Subunit Of Ca2 Research Articles

Critical Role of the Presynaptic Protein CAST in Maintaining the Photoreceptor Ribbon Synapse Triad.

The cytomatrix at the active zone-associated structural protein (CAST) and its homologue, named ELKS, being rich in glutamate (E), leucine (L), lysine (K), and serine (S), belong to a family of proteins that organize presynaptic active zones at nerve terminals. These proteins interact with other active zone proteins, including RIMs, Munc13s, Bassoon, and the β subunit of Ca2+ channels, and have various roles in neurotransmitter release. A previous study showed that depletion of CAST/ELKS in the retina causes morphological changes and functional impairment of this structure. In this study, we investigated the roles of CAST and ELKS in ectopic synapse localization. We found that the involvement of these proteins in ribbon synapse distribution is complex. Unexpectedly, CAST and ELKS, in photoreceptors or in horizontal cells, did not play a major role in ribbon synapse ectopic localization. However, depletion of CAST and ELKS in the mature retina resulted in degeneration of the photoreceptors. These findings suggest that CAST and ELKS play critical roles in maintaining neural signal transduction in the retina, but the regulation of photoreceptor triad synapse distribution is not solely dependent on their actions within photoreceptors and horizontal cells.

Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes

Mammalian voltage-activated L-type Ca2+ channels, such as Ca(v)1.2, control transmembrane Ca2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current-voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca2+ channels in excitable cells.

Mirogabalin ameliorates neuropathic pain by activating the descending noradrenergic system involving its binding to the α<sub>2</sub>δ-1 subunit of voltage-gated Ca<sup>2+</sup> channels

Mirogabalin ameliorates neuropathic pain by activating the descending noradrenergic system involving its binding to the α<sub>2</sub>δ-1 subunit of voltage-gated Ca<sup>2+</sup> channels

The Ca2+ channel subunit CaV β2a-subunit down-regulates voltage-activated ion current densities by disrupting actin-dependent traffic in chromaffin cells.

β-Subunits of the Ca2+ channel have been conventionally regarded as auxiliary subunits that regulate the expression and activity of the pore-forming α1 subunit. However, they comprise protein-protein interaction domains, such as a SRC homology 3 domain (SH3) domain, which make them potential signaling molecules. Here we evaluated the role of the β2a subunit of the Ca2+ channels (CaV β2a) and its SH3 domain (β2a-SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with CaV β2a or β2a-SH3 under different conditions, in order to acutely interfere with endogenous associations of these proteins. As assayed by whole-cell patch clamp recordings, Ca2+ currents were reduced by CaV β2a in the presence of exogenous α1-interaction domain. β2a-SH3, but not its dimerization-deficient mutant, also reduced Ca2+ currents. Na+ currents were also diminished following β2a-SH3 injection. Furthermore, β2a-SH3 was still able to reduce Ca2+ currents when dynamin-2 function was disrupted, but not when SNARE-dependent exocytosis or actin polymerization was inhibited. Together with the additional finding that both CaV β2a and β2a-SH3 diminished the incorporation of new actin monomers to cortical actin filaments, β2a-SH3 emerges as a signaling module that might down-regulate forward trafficking of ion channels by modulating actin dynamics.

Whole genome sequencing and rare variant analysis in essential tremor families.

Essential tremor (ET) is one of the most common movement disorders. The etiology of ET remains largely unexplained. Whole genome sequencing (WGS) is likely to be of value in understanding a large proportion of ET with Mendelian and complex disease inheritance patterns. In ET families with Mendelian inheritance patterns, WGS may lead to gene identification where WES analysis failed to identify the causative single nucleotide variant (SNV) or indel due to incomplete coverage of the entire coding region of the genome, in addition to accurate detection of larger structural variants (SVs) and copy number variants (CNVs). Alternatively, in ET families with complex disease inheritance patterns with gene x gene and gene x environment interactions enrichment of functional rare coding and non-coding variants may explain the heritability of ET. We performed WGS in eight ET families (n = 40 individuals) enrolled in the Family Study of Essential Tremor. The analysis included filtering WGS data based on allele frequency in population databases, rare SNV and indel classification and association testing using the Mixed-Model Kernel Based Adaptive Cluster (MM-KBAC) test. A separate analysis of rare SV and CNVs segregating within ET families was also performed. Prioritization of candidate genes identified within families was performed using phenolyzer. WGS analysis identified candidate genes for ET in 5/8 (62.5%) of the families analyzed. WES analysis in a subset of these families in our previously published study failed to identify candidate genes. In one family, we identified a deleterious and damaging variant (c.1367G>A, p.(Arg456Gln)) in the candidate gene, CACNA1G, which encodes the pore forming subunit of T-type Ca(2+) channels, CaV3.1, and is expressed in various motor pathways and has been previously implicated in neuronal autorhythmicity and ET. Other candidate genes identified include SLIT3 which encodes an axon guidance molecule and in three families, phenolyzer prioritized genes that are associated with hereditary neuropathies (family A, KARS, family B, KIF5A and family F, NTRK1). Functional studies of CACNA1G and SLIT3 suggest a role for these genes in ET disease pathogenesis.

