthermore, through mutational analysis of all 18 exposed cysteine residues in the Cch1p protein, we show that the four mutants C587A, C606A, C636A and C642A, which are clustered together in a common cytoplasmic loop region, were functionally defective for both fast and slow activations, and also showed reduced glutathionylation. These four cysteine residues are also conserved across phyla, suggesting a conserved mechanism of activation. Investigations into the enzymes involved in the activation reveal that the yeast glutathione S-transferase Gtt1p is involved in the glutathionylation of Cch1p, while the thioredoxin Trx2p plays a role in the Cch1p deglutathionylation.

Early Alterations of Hippocampal Neuronal Firing Induced by Abeta42.

We studied the effect of Amyloid ß 1-42 oligomers (Abeta42) on Ca2+ dependent excitability profile of hippocampal neurons. Abeta42 is one of the Amyloid beta peptides produced by the proteolytic processing of the amyloid precursor protein and participates in the initiating event triggering the progressive dismantling of synapses and neuronal circuits. Our experiments on cultured hippocampal network reveal that Abeta42 increases intracellular Ca2+ concentration by 46% and inhibits firing discharge by 19%. More precisely, Abeta42 differently regulates ryanodine (RyRs), NMDA receptors (NMDARs), and voltage gated calcium channels (VGCCs) by increasing Ca2+ release through RyRs and inhibiting Ca2+ influx through NMDARs and VGCCs. The overall increased intracellular Ca2+ concentration causes stimulation of K+ current carried by big conductance Ca2+ activated potassium (BK) channels and hippocampal network firing inhibition. We conclude that Abeta42 alters neuronal function by means of at least 4 main targets: RyRs, NMDARs, VGCCs, and BK channels. The development of selective modulators of these channels may in turn be useful for developing effective therapies that could enhance the quality of life of AD patients during the early onset of the pathology.

When muscle Ca2+ channels carry monovalent cations through gating pores: insights into the pathophysiology of type 1 hypokalaemic periodic paralysis.

Patients suffering from type 1 hypokalaemic periodic paralysis (HypoPP1) experience attacks of muscle paralysis associated with hypokalaemia. The disease arises from missense mutations in the gene encoding the α1 subunit of the dihydropyridine receptor (DHPR), a protein complex anchored in the tubular membrane of skeletal muscle fibres which controls the release of Ca2+ from sarcoplasmic reticulum and also functions as a Ca2+ channel. The vast majority of mutations consist of the replacement of one of the outer arginines in S4 segments of the α1 subunit by neutral residues. Early studies have shown that muscle fibres from HypoPP1 patients are abnormally depolarized at rest in low K+ to the point of inducing muscle inexcitability. The relationship between HypoPP1 mutations and depolarization has long remained unknown. More recent investigations conducted in the closely structurally related voltage-gated Na+ and K+ channels have shown that comparable S4 arginine substitutions gave rise to elevated inward currents at negative potentials called gating pore currents. Experiments performed in muscle fibres from different models revealed such an inward resting current through HypoPP1 mutated Ca2+ channels. In mouse fibres transfected with HypoPP1 mutated channels, the elevated resting current was found to carry H+ for the R1239H arginine-to-histidine mutation in a S4 segment and Na+ for the V876E HypoPP1 mutation, which has the peculiarity of not being located in S4 segments. Muscle paralysis probably results from the presence of a gating pore current associated with hypokalaemia for both mutations, possibly aggravated by external acidosis for the R1239H mutation.

Voltage-gated Ca2+ channel activity modulates smooth muscle cell calcium waves in hamster cremaster arterioles.

Cremaster muscle arteriolar smooth muscle cells (SMCs) display inositol 1,4,5-trisphosphate receptor-dependent Ca2+ waves that contribute to global myoplasmic Ca2+ concentration and myogenic tone. However, the contribution made by voltage-gated Ca2+ channels (VGCCs) to arteriolar SMC Ca2+ waves is unknown. We tested the hypothesis that VGCC activity modulates SMC Ca2+ waves in pressurized (80 cmH2O/59 mmHg, 34°C) hamster cremaster muscle arterioles loaded with Fluo-4 and imaged by confocal microscopy. Removal of extracellular Ca2+ dilated arterioles (32 ± 3 to 45 ± 3 µm, n = 15, P < 0.05) and inhibited the occurrence, amplitude, and frequency of Ca2+ waves ( n = 15, P < 0.05), indicating dependence of Ca2+ waves on Ca2+ influx. Blockade of VGCCs with nifedipine (1 µM) or diltiazem (10 µM) or deactivation of VGCCs by hyperpolarization of smooth muscle with the K+ channel agonist cromakalim (10 µM) produced similar inhibition of Ca2+ waves ( P < 0.05). Conversely, depolarization of SMCs with the K+ channel blocker tetraethylammonium (1 mM) constricted arterioles from 26 ± 3 to 14 ± 2 µm ( n = 11, P < 0.05) and increased wave occurrence (9 ± 3 to 16 ± 3 waves/SMC), amplitude (1.6 ± 0.07 to 1.9 ± 0.1), and frequency (0.5 ± 0.1 to 0.9 ± 0.2 Hz, n = 10, P < 0.05), effects that were blocked by nifedipine (1 µM, P < 0.05). Similarly, the VGCC agonist Bay K8644 (5 nM) constricted arterioles from 14 ± 1 to 8 ± 1 µm and increased wave occurrence (3 ± 1 to 10 ± 1 waves/SMC) and frequency (0.2 ± 0.1 to 0.6 ± 0.1 Hz, n = 6, P < 0.05), effects that were unaltered by ryanodine (50 µM, n = 6, P > 0.05). These data support the hypothesis that Ca2+ waves in arteriolar SMCs depend, in part, on the activity of VGCCs. NEW & NOTEWORTHY Arterioles that control blood flow to and within skeletal muscle depend on Ca2+ influx through voltage-gated Ca2+ channels and release of Ca2+ from internal stores through inositol 1,4,5-trisphosphate receptors in the form of Ca2+ waves to maintain pressure-induced smooth muscle tone.