Calcium Channel Complex Research Articles

Differential expression of ion channel coding genes in the endometrium of women experiencing recurrent implantation failures

Our study probed the differences in ion channel gene expression in the endometrium of women with Recurrent Implantation Failure (RIF) compared to fertile women. We analyzed the relative expression of genes coding for T-type Ca2+, ENaC, CFTR, and KCNQ1 channels in endometrial samples from 20 RIF-affected and 10 control women, aged 22–35, via microarray analysis and quantitative real-time PCR. Additionally, we examined DNA methylation in the regulatory region of KCNQ1 using ChIP real-time PCR. The bioinformatics component of our research included Gene Ontology analysis, protein–protein interaction networks, and signaling pathway mapping to identify key biological processes and pathways implicated in RIF. This led to the discovery of significant alterations in the expression of ion channel genes in RIF women’s endometrium, most notably an overexpression of CFTR and reduced expression of SCNN1A, SCNN1B, SCNN1G, CACNA1H, and KCNQ1. A higher DNA methylation level of KCNQ1’s regulatory region was also observed in RIF patients. Gene-set enrichment analysis highlighted a significant presence of genes involved with ion transport and membrane potential regulation, particularly in sodium and calcium channel complexes, which are vital for cation movement across cell membranes. Genes were also enriched in broader ion channel and transmembrane transporter complexes, underscoring their potential extensive role in cellular ion homeostasis and signaling. These findings suggest a potential involvement of ion channels in the pathology of implantation failure, offering new insights into the mechanisms behind RIF and possible therapeutic targets.

Identification of novel candidate genes associated with non-syndromic tooth agenesis in Mongolian families

ObjectivesThis study aimed to identify genetic variants associated with non-syndromic tooth agenesis (TA) in nine families from Mongolia using whole-exome sequencing (WES) and bioinformatics analysis.Material and methodsThe study enrolled 41 participants, including three inherited and six non-inherited families. WES analysis was performed on 14 saliva samples from individuals with non-syndromic TA. The potential candidate genes were identified through variant filtering and segregation analysis. The filtered variants were then analyzed in silico mutation impact analysis.ResultsWES analysis identified 21 variants associated with TA, and 5 of these variants met all filtering criteria. These variants were located in the exome region of MAST4, ITGA6, PITX2, CACNA1S, and CDON genes. The variant in PITX2 was found in eight participants from inherited and non-inherited families, while the MAST4 variant was identified in 6 participants from inherited families.ConclusionsThe study identified various genetic variant candidates associated with TA in different family groups, with PITX2 being the most commonly identified. Our findings suggest that MAST4 may also be a novel candidate gene for TA due to its association with the Wnt signaling pathway. Additionally, we found that five candidate genes related to focal adhesion and calcium channel complex were significant and essential in tooth development.Clinical relevanceIdentifying new pathogenic genes associated with TA can improve our understanding of the molecular mechanisms underlying the disease, leading to better diagnosis, prevention, and treatment. Early detection of TA based on biomarkers can improve dental management and facilitate orthodontic and prosthetic treatment.

O-166 Distinct molecular signatures of successful sperm in situ

Abstract Out of millions of ejaculated sperm, a few reach the fertilization site in mammals. Flagellar Ca2+ signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca2+ and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that spermatozoa past the utero-tubal junction possess the intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.

Molecular Mechanisms of Nuclear Transport of the Neuronal Voltage-gated Ca2+ Channel β3 Auxiliary Subunit

Previous studies have shown that in addition to its role within the voltage-gated calcium channel complex in the plasma membrane, the neuronal CaVβ subunit can translocate to the cell nucleus. However, little is known regarding the role this protein could play in the nucleus, nor the molecular mechanism used by CaVβ to enter this cell compartment. This report shows evidence that CaVβ3 has nuclear localization signals (NLS) that are not functional, suggesting that the protein does not use a classical nuclear import pathway. Instead, its entry into the nucleus could be associated with another protein that would function as a carrier, using a mechanism known as a piggyback. Mass spectrometry assays and bioinformatic analysis allowed the identification of proteins that could be participating in the entry of CaVβ3 into the nucleus. Likewise, through proximity ligation assays (PLA), it was found that members of the heterogeneous nuclear ribonucleoproteins (hnRNPs) and B56δ, a regulatory subunit of the protein phosphatase 2A (PP2A), could function as proteins that regulate this piggyback mechanism. On the other hand, bioinformatics and site-directed mutagenesis assays allowed the identification of a functional nuclear export signal (NES) that controls the exit of CaVβ3 from the nucleus, which would allow the completion of the nuclear transport cycle of the protein. These results reveal a novel mechanism for the nuclear transport cycle of the neuronal CaVβ3 subunit.

