Recent studies have confirmed the protective effects of nonsteroidal MR (mineralocorticoid receptor) antagonists in diabetic kidney disease. However, the physiological mechanisms underlying their albuminuria-reducing effects remain incompletely defined. We hypothesized that inhibition of the MR could protect podocytes by limiting excessive calcium influx via TRPC (transient receptor potential canonical) 5, thereby reducing albuminuria. We evaluated the effects of the nonsteroidal MR antagonist finerenone on albuminuria, podocyte morphology, and glomerular function in diabetic mice. Reactive oxygen species generation and single-nephron glomerular filtration rate were analyzed using in vivo imaging. Cultured podocytes were used to assess MR-TRPC5 signaling through measurements of Sgk1 (serum- and glucocorticoid-regulated kinase 1) and TRPC5 expression, intracellular calcium, and actin cytoskeletal organization. Finerenone significantly reduced albuminuria, ameliorated podocyte morphological abnormalities, and decreased glomerular reactive oxygen species production in diabetic mice. In cultured podocytes, aldosterone increased Sgk1 and TRPC5 expression, elevated intracellular calcium, and induced actin reorganization; these changes were attenuated by finerenone and by the AC1903 (TRPC5 inhibitor). Activation of MR-TRPC signaling was associated with increased calcium influx and features of podocyte injury. In vivo imaging further indicated that finerenone was associated with lower single-nephron glomerular filtration rate, consistent with an attenuation of glomerular hyperfiltration. Finerenone is associated with reductions in albuminuria in diabetic kidney disease, together with improvements in podocyte injury indices and glomerular hemodynamics. These benefits may be mediated in part through MR-TRPC5 signaling although additional pathways are likely to contribute.

Domoic acid (DA) is a neurotoxic terpenoid compound produced by certain marine algae. It accumulates through the food web and poses a significant threat to humans and animals by selectively targeting hippocampal neurons, leading to neuronal degeneration, necrosis, and subsequent memory impairment. The primary mechanism involves its potent agonism at glutamate receptors, which induces excessive calcium influx, resulting in excitotoxic cell swelling and death. Recent studies have further elucidated the critical role of downstream oxidative stress and other pathogenic factors in DA-induced neurotoxicity. These insights into its multifaceted mechanism have paved the way for novel therapeutic strategies, highlighting promising directions for future treatment development.

Alzheimer's disease is a neurodegenerative disorder characterized by impairments in cognitive functions such as thinking, behavior, and memory. The major pathological abnormalities associated with the disease include the formation of neurofibrillary tangles and amyloid plaques, which further cause neuroinflammation and nerve cell death. Currently, treatments for the disease focus on symptomatic management rather than addressing the root cause of neurological changes. Therefore, the current status of therapy highlights the need for more effective therapeutic substances that can either prevent abnormal deposition or slow neurodegeneration to preserve nerve cells. In this respect, ATP-sensitive potassium channel openers may have a potential role in prevention and protection. The present article focuses on several cellular mechanisms of this class, including the limitation of neuronal excitability, modulation of neurotransmitter release, prevention of aberrant protein buildup, reduction of excessive calcium influx, reduction of reactive oxygen species levels, and reduction of microglial activation.

Spinal cord injury (SCI) leads to devastating functional loss, partly due to a hostile post-injury microenvironment characterized by detrimental events like excessive calcium influx and apoptosis, which also limit the efficacy of reparative strategies such as neural tissue transplantation. It has been reported that electrical stimulation (ES) could modulate these events. Here, we investigated whether ES could enhance SCI repair, particularly when combined with allogeneic adult spinal cord tissue (aSCT) transplantation. Adult rats with complete thoracic SCI received aSCT grafts, with a subset also receiving continuous ES for two weeks. It found that locomotor function was significantly improved in rats receiving ES in conjunction with transplantation compared to those with transplantation alone. Histologically, ES treatment markedly enhanced neuronal survival and increased GAP-43+ axonal fiber density in the injured spinal cord. Mechanism studies revealed that ES downregulated L-type voltage-gated calcium channel (L-VGCC) and cleaved Caspase-3 expression. These results indicate that ES, when applied with aSCT transplantation, significantly promotes motor functional recovery and neural repair by mitigating calcium influx and apoptosis. Therefore, ES could be considered as a potent therapeutic strategy to augment reparative processes for SCI.

