Modulation Of Calcium Channels Research Articles

Transcriptomic imputation identifies tissue-specific genes associated with cervical myelopathy

Degenerative cervical myelopathy (DCM) is a progressive spinal condition that can lead to severe neurological dysfunction. Despite its degenerative pathophysiology, family history has shown to be a largely important factor in incidence and progression, suggesting that inherent genetic predisposition may play a role in pathophysiology. To determine the tissue-specific, functional genetic basis of hereditary predisposition to cervical myelopathy. Retrospective case-control study using patient genetics and matched EHR from the Mount Sinai BioMe Biobank. In a large, diverse, urban biobank of 32,031 individuals, with 558 individuals with cervical myopathy, we applied transcriptomic imputation to identify genetically regulated gene expression signatures associated with DCM. We performed drug-repurposing analysis using the CMAP database to identify candidate therapeutic interventions to reverse the cervical myelopathy-associated gene signature. We identified 16 genes significantly associated with DCM across five different tissues, suggesting tissue-specific manifestations of inherited genetic risk (upregulated: HES6, PI16, TMEM183A, BDH2, LINC00937, CLEC4D, USP43, SPATA1; downregulated: TTC12, CDK5, PAFAH1B2, RCSD1, KLHL29, PTPRG, RP11-620J15.3, C1RL). Drug repurposing identified 22 compounds with the potential to reverse the DCM-associated signature, suggesting points of therapeutic intervention. The inherited genetic risk for cervical myelopathy is functionally associated with genes involved in tissue-specific nociceptive and proliferative processes. These signatures may be reversed by candidate therapeutics with nociceptive, calcium channel modulating, and antiproliferative effects. Understanding the genetic basis of DCM provides critical insights into the hereditary factors contributing to the disease, allowing for more personalized and targeted therapeutic approaches. The identification of candidate drugs through transcriptomic imputation and drug repurposing analysis offers potential new treatments that could significantly improve patient outcomes and quality of life by addressing the underlying genetic mechanisms of DCM.

Over Activation of IL-6/STAT3 Signaling Pathway in Juvenile Dermatomyositis.

Juvenile dermatomyositis (JDM) is characterized by persistent non-purulent inflammation in the muscle and skin. The underlying mechanisms still remain uncertain. This study aims to elucidate the mechanism of interleukin-6 (IL-6) activation of Janus kinase/signal transducer and activator of transcription 3 pathway (JAK/STAT3), contributing to the pathogenesis of JDM. Serum IL-6 levels were compared between 72 newly diagnosed patients with JDM and the same patient cohort in treatment remission. Single-cell RNA sequencing (scRNA-seq) was employed to identify differential signaling pathway expression in muscle biopsy samples from two patients with JDM and healthy controls. Immunohistochemistry was used to examine differences in STAT3 phosphorylation between JDM and control muscle tissues. In vitro, skeletal muscle cell lines were stimulated with IL-6, and the transcription levels of genes related to mitochondrial calcium channels were quantified via reverse transcription-polymerase chain reaction (RT-PCR). Reactive oxygen species (ROS) production was measured in both IL-6 treated and untreated groups. ROS levels were then compared between IL-6 receptor antagonist pre-treated skeletal muscle cells and untreated cells. IL-6 levels in newly onset patients with JDM were significantly higher compared to the same patient cohort in remission states (p < 0.0001). Serum IL-6 was significantly increased in patients with negative myositis specific antibody (MSA), positive melanoma differentiation associated protein 5 (MDA5) and positive nuclear matrix protein 2 (NXP2), yet not for JDM with positive transcriptional intermediary factor γ(TIF1γ), based on subgroup analysis. ScRNA-seq analysis of muscle biopsies from patients with MDA5-positive JDM and patients with MSA negative JDM revealed abnormal activation of the JAK/STAT3 pathway in skeletal myocytes, macrophages, and vascular endothelial cells. The phosphorylation levels of STAT3 were elevated in active JDM cases. Transcription of the calcium channel modulation gene sarcolipin (SLN) was significantly higher in JDM primary skeletal muscle cells compared to normal cells. In vitro, IL-6 enhanced SLN transcription and induced ROS production, and blocking the IL-6 receptor resulted in decreased ROS generation in skeletal muscle cells. IL-6/JAK/STAT3 signaling pathway was abnormally activated in patients with JDM. IL-6 may be involved in the pathogenesis of muscle damage by triggering the development of calcium overload and production of ROS. Blockade of the IL-6/JAK/STAT3 pathway can be a potential treatment option for JDM, especially MDA5-positive patients and those who are negative for MSA.