Comprehensive Analysis of Transcriptomic Profiles Identified the Prediction of Prognosis and Drug Sensitivity of Aminopeptidase-Like 1 (NPEPL1) for Clear Cell Renal Cell Carcinoma.

Aminopeptidase-like 1 (NPEPL1) is a member of the aminopeptidase group that plays a role in the development and progression of various diseases. Expression of NPEPL1 has been reported to be involved in prostate, breast, and colorectal cancers. However, the role and mechanism of NPEPL1 in clear cell renal cell carcinoma (ccRCC) are unclear. The Cancer Genome Atlas (TCGA) and Human Protein Atlas (HPA) databases were used to predict the relationship between clinicopathological features and NPEPL1 expression. Changes in immune status and drug sensitivity with NPEPL1 expression were analyzed by the "CIBERSORT" function in R software. The results found that NPEPL1 expression was upregulated in ccRCC tissues, with expression progressively increasing with ccRCC stage and grade. Patients with high NPEPL1 expression presented with a poor prognosis across different clinicopathological features. Univariate and multivariate Cox regression analyses indicated that aberrant NPEPL1 expression was an independent risk factor for ccRCC. The nomogram showed that NPEPL1 expression improved the accuracy of predicting the prognosis of ccRCC patients. The Gene Ontology (GO) term enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that NPEPL1 may be involved in the development of ccRCC through the voltage-gated calcium channel complex, channel activity, cAMP signaling pathway, and oxytocin signaling pathway. The coexpression analysis found that NPEPL1 altered tumor characteristics by interacting with related genes. The "CIBERSORT" analysis showed that elevated NPEPL1 expression was followed by an enrichment of regulatory T cells and follicular helper T cells in the microenvironment. The drug sensitivity analysis found patients with high NPEPL1 expression had a higher benefit from axitinib, cisplatin, and GSK429286A. In conclusion, upregulation of NPEPL1 expression was involved in ccRCC prognosis and treatment. NPEPL1 could be used as a therapeutic target to guide clinical dosing.

β subunits of voltage-gated calcium channels in cardiovascular diseases.

Calcium signaling is required in bodily functions essential for survival, such as muscle contractions and neuronal communications. Of note, the voltage-gated calcium channels (VGCCs) expressed on muscle and neuronal cells, as well as some endocrine cells, are transmembrane protein complexes that allow for the selective entry of calcium ions into the cells. The α1 subunit constitutes the main pore-forming subunit that opens in response to membrane depolarization, and its biophysical functions are regulated by various auxiliary subunits-β, α2δ, and γ subunits. Within the cardiovascular system, the γ-subunit is not expressed and is therefore not discussed in this review. Because the α1 subunit is the pore-forming subunit, it is a prominent druggable target and the focus of many studies investigating potential therapeutic interventions for cardiovascular diseases. While this may be true, it should be noted that the direct inhibition of the α1 subunit may result in limited long-term cardiovascular benefits coupled with undesirable side effects, and that its expression and biophysical properties may depend largely on its auxiliary subunits. Indeed, the α2δ subunit has been reported to be essential for the membrane trafficking and expression of the α1 subunit. Furthermore, the β subunit not only prevents proteasomal degradation of the α1 subunit, but also directly modulates the biophysical properties of the α1 subunit, such as its voltage-dependent activities and open probabilities. More importantly, various isoforms of the β subunit have been found to differentially modulate the α1 subunit, and post-translational modifications of the β subunits further add to this complexity. These data suggest the possibility of the β subunit as a therapeutic target in cardiovascular diseases. However, emerging studies have reported the presence of cardiomyocyte membrane α1 subunit trafficking and expression in a β subunit-independent manner, which would undermine the efficacy of β subunit-targeting drugs. Nevertheless, a better understanding of the auxiliary β subunit would provide a more holistic approach when targeting the calcium channel complexes in treating cardiovascular diseases. Therefore, this review focuses on the post-translational modifications of the β subunit, as well as its role as an auxiliary subunit in modulating the calcium channel complexes.

