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Allergic inflammation is closely related to the activation of mast cells (MCs), which is regulated by its intracellular Ca2+ level, but the intake and effects of the intracellular Ca2+ remain unclear. The Ca2+ influx is controlled by members of Ca2+ channels, among which calcium voltage-gated channel subunit alpha1 C (Ca V 1.2) is the most robust. This study aimed to reveal the role and underlying mechanism of MC Ca V 1.2 in allergic inflammation. We found that Ca V 1.2 participated in MC activation and allergic inflammation. Nimodipine (Nim), as a strong Ca V 1.2-specific antagonist, ameliorated allergic inflammation in mice. Further, Ca V 1.2 activation in MC was triggered by phosphatizing at its Ser1928 through protein kinase C (PKC), which calcium/calmodulin-dependent protein kinase II (CaMKII) catalyzed. Overexpression or knockdown of MC Ca V 1.2 influenced MC activation. Importantly, Ca V 1.2 expression in MC had detrimental effects, while its deficiency ameliorated allergic pulmonary inflammation. Results provide novel insights into Ca V 1.2 function and a potential drug target for controlling allergic inflammation. [Display omitted] • MC Ca V 1.2 deficiency or inhibition ameliorates degranulation and allergic inflammation. • The opening of MC Ca V 1.2 does not require depolarization of membrane potential. • MC Ca V 1.2 activation is triggered by CaMKII/PKC/Ser1928 phosphorylation. [ABSTRACT FROM AUTHOR]

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• Cavβ 2a G113S, and Cavβ 2a S143F corresponding to human autistic Cavβ 2d mutants, mediate ET coupling via the Ras/ERK/CREB pathway similar to wtCavβ 2a. • The Cavβ 2a G113S, and Cavβ 2a S143F mutants, as opposed to wtCavβ 2a , elicit spontaneous activation of ERK1/2 and CREB. • Enhanced spontaneous channel activity appears a common molecular mechanism of Ca2+-channel-mediated neurodevelopmental disorders. • wt Cavβ 2a , Cavβ 2a G113S, Cavβ 2a S1143F and Cavβ 2a DDD/AAA selectively interact with RasGRF2 and RhoGEF • The triple-D -mutant within the guanylate kinase domain inhibits ET coupling. Membrane depolarization triggers gene expression through voltage-gated calcium channels (VGCC) in a process called Excitation-transcription (ET) coupling. Mutations in the channel subunits α 1 1.2, or β 2d , are associated with neurodevelopmental disorders such as ASD. Here, we found that two mutations S143F and G113S within the rat Cavβ 2a corresponding to autistic related mutations Cavβ 2d S197F and Cavβ 2d G167S in the human Cavβ 2d , activate ET-coupling via the RAS/ERK/CREB pathway. Membrane depolarization of HEK293 cells co-expressing α 1 1.2 and α 2δ with Cavβ 2a S143F or Cavβ 2a G113S triggers constitutive transcriptional activation, which is correlated with facilitated channel activity. Similar to the Timothy-associated autistic mutation α 1 1.2G406R, constitutive gene activation is attributed to a hyperpolarizing shift in the activation kinetics of Cav1.2. Pulldown of RasGRF2 and RhoGEF by wt and the Cavβ 2a autistic mutants is consistent with Cavβ 2 /Ras activation in ET coupling and implicates Rho signaling as yet another molecular pathway activated by Cavα 1 1.2/Cavβ2. Facilitated spontaneous channel activity preceding enhanced gene activation via the Ras/ERK/CREB pathway, appears a general molecular mechanism for Ca2+ channel mediated ASD and other neurodevelopmental disorders. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: We have recently shown that freshly isolated astrocytes from the mouse brain express mRNA for the L-channel gene Cav1.3 to at least the same degree (per mg mRNA) as corresponding neurons. The amount of extracellular Ca2+ actually entering cultured astrocytes by its opening is modest, but due to secondary Ca2+-mediated stimulation of the ryanodine receptor (RyR) the increase in free cytosolic Ca2+ [Ca2+]i is substantial. The other Cav1 subtype expressed in brain is Cav1.2, which is even expressed in higher density. Although the different primers used for the two genes preclude exact quantitative comparison, the present study suggests that this is also the case in the freshly isolated astrocytes and neurons, which express equal Cav1.2 densities. Again, most of the increase in [Ca2+]i occurred by RyR activity. In contrast to Cav1.3 the expression of Cav1.2 was greatly increased (doubled) after two weeks of treatment with fluoxetine hydrochloride (10mg/kg). Accordingly [Ca2+]i in cultured astrocytes exposed to the addition of 10–60mM KCl increased substantially in cultured astrocytes treated chronically with fluoxetine with the lag time until the effect was observed depending upon the fluoxetine concentration. This effect was inhibited by nifedipine or siRNA against Cav1.2. The increase in K+-induced rise in [Ca2+]i after fluoxetine treatment is directly opposite to a decrease in [Ca2+]i after treatment with any of the anti-bipolar drugs lithium, carbamazepine or valproic acid, due to reduced capacitative Ca2+ influx. We have previously shown a similar effect after fluoxetine treatment, but it becomes overridden by the Cav1.2 up-regulation. [Copyright &y& Elsevier]

