Calcium influx through L-type CaV1.2 Ca2+ channels regulates mandibular development.
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ABSTRACT:
SUBMITTER: Ramachandran KV
PROVIDER: S-EPMC3613930 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ca(2+) influx via L-type Ca(v)1.2 channels is essential for multiple physiological processes, including gene expression, excitability, and contraction. Amplification of the Ca(2+) signals produced by the opening of these channels is a hallmark of many intracellular signaling cascades, including excitation-contraction coupling in heart. Using optogenetic approaches, we discovered that Ca(v)1.2 channels form clusters of varied sizes in ventricular myocytes. Physical interaction between these channels via their C-tails renders them capable of coordinating their gating, thereby amplifying Ca(2+) influx and excitation-contraction coupling. Light-induced fusion of WT Ca(v)1.2 channels with Ca(v)1.2 channels carrying a gain-of-function mutation that causes arrhythmias and autism in humans with Timothy syndrome (Ca(v)1.2-TS) increased Ca(2+) currents, diastolic and systolic Ca(2+) levels, contractility and the frequency of arrhythmogenic Ca(2+) fluctuations in ventricular myocytes. Our data indicate that these changes in Ca(2+) signaling resulted from Ca(v)1.2-TS increasing the activity of adjoining WT Ca(v)1.2 channels. Collectively, these data support the concept that oligomerization of Ca(v)1.2 channels via their C termini can result in the amplification of Ca(2+) influx into excitable cells.
| S-EPMC3277143 | biostudies-literature
Project description:CaV1.2 interacts with the Ca(2+) sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, α1C. Ca(2+)/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca(2+) ions (Ca(2+)-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the α1C- subunit using Förster resonance energy transfer (FRET) and recording of Cav1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of α1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for α1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaM altered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15-20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated α1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that the NT is not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in α1C.
| S-EPMC3642314 | biostudies-literature
Project description:Ca2+-binding protein-1 (CaBP1) is a Ca2+-binding protein that is closely related to calmodulin (CaM) and localized in somatodendritic regions of principal neurons throughout the brain, but how CaBP1 participates in postsynaptic Ca2+ signaling is not known. Here, we describe a novel role for CaBP1 in the regulation of Ca2+ influx through Ca(v)1.2 (L-type) Ca2+ channels. CaBP1 interacts directly with the alpha1 subunit of Ca(v)1.2 at sites that also bind CaM. CaBP1 binding to one of these sites, the IQ domain, is Ca2+ dependent and competitive with CaM binding. The physiological significance of this interaction is supported by the association of Ca(v)1.2 and CaBP1 in postsynaptic density fractions purified from rat brain. Moreover, in double-label immunofluorescence experiments, CaBP1 and Ca(v)1.2 colocalize in numerous cell bodies and dendrites of neurons, particularly in pyramidal cells in the CA3 region of the hippocampus and in the dorsal cortex. In electrophysiological recordings of cells transfected with Ca(v)1.2, CaBP1 greatly prolonged Ca2+ currents, prevented Ca2+-dependent inactivation, and caused Ca2+-dependent facilitation of currents evoked by step depolarizations and repetitive stimuli. These effects contrast with those of CaM, which promoted strong Ca2+-dependent inactivation of Ca(v)1.2 with these same voltage protocols. Our findings reveal how Ca2+-binding proteins, such as CaM and CaBP1, differentially adjust Ca2+ influx through Ca(v)1.2 channels, which may specify diverse modes of Ca2+ signaling in neurons.
| S-EPMC6729388 | biostudies-literature
Project description:T-type Ca(2+) channels (TTCCs) are expressed in the developing heart, are not present in the adult ventricle, and are reexpressed in cardiac diseases involving cardiac dysfunction and premature, arrhythmogenic death. The goal of this study was to determine the functional role of increased Ca(2+) influx through reexpressed TTCCs in the adult heart. A mouse line with cardiac-specific, conditional expression of the alpha1G-TTCC was used to increase Ca(2+) influx through TTCCs. alpha1G hearts had mild increases in contractility but no cardiac histopathology or premature death. This contrasts with the pathological phenotype of a previously studied mouse with increased Ca(2+) influx through the L-type Ca(2+) channel (LTCC) secondary to overexpression of its beta2a subunit. Although alpha1G and beta2a myocytes had similar increases in Ca(2+) influx, alpha1G myocytes had smaller increases in contraction magnitude, and, unlike beta2a myocytes, there were no increases in sarcoplasmic reticulum Ca(2+) loading. Ca(2+) influx through TTCCs also did not induce normal sarcoplasmic reticulum Ca(2+) release. alpha1G myocytes had changes in LTCC, SERCA2a, and phospholamban abundance, which appear to be adaptations that help maintain Ca(2+) homeostasis. Immunostaining suggested that the majority of alpha1G-TTCCs were on the surface membrane. Osmotic shock, which selectively eliminates T-tubules, induced a greater reduction in L- versus TTCC currents. These studies suggest that T- and LTCCs are in different portions of the sarcolemma (surface membrane versus T-tubules) and that Ca(2+) influx through these channels induce different effects on myocyte contractility and lead to distinct cardiac phenotypes.
