Neuronal and astrocyte NCX isoform/splice variants: How do they participate in Na+ and Ca2+ signalling?

NCX1, NCX2, and NCX3 gene isoforms and their splice variants are characteristically expressed in different regions of the brain. The tissue-specific splice variants of NCX1-3 isoforms show specific expression profiles in neurons and astrocytes, whereas the relevant NCX isoform/splice variants exhibit diverse allosteric modes of Na+- and Ca2+-dependent regulation. In general, overexpression of NCX1-3 genes leads to neuroprotective effects, whereas their ablation gains the opposite results. At this end, the partial contributions of NCX isoform/splice variants to neuroprotective effects remain unresolved. The glutamate-dependent Na+ entry generates Na+ transients (in response to neuronal cell activities), whereas the Na+-driven Ca2+ entry (through the reverse NCX mode) raises Ca2+ transients. This special mode of signal coupling translates Na+ transients into the Ca2+ signals while being a part of synaptic neurotransmission. This mechanism is of general interest since disease-related conditions (ischemia, metabolic stress, and stroke among many others) trigger Na+ and Ca2+ overload with deadly outcomes of downstream apoptosis and excitotoxicity. The recently discovered mechanisms of NCX allosteric regulation indicate that some NCX variants might play a critical role in the dynamic coupling of Na+-driven Ca2+ entry. In contrast, the others are less important or even could be dangerous under altered conditions (e.g., metabolic stress). This working hypothesis can be tested by applying advanced experimental approaches and highly focused computational simulations. This may allow the development of structure-based blockers/activators that can selectively modulate predefined NCX variants to lessen the life-threatening outcomes of excitotoxicity, ischemia, apoptosis, metabolic deprivation, brain injury, and stroke.

NLRP14 Safeguards Calcium Homeostasis via Regulating the K27 Ubiquitination of Nclx in Oocyte-to-Embryo Transition.

Sperm-induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+ /Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.

Inhibition of Acid-Sensing Ion Channels by KB-R7943, a Reverse Na+/Ca2+ Exchanger Inhibitor.

KB-R7943, an isothiourea derivative, is widely used as a pharmacological inhibitor of reverse sodium-calcium exchanger (NCX). It has been shown to have neuroprotective and analgesic effects in animal models; however, the detailed molecular mechanisms remain elusive. In the current study, we investigated whether KB-R7943 modulates acid-sensing ion channels (ASICs), a group of proton-gated cation channels implicated in the pathophysiology of various neurological disorders, using the whole-cell patch clamp techniques. Our data show that KB-R7943 irreversibly inhibits homomeric ASIC1a channels heterologously expressed in Chinese Hamster Ovary (CHO) cells in a use- and concentration-dependent manner. It also reversibly inhibits homomeric ASIC2a and ASIC3 channels in CHO cells. Both the transient and sustained current components of ASIC3 are inhibited. Furthermore, KB-R7943 inhibits ASICs in primary cultured peripheral and central neurons. It inhibits the ASIC-like currents in mouse dorsal root ganglion (DRG) neurons and the ASIC1a-like currents in mouse cortical neurons. The inhibition of the ASIC1a-like current is use-dependent and unrelated to its effect on NCX since neither of the other two well-characterized NCX inhibitors, including SEA0400 and SN-6, shows an effect on ASIC. Our data also suggest that the isothiourea group, which is lacking in other structurally related analogs that do not affect ASIC1a-like current, may serve as a critical functional group. In summary, we characterize KB-R7943 as a new ASIC inhibitor. It provides a novel pharmacological tool for the investigation of the functions of ASICs and could serve as a lead compound for developing small-molecule drugs for treating ASIC-related disorders.

Silencing mouse circular RNA circSlc8a1 by circular antisense cA-circSlc8a1 induces cardiac hepatopathy.

Circular RNAs (circRNAs) are a group of non-coding RNAs with a unique circular structure generated by back-splicing. It is acknowledged that circRNAs play critical roles in cardiovascular diseases. However, functional studies of circRNAs were impeded due to lack of effective in vivo silencing approaches. Since most circRNAs are produced by protein-coding transcripts, gene editing typically affects the coding activity of the parental genes. In this study, we developed a circular antisense RNA (cA-circSlc8a1) that could silence the highly expressed circRNA circSlc8a1 in the mouse heart but not its parental Slc8a1 linear mRNA. Transgenic cA-circSlc8a1 mice developed congestive heart failure resulting in a significant increase in the body weight secondary to peripheral edema and congestive hepatopathy. To further test the role of circSlc8a1, we generated transgenic mice overexpressing circSlc8a1 and observed a protective effect of circSlc8a1 in a pressure overload model. Mechanistically, we found that circSlc8a1 translocated into mitochondria to drive ATP synthesis. While establishing a transgenic murine model for antisense-mediated circRNA silencing without interfering with the parental linear RNA, our finding revealed the essential role of circSlc8a1 in maintaining heart function and may lay the groundwork of using the circular antisense RNA as a potential gene therapy approach for cardiovascular diseases.

