The chemo-electrical transduction process in olfactory neurons is accompanied by a rapid and transient increase in intracellular calcium concentrations. The notion that Na+/Ca2+ exchanger activities may play a major role in extruding calcium ions out of the cell and maintaining Ca2+ homeostasis in olfactory receptor cells was assessed by means of laser scanning confocal microscopy in combination with the fluorescent indicators Fluo-3 and Fura-Red. The data indicate that high exchanger activity, which was inhibited by amiloride derivatives, is located in the dendritic knob and probably in the olfactory cilia. This result was supported by experiments using specific antiserum raised against retinal Na+/Ca2+ exchanger protein which labelled an immunoreactive protein of 230 kDa in Western blots from olfactory tissue and strongly stained the ciliary layer of the olfactory epithelium.

Nociception requires transduction and impulse electrogenesis in nerve fibers which innervate the body surface, including the skin. However, the molecular substrates for transduction and action potential initiation in nociceptors are incompletely understood. In this study, we examined the expression and distribution of Na+/Ca2+ exchanger (NCX) and voltage-gated sodium channel isoforms in intra-epidermal free nerve terminals.

Na+/Ca2+ exchangers (NCX) transport Ca2+ in or out of cells in exchange for Na+. They are ubiquitously expressed and play an essential role in maintaining cytosolic Ca2+ homeostasis. Although extensively studied, little is known about the global structural arrangement of eukaryotic NCXs and the structural mechanisms underlying their regulation by various cellular cues including cytosolic Na+ and Ca2+. Here we present the cryo-EM structures of human cardiac NCX1 in both inactivated and activated states, elucidating key structural elements important for NCX ion exchange function and its modulation by cytosolic Ca2+ and Na+. We demonstrate that the interactions between the ion-transporting transmembrane (TM) domain and the cytosolic regulatory domain define the activity of NCX. In the inward-facing state with low cytosolic [Ca2+], a TM-associated four-stranded β-hub mediates a tight packing between the TM and cytosolic domains, resulting in the formation of a stable inactivation assembly that blocks the TM movement required for ion exchange function. Ca2+ binding to the cytosolic second Ca2+-binding domain (CBD2) disrupts this inactivation assembly which releases its constraint on the TM domain, yielding an active exchanger. Thus, the current NCX1 structures provide an essential framework for the mechanistic understanding of the ion transport and cellular regulation of NCX family proteins.

Female rabbit hearts are more susceptible to torsade de pointes (TdP) in acquired long QT type 2 than males, in-part due to higher L-type Ca2+ current (ICa,L) at the base of the heart. In principle, higher Ca2+ influx via ICa,L should be balanced by higher efflux, perhaps mediated by parallel sex differences of sodium-calcium exchange (NCX) current (INCX). We now show that NCX1, like Cav1.2α, is greater at the base of female than male left ventricular epicardium and greater at the base than at the apex in both sexes. In voltage-clamp studies, inward (0, +20 mV, P < 0.04) and outward (-80, -60, -40, -20 mV, P < 0.01) INCX densities were significantly higher (1.5-2 fold) in female base compared to apex and male (base and apex) myocytes. Myocytes were incubated ±17β-oestradiol (E2 = 1 nm) and INCX was measured on days 0, 1, 2 and 3. Inward and outward INCX decreased over 2 days in female base myocytes becoming similar to INCX at the apex. E2 incubation (24 h) increased NCX1 (50%) and INCX (∼3-fold at 60 mV) in female base but not endocardium, apex or in male base myocytes. INCX upregulation by E2 was blunted by an oestrogen receptor (ER) antagonist (fulvestrant, 1 μm), and inhibition of transcription (actinomycin D, 5 μg ml-1) or translation (cycloheximide, 20 μg ml-1). Dofetilide (an IKr blocker) induced early afterdepolarizations (EADs) in female base myocytes cultured for 1 day if incubated with E2, but not without E2 or with E2+KB-R4973 (an INCX inhibitor), E2+fulvestrant or E2 with apex myocytes. Thus, E2 upregulates NCX1 by a genomic mechanism mediated by ERs, and de novo mRNA and protein biosynthesis, in a sex- and region-dependent manner which contributes to the enhanced propensity to EADs and TdP in female hearts.