Abstract 341: Prolonged Post-Differentiation Culture Influences the Biophysics of Na + - And Ca 2+ Channels in Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background: Cardiomyocytes (CM), derived from human induced-pluripotent stem cells (iPSC-CM), are promising tools for in vitro proarrhythmia screening for drug safety. However, among various challenges with interpretation of iPSC-CM experimental results, the influence of varied culture periods post-differentiation on the electrophysiological properties of iPSC-CMs is unclear. Objective: To test the hypothesis that prolonged post-differentiation culture of iPSC-CMs will influence the biophysical characteristics of Na + - and Ca 2+ -channels. Methods: Using whole-cell patch clamp, we compared the electrophysiological properties of differentiated ventricular-like iPSC-CMs (APD 30-40 /APD 70-80 >1.5) cultured for 10 to 15 days (D10-15) to those cultured for more than 30 days (≥ D30) both under current- and voltage- clamp configurations. Quantitative real-time PCR was used to quantify the mRNA expression of voltage-gated Na channel subunits SCN1A, SCN5A and Ca channel subunits CACNA1C, CACNA1G and CACNA1I. Fold change was calculated by 2 -ΔΔCt method. Data were analyzed by Student’s t test, and P value <0.05 was considered significant. Results: Post-differentiation prolonged culture imparts increased cellular excitability with high frequency spontaneous action potentials, robust increase in the magnitude of peak Na + -current level, relatively shallow inactivation kinetics of Na + channels, faster recovery from inactivation, and augmented Ca 2+ current density compared to early cultured iPSC-CM. This was associated with a greatly enhanced mRNA expression of α-subunit transcripts of SCN1A (7.5 fold) and SCN5A (38 fold) Na channel subtypes while less robust elevation in the mRNA expression of CACNA1C (0.5 fold), CACNA1G (2 fold) and CACNA1I (2 fold) subunits in prolonged culture compared to early cultured iPSC-CM. Conclusion: Prolonged culture of ventricular-like iPSC-CMs affects the excitability, single-cell electrophysiological properties and differential ion channels subunit expression. Standardization of post-differentiation period for ventricular-like iPSC-CMs culture is crucial for in vitro proarrhythmia screening, health/disease modelling to study cellular mechanisms or test high-throughput drugs’ efficacy and toxicity.

Medial preoptic circuit induces hunting-like actions to target objects and prey.

As animals forage, they must obtain useful targets by orchestrating appropriate actions that range from searching to chasing, biting and carrying. Here, we reveal that neurons positive for the α subunit of Ca2+/calmodulin-dependent kinase II (CaMKIIα) in the medial preoptic area (MPA) that send projections to the ventral periaqueductal gray (vPAG) mediate these target-directed actions in mice. During photostimulation of the MPA-vPAG circuit, mice vigorously engaged with 3D objects and chased moving objects. When exposed to a cricket, they hunted down the prey and bit it to kill. By applying a head-mounted object control with timely photostimulation of the MPA-vPAG circuit, we found that MPA-vPAG circuit-induced actions occurred only when the target was detected within the binocular visual field. Using this device, we successfully guided mice to navigate specified routes. Our study explains how the brain yields a strong motivation to acquire a target object along the continuum of hunting behavior.