Background: Dilated cardiomyopathy (DCM) is a primary disease of the heart muscle associated with sudden cardiac death secondary to ventricular tachyarrhythmias and asystole. However, the molecular pathways linking DCM to arrhythmias and sudden cardiac death are unknown. We previously identified a S196L mutation in exon 4 of LBD3-encoded ZASP in a family with DCM and sudden cardiac death. These findings led us to hypothesize that this mutation may precipitate both cytoskeletal and conduction abnormalities in vivo. Therefore, we investigated the role of the ZASP4 mutation S196L in cardiac cytoarchitecture and ion channel biology.Methods and Results: We generated and analyzed transgenic mice with cardiac-restricted expression of the S196L mutation. We also performed cellular electrophysiological analysis on isolated S196L cardiomyocytes and protein-protein interaction studies. Ten month-old S196L mice developed hemodynamic dysfunction consistent with DCM, whereas 3-month-old S196L mice presented with cardiac conduction defects and atrioventricular block. Electrophysiological analysis on isolated S196L cardiomyocytes demonstrated that the L-type Ca(2+) currents and Na(+) currents were altered. The pull-down assay demonstrated that ZASP4 complexes with both calcium (Ca(v)1.2) and sodium (Na(v)1.5) channels.Conclusions: Our findings provide new insight into the mechanisms by which mutations of a structural/cytoskeletal protein, such as ZASP, lead to cardiac functional and electric abnormalities. This work represents a novel framework to understand the development of conduction defects and arrhythmias in subjects with cardiomyopathies, including DCM. [ABSTRACT FROM AUTHOR]

Abstract: Activity-dependent increase in cytosolic calcium ([Ca2+]i) is a prerequisite for many neuronal functions. We previously reported a strong direct depolarization, independent of glutamate receptors, effectively caused a release of Ca2+ from ryanodine-sensitive stores and induced the synthesis of endogenous cannabinoids (eCBs) and eCB-mediated responses. However, the cellular mechanism that initiated the depolarization-induced Ca2+-release is not completely understood. In the present study, we optically recorded [Ca2+]i from CA1 pyramidal neurons in the hippocampal slice and directly monitored miniature Ca2+ activities and depolarization-induced Ca2+ signals in order to determine the source(s) and properties of [Ca2+]i-dynamics that could lead to a release of Ca2+ from the ryanodine receptor. In the absence of depolarizing stimuli, spontaneously occurring miniature Ca2+ events were detected from a group of hippocampal neurons. This miniature Ca2+ event persisted in the nominal Ca2+-containing artificial cerebrospinal fluid (ACSF), and increased in frequency in response to the bath-application of caffeine and KCl. In contrast, nimodipine, the antagonist of the L-type Ca2+ channel (LTCC), a high concentration of ryanodine, the antagonist of the ryanodine receptor (RyR), and thapsigargin (TG) reduced the occurrence of the miniature Ca2+ events. When a brief puff-application of KCl was given locally to the soma of individual neurons in the presence of glutamate receptor antagonists, these neurons generated a transient increase in the [Ca2+]i in the dendrosomal region. This [Ca2+]i-transient was sensitive to nimodipine, TG, and ryanodine suggesting that the [Ca2+]i-transient was caused primarily by the LTCC-mediated Ca2+-influx and a release of Ca2+ from RyR. We observed little contribution from N- or P/Q-type Ca2+ channels. The coupling between LTCC and RyR was direct and independent of synaptic activities. Immunohistochemical study revealed a cellular localization of LTCC and RyR in a juxtaposed configuration in the proximal dendrites and soma. We conclude in the hippocampal CA1 neuron that: (1) homeostatic fluctuation of the resting membrane potential may be sufficient to initiate functional coupling between LTCC and RyR; (2) the juxtaposed localization of LTCC and RyR has anatomical advantage of synchronizing a Ca2+-release from RyR upon the opening of LTCC; and (3) the synchronized Ca2+-release from RyR occurs immediately after the activation of LTCC and determines the peak amplitude of depolarization-induced global increase in dendrosomal [Ca2+]i. [Copyright &y& Elsevier]

The article discusses the study which focuses on the significance of K+ channel interacting protein 2 (KChIP2) as a direct regulator of the cardiac L-type Ca2+ current (ICa,L. It states that researchers found a direct interaction between KChIP2 and CaV1,2 a1c subunit N terminus using a biochemical analysis. In conclusion, it suggests that KChIP2 is a multimodal regulator of cardiac ionic currents.