Various types of sensory stimuli have been shown to induce Ca2+ elevations in glia. However, a mechanistic understanding of the signaling pathways mediating sensory-evoked activity in glia in intact animals is still emerging. During early development of the Xenopus laevis visual system, radial astrocytes in the optic tectum are highly responsive to sensory stimulation. Ca2+ transients occur spontaneously in radial astrocytes at rest and are abolished by silencing neuronal activity with tetrodotoxin. Visual stimulation drives temporally correlated increases in the activity patterns of neighboring radial astrocytes. Following blockade of all glutamate receptors (gluRs), visually evoked Ca2+ activity in radial astrocytes persists, while neuronal activity is suppressed. The additional blockade of either glu transporters or sodium-calcium exchangers (NCX) abolishes visually evoked responses in glia. Finally, we demonstrate that blockade of NCX alone is sufficient to prevent visually evoked responses in radial astrocytes, highlighting a pivotal role for NCX in glia during development.

Repolarization alternans, a periodic oscillation of long-short action potential duration, is an important source of arrhythmogenic substrate, although the mechanisms driving it are insufficiently understood. Despite its relevance as an arrhythmia precursor, there are no successful therapies able to target it specifically. We hypothesized that blockade of the sodium‑calcium exchanger (NCX) could inhibit alternans. The effects of the selective NCX blocker ORM-10962 were evaluated on action potentials measured with microelectrodes from canine papillary muscle preparations, and calcium transients measured using Fluo4-AM from isolated ventricular myocytes paced to evoke alternans. Computer simulations were used to obtain insight into the drug's mechanisms of action. ORM-10962 attenuated cardiac alternans, both in action potential duration and calcium transient amplitude. Three morphological types of alternans were observed, with differential response to ORM-10962 with regards to APD alternans attenuation. Analysis of APD restitution indicates that calcium oscillations underlie alternans formation. Furthermore, ORM-10962 did not markedly alter APD restitution, but increased post-repolarization refractoriness, which may be mediated by indirectly reduced L-type calcium current. Computer simulations reproduced alternans attenuation via ORM-10962, suggesting that it is acts by reducing sarcoplasmic reticulum release refractoriness. This results from the ORM-10962-induced sodium‑calcium exchanger block accompanied by an indirect reduction in L-type calcium current. Using a computer model of a heart failure cell, we furthermore demonstrate that the anti-alternans effect holds also for this disease, in which the risk of alternans is elevated. Targeting NCX may therefore be a useful anti-arrhythmic strategy to specifically prevent calcium driven alternans.

Changes in intracellular Ca2+ concentrations ([Ca2+]i) are an important signal for various physiological activities. The Na+/Ca2+ exchangers (NCX) at the plasma membrane transport Ca2+ into or out of the cell according to the electrochemical gradients of Na+ and Ca2+ to modulate [Ca2+]i homeostasis. Calmodulin (CaM) senses [Ca2+]i changes and relays Ca2+ signals by binding to target proteins such as channels and transporters. However, it is not clear how calmodulin modulates NCX activity. Using CaM as a bait, we pulled down the intracellular loops subcloned from the NCX1 splice variants NCX1.1 and NCX1.3. This interaction requires both Ca2+ and a putative CaM-binding segment (CaMS). To determine whether CaM modulates NCX activity, we co-expressed NCX1 splice variants with CaM or CaM1234 (a Ca2+-binding deficient mutant) in HEK293T cells and measured the increase in [Ca2+]i contributed by the influx of Ca2+ through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activity and decreased membrane localization. Without the mutually exclusive exon, the exchange activity was decreased and could be partially rescued by CaM1234. Point-mutations at any of the 4 conserved a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3rd or 4th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2nd and 3rd conserved a.a. residues in NCX1.3 decreased exchange activity. Taken together, our results demonstrate that CaM senses changes in [Ca2+]i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.