Mitochondrial sodium/calcium exchanger NCLX regulates glycolysis in astrocytes, impacting on cognitive performance.

Intracellular Ca2+ concentrations are strictly controlled by plasma membrane transporters, the endoplasmic reticulum, and mitochondria, in which Ca2+ uptake is mediated by the mitochondrial calcium uniporter complex (MCUc), while efflux occurs mainly through the mitochondrial Na+ /Ca2+ exchanger (NCLX). RNAseq database repository searches led us to identify the Nclx transcript as highly enriched in astrocytes when compared with neurons. To assess the role of NCLX in mouse primary culture astrocytes, we inhibited its function both pharmacologically or genetically. This resulted in re-shaping of cytosolic Ca2+ signaling and a metabolic shift that increased glycolytic flux and lactate secretion in a Ca2+ -dependent manner. Interestingly, in vivo genetic deletion of NCLX in hippocampal astrocytes improved cognitive performance in behavioral tasks, whereas hippocampal neuron-specific deletion of NCLX impaired cognitive performance. These results unveil a role for NCLX as a novel modulator of astrocytic glucose metabolism, impacting on cognition.

Real time monitoring of cold Ca2+ dependent transcription and its modulation by NCX inhibitors.

Real-time monitoring of cellular temperature responses is an important technique in thermal biology and drug development. Recent study identified that Na+/Ca2+ exchanger (NCX)-dependent Ca2+ influx transduces cold signals to circadian clock in mammalian cultured cells. The finding raised an idea that cellular responses to the cold signals can be analyzed by monitoring of clock gene expression. We found that Per1 and Per2 were up-regulated after culture at 27 °C compared to 37 °C in Rat-1 fibroblasts. In order to monitor cold-Ca2+-dependent transcription in living cells, we developed a luciferase-based real-time reporting system by using Per1 promoter, Per2 promoter, Ca2+/cAMP-response elements (CRE) or NFAT-binding elements. We found that benzyloxyphenyl NCX inhibitor KB-R7943 and SN-6, but not SEA-0400 or YM-244769 inhibited the cold induction of Per2. Our study established a real-time monitoring system for cold Ca2+ signaling which can be applied to evaluation of drugs.

Silencing lncRNA 93358 Inhibits the Apoptosis of Myocardial Cells in Myocardial Infarction Rats by Inducing the Expression of SLC8A1.

Objective: To explore the inhibitor effects and mechanism of lncRNA 93358 against the apoptosis of myocardial cells in rats with myocardial infarction. Methods: The myocardial infarction model was established in rats, which were identified by cardiac ultrasound. TTC staining was used to evaluate the degree of heart infarction, and HE staining was utilized to determine the pathological state in myocardial tissues. The apoptotic state in myocardial tissues was confirmed by TUNEL assay. lncRNA 93358 was screened out using a high-throughput sequencing which was confirmed by RT-qPCR. The interaction between miR-466c-3p and SLC8A1 was identified using the dual-luciferase reporter assay. The expression level of Bax, Bcl-2, and SLC8A1 was determined in lncRNA 93358 knockdown cells using RT-qPCR and Western blotting. Results: Massive myocardial necrosis was observed in model rats according to the results of TTC staining, HE staining, and TUNEL assay. lncRNA 93358 and Bax were found significantly upregulated, and Bcl-2 and SLC8A1 were greatly downregulated in model rats, which were dramatically reversed by the knockdown of lncRNA 93358, accompanied by the decline area of myocardial necrosis and decreased apoptotic myocardial cells. Conclusion: Silencing lncRNA 93358 inhibits the apoptosis of myocardial cells in rats with myocardial infarction by inducing the expression of SLC8A1.

Abnormal levels of mitochondrial Ca2+ channel proteins in plasma neuron-derived extracellular vesicles of early schizophrenia.