Abstract: Homers are scaffolding proteins that bind Ca2+ signaling proteins in cellular microdomains. The Homers participate in targeting and localization of Ca2+ signaling proteins in signaling complexes. However, recent work showed that the Homers are not passive scaffolding proteins, but rather they regulate the activity of several proteins within the Ca2+ signaling complex in an isoform-specific manner. Homer2 increases the GAP activity of RGS proteins and PLCβ that accelerate the GTPase activity of Gα subunits. Homer1 gates the activity of TRPC channels, controls the rates of their translocation and retrieval from the plasma membrane and mediates the conformational coupling between TRPC channels and IP3Rs. Homer1 stimulates the activity of the cardiac and neuronal L-type Ca2+ channels Cav1.2 and Cav1.3. Homer1 also mediates the communication between the cardiac and smooth muscle ryanodine receptor RyR2 and Cav1.2 to regulate E–C coupling. In many cases the Homers function as a buffer to reduce the intensity of Ca2+ signaling and create a negative bias that can be reversed by the immediate early gene form of Homer1. Hence, the Homers should be viewed as the buffers of Ca2+ signaling that ensure a high spatial and temporal fidelity of the Ca2+ signaling and activation of downstream effects. [Copyright &y& Elsevier]

Abstract: l-type (Cav1.2) voltage-gated calcium channels play an essential role in muscle contraction in the cardiovascular system. Alternative splicing of the pore-forming Cav1.2 subunit provides potent means to enrich the functional diversity of the channels. There are 11 alternatively spliced exons identified in rat Cav1.2 gene and random rearrangements may generate up to hundreds of combinatorial splicing profiles. Due to such complexity, the real combinatorial splicing profiles of Cav1.2 have not been solved. This study investigated whether the 11 alternatively spliced exons are spliced randomly or linked and if linked, how many combinatorial splicing profiles can be arranged in cardiac- and smooth-muscle cells. By examining three full-length cDNA libraries of the Cav1.2 transcripts isolated from rat heart and aorta, our results showed that the arrangements of some of the alternatively spliced exons are tissue-specific and tightly linked, giving rise to only 41 alternative combinatorial profiles, of which 29 have not been reported. Interestingly, the 41 combinatorial profiles were distinctively distributed in the three Cav1.2 libraries and the one named “heart 1–50” contained unexpected splice variants. Significantly, the tissue-specific cardiac- and smooth-muscle combinatorial splicing profiles of Cav1.2 channels demonstrated distinct electrophysiological properties that may help rationalize the differences observed in native currents. The unique sequences in these tissue-specific splice variants may provide the potential targets for drug design and screening. [Copyright &y& Elsevier]

• TRPC3 protein affects hypertrophy of neonatal cardiomyocytes. • Ca2+ level and influx decrease in Trpc3−/− cardiomyocytes. • Ca V 1.2 protein levels and NFAT activity are regulated by TRPC3. • Cell size increase depends on TRPC3-controlled Ca V 1.2 expression. Ca V 1.2 and transient receptor potential canonical channel 3 (TRPC3) are two proteins known to have important roles in pathological cardiac hypertrophy; however, such roles still remain unclear. A better understanding of these roles is important for furthering the clinical understanding of heart failure. We previously reported that Trpc3-knockout (KO) mice are resistant to pathologic hypertrophy and that their Ca V 1.2 protein expression is reduced. In this study, we aimed to examine the relationship between these two proteins and characterize their role in neonatal cardiomyocytes. We measured Ca V 1.2 expression in the hearts of wild-type (WT) and Trpc3−/− mice, and examined the effects of Trpc3 knockdown and overexpression in the rat cell line H9c2. We also compared the hypertrophic responses of neonatal cardiomyocytes cultured from Trpc3−/− mice to a representative hypertrophy-causing drug, isoproterenol (ISO), and measured the activity of nuclear factor of activated T cells 3 (NFAT3) in neonatal cardiomyocytes (NCMCs). We inhibited the L-type current with nifedipine, and measured the intracellular calcium concentration using Fura-2 with 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ba2+ influx. When using the Trpc3-mediated Ca2+ influx, both intracellular calcium concentration and calcium influx were reduced in Trpc3-KO myocytes. Not only was the expression of Ca V 1.2 greatly reduced in Trpc3-KO cardiac lysate, but the size of the Ca V 1.2 currents in NCMCs was also greatly reduced. When NCMCs were treated with Trpc3 siRNA, it was confirmed that the expression of Ca V 1.2 and the intracellular nuclear transfer activity of NFAT decreased. In H9c2 cells, the ISO activated- and verapamil inhibited- Ca2+ influxes were dramatically attenuated by Trpc3 siRNA treatment. In addition, it was confirmed that both the expression of Ca V 1.2 and the size of H9c2 cells were regulated according to the expression and activation level of TRPC3. We found that after stimulation with ISO, cell hypertrophy occurred in WT myocytes, while the increase in size of Trpc3-KO myocytes was greatly reduced. These results suggest that not only the cell hypertrophy process in neonatal cardiac myocytes and H9c2 cells were regulated according to the expression level of Ca V 1.2, but also that the expression level of Ca V 1.2 was regulated by TRPC3 through the activation of NFAT. [ABSTRACT FROM AUTHOR]