The transfer of information and metabolites between the mitochondria and the endoplasmic reticulum (ER) is mediated by mitochondria-ER contact sites (MERCS), allowing adaptations in response to changes in cellular homeostasis. MERCS are dynamic structures essential for maintaining cell homeostasis through the modulation of calcium transfer, redox signalling, lipid transfer, autophagy and mitochondrial dynamics. Under stress conditions such as ER protein misfolding, the Unfolded Protein Response (UPRER) mediates PERK and IRE1 activation, both of which localise at MERCS. Adaptive UPRER signalling enhances mitochondrial function and calcium import, whereas maladaptive responses lead to excessive calcium influx and apoptosis. In this study, induction of mild acute ER stress with tunicamycin (TM) in myoblasts promoted myogenesis that required PERK for increased MERCS assembly, mitochondrial turnover and function. Similarly, treatment of C. elegans embryos with an acute low concentration of TM, promoted an extension in lifespan and health-span. The adaptive ER stress response following a low dose of TM in both myoblasts and C. elegans, increased MERCS assembly and activated autophagy machinery, ultimately promoting an increase in mitochondrial remodelling. However, these beneficial adaptations were dependent on the developmental stage, as treatment of myotubes or adult C. elegans resulted in a maladaptive response. In both models the adaptations to UPRER activation were dependent on PERK signalling and its interaction with the UPRmt. The results demonstrate PERK is required for the increased mitochondrial ER communication in response to adaptive UPR signalling, promoting mitochondrial remodelling and improved physiological function.

Traumatic brain injury (TBI) is a significant global public health issue, affecting millions annually. Excessive calcium influx in neurons and astrocytes triggers a cascade of neurotoxic events, including mitochondrial dysfunction, increased production of reactive oxygen species, and hypometabolism, all of which contribute to impaired neurological function. Following TBI, alterations in presynaptic voltage-gated calcium channels (VGCCs) and the formation of plasma membrane pores facilitate Ca2+ influx, membrane depolarization, and an increased vesicular release of glutamate and Ca2+ into the synaptic cleft. This leads to the overactivation of NMDA receptors and the propagation of neurotoxic Ca2+ signals to neighboring neurons, further spreading neurobiochemical disruptions. Given this, blocking Ca2+ influx may mitigate excitotoxicity, and mitochondrial alterations caused by TBI. Among the pathways involved in Ca2+ cytotoxicity, the alpha-2-delta (α2δ(1-2)) subunit of VGCCs, located at the presynaptic terminal, remains the least explored. In this review, we briefly examine the pathophysiological hallmarks of TBI and their connection to Ca2+ dysregulation, while exploring the distribution of VGCC subtypes in the brain. Additionally, we highlight pregabalin, an analog of gabapentin and a selective antagonist of the α2δ(1-2) subunit, as a promising therapeutic strategy to counteract Ca2+-induced neurotoxicity following TBI.