A novel calcium channel Cavβ2 splice variant with unique properties predominates in the retina

Cavβ subunits are essential for surface expression of voltage-gated calcium channel complexes and crucially modulate biophysical properties like voltage-dependent inactivation. Here, we describe the discovery and characterization of a novel Cavβ2 variant with distinct features that predominates in the retina. We determined spliced exons in retinal transcripts of the Cacnb2 gene, coding for Cavβ2, by RNA-Seq data analysis and quantitative PCR. We cloned a novel Cavβ2 splice variant from mouse retina, which we are calling β2i, and investigated biophysical properties of calcium currents with this variant in a heterologous expression system as well as its intrinsic membrane interaction when expressed alone. Our data showed that β2i predominated in the retina with expression in photoreceptors and bipolar cells. Furthermore, we observed that the β2i N-terminus exhibited an extraordinary concentration of hydrophobic residues, a distinct feature not seen in canonical variants. The biophysical properties resembled known membrane-associated variants, and β2i exhibited both a strong membrane association and a propensity for clustering, which depended on hydrophobic residues in its N-terminus. We considered available Cavβ structure data to elucidate potential mechanisms underlying the observed characteristics but resolved N-terminus structures were lacking and thus, precluded clear conclusions. With this description of a novel N-terminus variant of Cavβ2, we expand the scope of functional variation through N-terminal splicing with a distinct form of membrane attachment. Further investigation of the molecular mechanisms underlying the features of β2i could provide new angles on the way Cavβ subunits modulate Ca2+ channels at the plasma membrane.

Constitutive activation of a nuclear-localized calcium channel complex in Medicago truncatula

Nuclear Ca2+ oscillations allow symbiosis signaling, facilitating plant recognition of beneficial microsymbionts, nitrogen-fixing rhizobia, and nutrient-capturing arbuscular mycorrhizal fungi. Two classes of channels, DMI1 and CNGC15, in a complex on the nuclear membrane, coordinate symbiotic Ca2+ oscillations. However, the mechanism of Ca2+ signature generation is unknown. Here, we demonstrate spontaneous activation of this channel complex, through gain-of-function mutations in DMI1, leading to spontaneous nuclear Ca2+ oscillations and spontaneous nodulation, in a CNGC15-dependent manner. The mutations destabilize a hydrogen-bond or salt-bridge network between two RCK domains, with the resultant structural changes, alongside DMI1 cation permeability, activating the channel complex. This channel complex was reconstituted in human HEK293T cell lines, with the resultant calcium influx enhanced by autoactivated DMI1 and CNGC15s. Our results demonstrate the mode of activation of this nuclear channel complex, show that DMI1 and CNGC15 are sufficient to create oscillatory Ca2+ signals, and provide insights into its native mode of induction.

Sex-heterogeneous SNPs disproportionately influence gene expression and health.

Phenotypic differences across sexes are pervasive, but the genetic architecture of sex differences within and across phenotypes is mostly unknown. In this study, we aimed to improve detection power for sex-differentially contributing SNPs previously demonstrated to be enriched in disease association, and we investigate their functions in health, pathophysiology, and genetic function. We leveraged GIANT and UK Biobank summary statistics and defined a set of 2,320 independent SNPs having sexually dimorphic effects within and across biometric traits (MAF > 0.001, P < 5x10-8). Biometric trait sex-heterogeneous SNPs (sex-het SNPs) showed enrichment in association signals for 20 out of 33 diseases/traits at 5% alpha compared to sex-homogeneous matched SNPs (empP < 0.001), and were significantly overrepresented in muscle, skeletal and stem cell development processes, and in calcium channel and microtubule complexes (FDR < 0.05, empP < 0.05). Interestingly, we found that sex-het SNPs significantly map to predicted expression quantitative trait loci (Pr-eQTLs) across brain and other tissues, methylation quantitative trait loci (meQTLs) during development, and transcription start sites, compared to sex-homogeneous SNPs. Finally, we verified that the sex-het disease/trait enrichment was not explained by Pr-eQTL enrichment alone, as sex-het Pr-eQTLs were more enriched than matched sex-homogeneous Pr-eQTLs. We conclude that genetic polymorphisms with sexually dimorphic effects on biometric traits not only contribute to fundamental embryogenic processes, but later in life play an outsized role in disease risk. These sex-het SNPs disproportionately influence gene expression and have a greater influence on disorders of body and brain than other expression-regulatory variation. Together, our data emphasize the genetic underpinnings of sexual dimorphism and its role in human health.