Abstract 1881: STIM1 and Orai1 control non-Hodgkin lymphoma cells migration

Abstract Non-Hodgkin lymphoma is one of the most common cancer in United States representing 4% of all cancer. Diffuse large B cell lymphoma (DLBCL) is the most common and aggressive non-Hodgkin B lymphoma. One of the characteristic features of this disease is the dissemination of the cancer cells, through the lymphatic system in the secondary lymphoid organs and extranodal sites, leading to the death of patients. Chemokines such as the Stromal Derived Factor 1 (SDF1) control the spread and the homing of the cancer cells. It is known that SDF1 activates various signalling pathways involved in cell proliferation, transcription or migration. Moreover, SDF1 induces an increase in intracellular calcium concentration whose role is still unknown in B cells. Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway in this type of cells. By definition, SOCE is activated by Ca2+ release from the endoplasmic reticulum. Two genes are responsible for SOCE activity: Stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor that detects store depletion and ORAI1, the pore-forming subunit of Ca2+ release-activated Ca2+ (CRAC) channel. Several studies performed on adherent cells showed that Orai1/STIM1 proteins are involved in cancer cell migration. However, the molecular mechanisms involved in cell migration differ widely between adherent and non-adherent cells. We studied the role of both actors of calcium entry : Orai1 and STIM1 in DLBCL pathology and more precisely in basal and SDF1-induced of DLBCL cell migration. Using Tissue MicroArrays approach we revealed that both Orai1 and STIM1 are under-expressed in DLBCL tumoral tissue compared to normal lymphoid tissue. Next, using calcium imaging experiments we confirmed that SDF-1 triggered Ca2+ responses in two DLBCL cell lines (SUDHL4 and HLY1) involving intracellular Ca2+ store mobilization and extracellular Ca2+ influx. Based on these observations, we investigated the role of Orai1 and STIM1 on SDF1-induced Ca2+ influx using pharmacological and RNA interference approaches. The inhibition of Orai1 by BTP2 as well as the under-expression of Orai1 or STIM1 by shRNA, prevented Ca2+ influx induced by SDF1 suggesting the involvement of Orai1 and STIM1 in this process. Regarding this results , we studied the role of Orai1 and STIM1 on DLBCL cell migration in vitro and in vivo. Our results show that basal or SDF1-induced cell migration was significantly inhibited by underexpression of STIM1 or Orai1 in SUDHL4 and HLY1 cell lines. These results suggest that STIM1 and Orai1 play a key role in the DLBCL cell migration. The identification of STIM1 and Orai1 proteins as key players in the migration of DLBCL cells might provide new therapeutic targets for the treatment of this pathology. Citation Format: Simon Latour, Isabelle Mahouche, Floriane Cherrier, Jean-Philippe Merlio, Sandrine Poglio, Laurence Bresson Bepoldin. STIM1 and Orai1 control non-Hodgkin lymphoma cells migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1881. doi:10.1158/1538-7445.AM2017-1881

Autolysosome biogenesis and developmental senescence are regulated by both Spns1 and v-ATPase

ABSTRACTSpns1 (Spinster homolog 1 [Drosophila]) in vertebrates, as well as Spin (Spinster) in Drosophila, is a hypothetical lysosomal H+-carbohydrate transporter, which functions at a late stage of macroautophagy (hereafter autophagy). The Spin/Spns1 defect induces aberrant autolysosome formation that leads to developmental senescence in the embryonic stage and premature aging symptoms in adulthood. However, the molecular mechanism by which loss of Spin/Spns1 leads to the specific pathogenesis remains to be elucidated. Using chemical, genetic and CRISPR/Cas9-mediated genome-editing approaches in zebrafish, we investigated and determined a mechanism that suppresses embryonic senescence as well as autolysosomal impairment mediated by Spns1 deficiency. Unexpectedly, we found that a concurrent disruption of the vacuolar-type H+-ATPase (v-ATPase) subunit gene, atp6v0ca (ATPase, H+ transporting, lysosomal, V0 subunit ca) led to suppression of the senescence induced by the Spns1 defect, whereas the sole loss of Atp6v0ca led to senescent embryos similar to the single spns1 mutation. Moreover, we discovered that the combined stable defect seen in the presence of both the spns1 and atp6v0ca mutant genes still subsequently induced premature autophagosome-lysosome fusion marked by insufficient acidity, while extending developmental life span, compared with the solely mutated spns1 defect. Our data suggest that Spns1 and the v-ATPase orchestrate proper autolysosomal biogenesis with optimal acidification that is critically linked to developmental senescence and survival.