Importance of Zinc Homeostasis for Normal Cardiac Rhythm

&lt;p&gt;Current arrhythmia therapies such as ion channel blockers, catheter ablation, or implantable cardioverter defibrillators have limitations and side effects, and given the proarrhythmic risk associated with conventional, ion channel-targeted anti-arrhythmic drug therapies, a new approach to arrhythmias may be warranted. Measuring and adjusting the level of particular ions that impact heart rhythm can be a simple and low-complication strategy for preventing or treating specific arrhythmias. In addition, new medicines targeting these ions may effectively treat arrhythmias. Numerous studies have shown that intracellular and extracellular zinc concentrations impact the heart's electrical activity. Zinc has been observed to affect cardiac rhythm through a range of mechanisms. These mechanisms encompass the modulation of sodium, calcium, and potassium ion channels, as well as the influence on beta-adrenergic receptors and the enzyme adenylate cyclase. &lt;/p&gt;&lt;p&gt; Moreover, zinc can either counteract or induce oxidative stress, hinder calmodulin or the enzyme Ca (2+)/calmodulin-dependent protein kinase II (CaMKII), regulate cellular ATP levels, affect the processes of aging and autophagy, influence calcium ryanodine receptors, and control cellular inflammation. Additionally, zinc has been implicated in the modulation of circadian rhythm. Additionally, zinc has been implicated in the modulation of circadian rhythm. In all the above cases, the effect of zinc largely depends on the normal or increased cellular level of zinc, which shows the importance of maintaining the serum and intracellular levels of zinc within the normal range.&lt;/p&gt;

L-DOPA ELICITATION THROUGH CALCIUM ION MEDIATED CHANNEL IN CALLUS CULTURES OF MUCUNA PRURIENS

Objective: The production of L-Dopa in callus cultures of Mucuna pruriens is influenced by substances that regulate calcium channels. Methods: M. pruriens seeds were gathered from the Western Ghats area in Tamil Nadu. L-Dopa was obtained by the Brain (1976) method, A. niger cultures were used for fungal elicitor preparation, and the assay of Ca2+ATPase was studied. Results: The use of fungal triggers resulted in increased L-Dopa levels on the 9th day but reduced levels on the 24th day of culture. When calcium channel modulators like verapamil and chlorpromazine were added, it decreased the growth of calli and L-Dopa production, suggesting the role played by calcium ion channels in L-Dopa synthesis. The calcium ionophore A23187 enhanced calli growth and L-Dopa production, increasing Ca2+ATPase activity. Particularly on the 12th d, Ca2+ATPase was notably active, while the presence of calcium ionophore boosted L-Dopa biosynthesis. Conclusion: The use of fungal elicitors in in vitro cultures of Mucuna pruriens is recommended as a potential method to increase the production of secondary metabolites.

The Multifunctional Role of KCNE2: From Cardiac Arrhythmia to Multisystem Disorders.

The KCNE2 protein is encoded by the kcne2 gene and is a member of the KCNE protein family, also known as the MinK-related protein 1 (MiRP1). It is mostly present in the epicardium of the heart and gastric mucosa, and it is also found in the thyroid, pancreatic islets, liver and lung, among other locations, to a lesser extent. It is involved in numerous physiological processes because of its ubiquitous expression and partnering promiscuity, including the modulation of voltage-dependent potassium and calcium channels involved in cardiac action potential repolarization, and regulation of secretory processes in multiple epithelia, such as gastric acid secretion, thyroid hormone synthesis, generation and secretion of cerebrospinal fluid. Mutations in the KCNE2 gene or aberrant expression of the protein may play a critical role in cardiovascular, neurological, metabolic and multisystem disorders. This article provides an overview of the advancements made in understanding the physiological functions in organismal homeostasis and the pathophysiological consequences of KCNE2 in multisystem diseases.

CaM-dependent modulation of human CaV1.3 whole-cell and single-channel currents by C-terminal CaMKII phosphorylation site S1475.