Background: Blockage of the Na+/Ca2+ exchanger (NCX) is used to determine the role of NCX in arrhythmogenesis. Trisulfated heparin disaccharide (TD) and Low Molecular Weight Heparins (LMWHs) can directly interact with the NCX and accelerate its activity. Objective: In this work, we investigated the antiarrhythmic effect of heparin oligosaccharides related to the NCX activity. Methods: The effects of heparin oligosaccharides were tested on the NCX current (patch clamping) and intracellular calcium transient in rat cardiomyocytes. The effects of heparin oligosaccharides were further investigated in arrhythmia induced in isolated rat atria and rats in vivo. Results: The intracellular Ca2+ concentration decreases upon treatment with either enoxaparin or ardeparin. These drugs abolished arrhythmia induction in isolated atria. The NCX antagonist KB-R7943 abolished the enoxaparin or ardeparin antiarrhythmic effects in isolated atria. In the in vivo measurements, injection of TD 15 min both before coronary occlusion or immediately after reperfusion, significantly prevented the occurrence of reperfusion-induced arrhythmias (ventricular arrhythmia and total AV block) and reduced the lethality rate. The patch clamping experiments showed that, mechanistically, TD increases the forward mode NCX current. Conclusion: Together, the data shows that heparin oligosaccharides may constitute a new class of antiarrhythmic drug that acts by accelerating the forward mode NCX under calcium overload.

Ziram is a dimethyldithiocarbamate fungicide which can cause intraneuronal calcium (Ca(2+)) dysregulation and subsequently neuronal death. The signaling mechanisms underlying ziram-induced Ca(2+) dyshomeostasis and neurotoxicity are not fully understood. NCX3 is the third isoform of the sodium-calcium exchanger (NCX) family and plays an important role in regulating Ca(2+) homeostasis in excitable cells. We previously generated a mouse model deficient for the sodium-calcium exchanger 3 and showed that NCX3 is protective against ischemic damage. In the present study, we aim to examine whether NCX3 exerts a similar role against toxicological injury caused by the pesticide ziram. Our data show baby hamster kidney (BHK) cells stably transfected with NCX3 (BHK-NCX3) are more susceptible to ziram toxicity than cells transfected with the empty vector (BHK-WT). Increased toxicity in BHK-NCX3 was associated with a rapid rise in cytosolic Ca(2+) concentration [Ca(2+)]i induced by ziram. Profound mitochondrial dysfunction and ATP depletion were also observed in BHK-NCX3 cells following treatment with ziram. Lastly, primary dopaminergic neurons lacking NCX3 (NCX3(-/-)) were less sensitive to ziram neurotoxicity than wildtype control dopaminergic neurons. These results demonstrate that NCX3 genetic deletion protects against ziram-induced neurotoxicity and suggest NCX3 and its downstream molecular pathways as key factors involved in ziram toxicity. Our study identifies new molecular events through which pesticides (e.g. ziram) can lead to pathological features of degenerative diseases such as Parkinson's disease and indicates new targets to slow down neuronal degeneration.

Myelination in the central nervous system depends on interactions between axons and oligodendrocyte precursor cells (OPCs). Action potentials in an axon can be followed by release of biologically active substances, like glutamate, which can instruct OPCs to start myelination. Myelin Basic Protein (MBP) is an "executive molecule of myelin" required for the formation of compact myelin. As cells of the oligodendrocyte lineage (OLCs) are capable of producing MBP in pure oligodendrocyte cultures, i.e. without neurons, we investigated Ca2+ signaling in developing OLCs in cultures. We show that spontaneous Ca2+ transients (CTs) occur at very low frequency in both bipolar OPCs and mature oligodendrocytes. In contrast immature OLCs (imOLCs), cells with several thick processes, demonstrate a relatively high frequency of CTs. Moreover, CT frequency in imOLC processes is much higher as compared with the somatic CT frequency. Somatic CTs are almost completely blocked by thapsigargin, an antagonist of sarco-(endo-) plasmic reticulum Ca2+ ATPase, and ryanodine, a blocker of ryanodine receptors, indicating an involvement of Ca2+ release from the endoplasmic reticulum. Ryanodine strongly reduces CT frequency in imOLC processes. Ouabain, an antagonist of Na+, K+-ATPase (NKA), applied at low concentration increases CT frequency, while KB-R7943, a blocker of reverse mode of Na+, Ca2+ exchanger (NCX), decreases CT frequency. We suggest that local RyR-NCX-(NKA?) interaction might underlie the generation of CTs in imOLC in the absence of neurons, and this activity influences oligodendrocyte maturation.