Structural alterations or quantitative abnormalities of some mitochondrial ion channels and exchangers are associated with altered neuronal functions and increased susceptibility to mental illness. Here we have assessed levels of functionally prominent mitochondrial calcium ion channel proteins in plasma neuron-derived extracellular vesicles (NDEVs) of living patients with first episodes of psychosis (FP) and matched controls (Cs). NDEVs were enriched with an established method of precipitation and immunoabsorption by anti-human CD171 neural adhesion protein (L1CAM) antibody and extracted proteins quantified with ELISAs. CD81 exosome marker-normalized NDEV levels of leucine zipper EF-hand containing transmembrane 1 protein (LETM1), transient receptor potential cation channel subfamily M, member 4 (TRPM4), and solute carrier family 8 member B1 (SLC24A6) or mitochondrial Na+ /Ca2+ exchanger (NCLX) were significantly lower for FP patients (n = 10) than Cs (n = 10), whereas NDEV levels of voltage-dependent L-type calcium channel subunit α-1C (CACNA-1C) were significantly higher for FP patients than Cs. Abnormal structures or mitochondrial levels of LETM1, NCLX, and CACNA-1C have been linked through analyses of individual proteins, genome-wide association studies, and whole exome protein-coding sequence studies to neurodevelopmental disorders, mental retardation, schizophrenia, and major depressive diseases. A greater understanding of the altered calcium homeostasis in schizophrenia, that is attributable to underlying mitochondrial calcium channel abnormalities, will lead to improved diagnosis and treatment.

The airway smooth muscle sodium/calcium exchanger NCLX is critical for airway remodeling and hyperresponsiveness in asthma.

The structural changes of airway smooth muscle (ASM) that characterize airway remodeling (AR) are crucial to the pathogenesis of asthma. During AR, ASM cells dedifferentiate from a quiescent to a proliferative, migratory, and secretory phenotype. Calcium (Ca2+) is a ubiquitous second messenger that regulates many cellular processes, including proliferation, migration, contraction, and metabolism. Furthermore, mitochondria have emerged as major Ca2+ signaling organelles that buffer Ca2+ through uptake by the mitochondrial Ca2+ uniporter and extrude it through the Na+/Ca2+ exchanger (NCLX/Slc8b1). Here, we show using mitochondrial Ca2+-sensitive dyes that NCLX only partially contributes to mitochondrial Ca2+ extrusion in ASM cells. Yet, NCLX is necessary for ASM cell proliferation and migration. Through cellular imaging, RNA-Seq, and biochemical assays, we demonstrate that NCLX regulates these processes by preventing mitochondrial Ca2+ overload and supporting store-operated Ca2+ entry, activation of Ca2+/calmodulin-dependent kinase II, and transcriptional and metabolic reprogramming. Using small animal respiratory mechanic measurements and immunohistochemistry, we show that smooth muscle-specific NCLX KO mice are protected against AR, fibrosis, and hyperresponsiveness in an experimental model of asthma. Our findings support NCLX as a potential therapeutic target in the treatment of asthma.

K+-Dependent Na+/Ca2+ Exchanger Isoform 2, Nckx2, Takes Part in the Neuroprotection Elicited by Ischemic Preconditioning in Brain Ischemia.

Sodium/Calcium exchangers are neuronal plasma membrane antiporters which, by coupling Ca2+ and Na+ fluxes across neuronal membranes, play a relevant role in brain ischemia. The most brain-expressed isoform among the members of the K+-dependent Na+/Ca2+ exchanger family, NCKX2, is involved in the progression of the ischemic lesion, since both its knocking-down and its knocking-out worsens ischemic damage. The aim of this study was to elucidate whether NCKX2 functions as an effector in the neuroprotection evoked by ischemic preconditioning. For this purpose, we investigated: (1) brain NCKX2 expression after preconditioning and preconditioning + ischemia; (2) the contribution of AKT and calpain to modulating NCKX2 expression during preconditioning; and (3) the effect of NCKX2 knocking-out on the neuroprotection mediated by ischemic preconditioning. Our results showed that NCKX2 expression increased in those brain regions protected by ischemic preconditioning. These changes were p-AKT-mediated since its inhibition prevented NCKX2 up-regulation. More interestingly, NCKX2 knocking-out significantly prevented the protection exerted by ischemic preconditioning. Overall, our results suggest that NCKX2 plays a fundamental role in the neuroprotective effect mediated by ischemic preconditioning and support the idea that the enhancement of its expression and activity might represent a reasonable strategy to reduce infarct extension after stroke.

Sodium-calcium exchanger-3 regulates pain "wind-up": From human psychophysics to spinal mechanisms.