Phosphorylation enables rapid modulation of voltage-gated calcium channels (VGCC) in physiological and pathophysiological conditions. How phosphorylation modulates human CaV1.3 VGCC, however, is largely unexplored. We characterized modulation of CaV1.3 gating via S1475, the human equivalent of a phosphorylation site identified in the rat. S1475 is highly conserved in CaV1.3 but absent from all other high-voltage activating calcium channel types co-expressed with CaV1.3 in similar tissues. Further, it is located in the C-terminal EF-hand motif, which binds calmodulin (CaM). This is involved in calcium-dependent channel inactivation (CDI). We used amino acid exchanges that mimic either sustained phosphorylation (S1475D) or phosphorylation resistance (S1475A). Whole-cell and single-channel recordings of phosphorylation state imitating CaV1.3 variants in transiently transfected HEK-293 cells revealed functional relevance of S1475 in human CaV1.3. We obtained three main findings: (1) CaV1.3_S1475D, imitating sustained phosphorylation, displayed decreased current density, reduced CDI and (in-) activation kinetics shifted to more depolarized voltages compared with both wildtype CaV1.3 and the phosphorylation-resistant CaV1.3_S1475A variant. Corresponding to the decreased current density, we find a reduced open probability of CaV1.3_S1475D at the single-channel level. (2) Using CaM overexpression or depletion, we find that CaM is necessary for modulating CaV1.3 through S1475. (3) CaMKII activation led to CaV1.3_WT-current properties similar to those of CaV1.3_S1475D, but did not affect CaV1.3_S1475A, confirming that CaMKII modulates human CaV1.3 via S1475. Given the physiological and pathophysiological importance of CaV1.3, our findings on the S1475-mediated interplay of phosphorylation, CaM interaction and CDI provide hints for approaches on specific CaV1.3 modulation under physiological and pathophysiological conditions. KEY POINTS: Phosphorylation modulates activity of voltage-gated L-type calcium channels for specific cellular needs but is largely unexplored for human CaV1.3 channels. Here we report that S1475, a CaMKII phosphorylation site identified in rats, is functionally relevant in human CaV1.3. Imitating phosphorylation states at S1475 alters current density and inactivation in a calmodulin-dependent manner. In wildtype CaV1.3 but not in the phosphorylation-resistant variant S1475A, CaMKII activation elicits effects similar to constitutively mimicking phosphorylation at S1475. Our findings provide novel insights on the interplay of modulatory mechanisms of human CaV1.3 channels, and present a possible target for CaV1.3-specific gating modulation in physiological and pathophysiological conditions.

Sustainable Dynamic Wrinkle Efficacy: Non-Invasive Peptides as the Future of Botox Alternatives

Dynamic wrinkle reduction continues to challenge aesthetic dermatology, predominantly addressed through Botulinumtoxin (Botox) injections. Despite Botox’s robust efficacy with up to an 80% reduction in wrinkle visibility within just one week, its invasive administration and specific mechanism of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex inhibition prompt the exploration of safer, non-invasive alternatives. This review critically assesses recent innovations in non-invasive effects, with a focus on peptides and botanical extracts that exhibit a diverse array of mechanisms including SNARE complex inhibition, modulation of calcium and sodium channels, and interactions with acetylcholine receptors, contributing to their effectiveness in muscle relaxation on dynamic wrinkle approaches. Noteworthy peptides such as Argireline and SYN-Ake replicate the neuromodulatory effects of Botox, achieving up to a 52% reduction in wrinkles within four weeks without injections. Moreover, botanical extracts meet the rising demand for clean beauty solutions by enhancing skin elasticity and health through gentle yet potent mechanisms. However, the main concern with peptides is their low absorption rate, with only six clinical validations regarding Botox-like peptide anti-wrinkle efficacy available. These advancements not only deepen our understanding of cosmetic dermatology but also significantly influence market dynamics and consumer behavior, underscoring their pivotal role in redefining the future landscape of anti-aging effects.

Urolithin A's Role in Alleviating Severe Acute Pancreatitis via Endoplasmic Reticulum-Mitochondrial Calcium Channel Modulation.