Pathophysiological Significance of Store-Operated Calcium Entry in Megakaryocyte Function: Opening New Paths for Understanding the Role of Calcium in Thrombopoiesis.

Store-Operated Calcium Entry (SOCE) is a universal calcium (Ca2+) influx mechanism expressed by several different cell types. It is now known that Stromal Interaction Molecule (STIM), the Ca2+ sensor of the intracellular compartments, together with Orai and Transient Receptor Potential Canonical (TRPC), the subunits of Ca2+ permeable channels on the plasma membrane, cooperate in regulating multiple cellular functions as diverse as proliferation, differentiation, migration, gene expression, and many others, depending on the cell type. In particular, a growing body of evidences suggests that a tight control of SOCE expression and function is achieved by megakaryocytes along their route from hematopoietic stem cells to platelet production. This review attempts to provide an overview about the SOCE dynamics in megakaryocyte development, with a focus on most recent findings related to its involvement in physiological and pathological thrombopoiesis.

In vivo and in vitro effects of vortioxetine on molecules associated with neuroplasticity.

Neuroplasticity is fundamental for brain functions, abnormal changes of which are associated with mood disorders and cognitive impairment. Neuroplasticity can be affected by neuroactive medications and by aging. Vortioxetine, a multimodal antidepressant, has shown positive effects on cognitive functions in both pre-clinical and clinical studies. In rodent studies, vortioxetine increases glutamate neurotransmission, promotes dendritic branching and spine maturation, and elevates hippocampal expression of the activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) at the transcript level. The present study aims to assess the effects of vortioxetine on several neuroplasticity-related molecules in different experimental systems. Chronic (1 month) vortioxetine increased Arc/Arg3.1 protein levels in the cortical synaptosomes of young and middle-aged mice. In young mice, this was accompanied by an increase in actin-depolymerizing factor (ADF)/cofilin serine 3 phosphorylation without altering the total ADF/cofilin protein level, and an increase in the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor phosphorylation at serine 845 (S845) without altering serine 831 (S831) GluA1 phosphorylation nor the total GluA1 protein level. Similar effects were detected in cultured rat hippocampal neurons: Acute vortioxetine increased S845 GluA1 phosphorylation without changing S831 GluA1 phosphorylation or the total GluA1 protein level. These changes were accompanied by an increase in α subunit of Ca2+/calmodulin-dependent kinase (CaMKIIα) phosphorylation (at threonine 286) without changing the total CaMKIIα protein level in cultured neurons. In addition, chronic (1 month) vortioxetine, but not fluoxetine, restored the age-associated reduction in Arc/Arg3.1 and c-Fos transcripts in the frontal cortex of middle-aged mice. Taken together, these results demonstrated that vortioxetine modulates molecular targets that are related to neuroplasticity.

Studies of the expression of subunits α2 and β1 of Na+/K+-ATPase, α1S (L-type) Ca2+-channel, and SERCA 1/2/3 of Ca2+-ATPase of phasic and postural rat muscles in a model of hypogravity using the method of fluorescent microscopy

Using fluorescent microscopy, we found decreased expression of the β1 subunit of Na+/K+-ATPase and subunits of Ca2+-ATPase, increased expression of the α1S subunit of the L-type Ca2+-channel, and no changes in the expression of the α2 subunit of Na+/K+-ATPase in rat postural muscle under the conditions of modeled hypogravity. In the phasic muscle, we observed decreased expression of the β1 subunit, which was similar to that found in the postural muscle, whereas the other studied parameters remained without alterations. However, a decrease in the fluorescence intensity of the β1 subunit was insignificant due to a high variability of data. Thus, hypogravity negatively influenced primarily those skeletal muscles that are responsible for static load.

5α-Dihydrotestosterone regulates the expression of L-type calcium channels and calcium-binding protein regucalcin in human breast cancer cells with suppression of cell growth.