Sodium/calcium(-potassium) exchangers (NCX and NCKX) are critical for the rapid extrusion of calcium, which follows the stimulation of a variety of excitable cells. To further understand the mechanisms of calcium regulation in signaling, we have cloned a Drosophila sodium/calcium-potassium exchanger, Nckx30C. The overall deduced protein topology for NCKX30C is similar to that of mammalian NCKX, having five membrane-spanning domains in the NH(2) terminus separated from six at the COOH-terminal end by a large intracellular loop. We show that NCKX30C functions as a potassium-dependent sodium/calcium exchanger, and is not only expressed in adult neurons as was expected, but is also expressed during ventral nerve cord development in the embryo and in larval imaginal discs. Nckx30C is expressed in a dorsal-ventral pattern in the eye-antennal disc in a pattern that is similar to, but broader than that of wingless, suggesting that large fluxes of calcium may be occurring during imaginal disc development. Nckx30C may not only function in the removal of calcium and maintenance of calcium homeostasis during signaling in the adult, but may also play a critical role in signaling during development.

Calcium influx plays important roles in the pathophysiology of seizures, including acoustically evoked alcohol withdrawal-induced seizures (AWSs). One Ca2+ influx route of interest is the Na+/Ca2+ exchanger (NCX) that, when operating in its reverse mode (NCXrev) activity, can facilitate Ca2+ entry into neurons, possibly increasing neuronal excitability that leads to enhanced seizure susceptibility. Here, we probed the involvement of NCXrev activity on AWS susceptibility by quantifying the effects of SN-6 and KB-R7943, potent blockers of isoform type 1 (NCX1rev) and 3 (NCX3rev), respectively. Male, adult Sprague-Dawley rats were used. Acoustically evoked AWSs consisted of wild running seizures (WRSs) that evolved into generalized tonic-clonic seizures (GTCSs). Quantification shows that acute SN-6 treatment at a relatively low dose suppressed the occurrence of the GTCSs (but not WRSs) component of AWSs and markedly reduced the seizure severity. However, administration of KB-R7943 at a relatively high dose only reduced the incidence of GTCSs. These findings demonstrate that inhibition of NCX1rev activity is a putative mechanism for the suppression of alcohol withdrawal-induced GTCSs.

Numerous previous studies have found that C-reactive protein (CRP) is associated with cardiac arrhythmia and cardiac remodeling. However, the underlying mechanisms of this association remain unclear. Sodium-calcium exchanger 1 (NCX1) serves an important role in the regulation of intracellular calcium concentration, which is closely related with cardiac arrhythmia and cardiac remodeling. The present study aimed to evaluate the effects of CRP on NCX1 and intracellular calcium concentration in cardiomyocytes. Primary neonatal mouse ventricular cardiomyocytes were cultured and treated with varying concentrations of CRP (0, 5, 10, 20 and 40 µg/ml). The cardiomyocytes were also treated with NF-κB-specific inhibitor PTDC and a specific inhibitor of the reverse NCX1 KB-R7943 before their intracellular calcium concentrations were measured. mRNA and protein expression levels of NCX1 were detected by reverse transcription-quantitative PCR and western blotting, respectively and intracellular calcium concentration was evaluated by flow cytometry. CRP treatment significantly increased mRNA and protein expression levels of NCX1 in myocytes (P=0.024), as well as intracellular calcium concentration (P=0.01). These results were significantly attenuated by the NF-κB-specific inhibitor PDTC and a specific inhibitor of the reverse NCX1, KB-R7943. CRP significantly upregulated NCX1 expression and increased intracellular calcium concentration in cardiomyocytes via the NF-κB pathway, suggesting that CRP may serve a pro-arrhythmia role via direct influence on the calcium homeostasis of cardiomyocytes.

