-
1.
Neocortical High Probability Release Sites Are Formed by Distinct Ca2+ Channel-to-Release Sensor Topographies during Development.
Bornschein, G, Eilers, J, Schmidt, H
Cell reports. 2019;(6):1410-1418.e4
Abstract
Coupling distances between Ca2+ channels and release sensors regulate vesicular release probability (pv). Tight coupling is thought to provide a framework for high pv and loose coupling for high plasticity at low pv. At synapses investigated during development, coupling distances decrease, thereby increasing pv and transmission fidelity. We find that neocortical high-fidelity synapses deviate from these rules. Paired recordings from pyramidal neurons with "slow" and "fast" Ca2+ chelators combined with experimentally constrained simulations suggest that coupling tightens significantly during development. However, fluctuation analysis revealed that neither pv (∼0.63) nor the number of release sites (∼8) changes concomitantly. Moreover, the amplitude and time course of presynaptic Ca2+ transients are not different between age groups. These results are explained by high-pv release sites with Ca2+ microdomains in young synapses and nanodomains in mature synapses. Thus, at neocortical synapses, a developmental reorganization of the active zone leaves pv unaffected, emphasizing developmental and functional synaptic diversity.
-
2.
Mibefradil, a T-type Ca2+ channel blocker also blocks Orai channels by action at the extracellular surface.
Li, P, Rubaiy, HN, Chen, GL, Hallett, T, Zaibi, N, Zeng, B, Saurabh, R, Xu, SZ
British journal of pharmacology. 2019;(19):3845-3856
-
-
Free full text
-
Abstract
BACKGROUND AND PURPOSE Mibefradil, a T-type Ca2+ channel blocker, has been investigated for treating solid tumours. However, its underlying mechanisms are still unclear. Here, we have investigated the pharmacological actions of mibefradil on Orai store-operated Ca2+ channels. EXPERIMENTAL APPROACH Human Orai1-3 cDNAs in tetracycline-regulated pcDNA4/TO vectors were transfected into HEK293 T-REx cells with stromal interaction molecule 1 (STIM1) stable expression. The Orai currents were recorded by whole-cell and excised-membrane patch clamp. Ca2+ influx or release was measured by Fura-PE3/AM. Cell growth and death were monitored by WST-1, LDH assays and flow cytometry. KEY RESULTS Mibefradil inhibited Orai1, Orai2, and Orai3 currents dose-dependently. The IC50 for Orai1, Orai2, and Orai3 channels was 52.6, 14.1, and 3.8 μM respectively. Outside-out patch demonstrated that perfusion of 10-μM mibefradil to the extracellular surface completely blocked Orai3 currents and single channel activity evoked by 2-APB. Intracellular application of mibefradil did not alter Orai3 channel activity. Mibefradil at higher concentrations (>50 μM) inhibited Ca2+ release but had no effect on cytosolic STIM1 translocation evoked by thapsigargin. Inhibition on Orai channels by mibefradil was structure-related, as other T-type Ca2+ channel blockers with different structures, such as ethosuximide and ML218, had no or minimal effects on Orai channels. Moreover, mibefradil inhibited cell proliferation, induced apoptosis, and arrested cell cycle progression. CONCLUSIONS AND IMPLICATIONS Mibefradil is a potent cell surface blocker of Orai channels, demonstrating a new pharmacological action of this compound in regulating cell growth and death, which could be relevant to its anti-cancer activity.
-
3.
Three-Dimensional and Chemical Mapping of Intracellular Signaling Nanodomains in Health and Disease with Enhanced Expansion Microscopy.
Sheard, TMD, Hurley, ME, Colyer, J, White, E, Norman, R, Pervolaraki, E, Narayanasamy, KK, Hou, Y, Kirton, HM, Yang, Z, et al
ACS nano. 2019;(2):2143-2157
Abstract
Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a β-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.
-
4.
Ca2+ to the rescue - Ca2+channels and signaling in plant immunity.