Androgens have been associated with the development of normal breast, and their role in mammary gland carcinogenesis has also been described. Several studies reported that androgens inhibit breast cancer cell growth, whereas others linked their action with the modulation of calcium (Ca(2+)) pumps, Ca(2+) channels and Ca(2+)-binding proteins. Also, it is known that deregulated Ca(2+) homeostasis has been implicated in the pathophysiology of breast. The L-type Ca(2+) channels (LTCCs) were found to be up-regulated in colon, colorectal and prostate cancer, but their presence in breast tissues remains uncharacterized. On the other hand, regucalcin (RGN) is a Ca(2+)-binding protein involved in the control of mammary gland cell proliferation, which has been identified as an androgen target gene in distinct tissues except breast. This study aimed to confirm the expression and activity of LTCCs in human breast cancer cells and investigate the effect of androgens in regulating the expression of α1C subunit (Cav1.2) of LTCCs and Ca(2+)-binding protein RGN. PCR, Western blot, immunofluorescence and electrophysiological experiments demonstrated the expression and activity of Cav1.2 subunit in MCF-7 cells. The MCF-7 cells were treated with 1, 10 or 100nM of 5α-dihydrotestosterone (DHT) for 24-72h. The obtained results showed that 1nM DHT up-regulated the expression of Cav1.2 subunit while diminishing RGN protein levels, which was underpinned by reduced cell viability. These findings first confirmed the presence of LTCCs in breast cancer cells and opened new perspectives for the development of therapeutic approaches targeting Ca(2+) signaling.

Piezo1 Integration of Vascular Architecture with Physiological Force

The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca2+‐permeable non‐selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial‐specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear‐stress‐evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology. This work was supported by Welcome trust and Medical Research Council.

Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel β4 subunit: a low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents.

Rem, Rad, Kir/Gem (RGK) proteins, including Rem2, mediate profound inhibition of high-voltage activated Ca(2+) channels containing intracellular regulatory β subunits. All RGK proteins bind to voltage-gated Ca(2+) channel β subunit (Cavβ) subunits in vitro, but the necessity of the interaction for current inhibition remains controversial. This study applies NMR and calorimetric techniques to map the binding site for Rem2 on human Cavβ4a and measure its binding affinity. Our experiments revealed 2 binding surfaces on the β4 guanylate kinase domain contributing to a 156 ± 18 µM Kd interaction: a hydrophobic pocket lined by 4 critical residues (L173, N261, H262, and V303), mutation of any of which completely disrupted binding, and a nearby surface containing 3 residues (D206, L209, and D258) that when individually mutated decreased affinity. Voltage-gated Ca(2+) channel α1A subunit (Cav2.1) Ca(2+) currents were completely inhibited by Rem2 when co-expressed with wild-type Cavβ4a, but were unaffected by Rem2 when coexpressed with a Cavβ4a site 1 (L173A/V303A) or site 2 (D258A) mutant. These results provide direct evidence for a low-affinity Rem2/Cavβ4 interaction and show definitively that the interaction is required for Cav2.1 inhibition.

Opposite regulation of inhibitory synaptic plasticity by α and β subunits of Ca(2+)/calmodulin-dependent protein kinase II.

Induction of several forms of synaptic plasticity, a cellular basis for learning and memory, depends on the activation of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII). CaMKII acts as a holoenzyme consisting of α and β subunits (α- and βCaMKII). However, it remains elusive how the subunit composition of a CaMKII holoenzyme affects its activation and hence synaptic plasticity. We addressed this issue by focusing on long-term potentiation (LTP) at inhibitory synapses on cerebellar Purkinje neurons (PNs) (called rebound potentiation, RP). The contribution of each subunit to RP was examined by selective knock-down or overexpression of that subunit. Electrophysiological recording from a rat cultured PN demonstrated that βCaMKII is essential for RP induction, whereas αCaMKII suppresses it. Thus, RP was negatively regulated due to the greater relative abundance of αCaMKII compared to βCaMKII, suggesting a critical role of CaMKII subunit composition in RP. The higher affinity of βCaMKII to Ca(2+)/CaM compared with αCaMKII was responsible for the predominant role in RP induction. Live-cell imaging of CaMKII activity based on the Förster resonance energy transfer (FRET) technique revealed that βCaMKII enrichment enhances the total CaMKII activation upon a transient conditioning depolarization. Taken together, these findings clarified that α- and βCaMKII oppositely regulate CaMKII activation, controlling the induction of inhibitory synaptic plasticity in a PN, which might contribute to the adaptive information processing of the cerebellar cortex.