IntroductionDuration of untreated psychosis (DUP) is defined as the time from manifestation of the first psychotic symptom to initiation of adequate antipsychotic drug treatment. It is associated with poorer response rates to antipsychotic medications and impaired cognition, which traslates in worse positive and negative symtomps. Various hypotheses for how untreated psychosis could impact brain function have been proposed. Dopaminergic hyperactivity leading to a progressive reduction in regional brain volumes and oxidative injury due to persistent catecholaminergic activity and prolonged activation of the hypothalamic–pituitary–adrenal axis provide possible explanations for how chronic psychosis could be neurotoxic. In addition, glutamate-mediated excitotoxicity may also contribute to these effects through neuronal overstimulation that leads to an excessive influx of calcium and subsequent excitotoxicity and, ultimately, cell death via apoptosis.ObjectivesTo analyze the advantages of long-acting injectable therapy in the treatment of psychotic disorders in patients with prolongued duration of untreated psychosis with a case communication.MethodsWe present a 61 year old female patient, native of a rural area of Peru who moved to Spain and started treatment in our outpatient department. She does not provide any medical report. The family reports that she began to present symptoms from her first pregnancy, and has had to be admitted to hospital several times for suicide attempts. She had never taken any antipsychotic drug. During the last 30 years she developed symptoms consisting of erotomaniac and paranoid delusional ideation, cenesthetic and auditory hallucinations, soliloquy, self care deficit and disorganised speech and behaviour. She presented periods of maniac mood. As a result, she ended up isolated, taken care by his father and highly dependent on instrumental activities of daily living.ResultsTreatment with Risperidone was started, although the patient presented poor adherence to it. She was finally admitted to hospital for a month and we introduced once-monthly Risperidone and Valproate with significant clinical improvement.ConclusionsLong acting injectables are a good therapeutic option in patients with psychotic disorders, even with prolongued duration of untreated psychosis.Disclosure of InterestNone Declared

Targeting TRPC6 in podocytopathies: Why clinical translation remains a challenge?

Opioid use for pain management and illicit consumption has been associated with adverse cardiovascular and cardiorenal outcomes. Despite these associations, the mechanisms underlying opioid-induced kidney damage remain poorly understood. Recently, we demonstrated that stimulation of kappa opioid receptors (KOR) is implicated in the aggravation of salt-sensitive hypertension, glomerular injury, and podocyte damage through excessive podocyte calcium influx. This study aims to elucidate the KOR signaling and renal outcomes underlying opioid use in Sprague-Dawley (SD) rats. Here, we employed freshly isolated glomeruli from SD male rats and immortalized human podocyte cell cultures to investigate the role of KORs in podocyte calcium regulation and overall glomerular function. A glomerular permeability assay was used to evaluate the impact of KORs on glomerular filter integrity. Additionally, the long-term effects of KOR activation were assessed in vivo by chronic intravenous infusion of selective KOR agonist BRL 52537 in SD rats. We found that acute application of BRL 52537 resulted in increased plasma membrane ion channel activity in immortalized human podocytes. Significant calcium influx in response to BRL 52537 was detected in podocytes of the isolated SD rat glomeruli. Further, glomerular permeability analysis revealed increased permeability and impaired filter integrity, indicating altered glomerular function. Lastly, prolonged KOR activation in SD rats results in an increase in blood pressure, an elevation of basal calcium levels in podocytes, and albuminuria. In conclusion, this study identifies novel renal physiological mechanisms through which opioid-induced KOR activation contributes to podocyte injury and glomerular damage in SD rats.