Repeated application of noxious stimuli leads to a progressively increased pain perception; this temporal summation is enhanced in and predictive of clinical pain disorders. Its electrophysiological correlate is "wind-up," in which dorsal horn spinal neurons increase their response to repeated nociceptor stimulation. To understand the genetic basis of temporal summation, we undertook a GWAS of wind-up in healthy human volunteers and found significant association with SLC8A3 encoding sodium-calcium exchanger type 3 (NCX3). NCX3 was expressed in mouse dorsal horn neurons, and mice lacking NCX3 showed normal, acute pain but hypersensitivity to the second phase of the formalin test and chronic constriction injury. Dorsal horn neurons lacking NCX3 showed increased intracellular calcium following repetitive stimulation, slowed calcium clearance, and increased wind-up. Moreover, virally mediated enhanced spinal expression of NCX3 reduced central sensitization. Our study highlights Ca2+ efflux as a pathway underlying temporal summation and persistent pain, which may be amenable to therapeutic targeting.

Multiomics Integrated Analysis Identifies SLC24A2 as a Potential Link between Type 2 Diabetes and Cancer.

Background: So far, type 2 diabetes (T2D) is considered as an independent risk factor for various cancers, but the underlying mechanism remains unclear. Methods. SLC24A2 was first identified as a key gene strongly associated with fasting plasma glucose (FPG). Then, overlapped differentially expressed genes (DEGs) between T2D verse control and SLC24A2-high verse SLC24A2-low were extracted and imported into weighted correlation network analysis. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and gene set enrichment analysis were used for functional enrichment analysis of DEGs. Least absolute shrinkage and selection operator was utilized to build a T2D prediction model. Timer and K-M plotters were employed to find the expression and prognosis of SLC24A2 in pan cancer. Results: Interestingly, both DEGs between T2D verse control and SLC24A2-high verse SLC24A2-low enriched in cancer-related pathways. Moreover, a total of 3719 overlapped DEGs were divided into 8 functional modules. Grey module negatively correlated with T2D and FPG and was markedly involved in ribosome biogenesis. Ten SLC24A2-related genes (RRP36, RPF1, GRWD1, FBL, EXOSC5, BCCIP, UTP14A, TWISTNB, TBL3, and SKIV2L) were identified as hub genes, based on which the LASSO model accurately predicts the occurrence of T2D (AUC = 0.841). In addition, SLC24A2 was only expressed in islet ß cells and showed abnormal expression in 17 kinds of cancers and significantly correlated with the prognosis of 10 kinds of cancers. Conclusion: Taken together, SLC24A2 may link T2D and cancer by influencing the ribosome function of islet ß cells and play different prognostic roles in different cancers.

Enhanced NCLX-dependent mitochondrial Ca2+ efflux attenuates pathological remodeling in heart failure.

Mitochondrial calcium (mCa2+) uptake couples changes in cardiomyocyte energetic demand to mitochondrial ATP production. However, excessive mCa2+ uptake triggers permeability transition and necrosis. Despite these established roles during acute stress, the involvement of mCa2+ signaling in cardiac adaptations to chronic stress remains poorly defined. Changes in NCLX expression are reported in heart failure (HF) patients and models of cardiac hypertrophy. Therefore, we hypothesized that altered mCa2+ homeostasis contributes to the hypertrophic remodeling of the myocardium that occurs upon a sustained increase in cardiac workload. The impact of mCa2+ flux on cardiac function and remodeling was examined by subjecting mice with cardiomyocyte-specific overexpression (OE) of the mitochondrial Na+/Ca2+ exchanger (NCLX), the primary mediator of mCa2+ efflux, to several well-established models of hypertrophic and non-ischemic HF. Cardiomyocyte NCLX-OE preserved contractile function, prevented hypertrophy and fibrosis, and attenuated maladaptive gene programs in mice subjected to chronic pressure overload. Hypertrophy was attenuated in NCLX-OE mice, prior to any decline in cardiac contractility. NCLX-OE similarly attenuated deleterious cardiac remodeling in mice subjected to chronic neurohormonal stimulation. However, cardiomyocyte NCLX-OE unexpectedly reduced overall survival in mice subjected to severe neurohormonal stress with angiotensin II + phenylephrine. Adenoviral NCLX expression limited mCa2+ accumulation, oxidative metabolism, and de novo protein synthesis during hypertrophic stimulation of cardiomyocytes in vitro. Our findings provide genetic evidence for the contribution of mCa2+ to early pathological remodeling in non-ischemic heart disease, but also highlight a deleterious consequence of increasing mCa2+ efflux when the heart is subjected to extreme, sustained neurohormonal stress.