The evolution of the four subunits of voltage-gated calcium channels: ancient roots, increasing complexity, and multiple losses.

The alpha subunits of voltage-gated calcium channels (Cavs) are large transmembrane proteins responsible for crucial physiological processes in excitable cells. They are assisted by three auxiliary subunits that can modulate their electrical behavior. Little is known about the evolution and roles of the various subunits of Cavs in nonbilaterian animals and in nonanimal lineages. For this reason, we mapped the phyletic distribution of the four channel subunits and reconstructed their phylogeny. Although alpha subunits have deep evolutionary roots as ancient as the split between plants and opistokonths, beta subunits appeared in the last common ancestor of animals and their close-relatives choanoflagellates, gamma subunits are a bilaterian novelty and alpha2/delta subunits appeared in the lineage of Placozoa, Cnidaria, and Bilateria. We note that gene losses were extremely common in the evolution of Cavs, with noticeable losses in multiple clades of subfamilies and also of whole Cav families. As in vertebrates, but not protostomes, Cav channel genes duplicated in Cnidaria. We characterized by in situ hybridization the tissue distribution of alpha subunits in the sea anemone Nematostella vectensis, a nonbilaterian animal possessing all three Cav subfamilies common to Bilateria. We find that some of the alpha subunit subtypes exhibit distinct spatiotemporal expression patterns. Further, all six sea anemone alpha subunit subtypes are conserved in stony corals, which separated from anemones 500 MA. This unexpected conservation together with the expression patterns strongly supports the notion that these subtypes carry unique functional roles.

Piezo1 integration of vascular architecture with physiological force.

The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic1-5. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca2+-permeable non-selective cationic channels for detection of noxious mechanical impact6-8. Here we show Piezo1 (FAM38A) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. Importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx was protease activity and spatial organization of endothelial cells to the polarity of the applied force. The data suggest Piezo1 channels as pivotal integrators in vascular biology.

Molecular imaging reveals epileptogenic Ca2+-channel promoter activation in hippocampi of living mice.

Focal epilepsies often originate in the hippocampal formation of the temporal lobe (temporal lobe epilepsy) and are generally acquired after transient brain insults. Such insults induce cellular and structural reorganization processes of the hippocampus, referred to as epileptogenesis that finally convert the brain spontaneous epileptic. Here, we developed a new molecular imaging strategy in a state-of-the-art animal model to provide insights into key epileptogenic mechanisms. Our new approach combines recombinant adeno-associated virus (rAAV) gene delivery with in vivo bioluminescence imaging. rAAV particles harboring the luciferase reporter gene under control of the minimal T type Ca(2+)-channel subunit Ca V 3.2-promoter were generated and injected stereotaxically in the hippocampal region of mice. Bioluminescent signals, corresponding to Ca V 3.2 promoter activation, were imaged in vivo in the pilocarpine model of status epilepticus (SE). We detected activation of key Ca V 3.2 promoter motifs at 3 and 10 days after SE but not after the onset of chronic seizures. These data suggest Ca V 3.2 promoter activation as novel anti-epileptogenic target. In more general terms, we have established an experimental approach that allows to follow cerebral gene promoter dynamics longitudinally and to correlate this activity to behavioral parameters in the same mice.

Crotoxin from Crotalus durissus terrificus snake venom induces the release of glutamate from cerebrocortical synaptosomes via N and P/Q calcium channels

Crotoxin (Crtx), the main toxin in the venom of Crotalus durissus terrificus snake, is a heterodimer with a basic subunit, CB, and an acidic subunit, CA. CB is a phospholipase A2 that depends on CA to specifically bind to the cell membrane. This toxin acts in the central nervous system (CNS) causing chronic seizure effects and other cytotoxic effects. Here, we report its action on glutamate release in rat cerebral cortex synaptosomes. Aiming at a better understanding of the mechanism of action of Crtx, calcium channel blockers were used and internalization studies were performed in cerebellar granule neurons. Our results show that Crtx induces calcium-dependent glutamate release via N and P/Q calcium channels. In addition, the CB subunit of Crtx is shown to be internalized. This internalization does not depend on the presence of CA subunit neither on the PLA2 activity of CB. A correlation between CB internalization and glutamate release remains to be established.