Intracerebral hemorrhage alters α2δ1 and thrombospondin expression in rats

Calcium voltage-gated channel auxiliary subunit (α2δ1) is a non-essential subunit of the voltage-gated calcium channel complex and is ubiquitously expressed in a number of tissues, including the brain. Thrombospondin (TSP)1 and TSP2 are extracellular matrix proteins and belong to the multi-domain glycoprotein family of macromolecular oligomers. TSP1/2 and α2δ1 promote synapse formation and functional recovery following cerebral infarction. However, to the best of our knowledge, the expression levels of α2δ1 and TSP1/2 in brain tissues, and the effects of intracerebral hemorrhage (ICH) on these levels have not yet been elucidated. The present study established a rat model of hemorrhage induced by injecting collagenase IV into the striatum to determine the changes in α2δ1 and TSP1/2 expression following ICH. The protein expression levels of α2δ1 and TSP1 in the striatum after hemorrhage were significantly increased on day 5 and returned to baseline levels on day 21; however, the protein expression levels of TSP2 were decreased on day 5, whereas they increased on day 14, subsequently returning to baseline levels. In addition, using proteomics analysis of tissues from the sham group (saline injection) and at 24 h post-ICH, it was found that both α2δ1 and TSP1 interacted with neural EGFL like 2. Taken together, these findings suggested that the expression levels of α2δ1 and TSP1/2 were altered in brain tissues in response to ICH.

CRACR2A associated immunodeficiency – the supporting factor takes center stage

Loss of function mutations in one of the core components of the calcium release-activated calcium (CRAC) channel complex, STIM1 and ORAI1, lead to severe immunodeficiency. Whether changes in regulatory components of the CRAC channel may also contribute to a disease state remained unknown. In a recent study, Wu et al. report a case of late onset immunodeficiency in a patient with bi-allelic mutation in CRACR2A, a regulatory component of the CRAC channel.

C2CD6 regulates targeting and organization of the CatSper calcium channel complex in sperm flagella.

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here, we report identification of an uncharacterized protein, C2CD6, as a subunit of the mouse CatSper complex. C2CD6 contains a calcium-dependent, membrane-targeting C2 domain. C2CD6 associates with the CatSper calcium-selective, core-forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. CatSper currents are present but at a significantly lower level in C2CD6-deficient sperm. Transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 facilitates incorporation of the CatSper complex into the flagellar plasma membrane and may function as a calcium sensor. The identification of C2CD6 may enable the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

Network Pharmacology-Based Strategy and Molecular Docking to Explore the Potential Mechanism of Jintiange Capsule for Treating Osteoporosis.

Background With the advent of ageing population, osteoporosis (OP) has already become a global challenge. Jintiange capsule is extensively applied to treat OP in China. Although recent studies demonstrate that it generates significant effects on strengthening bone, the exact mechanism of the jintiange capsule for treating OP remains unknown. Purpose To understand the main ingredients of the jintiange capsule, predict the possible targets and the relevant signal transduction pathways, and explore the mechanism of the jintiange capsule for the treatment of OP. Methods Main ingredients of the jintiange capsule, drug targets, and potential disease targets for OP were obtained from public databases. Molecular biological processes and signaling pathways were determined via bioinformatic analysis, containing protein-protein interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG). Subsequently, the disease-drug-ingredient-targets-pathways networks were constructed using Cytoscape. According to CytoNCA, core targets were acquired. Finally, the present study conducted molecular docking for better testing the abovementioned results. Results In the current work, we found that 4 main ingredients of the jintiange capsule, 33 drug targets, 4745 potential disease targets for OP, and 12 overlapping targets were identified. PPI network containing 12 nodes and 25 edges proved that there existed a complex relationship. As revealed by GO functional annotation, the intersected targets were mostly associated with BP, CC, and MF. The targets were enriched to 368 items in BP, 27 items in CC, and 42 items in MF. They mainly included calcium ion homeostasis, calcium channel complex, and calcium channel regulator activity. According to KEGG pathway analysis, the intersected targets were mostly associated with Rap 1, cGMP-PKG, Ras, cAMP, calcium pathways, and so on. Based on the analysis with CytoNCA, we acquired 4 core targets, respectively—CALR, SPARC, CALM1, and CALM2. Besides, 2 core targets, CALR and CALM1, were selected for molecular docking experiments. Molecular docking revealed that the main ingredient, calcium phosphate, had good binding with the CALR protein and CALM1 protein. Conclusion To conclude, the main ingredient of the jintiange capsule, particularly calcium phosphate, may interact with 2 targets, CALR and CALM1, and regulate multiple signaling pathways to treat OP. Additionally, this also benefits us in further understanding the mechanism of the jintiange capsule for treating OP.