Identification and characterization of the promoter and transcription factors regulating the expression of cerebral sodium/calcium exchanger 2 (NCX2) gene.

The isoform 2 of sodium-calcium exchanger family (NCX2) is selectively expressed in neuronal and glial cells where it participates in Ca2+-clearance following neuronal depolarization, synaptic plasticity, hippocampal-dependent learning and memory consolidation processes. On the other hand, NCX2 is also involved in a neuroprotective effect following stroke. Despite the relevance of this antiporter under physiological and pathophysiological conditions, no studies have been reported on its genetic/epigenetic regulation. Therefore, we identified, cloned, and characterized a transcriptional regulatory region (R3) of rat Slc8a2 gene encoding for NCX2. In particular, R3 sequence displayed a promoter activity in PC12, SH-SY5Y and U87MG cell lines consistent with their endogenous NCX2 expression levels. On the other hand, R3 failed to induce detectable luciferase activity in BHK cell line that does not express NCX2 under control conditions. These data support the hypothesis that R3 represents the promoter region of NCX2. Moreover, among several conserved binding sequences for transcription factors identified by in-silico analysis, we evaluated the transcriptional regulation and the binding sites of Sp1, Sp4, NFkB1, GATA2 and CREB1 on R3 sequence by using site-direct mutagenesis and ChIP assays. In particular, transfection of Sp1, Sp4, and CREB1 enhanced both R3 promoter activity and NCX2 transcription in PC12 cell line. More important, CREB1 transfection also enhanced NCX2 protein levels and NCX reverse mode activity in PC12 cells. Altogether, these data suggested that: (i) the identified region contained the regulatory promoter of the antiporter; (ii) NCX2 might represent a downstream effector of transcription factors involved in synaptic plasticity and neuronal survival.

Na+/Ca2+ exchanger isoform 1 (NCX1) and canonical transient receptor potential channel 6 (TRPC6) are recruited by STIM1 to mediate Store-Operated Calcium Entry in primary cortical neurons.

Excessive calcium (Ca2+) release from the endoplasmic reticulum (ER) represents an important hallmark of several neurodegenerative diseases. ER is recharged from Ca2+ through the so-called Store-Operated Calcium Entry (SOCE) thus providing Ca2+ signals to regulate critical cell functions. Single transmembrane-spanning domain protein stromal interacting molecule 1 (STIM1), mainly residing in the ER, and plasmalemmal channel Orai1 represent the SOCE key components at neuronal level. However, many other proteins participate to ER Ca2+ refilling including the Na+/Ca2+ exchanger isoform 1 (NCX1), whose regulation by ER remains unknown. In this study, we tested the possibility that neuronal NCX1 may take part to SOCE through the interaction with STIM1. In rat primary cortical neurons and in nerve growth factor (NGF)-differentiated PC12 cells NCX1 knocking down by siRNA strategy significantly prevented SOCE as well as SOCE pharmacological inhibition by SKF-96365 and 2-APB. A significant reduction of SOCE was recorded also in synaptosomes from ncx1-/- mice brain compared with ncx1+/+ mice. Double labeling confocal experiments showed a large co-localization between NCX1 and STIM1 in rat primary cortical neurons. Accordingly, NCX1 and STIM1 co-immunoprecipitated and functionally interacted each other during ischemic preconditioning, a phenomenon inducing ischemic tolerance. However, STIM1 knocking down reduced NCX1 activity recorded by either patch-clamp electrophysiology or Fura-2 single-cell microfluorimetry. Furthermore, canonical transient receptor potential channel 6 (TRPC6) was identified as the mechanism mediating local increase of sodium (Na+) useful to drive NCX1 reverse mode and, therefore, NCX1-mediated Ca2+ refilling. In fact, TRPC6 not only interacted with STIM1, as shown by the co-localization and co-immunoprecipitation with the ER Ca2+ sensor, but it also mediated 1,3-Benzenedicarboxylic acid, 4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester (SBFI)-monitored Na+ increase elicited by thapsigargin in primary cortical neurons. Accordingly, efficient TRPC6 knockdown prevented thapsigargin-induced intracellular Na+ elevation and SOCE. Collectively, we identify NCX1 as a new partner of STIM1 in mediating SOCE, whose activation in the reverse mode may be facilitated by the local increase of Na+ concentration due to the interaction between STIM1 and TRPC6 in primary cortical neurons.