Moeder, W, Phan, V, Yoshioka, K
Plant science : an international journal of experimental plant biology. 2019;:19-26
Abstract
Ca2+ is a universal second messenger in many signaling pathways in all eukaryotes including plants. Transient changes in [Ca2+]cyt are rapidly generated upon a diverse range of stimuli such as drought, heat, wounding, and biotic stresses (infection by pathogenic and symbiotic microorganisms), as well as developmental cues. It has been known for a while that [Ca2+]cyt transient signals play crucial roles to activate plant immunity and recently significant progresses have been made in this research field. However the identity and regulation of ion channels that are involved in defense related Ca2+ signals are still enigmatic. Members of two ligand gated ion channel families, glutamate receptor-like channels (GLRs) and cyclic nucleotide-gated channels (CNGCs) have been implicated in immune responses; nevertheless more precise data to understand their direct involvement in the creation of Ca2+ signals during immune responses is necessary. Furthermore, the study of other ion channel groups is also required to understand the whole picture of the intra- and inter-cellular Ca2+ signalling network. In this review we summarize Ca2+ signals in plant immunity from an ion channel point of view and discuss future challenges in this exciting research field.
-
5.
Malan syndrome in a patient with 19p13.2p13.12 deletion encompassing NFIX and CACNA1A genes: Case report and review of the literature.
Bellucco, FT, de Mello, CB, Meloni, VA, Melaragno, MI
Molecular genetics & genomic medicine. 2019;(12):e997
Abstract
BACKGROUND Malan syndrome is a recently introduced overgrowth disorder described in a limited number of individuals. Haploinsufficiency and also point mutations of NFIX gene have been proposed as its leading causative mechanism, however, due to the limited number of cases and different deletion sizes, genotype/phenotype correlations are still limited. METHODS Here, we report the first Brazilian case of Malan syndrome caused by a 990 kb deletion in 19p13.2p13.12, focusing on clinical and behavioral aspects of the syndrome. RESULTS The patient presented with macrocephaly, facial dysmorphisms, hypotonia, developmental delay, moderate thoracolumbar scoliosis, and seizures. The intellectual and behavioral assessments showed severe cognitive, language, and adaptive functions impairments. The 19p deleted region of our patient encompasses NFIX, CACNA1A, which seems to be related to a higher frequency of seizures among individuals with microdeletions in 19p13.2, and 15 other coding genes, including CC2D1A and NACC1, both known to be involved in neurobiological process and pathways. CONCLUSION Deletions involving NFIX gene should be considered in patients with overgrowth during childhood, macrocephaly, developmental delay, and seizures, as well as severe intellectual disability.
-
6.
Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation.
Brukman, NG, Nuñez, SY, Puga Molina, LDC, Buffone, MG, Darszon, A, Cuasnicu, PS, Da Ros, VG
Journal of cellular physiology. 2019;(4):5276-5288
Abstract
Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca2+ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca 2+ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca 2+ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca 2+ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca 2+ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca 2+ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH 4 Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca 2+ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.
-
7.
Calcium signalling in T cells.
Trebak, M, Kinet, JP
Nature reviews. Immunology. 2019;(3):154-169
-
-
Free full text
-
Abstract
Calcium (Ca2+) signalling is of paramount importance to immunity. Regulated increases in cytosolic and organellar Ca2+ concentrations in lymphocytes control complex and crucial effector functions such as metabolism, proliferation, differentiation, antibody and cytokine secretion and cytotoxicity. Altered Ca2+ regulation in lymphocytes leads to various autoimmune, inflammatory and immunodeficiency syndromes. Several types of plasma membrane and organellar Ca2+-permeable channels are functional in T cells. They contribute highly localized spatial and temporal Ca2+ microdomains that are required for achieving functional specificity. While the mechanistic details of these Ca2+ microdomains are only beginning to emerge, it is evident that through crosstalk, synergy and feedback mechanisms, they fine-tune T cell signalling to match complex immune responses. In this article, we review the expression and function of various Ca2+-permeable channels in the plasma membrane, endoplasmic reticulum, mitochondria and endolysosomes of T cells and their role in shaping immunity and the pathogenesis of immune-mediated diseases.
-
8.
CACNG2 polymorphisms associate with chronic pain after mastectomy.