The Negative Effect of Time and Air Exposure on HSC/HSPC Populations

Background: Hematopoietic stem and progenitor cells (HSC/HSPCs) inhabit niches within bone marrow that have significantly lower oxygen tension (1-4% O2, LowO2) than ambient air (21% O2) and provide signals regulating HSC signaling/function/self-renewal. Despite this knowledge, the majority of HSC/HSPC experiments are performed in ambient air, not replicating the endogenous environment. Our previous studies of HSC/HSPCs in LowO2 demonstrated enhanced phenotypic (CD150) marker expression, and frequency, of HSC/HSPC in live cells (~3fold LSK/LSKCD150 p=0.04) relative to historic data (fixed cells) as well as alterations in novel signaling/functional pathways. &#x0D; &#x0D; Methods: Our novel technology facilitates the isolation/sorting, and analysis of rare HSC populations, in live cells, continuously at their native LowO2 environments (3% O2). All assays have been adapted and validated in LowO2. &#x0D; &#x0D; Results: Time course (0 min, 15 min, 40 min, 60 min, 180 min) analysis comparing phenotypic markers of HSC/HSPC populations at LowO2 and ambient air, showed HSC/HSPC enhancement at baseline in LowO2 that were significantly blunted by exposure to air (p-value =.00007) and time beginning at 40 min (p-value =.00003). Phenotypic expression, as shown by the marker CD150, was also diminished by exposure to air (p-value =.0008) and via time (p-value =.05). Analysis via mRNA sequencing showed enhancement in 324 and 73 (LSK, LSKCD150) pathways including calcium channel complex and calcium ion binding when HSC/HSPC remained in LowO2 compared to air. Analysis of calcium regulation via FURA staining correlated with these data confirming increased calcium influx in LowO2 HSC/HSPC populations. &#x0D; &#x0D; Potential Impact: Our findings demonstrate significant negative impacts of ambient air, fixation, and time from harvest on HSC/HSPC phenotype and function. These findings suggest the critical importance of studying HSC/HSPC, live, quickly, and in their native environment, highlight potential caveats of current approaches, and rationale for lack of comparable results for studies of HSC/HSPC in the literature.

3D in situ imaging of the female reproductive tract reveals molecular signatures of fertilizing spermatozoa in mice.

Out of millions of ejaculated sperm, a few reach the fertilization site in mammals. Flagellar Ca2+ signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca2+ and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that spermatozoa past the utero-tubal junction possess the intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.

Metabolic insights from a GHSR-A203E mutant mouse model

ObjectiveBinding of ghrelin to its receptor, growth hormone secretagogue receptor (GHSR), stimulates GH release, induces eating, and increases blood glucose. These processes may also be influenced by constitutive (ghrelin-independent) GHSR activity, as suggested by findings in short people with naturally occurring GHSR-A204E mutations and reduced food intake and blood glucose in rodents administered GHSR inverse agonists, both of which impair constitutive GHSR activity. In this study, we aimed to more fully determine the physiologic relevance of constitutive GHSR activity. MethodsWe generated mice with a GHSR mutation that replaces alanine at position 203 with glutamate (GHSR-A203E), which corresponds to the previously described human GHSR-A204E mutation, and used them to conduct ex vivo neuronal electrophysiology and in vivo metabolic assessments. We also measured signaling within COS-7 and HEK293T cells transfected with wild-type GHSR (GHSR-WT) or GHSR-A203E constructs. ResultsIn COS-7 cells, GHSR-A203E resulted in lower baseline IP3 accumulation than GHSR-WT; ghrelin-induced IP3 accumulation was observed in both constructs. In HEK293T cells co-transfected with voltage-gated CaV2.2 calcium channel complex, GHSR-A203E had no effect on basal CaV2.2 current density while GHSR-WT did; both GHSR-A203E and GHSR-WT inhibited CaV2.2 current in the presence of ghrelin. In cultured hypothalamic neurons from GHSR-A203E and GHSR-deficient mice, native calcium currents were greater than those in neurons from wild-type mice; ghrelin inhibited calcium currents in cultured hypothalamic neurons from both GHSR-A203E and wild-type mice. In brain slices, resting membrane potentials of arcuate NPY neurons from GHSR-A203E mice were hyperpolarized compared to those from wild-type mice; the same percentage of arcuate NPY neurons from GHSR-A203E and wild-type mice depolarized upon ghrelin exposure. The GHSR-A203E mutation did not significantly affect body weight, body length, or femur length in the first ∼6 months of life, yet these parameters were lower in GHSR-A203E mice after 1 year of age. During a 7-d 60% caloric restriction regimen, GHSR-A203E mice lacked the usual marked rise in plasma GH and demonstrated an exaggerated drop in blood glucose. Administered ghrelin also exhibited reduced orexigenic and GH secretagogue efficacies in GHSR-A203E mice. ConclusionsOur data suggest that the A203E mutation ablates constitutive GHSR activity and that constitutive GHSR activity contributes to the native depolarizing conductance of GHSR-expressing arcuate NPY neurons. Although the A203E mutation does not block ghrelin-evoked signaling as assessed using in vitro and ex vivo models, GHSR-A203E mice lack the usual acute food intake response to administered ghrelin in vivo. The GHSR-A203E mutation also blunts GH release, and in aged mice leads to reduced body length and femur length, which are consistent with the short stature of human carriers of the GHSR-A204E mutation.