| S-EPMC2678411 | biostudies-literature
Project description:Chronic intermittent hypoxia (CIH) is a hallmark manifestation of sleep apnea. A heightened carotid body activity and the resulting chemosensory reflex mediate increased sympathetic nerve activity by CIH. However, the mechanisms underlying heightened carotid body activity by CIH are not known. An elevation of intracellular calcium ion concentration ([Ca(2+)]i) in glomus cells, the primary oxygen-sensing cells, is an essential step for carotid body activation by hypoxia. In the present study, we examined the effects of CIH on the glomus cell [Ca(2+)]i response to hypoxia and assessed the underlying mechanisms. Glomus cells were harvested from adult rats or wild-type mice treated with 10 days of either room air (control) or CIH (alternating cycles of 15 s of hypoxia and 5 min of room air; 9 episodes/h; 8 h/day). CIH-treated glomus cells exhibited an enhanced [Ca(2+)]i response to hypoxia, and this effect was absent in the presence of 2-(4-cyclopropylphenyl)-N-((1R)-1-[5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl]ethyl)acetamide (TTA-A2), a specific inhibitor of T-type Ca(2+) channels, and in voltage-gated calcium channel, type 3.2 (CaV3.2), null glomus cells. CaV3.2 knockout mice exhibited an absence of CIH-induced hypersensitivity of the carotid body. CIH increased reactive oxygen species (ROS) levels in glomus cells. A ROS scavenger prevented the exaggerated TTA-A2-sensitive [Ca(2+)]i response to hypoxia. CIH had no effect on CaV3.2 mRNA levels. CIH augmented Ca(2+) currents and increased CaV3.2 protein in plasma membrane fractions of human embryonic kidney-293 cells stably expressing CaV3.2, and either a ROS scavenger or brefeldin-A, an inhibitor of protein trafficking, prevented these effects. These findings suggest that CIH leads to an augmented Ca(2+) influx via ROS-dependent facilitation of CaV3.2 protein trafficking to the plasma membrane.
| S-EPMC4760493 | biostudies-literature
Project description:Communication between neurons and developing oligodendrocytes (OLs) leading to OL Ca2+ rise is critical for axon myelination and OL development. Here, we investigate signaling factors and sources of Ca2+ rise in OLs in the mouse brainstem. Glutamate puff or axon fiber stimulation induces a Ca2+ rise in pre-myelinating OLs, which is primarily mediated by Ca2+ -permeable AMPA receptors. During glutamate application, inward currents via AMPA receptors and elevated extracellular K+ caused by increased neuronal activity collectively lead to OL depolarization, triggering Ca2+ influx via P/Q- and L-type voltage-gated Ca2+ (Cav ) channels. Thus, glutamate is a key signaling factor in dynamic communication between neurons and OLs that triggers Ca2+ transients via AMPARs and Cav channels in developing OLs. The results provide a mechanism for OL Ca2+ dynamics in response to neuronal input, which has implications for OL development and myelination.
| S-EPMC6771819 | biostudies-literature
Project description:We previously reported that INS-1 cells expressing the intracellular II-III loop of the L-type Ca(2+) channel Cav1.2 (Cav1.2/II-III cells) are deficient in Ca(2+)-induced Ca(2+) release (CICR). Here we show that glucose-stimulated ERK 1/2 phosphorylation (GSEP) is slowed and reduced in Cav1.2/II-III cells compared to INS-1 cells. This parallels a decrease in glucose-stimulated cAMP accumulation (GS-cAMP) in Cav1.2/II-III cells. Influx of Ca(2+) via L-type Ca(2+) channels and CICR play roles in both GSEP and GS-cAMP in INS-1 cells since both are inhibited by nicardipine or ryanodine. Further, the Epac1-selective inhibitor CE3F4 abolishes glucose-stimulated ERK activation in INS-1 cells, as measured using the FRET-based sensor EKAR. The non-selective Epac antagonist ESI-09 but not the Epac2-selective antagonist ESI-05 nor the PKA antagonist Rp-cAMPs inhibits GSEP in both INS-1 and Cav1.2/II-III cells. We conclude that L-type Ca(2+) channel-dependent cAMP accumulation, that's amplified by CICR, activates Epac1 and drives GSEP in INS-1 cells.
| S-EPMC4684454 | biostudies-literature
Project description:During the early postnatal period, GABA(B) receptor activation facilitates L-type calcium current in rat hippocampus. One developmental process that L-type current may regulate is the change in expression of the K(+)Cl(-) co-transporter (KCC2) and N(+)K(+)2Cl(-) co-transporter (NKCC1), which are involved in the maturation of the GABAergic system. The present study investigated the connection between L-type current, GABA(B) receptors, and expression of chloride transporters during development. The facilitation of L-type current by GABA(B) receptors is more prominent in the second week of development, with the highest percentage of cells exhibiting facilitation in cultures isolated from 7 day old rats (37.5%). The protein levels of KCC2 and NKCC1 were investigated to determine the developmental timecourse of expression as well as expression following treatment with an L-type channel antagonist and a GABA(B) receptor agonist. The time course of both chloride transporters in culture mimics that seen in hippocampal tissue isolated from various ages. KCC2 levels increased drastically in the first two postnatal weeks while NKCC1 remained relatively stable, suggesting that the ratio of the chloride transporters is important in mediating the developmental change in chloride reversal potential. Treatment of cultures with the L-type antagonist nimodipine did not affect protein levels of NKCC1, but significantly decreased the upregulation of KCC2 during the first postnatal week. In addition, calcium current facilitation occurs slightly before the large increase in KCC2 expression. These results suggest that the expression of KCC2 is regulated by calcium influx through L-type channels in the early postnatal period in hippocampal neurons.