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.

Mammalian Na+/Ca2+ exchangers, NCX1 and NCX3, generate splice variants, whereas NCX2 does not. The CBD1 and CBD2 domains form a regulatory tandem (CBD12), where Ca2+ binding to CBD1 activates and Ca2+ binding to CBD2 (bearing the splicing segment) alleviates the Na+-induced inactivation. Here, the NCX2-CBD12, NCX3-CBD12-B, and NCX3-CBD12-AC proteins were analyzed by small-angle X-ray scattering (SAXS) and hydrogen-deuterium exchange mass-spectrometry (HDX-MS) to resolve regulatory variances in the NCX2 and NCX3 variants. SAXS revealed the unified model, according to which the Ca2+ binding to CBD12 shifts a dynamic equilibrium without generating new conformational states, and where more rigid conformational states become more populated without any global conformational changes. HDX-MS revealed the differential effects of the B and AC exons on the folding stability of apo CBD1 in NCX3-CBD12, where the dynamic differences become less noticeable in the Ca2+-bound state. Therefore, the apo forms predefine incremental changes in backbone dynamics upon Ca2+ binding. These observations may account for slower inactivation (caused by slower dissociation of occluded Ca2+ from CBD12) in the skeletal vs the brain-expressed NCX2 and NCX3 variants. This may have physiological relevance, since NCX must extrude much higher amounts of Ca2+ from the skeletal cell than from the neuron.

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.

Mild traumatic brain injury (mTBI) is a frequently overlooked public health concern that is difficult to diagnose and treat. Diffuse axonal injury (DAI) is a common mTBI neuropathology in which axonal shearing and stretching induces breakdown of the cytoskeleton, impaired axonal trafficking, axonal degeneration, and cognitive dysfunction. DAI is becoming recognized as a principal neuropathology of mTBI with supporting evidence from animal model, human pathology, and neuroimaging studies. As mitochondrial dysfunction and calcium overload are critical steps in secondary brain and axonal injury, we investigated changes in protein expression of potential targets following mTBI using an in vivo controlled cortical impact model. We show upregulated expression of sodium calcium exchanger1 (NCX1) in the hippocampus and cortex at distinct time points post-mTBI. Expression of dynamin-related protein1 (Drp1), a GTPase responsible for regulation of mitochondrial fission, also changes differently post-injury in the hippocampus and cortex. Using an in vitro model of DAI previously reported by our group, we tested whether pharmacological inhibition of NCX1 by SN-6 and of dynamin1, dynamin2, and Drp1 by dynasore mitigates secondary damage. Dynasore and SN-6 attenuate stretch injury-induced swelling of axonal varicosities and mitochondrial fragmentation. In addition, we show that dynasore, but not SN-6, protects against H2O2-induced damage in an organotypic oxidative stress model. As there is currently no standard treatment to mitigate cell damage induced by mTBI and DAI, this work highlights two potential therapeutic targets for treatment of DAI in multiple models of mTBI and DAI.