Severe acute pancreatitis (SAP), characterized by pancreatic acinar cell death, currently lacks effective targeted therapies. Ellagic acid (EA), rich in pomegranate, shows promising anti-inflammatory and antioxidant effects in SAP treatment. However, the roles of other forms of EA, such as plant extracellular vesicles (EVs) extracted from pomegranate, and Urolithin A (UA), converted from EA through gut microbiota metabolism in vivo, have not been definitively elucidated. Our research aimed to compare the effects of pomegranate-derived EVs (P-EVs) and UA in the treatment of SAP to screen an effective formulation and to explore its mechanisms in protecting acinar cells in SAP. By comparing the protective effects of P-EVs and UA on injured acinar cells, UA showed superior therapeutic effects than P-EVs. Subsequently, we further discussed the mechanism of UA in alleviating SAP inflammation. In vivo animal experiments found that UA could not only improve the inflammatory environment of pancreatic tissue and peripheral blood circulation in SAP mice but also revealed that the mechanism of UA in improving SAP might be related to mitochondria and endoplasmic reticulum (ER) through the results including pancreatic tissue transcriptomics and transmission electron microscopy. Further research found that UA could regulate ER-mitochondrial calcium channels and reduce pancreatic tissue necroptosis. In vitro experiments of mouse pancreatic organoids and acinar cells also confirmed that UA could improve pancreatic inflammation by regulating the ER-mitochondrial calcium channel and necroptosis pathway proteins. This study not only explored the therapeutic effect of plant EVs on SAP but also revealed that UA could alleviate SAP by regulating ER-mitochondrial calcium channel and reducing acinar cell necroptosis, providing insights into the pathogenesis and potential treatment of SAP.

Protein kinase C epsilon-mediated modulation of T-type calcium channels underlies alcohol withdrawal hyperexcitability in the midline thalamus.

Millions of people struggle with alcohol use disorder (AUD). Abrupt abstinence after a period of chronic alcohol use can precipitate the alcohol withdrawal syndrome (AWS), which includes hyperexcitability and, potentially, seizures. We have shown that T-type Ca2+ channels are novel, sensitive targets of alcohol, an effect that is dependent upon protein kinase C (PKC). The purpose of this study was to (1) understand midline thalamic neuronal hyperexcitability during alcohol withdrawal and its dependence on PKC; (2) characterize T channel functional changes using both current clamp and voltage clamp methods; and (3) determine which PKC isoform may be responsible for alcohol withdrawal (WD) effects. Whole-cell patch clamp recordings were performed in midline thalamic neurons in brain slices prepared from C57bl/6 mice that underwent chronic intermittent alcohol exposure in a standard vapor chamber model. The recordings were compared to those from air-exposed controls. T-channel inactivation curves and burst responses were acquired through voltage-clamp and current-clamp recordings, respectively. Whole-cell voltage clamp recordings of native T-type current exhibited a depolarizing shift in the voltage-dependency of inactivation during alcohol withdrawal compared to air-exposed controls. A PKCε translocation inhibitor peptide mitigated this change. Current clamp recordings demonstrated more spikes per burst during alcohol withdrawal. Consistent with voltage clamp findings, the PKCɛ translocation inhibitor peptide reduced the number of spikes per burst after WD. We found that alcohol WD produces T channel-mediated hyperexcitability in the midline thalamus, produced in part by a shift in the inactivation curve consistent with greater availability of T current. WD effects on T current inactivation were reduced to control levels by blocking PKCε translocation. Our results demonstrate that PKCε translocation plays an important role in the regulation of alcohol withdrawal-induced hyperexcitability in midline thalamic circuitry.

Comparative study of pregabalin and carbamazepine in painful diabetic peripheral neuropathy at the tertiary care hospital

This study aimed to examine the effectiveness of carbamazepine as well as pregabalin in lowering the degree of pain associated with diabetic peripheral neuropathy. The results revealed noteworthy insights into the comparative effectiveness of these medications. Pregabalin, as a calcium channel modulator, has demonstrated efficacy in numerous neuropathic pain conditions. Beyond pain reduction, these drugs influence various aspects of daily functioning, emotional well-being, and social interactions is crucial for tailoring treatment approaches. The discussion explores Pregabalin medication stands out in improving overall quality of life of the patients. The results presented here indicated that among diabetic patients, age, HbA1c, DR, and the duration of their diabetes are related to considerably higher risks of DPN; however, BMI, smoking, TG, as well as TC did not show any evidence of an increased risk of DPN. The results of the study offer a scientific foundation for a deeper comprehension of the causes of peripheral neuropathy-complicated type 2 diabetes and the outcomes of preventative measures.Bottom of Form

Expert Consensus on Ion Channel Drugs for Chronic Pain Treatment in China.