| S-EPMC2989739 | biostudies-literature
Project description:BackgroundMultiple myeloma (MM) cell motility is a critical step during MM dissemination throughout the body, but how it is regulated remains largely unknown. As hypercalcemia is an important clinical feature of MM, high calcium (Ca2+) and altered Ca2+ signaling could be a key contributing factor to the pathological process.MethodsBioinformatics analyses were employed to assess the clinical significance of Ca2+ influx channels in clinical specimens of smoldering and symptomatic MM. Functional and regulatory roles of influx channels and downstream signaling in MM cell migration and invasion were conducted and experimental MM dissemination was examined in a xenograft mouse model using in vivo live imaging and engraftment analysis.ResultsInhibition of TRPM7, ORAI1, and STIM1 influx channels, which are highly expressed in MM patients, and subsequent blockage of Ca2+ influx by CRISPR/Cas9 and small molecule inhibitors, effectively inhibit MM cell migration and invasion, and attenuate the experimental MM dissemination. Mechanistic studies reveal a nutrient sensor O-GlcNAcylation as a downstream regulator of Ca2+ influx that specifically targets cell adhesion molecules. Hyper-O-GlcNAcylation following the inhibition of Ca2+ influx channels induces integrin α4 and integrin β7 downregulation via ubiquitin-proteasomal degradation and represses the aggressive MM phenotype.ConclusionsOur findings unveil a novel regulatory mechanism of MM cell motility via Ca2+ influx/O-GlcNAcylation axis that directly targets integrin α4 and integrin β7, providing mechanistic insights into the pathogenesis and progression of MM and demonstrating potential predictive biomarkers and therapeutic targets for advanced MM.
| S-EPMC7968185 | biostudies-literature
Project description:Action potential-evoked neurotransmitter release is impaired in knock-out neurons lacking synaptic cell-adhesion molecules ?-neurexins (?Nrxns), the extracellularly longer variants of the three vertebrate Nrxn genes. Ca2+ influx through presynaptic high-voltage gated calcium channels like the ubiquitous P/Q-type (CaV2.1) triggers release of fusion-ready vesicles at many boutons. ?2? Auxiliary subunits regulate trafficking and kinetic properties of CaV2.1 pore-forming subunits but it has remained unclear if this involves ?Nrxns. Using live cell imaging with Ca2+ indicators, we report here that the total presynaptic Ca2+ influx in primary hippocampal neurons of ?Nrxn triple knock-out mice of both sexes is reduced and involved lower CaV2.1-mediated transients. This defect is accompanied by lower vesicle release, reduced synaptic abundance of CaV2.1 pore-forming subunits, and elevated surface mobility of ?2?-1 on axons. Overexpression of Nrxn1? in ?Nrxn triple knock-out neurons is sufficient to restore normal presynaptic Ca2+ influx and synaptic vesicle release. Moreover, coexpression of Nrxn1? together with ?2?-1 subunits facilitates Ca2+ influx further but causes little augmentation together with a different subunit, ?2?-3, suggesting remarkable specificity. Expression of defined recombinant CaV2.1 channels in heterologous cells validates and extends the findings from neurons. Whole-cell patch-clamp recordings show that Nrxn1? in combination with ?2?-1, but not with ?2?-3, facilitates Ca2+ currents of recombinant CaV2.1 without altering channel kinetics. These results suggest that presynaptic Nrxn1? acts as a positive regulator of Ca2+ influx through CaV2.1 channels containing ?2?-1 subunits. We propose that this regulation represents an important way for neurons to adjust synaptic strength.SIGNIFICANCE STATEMENT Synaptic transmission between neurons depends on the fusion of neurotransmitter-filled vesicles with the presynaptic membrane, which subsequently activates postsynaptic receptors. Influx of calcium ions into the presynaptic terminal is the key step to trigger vesicle release and involves different subtypes of voltage-gated calcium channels. We study the regulation of calcium channels by neurexins, a family of synaptic cell-adhesion molecules that are essential for many synapse properties. Using optical measurements of calcium influx in cultured neurons and electrophysiological recordings of calcium currents from recombinant channels, we show that a major neurexin variant facilitates calcium influx through P/Q-type channels by interacting with their ?2?-1 auxiliary subunits. These results propose a novel way how neurons can modulate the strength of distinct synapses.
| S-EPMC6596161 | biostudies-literature