Diabetic nephropathy (DN) stands as the primary cause of end-stage renal failure. The pathological changes associated with DN, such as albuminuria and glomerular damage, result in progressive loss of renal function. Despite existing treatments, DN continues to worsen in many patients, requiring novel therapeutic strategies targeting disease progression mechanisms. Our recent studies have demonstrated that elevated levels of serine proteases during the progression of DN lead to overactivation of protease-activated receptor 1 (PAR1) signaling mechanisms. This overactivation drives excessive calcium influx in podocytes, resulting in the onset of albuminuria, perturbations in glomerular hemodynamics, and kidney damage. Therefore, pharmacological inhibition of PAR1 may blunt the effect of PAR1 signaling on DN progression. To test this hypothesis, we used a selective PAR1 antagonist Vorapaxar (SCH 530348, 10 mg/kg of diet). Vorapaxar is an FDA-approved medication used as antiplatelet therapy to reduce the risk of thrombotic cardiovascular events. 16-week-old male type 2 diabetic nephropathy (T2DN) rats were treated for 4 months to determine the effect of pharmacological systemic PAR1 inhibition on the development of DN. After 4 months of treatment, the glomerular filtration rate (GFR) was better preserved in the treated animals compared to the control group, which showed a decline (0.76 ± 0.19 vs 1.11 ± 0.29 mL/min/100g b.w., p=0.032, N≥6). The development of albuminuria was evident in both groups, confirming the progression of DN. However, a trend toward decline in the Alb/Cre ratio in the treated group was observed (12.4 ± 6.3 vs 8.1 ± 3.8, p=0.08, N≥6). Freshly isolated glomeruli from the vorpaxar-treated animals exhibit a significant decline in thrombin-induced calcium response and basal calcium levels within podocytes (240 ± 147 vs 145 ± 80 nM, p=0.035, n≥13, N≥6). Additionally, PAR1 inhibition influenced inflammation-resolution mediators in both the kidney and heart. In the kidney, Maresin Conjugates in Tissue Regeneration (MCTR1) were significantly upregulated, while hydroxy-docosahexaenoic acids (7-, 14-, and 17-HDHA) were downregulated. In the heart, significant downregulation was observed for Resolvine D4 (RvD4), Protectin D1 (PD1), and 17-HDHA. The inhibition of thrombin-induced calcium signaling and reduced basal calcium levels highlight a potential protective effect of PAR1 inhibition on podocytes. Changes in GFR may be linked to reduced glomerular damage, indicating that the structural integrity of the glomeruli is better maintained in treated animals. These findings point to PAR1 inhibition as a potential therapeutic strategy for protecting kidney function and modulating inflammatory and pro-resolving pathways in DN. The observed changes in lipid mediators and podocyte calcium dynamics suggest both direct and systemic effects, which may help mitigate the progression of kidney and heart complications commonly associated with diabetes. This work is supported by NIH grants R01 DK135644 (to A.S.) and R01 DK129227 (to A.S. and O.P.), and the Vascular Inflammation and Injury Training Program T32 HL160529 (to R.B.). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Therapeutic modulation of mitochondrial dynamics by agmatine in neurodegenerative disorders.

The Shaker/Kv1 subfamily of voltage-gated potassium (K+) channels is essential for modulating membrane excitability. Their loss results in prolonged depolarization and excessive calcium influx. These channels have also been implicated in a variety of other cellular processes, but the underlying mechanisms remain poorly understood. Through comprehensive screening of K+ channel mutants in C. elegans, we discovered that shk-1 mutants are highly susceptible to bacterial pathogen infection and oxidative stress. This vulnerability is associated with reduced glycogen levels and substantial mitochondrial dysfunction, including decreased ATP production and dysregulated mitochondrial membrane potential under stress conditions. SHK-1 is predominantly expressed and functions in body wall muscle to maintain glycogen storage and mitochondrial homeostasis. RNA-sequencing data reveal that shk-1 mutants have decreased expression of a set of cation-transporting ATPases (CATP), which are crucial for maintaining electrochemical gradients. Intriguingly, overexpressing catp-3, but not other catp genes, restores the depolarization of mitochondrial membrane potential under stress and enhances stress tolerance in shk-1 mutants. This finding suggests that increased catp-3 levels may help restore electrochemical gradients disrupted by shk-1 deficiency, thereby rescuing the phenotypes observed in shk-1 mutants. Overall, our findings highlight a critical role for SHK-1 in maintaining stress tolerance by regulating glycogen storage, mitochondrial homeostasis, and gene expression. They also provide insights into how Shaker/Kv1 channels participate in a broad range of cellular processes.