Ion channel drugs have been increasing used for chronic pain management with progress in the development of selective calcium channel modulators. Although ion channel drugs have been proven safe and effective in clinical practice, uncertainty remains regarding its use to treat chronic pain. To standardize the clinical practice of ion channel drug for the treatment of chronic pain, the National Health Commission Capacity Building and Continuing Education Center for Pain Diagnosis and Treatment Special Ability Training Project established an expert group to form an expert consensus on the use of ion channel drugs for the treatment of chronic pain after repeated discussions on existing medical evidence combined with the well clinical experience of experts. The consensus provided information on the mechanism of action of ion channel drugs and their recommendations, caution use, contraindications, and precautions for their use in special populations to support doctors in their clinical decision-making.

Combinatorial effects of ion channel mis-splicing as a cause of myopathy in myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder caused by an unstable expanded CTG repeat located in the 3'-UTR of the DM1 protein kinase (DMPK) gene. The pathogenic mechanism results in misregulated alternative splicing of hundreds of genes, creating the dilemma of establishing which genes contribute to the mechanism of DM1 skeletal muscle pathology. In this issue of the JCI, Cisco and colleagues systematically tested the combinatorial effects of DM1-relevant mis-splicing patterns in vivo and identified the synergistic effects of mis-spliced calcium and chloride channels as a major contributor to DM1 skeletal muscle impairment. The authors further demonstrated the therapeutic potential for calcium channel modulation to block the synergistic effects and rescue myopathy.

Mitochondrial calcium function is altered by lithium in hippocampal cell culture and triple transgenic for Alzheimer’s disease

AbstractBackgroundCalcium (Ca2+) regulate many functions in neuronal and glial cells. The Mitochondrial Calcium Uniporter (Mcu) complex is thought to be the primary pathway for Ca2+ entry into mitochondria. The Mcu protein has an important role in regulating mitochondrial Ca2+ homeostasis. Dysregulation of mitochondrial Ca2+ homeostasis has been implicated in various pathological conditions, such as Alzheimer disease. AD pathology is characterized by the progressive accumulation of amyloid β‐peptide and neurofibrillary tangles. In AD patients a significant increase in an oxidative damage and decrease in energy metabolism resulting in a mitochondrial calcium disbalance. Lithium has been used for decades to treat neurodegenerative diseases, as it has an action on several intracellular organelles, including mitochondria. The lithium regulates calcium channels, decreases oxidative stress and increases autophagic processes. Therefore, we aim to analyze whether chronic treatment with lithium in primary cultures of hippocampal neurons and in the hippocampus of triple transgenic animals for Alzheimer’s disease (3xtgAD).MethodThe cultures were treated for 7 days (dose of 0.02; 0.2 and 2mM) and 3xtgAD for 8 months (doses of 1mM and 2mM). The hippocampus were analysed by proteomic and transcriptomics.ResultOur results showed that lithium increases Mcu and Micu 3 proteins, showing that lithium has an effect on the modulation of mitochondrial calcium channels that should be explored.ConclusionMitochondrial calcium is an important mechanism for understanding neurodegenerative diseases and lithium can reverse Alzheimer’s disease‐related dysfunction.

Repurposing phenothiazines for cancer therapy: compromising membrane integrity in cancer cells

The limitations of current cancer therapies, including the increasing prevalence of multidrug resistance, underscore the urgency for more effective treatments. One promising avenue lies in the repurposing of existing drugs. This review explores the impact of phenothiazines, primarily used as antipsychotic agents, on key mechanisms driving tumor growth and metastasis. The cationic and amphiphilic nature of phenothiazines allows interaction with the lipid bilayer of cellular membranes, resulting in alterations in lipid composition, modulation of calcium channels, fluidity, thinning, and integrity of the plasma membrane. This is especially significant in the setting of increased metabolic activity, a higher proliferative rate, and the invasiveness of cancer cells, which often rely on plasma membrane repair. Therefore, properties of phenothiazines such as compromising plasma membrane integrity and repair, disturbing calcium regulation, inducing cytosolic K-RAS accumulation, and sphingomyelin accumulation in the plasma membrane might counteract multidrug resistance by sensitizing cancer cells to membrane damage and chemotherapy. This review outlines a comprehensive overview of the mechanisms driving the anticancer activities of phenothiazines derivates such as trifluoperazine, prochlorperazine, chlorpromazine, promethazine, thioridazine, and fluphenazine. The repurposing potential of phenothiazines paves the way for novel approaches to improve future cancer treatment.