Intracellular Na(+) and H(+) inhibit Na(+)-Ca(2+) exchange. ATP regulates exchange activity by altering kinetic parameters for Ca(2+)(i), Na(+)(i) and Na(+)(o). The role of the Ca(2+)(i)regulatory site on Na(+)(i)-H(+)(i)-ATP interactions was explored by measuring the Na(+)(o)-dependent (45)Ca(2+) efflux (Na(+)(o)-Ca(2+)(i) exchange) and Ca(2+)(i)-dependent (22)Na(+) efflux (Na(+)(o)-Na(+)(i) exchange) in intracellular-dialysed squid axons. Our results show that: (1) without ATP, inhibition by Na(+)(i) is strongly dependent on H(+)(i). Lowering the pH(i) by 0.4 units from its physiological value of 7.3 causes 80 % inhibition of Na(+)(o)-Ca(2+)(i) exchange; (2) in the presence of MgATP, H(+)(i) and Na(+)(i) inhibition is markedly diminished; and (3) experiments on Na(+)(o)-Na(+)(i) exchange indicate that the drastic changes in the Na(+)(i)-H(+)(i)-ATP interactions take place at the Ca(2+)(i) regulatory site. The increase in Ca(2+)(i) affinity induced by ATP at acid pH (6.9) can be mimicked by a rise in pH(i) from 6.9 to 7.3 in the absence of the nucleotide. We conclude that ATP modulation of the Na(+)-Ca(2+) exchange occurs by protection from intracellular proton and sodium inhibition. These findings are predicted by a model where: (i) the binding of Ca(2+) to the regulatory site is essential for translocation but not for the binding of Na(+)(i) or Ca(2+)(i) to the transporting site; (ii) H(+)(i) competes with Ca(2+)(i) for the same form of the exchanger without an effect on the Ca(2+)(i) transporting site; (iii) protonation of the carrier increases the apparent affinity and changes the cooperativity for Na(+)(i) binding; and (iv) ATP prevents both H(+)(i) and Na(+)(i)-effects. The relief of H(+) and Na(+) inhibition induced by ATP could be important in cardiac ischaemia, in which a combination of acidosis and rise in [Na(+)](i) occurs.

Stretch and tachycardia are common triggers for cardiac remodelling in various conditions, but a comparative characterization of their role in the excitation-transcription coupling (ETC) and early regulation of gene expression and structural changes is lacking. Here, we show that stretch and tachycardia directly induced hypertrophy of neonatal rat cardiac myocytes and also of non-myocytes. Both triggers induced similar patterns of hypertrophy but had largely distinct gene expression profiles. ACTA1 served as good hypertrophy marker upon stretch, while RCAN1 was found increased in response to tachycardia in a rate-dependent fashion. Mechanistically, several calcium-handling proteins, including the sodium-calcium exchanger (NCX), contributed to ETC. Phosphorylation of the calcium/calmodulin-dependent protein kinase II (CaMKII) was elevated and occurred downstream of NCX activation upon tachycardia, but not stretch. Microarray profiling revealed that stretch and tachycardia regulated around 33% and 20% genes in a NCX-dependent manner, respectively. In conclusion, our data show that hypertrophy induction by stretch and tachycardia is associated with different gene expression profiles with a significant contribution of the NCX.

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN) is a frequent adverse event of colorectal cancer treatment. OIPN encompasses a chronic and an acute syndrome. The latter consists of transient axonal hyperexcitability, due to unbalance in Na+ voltage-operated channels (Na+VOC). This leads to sustained depolarisation which can activate the reverse mode of the Na+/Ca2+ exchanger 2 (NCX2), resulting in toxic Ca2+ accumulation and axonal damage (ADa). We explored the role of NCX2 in in vitro and in vivo settings. Embryonic rat Dorsal Root Ganglia (DRG) organotypic cultures treated with SEA0400 (SEA), a NCX inhibitor, were used to assess neuroprotection in a proof-of-concept and pilot study to exploit NCX modulation to prevent ADa. In vivo, OHP treated mice (7 mg/Kg, i.v., once a week for 8 weeks) were compared with a vehicle-treated group (n = 12 each). Neurophysiological and behavioural testing were performed to characterise acute and chronic OIPN, and morphological analyses were performed to detect ADa. Immunohistochemistry, immunofluorescence, and western blotting (WB) analyses were also performed to demonstrate changes in NCX2 immunoreactivity and protein expression. In vitro, NCX inhibition was matched by ADa mitigation. In the in vivo part, after verifyingboth acute and chronic OIPN had ensued, we confirmed via immunohistochemistry, immunofluorescence, and WB that a significant NCX2 alteration had ensued in the OHP group. Our data suggest NCX2 involvement in ADa development, paving the way to a new line of research to prevent OIPN.