BACKGROUNDObstructed defecation syndrome (ODS) represents the most prevalent form of chronic constipation, affecting a diverse patient population, leading to numerous complications, and imposing a significant burden on healthcare resources. Most ODS patients have insufficient rectal propulsion, but the exact mechanism underlying the pathogenesis of ODS remains unclear.AIMTo explore the molecular mechanism underlying the pathogenesis of ODS.METHODSA total of 30 pairs of rectal samples were collected from patients with ODS (ODS group) or grade IV prolapsed hemorrhoids without constipation (control group) for quantitative proteomic and bioinformatic analysis. Subsequently, 50 pairs of paraffin-embedded rectal specimens were selected for immunohistochemistry and immunofluorescence studies to validate the analysis results. Human intestinal smooth cell contractile function experiments and electrophysiological experiments were conducted to verify the physiological functions of target proteins. Cellular ultrastructure was detected using transmission electron microscopy.RESULTSIn comparison to the control group, the expression level of dystrophin (DMD) in rectal specimens from ODS patients was markedly reduced. This finding was corroborated using immunohistochemistry and immunofluorescence techniques. The diminished expression of DMD compromised the contractile function of intestinal smooth muscle cells. At the molecular level, nucleoporin protein 153 and L-type voltage-gated calcium channel were found to be overexpressed in intestinal smooth muscle cells exhibiting downregulated DMD expression. Electrophysiological experiments confirmed an excessive influx of calcium ions into these cells. Moreover, vacuolar-like structures which may be associated with excessive calcium influx were observed in the cells by transmission electron microscopy.CONCLUSIONDecreased DMD expression in intestinal smooth muscle may upregulate L-type voltage-gated calcium channel expression, leading to excessive calcium influx which may cause a decrease in rectal propulsion, thereby contributing to the pathogenesis of ODS.

Neurodegenerative diseases pose significant challenges to healthcare systems globally due to their complex etiology and relentless progression, often rendering conventional treatments ineffective. Recent advances have spotlighted excitatory amino acids, particularly D-amino acids, once considered as products of metabolism of the microbiota or deriving from food intake. This review explores the role of D-amino acids in mitigating excitotoxicity-a process characterized by excessive calcium influx through aberrant N-methyl-D-aspartate receptor (NMDAR) activation, which is implicated in the pathogenesis of diseases like Alzheimer's disease. By providing alternative pathways for neuronal signaling and protecting against excitotoxic damage, D-amino acids offer a novel approach to reversing neurodegenerative trajectories. Future research should focus on elucidating the detailed mechanisms of action of these compounds, evaluating their therapeutic potential through rigorous preclinical and clinical trials, and developing effective delivery systems to optimize their neuroprotective effects. This emerging field holds promise for developing innovative treatment strategies that could significantly improve outcomes for patients with neurodegenerative disorders.

Traumatic spinal cord injury (SCI) results in the disruption of physiological systems below the level of the spinal lesion. Connexin hemichannels (CxHCs) are membrane-bound, non-selective pore proteins that are lost in mature myofibers but reappear de novo on the sarcolemma after peripheral denervation, chronic SCI, diabetes, and severe systemic stress such as sepsis. Cx43 and Cx45 have been implicated as the major CxHCs present in diseased muscle, and muscle-restricted knockout of these genes reduces muscle atrophy after denervation, likely by reducing excess calcium influx with resultant inflammasome activation. A muscle-restricted Cx43/45 conditional knockout (mKO) mouse model was developed and tested to check whether it would improve outcomes following either a complete spinal cord transection at the level of thoracic vertebrae-9 (T9) or a motor-incomplete T9 impact-contusion SCI. mKO had no effect on the body mass after complete T9 transection. There was reduced atrophy of the plantaris 15 days post-SCI that was not associated with molecular markers of inflammation, hypertrophic/atrophic protein signaling, or protein and mRNA expression related to mitochondrial integrity and function. mKO mice had faster and greater locomotor recovery across 28 days after a motor-incomplete contusion SCI with no differences in spared white matter; male mKO mice generally had greater muscle mass than genotype controls post-injury, but muscle sparing was not observed in female mKO mice post-injury. The data establish a new paradigm where muscle Cx43/45 may contribute to the tissue crosstalk that determines the neuromuscular function of sub-lesional musculature after motor-incomplete SCI in a sex-dependent manner. Our novel findings should promote investigation to develop innovative treatment strategies to improve the function and quality of life for persons with SCI.

The pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the demise of motor neurons has been linked to excitotoxicity caused by excessive calcium influx via N-methyl-D-aspartate receptors (NMDARs), suggesting that uncompetitive NMDAR antagonism could be a strategy to attenuate motor neuron degeneration. REL-1017, the dextro-isomer of racemic methadone, is a low-affinity uncompetitive NMDAR antagonist. Importantly, in humans REL-1017 has shown excellent tolerability in clinical trials for major depression. Here, we tested if REL-1017 improves the disease phenotypes in the G93A SOD1 mouse, a well-established model of familial ALS, by examining survival and motor functions, as well as the expression of genes and proteins involved in neuroplasticity. We found a sex-dependent effect of REL-1017 in G93A SOD1 mice. A delay of ALS symptom onset, assessed as 10%-decrease of body weight (p < 0.01 vs. control untreated mice) and an extension of lifespan (p < 0.001 vs. control untreated mice) was observed in male G93A SOD1 mice. Female G93A SOD1 mice treated with REL-1017 showed an improvement of muscle strength (p < 0.01 vs. control untreated mice). Both males and females treated with REL-1017 showed a decrease in hind limb clasping. Sex-dependent effects of REL-1017 were also detected in molecular markers of neuronal plasticity (PSD95 and SYN1) in the spinal cord and in the GluN1 NMDAR subunit in quadricep muscles. In conclusion, this study provides preclinical in vivo evidence supporting the clinical evaluation of REL-1017 in ALS.

Ultraviolet B (UVB) radiation causes skin injury by trigging excessive calcium influx and signaling cascades in the skin keratinocytes. The heat-sensitive Ca2+ -permeable transient receptor potential vanilloid 3 (TRPV3) channels robustly expressed in the keratinocytes play an important role in skin barrier formation and wound healing. Here, we report that inhibition of cutaneous TRPV3 alleviates UVB radiation-induced skin lesions. In mouse models of ear swelling and dorsal skin injury induced by a single exposure of weak UVB radiation, TRPV3 genes and proteins were upregulated in quantitative real-time PCR and Western blot assays. In accompany with TRPV3 upregulations, the expressions of proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were also increased. Knockout of the TRPV3 gene alleviates UVB-induced ear swelling and dorsal skin inflammation. Furthermore, topical applications of two selective TRPV3 inhibitors, osthole and verbascoside, resulted in a dose-dependent attenuation of skin inflammation and lesions. Taken together, our findings demonstrate the causative role of overactive TRPV3 channel function in the development of UVB-induced skin injury. Therefore, topical inhibition of TRPV3 may hold potential therapy or prevention of UVB radiation-induced skin injury.

There is an urgent need to find common targets for precision therapy, as there are no effective preventive therapeutic measures for combined clinical heart-brain organ protection and common pathways associated with glutamate receptors are involved in heart-brain injury, but current glutamate receptor-related clinical trials have failed. Ischemia-reperfusion injury (IRI) is a common pathological condition that occurs in multiple organs, including the heart and brain, and can lead to severe morbidity and mortality. N-methyl-D-aspartate receptor (NMDAR), a type of ionotropic glutamate receptor, plays a crucial role in the pathogenesis of IRI. NMDAR activity is mainly regulated by endogenous activators, agonists, antagonists, and voltage-gated channels, and activation leads to excessive calcium influx, oxidative stress, mitochondrial dysfunction, inflammation, apoptosis, and necrosis in ischemic cells. In this review, we summarize current research advances regarding the role of NMDAR in myocardial and cerebral IRI and discuss potential therapeutic strategies to modulate NMDAR signaling to prevent and treat IRI.