A homozygous missense variant in CACNB4 encoding the auxiliary calcium channel beta4 subunit causes a severe neurodevelopmental disorder and impairs channel and non-channel functions.

P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 α1 subunit and the auxiliary α2δ-2 and β4 subunits. β4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat β4b cDNA. Heterologously expressed wild-type β4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the β4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, β4b and β4b-L125P augmented the calcium current amplitudes, however, β4b-L125P failed to stably complex with α1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of β4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype β4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of β4b in cultured myotubes and hippocampal neurons. While binding of β4b to the known interaction partner PPP2R5D (B56δ) was not affected by the mutation, complex formation between β4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of β4b.

Potassium and Calcium Channel Complexes as Novel Targets for Cancer Research.

The intracellular Ca2+ concentration is mainly controlled by Ca2+ channels. These channels form complexes with K+ channels, which function to amplify Ca2+ flux. In cancer cells, voltage-gated/voltage-dependent Ca2+ channels and non-voltage-gated/voltage-independent Ca2+ channels have been reported to interact with K+ channels such as Ca2+-activated K+ channels and voltage-gated K+ channels. These channels are activated by an increase in cytosolic Ca2+ concentration or by membrane depolarization, which induces membrane hyperpolarization, increasing the driving force for Ca2+ flux. These complexes, composed of K+ and Ca2+ channels, are regulated by several molecules including lipids (ether lipids and cholesterol), proteins (e.g. STIM), receptors (e.g. S1R/SIGMAR1), and peptides (e.g. LL-37) and can be targeted by monoclonal antibodies, making them novel targets for cancer research.

CACNA2D3 Enhances the Chemosensitivity of Esophageal Squamous Cell Carcinoma to Cisplatin via Inducing Ca2+-Mediated Apoptosis and Suppressing PI3K/Akt Pathways.

Resistance to platinum-based combination chemotherapy is the main cause of poor prognosis in patients with advanced esophageal squamous cell carcinoma (ESCC). Previously, we showed that CACNA2D3 (voltage-dependent subunit alpha 2 delta 3 of a calcium channel complex) was significantly downregulated and functioned as a tumor suppressor in ESCC, but its role in the chemosensitivity of ESCC to cisplatin remained unknown. Here, we found that the expression of CACNA2D3 was significantly associated with poor platinum response in ESCC patients from the Gene Expression Omnibus database. Overexpression of CACNA2D3 increased sensitivity to cisplatin in ESCC in vitro, whereas knockdown of CACNA2D3 increased cisplatin resistance. CACNA2D3 promoted cisplatin-induced apoptosis by modulating intracellular Ca2+ stores. In vivo experiments further showed that overexpression of CACNA2D3 enhanced cisplatin anti-tumor effects in a xenograft mouse model. CACNA2D3 overexpression also resulted in the attenuation of PI3K and Akt phosphorylation. Treatment with the PI3K/Akt inhibitor LY294002 restored the chemosensitivity of CACAN2D3-knockdown cells to cisplatin. In conclusion, the results of the current study indicate that CACAN2D3 enhances the chemosensitivity of ESCC to cisplatin via inducing Ca2+-mediated apoptosis and suppressing PI3K/Akt pathways. Therefore, regulating the expression of CACNA2D3 is a potential new strategy to increase the efficacy of cisplatin in ESCC patients.

Role of Store Operated Calcium Channel Complex in the Inflammatory Signaling in Peripheral Sensory Neurons

Role of Store Operated Calcium Channel Complex in the Inflammatory Signaling in Peripheral Sensory Neurons