Structure-Based Function and Regulation of NCX Variants: Updates and Challenges.

The plasma-membrane homeostasis Na+/Ca2+ exchangers (NCXs) mediate Ca2+ extrusion/entry to dynamically shape Ca2+ signaling/in biological systems ranging from bacteria to humans. The NCX gene orthologs, isoforms, and their splice variants are expressed in a tissue-specific manner and exhibit nearly 104-fold differences in the transport rates and regulatory specificities to match the cell-specific requirements. Selective pharmacological targeting of NCX variants could benefit many clinical applications, although this intervention remains challenging, mainly because a full-size structure of eukaryotic NCX is unavailable. The crystal structure of the archaeal NCX_Mj, in conjunction with biophysical, computational, and functional analyses, provided a breakthrough in resolving the ion transport mechanisms. However, NCX_Mj (whose size is nearly three times smaller than that of mammalian NCXs) cannot serve as a structure-dynamic model for imitating high transport rates and regulatory modules possessed by eukaryotic NCXs. The crystal structures of isolated regulatory domains (obtained from eukaryotic NCXs) and their biophysical analyses by SAXS, NMR, FRET, and HDX-MS approaches revealed structure-based variances of regulatory modules. Despite these achievements, it remains unclear how multi-domain interactions can decode and integrate diverse allosteric signals, thereby yielding distinct regulatory outcomes in a given ortholog/isoform/splice variant. This article summarizes the relevant issues from the perspective of future developments.

Regulation of K+-Dependent Na+/Ca2+-Exchangers (NCKX).

Potassium-dependent sodium-calcium exchangers (NCKX) have emerged as key determinants of calcium (Ca2+) signaling and homeostasis, especially in environments where ion concentrations undergo large changes, such as excitatory cells and transport epithelia. The regulation of NCKX transporters enables them to respond to the changing cellular environment thereby helping to shape the extent and kinetics of Ca2+ signals. This review examines the current knowledge of the different ways in which NCKX activity can be modulated. These include (i) cellular and dynamic subcellular location (ii); changes in protein expression mediated at the gene, transcript, or protein level (iii); genetic changes resulting in altered protein structure or expression (iv); regulation via changes in substrate concentration (v); and post-translational modification, partner protein interactions, and allosteric regulation. Detailed mechanistic understanding of NCKX regulation is an emerging area of research with the potential to provide important new insights into transporter function, the control of Ca2+ signals, and possible interventions for dysregulated Ca2+ homeostasis.

Pan-phylum genome-wide identification of sodium calcium exchangers reveal heterogeneous expansions and possible roles in nematode parasitism.

Calcium signaling is ubiquitous in nematode development from fertilization to cell specification to apoptosis. Calcium also regulates dauer entry in Caenorhabditis elegans, which corresponds to the infective stage of parasitic nematodes. In diverse parasites such as Trypanosoma cruzi and Toxoplasma gondii calcium has been shown to regulate host cell entry and egress, and perturbing calcium signaling represents a possible route to inhibit infection and parasitism in these species. Sodium calcium exchangers are considered the most important mechanism of calcium efflux, and our lab has previously characterized the sodium calcium exchanger gene family in C. elegans and studied the diversity of this family across a subset of specific nematode species. Here we build upon these data and explore sodium calcium exchangers across 108 species of nematodes. Our data reveal substantial differences in sodium calcium exchanger counts across the Phylum and detail expansions and contractions of specific exchanger subtypes within certain nematode clades. Finally, we also provide evidence for a role of sodium calcium exchangers in parasite activation by examining differentially expressed genes in non-activated versus activated infective stage larvae. Taken together our findings paint a heterogeneous picture of sodium calcium exchanger evolution across the Phylum Nematoda that may reflect unique adaptations to free-living and parasitic lifestyles.

Characteristic attributes limiting the transport rates in NCX orthologs.

The Na+/Ca2+ exchangers (NCXs) modulate the Ca2+ signaling and homeostasis in health and disease. The transport cycle turnover rates (kcat) and the kcat/Km values of eukaryotic NCXs are ~104-times higher than those of prokaryotic NCXs. Three ion-coordinating residues (out of twelve) differ between eukaryotic NCXs and NCX_Mj. The replacement of three ion-coordinating residues in NCX_Mj does not increase kcat, probably due to the structural rigidity of NCX_Mj. Phospholipids and cholesterol increase (up to 10-fold) the transport rates in the cardiac NCX1.1, but not in NCX_Mj. A lipid environment can partially contribute to the huge kinetic variances among NCXs.