<b>Abstract ID 16706</b> <b>Poster Board 248</b> Triple-negative breast cancer (TNBC) is one of the most aggressive forms of breast cancer that hormonal therapies cannot treat. Current standards of care include surgical removal of the tumor and aggressive chemotherapy. Consequently, there is an urgent need for new, molecular-based therapies to treat these patients effectively^1,2. On a molecular level, two proteins facilitate finely tuned calcium entry: Orai1, a pore-forming calcium channel, and stromal interaction molecule 1 (STIM1), a calcium sensor embedded in the ER membrane. Upon ER calcium store depletion, STIM1 undergoes a conformational change to bind to Orai1, which triggers extracellular calcium entry to achieve homeostasis³. Recently, it has been discovered that a third protein is implicated in this process: adenylyl cyclase type 8 (AC8)⁴. The N-terminus of AC8 can bind to the N-terminus of Orai1 to prolong calcium influx, which ceases upon Orai1 phosphorylation⁴. In normal breast epithelial cells, AC8 is expressed at a basal level and does not interfere with Orai1-STIM1 calcium entry. However, in TNBC cells, AC8 is grossly overexpressed so that all Orai1 channels become bound^4,5. As AC8 binds to Orai1 on its phosphorylation site, the Orai1 channel cannot be inactivated, leading to excess calcium influx and adverse cellular effects such as proliferation and migration. Thus, <b>we hypothesize</b> that if we inhibit the interaction between AC8 and Orai1 with a therapeutic small molecule, we will observe a decrease in proliferation in TNBC cells. We also expect that inhibiting the interaction between Orai1 and STIM1 will produce similar effects. Ultimately, we aim to screen a library of small molecules against our protein-protein interactions to find therapeutic inhibitors. We will use the NanoBiT system to study each different protein-protein interaction. Briefly, the NanoBiT system tags each protein with a split luciferase, allowing for the generation of a luminescent signal upon protein binding. Using this method, <b>we have found</b> that the interaction between STIM1 and Orai1 is strictly governed by calcium, whereas the interaction between AC8 and Orai1 is more constitutive. Interestingly, we found that forskolin, an AC8 activator, prevents AC8 binding to Orai1, as evident through loss of luminescence. This suggests that AC8 must be inactive to interact with Orai1 and that phosphorylated Orai1 cannot bind to AC8. These findings have allowed us to conduct Z-Score studies to prime for initial screening, which fall within a 0.6-0.8 range for each protein-protein interaction. To date, we have accomplished screening two libraries of small molecule compounds; NCI Diversity Set VI and NCI Approved Oncology Set X, which will be extended further to other diversity libraries. In addition, we plan to test any inhibitors in different TNBC cell lines (MDA-MB-231 and MDA-MB-157) to observe effects on cancer cell migration. <b>In conclusion</b>, understanding the unique interplay between AC8, Orai1, and STIM1 is critical to understanding how to prevent TNBC cell proliferation and migration. <b>References</b> Stovgaard, et. al. Triple-negative breast cancer… <i>Acta Onc</i>. 2018; 57:1, 74-82, Bose. Triple-negative Breast Carcinoma… <i>Adv. in Ana. Path</i>. 2015; 22: 306-313 Lunz, et. al. STIM1 activation of Orai1. <i>Cell Cal</i>. 2019; 77, 29-38 Sanchez-Collado, et&nbsp;al. Adenylyl Cyclase Type 8 Overexpression… <i>Cancers</i>. 2019; 11, 1624 Derler, et. al. Structure, regulation and biophysics of I(CRAC), STIM/Orai1. <i>Adv. Exp. Med. Biol</i>. 2012; 740, 383–410

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacidreceptors (AMPARs) are neurotransmitter-activated cation channelsubiquitously expressed in vertebrate brains. The regulation of calciumflux through the channel pore by RNA-editing is linked to synapticplasticity while excessive calcium influx poses a risk for neurodegeneration.Unfortunately, the molecular mechanisms underlying this key processare mostly unknown. Here, we investigated calcium conduction in calcium-permeableAMPAR using Molecular Dynamics (MD) simulations with recently introducedmultisite force-field parameters for Ca2+. Our calculationsare consistent with experiment and explain the distinct calcium permeabilityin different RNA-edited forms of GluA2. For one of the identifiedmetal binding sites, multiscale Quantum Mechanics/Molecular Mechanics(QM/MM) simulations further validated the results from MD and revealedsmall but reproducible charge transfer between the metal ion and itsfirst solvation shell. In addition, the ion occupancy derived fromMD simulations independently reproduced the Ca2+ bindingprofile in an X-ray structure of an NaK channel mimicking the AMPARselectivity filter. This integrated study comprising X-ray crystallography,multisite MD, and multiscale QM/MM simulations provides unprecedentedinsights into Ca2+ permeation mechanisms in AMPARs, andpaves the way for studying other biological processes in which Ca2+ plays a pivotal role.