Bortsov, AV, Devor, M, Kaunisto, MA, Kalso, E, Brufsky, A, Kehlet, H, Aasvang, E, Bittner, R, Diatchenko, L, Belfer, I
Pain. 2019;(3):561-568
-
-
Free full text
-
Abstract
Chronic postmastectomy pain (PMP) imposes a major burden on the quality of life of the ever-increasing number of long-term survivors of breast cancer. An earlier report by Nissenbaum et al. claimed that particular polymorphisms in the gene CACNG2 are associated with the risk of developing chronic PMP after breast surgery (Nissenbaum J, Devor M, Seltzer Z, Gebauer M, Michaelis M, Tal M, Dorfman R, Abitbul-Yarkoni M, Lu Y, Elahipanah T, delCanho S, Minert A, Fried K, Persson AK, Shpigler H, Shabo E, Yakir B, Pisante A, Darvasi A. Susceptibility to chronic pain following nerve injury is genetically affected by CACNG2. Genome Res 2010;20:1180-90). This information is important because in principle, it can inform the surgical, radiological, and chemotherapeutic decision-making process in ways that could mitigate the increased risk of chronic pain. In this study, we revisited this claim by independently evaluating the proposed marker haplotype using 2 different patient cohorts recruited in different research settings. Meta-analysis of these new postmastectomy cohorts and the original cohort confirmed significant association of the CACNG2 haplotype with PMP. In addition, we tested whether the same markers would predict chronic postsurgical pain in men who underwent surgery for inguinal hernia repair, and whether there is significant genetic association with cutaneous thermal sensitivity in postmastectomy and postherniotomy patients. We found that the biomarker is selective because it did not predict pain after laparoscopic hernia repair and was not associated with pain sensitivity to experimentally applied noxious thermal stimuli. We conclude that the A-C-C haplotype at the 3 single-nucleotide polymorphisms (rs4820242, rs2284015, and rs2284017) in the CACNG2 gene is associated with increased risk of developing PMP. This information may advance current knowledge on pathophysiology of PMP and serve as a step forward in the prediction of clinical outcomes and personalized pain management.
-
9.
Host Calcium Channels and Pumps in Viral Infections.
Chen, X, Cao, R, Zhong, W
Cells. 2019;(1)
Abstract
Ca2+ is essential for virus entry, viral gene replication, virion maturation, and release. The alteration of host cells Ca2+ homeostasis is one of the strategies that viruses use to modulate host cells signal transduction mechanisms in their favor. Host calcium-permeable channels and pumps (including voltage-gated calcium channels, store-operated channels, receptor-operated channels, transient receptor potential ion channels, and Ca2+-ATPase) mediate Ca2+ across the plasma membrane or subcellular organelles, modulating intracellular free Ca2+. Therefore, these Ca2+ channels or pumps present important aspects of viral pathogenesis and virus-host interaction. It has been reported that viruses hijack host calcium channels or pumps, disturbing the cellular homeostatic balance of Ca2+. Such a disturbance benefits virus lifecycles while inducing host cells' morbidity. Evidence has emerged that pharmacologically targeting the calcium channel or calcium release from the endoplasmic reticulum (ER) can obstruct virus lifecycles. Impeding virus-induced abnormal intracellular Ca2+ homeostasis is becoming a useful strategy in the development of potent antiviral drugs. In this present review, the recent identified cellular calcium channels and pumps as targets for virus attack are emphasized.
-
10.
Electrophysiological properties of human beta-cell lines EndoC-βH1 and -βH2 conform with human beta-cells.
Hastoy, B, Godazgar, M, Clark, A, Nylander, V, Spiliotis, I, van de Bunt, M, Chibalina, MV, Barrett, A, Burrows, C, Tarasov, AI, et al
Scientific reports. 2018;(1):16994
Abstract
Limited access to human islets has prompted the development of human beta cell models. The human beta cell lines EndoC-βH1 and EndoC-βH2 are increasingly used by the research community. However, little is known of their electrophysiological and secretory properties. Here, we monitored parameters that constitute the glucose-triggering pathway of insulin release. Both cell lines respond to glucose (6 and 20 mM) with 2- to 3-fold stimulation of insulin secretion which correlated with an elevation of [Ca2+]i, membrane depolarisation and increased action potential firing. Similar to human primary beta cells, KATP channel activity is low at 1 mM glucose and is further reduced upon increasing glucose concentration; an effect that was mimicked by the KATP channel blocker tolbutamide. The upstroke of the action potentials reflects the activation of Ca2+ channels with some small contribution of TTX-sensitive Na+ channels. The repolarisation involves activation of voltage-gated Kv2.2 channels and large-conductance Ca2+-activated K+ channels. Exocytosis presented a similar kinetics to human primary beta cells. The ultrastructure of these cells shows insulin vesicles composed of an electron-dense core surrounded by a thin clear halo. We conclude that the EndoC-βH1 and -βH2 cells share many features of primary human β-cells and thus represent a useful experimental model.