AHNAKs roles in physiology and malignant tumors.

The AHNAK family currently consists of two members, namely AHNAK and AHNAK2, both of which have a molecular weight exceeding 600 kDa. Homologous sequences account for approximately 90% of their composition, indicating a certain degree of similarity in terms of molecular structure and biological functions. AHNAK family members are involved in the regulation of various biological functions, such as calcium channel modulation and membrane repair. Furthermore, with advancements in biological and bioinformatics technologies, research on the relationship between the AHNAK family and tumors has rapidly increased in recent years, and its regulatory role in tumor progression has gradually been discovered. This article briefly describes the physiological functions of the AHNAK family, and reviews and analyzes the expression and molecular regulatory mechanisms of the AHNAK family in malignant tumors using Pubmed and TCGA databases. In summary, AHNAK participates in various physiological and pathological processes in the human body. In multiple types of cancers, abnormal expression of AHNAK and AHNAK2 is associated with prognosis, and they play a key regulatory role in tumor progression by activating signaling pathways such as ERK, MAPK, Wnt, and MEK, as well as promoting epithelial-mesenchymal transition.

Glia instruct axon regeneration via a ternary modulation of neuronal calcium channels in Drosophila

A neuron’s regenerative capacity is governed by its intrinsic and extrinsic environment. Both peripheral and central neurons exhibit cell-type-dependent axon regeneration, but the underlying mechanism is unclear. Glia provide a milieu essential for regeneration. However, the routes of glia-neuron signaling remain underexplored. Here, we show that regeneration specificity is determined by the axotomy-induced Ca2+ transients only in the fly regenerative neurons, which is mediated by L-type calcium channels, constituting the core intrinsic machinery. Peripheral glia regulate axon regeneration via a three-layered and balanced modulation. Glia-derived tumor necrosis factor acts through its neuronal receptor to maintain calcium channel expression after injury. Glia sustain calcium channel opening by enhancing membrane hyperpolarization via the inwardly-rectifying potassium channel (Irk1). Glia also release adenosine which signals through neuronal adenosine receptor (AdoR) to activate HCN channels (Ih) and dampen Ca2+ transients. Together, we identify a multifaceted glia-neuron coupling which can be hijacked to promote neural repair.

OR11-05 High-throughput Screening Identifies TR4 Modulators For Potential Therapeutic Application

Abstract Disclosure: S. Khowal: None. D. Zhang: None. R. Damoiseaux: None. K. Javaherian: None. A.P. Heaney: None. Cushing Disease (CD), due to an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, leads to excess cortisol and is a life-threatening “orphan disease”. Although surgical removal can be effective, CD frequently recurs. Thereafter, repeat surgery, radiation therapy and/or bilateral adrenalectomy are not always successful and cause significant morbidity. Medical therapy has significantly improved but there is a clear unmet need for efficacious and safe therapies that target the pituitary corticotroph tumor itself to offer biochemical and tumor control. We previously demonstrated that the orphan testicular receptor 4 (TR4) regulates pro-opiomelanocortin gene transcription and ACTH secretion. Herein, we used the Flexi Vector system to identify TR4 modulators by fusing the TR4 LBD (380-615aa) to a 3’ GAL4 DBD to generate a chimeric fusion plasmid, pBIND-TR4-LBD. The pBIND-TR4-LBD construct and an exogenous luciferase reporter construct pGL4.35 (luc2P/9XGAL4UAS), containing 9 copies of a GAL4 responsive upstream activating sequence (UAS), were then transiently transfected into human embryonic kidney (HEK293) cells. Following treatment with TR4 ligands or modulators, our TR4-LBD undergoes a conformational change and recruits additional cofactors and RNA polymerase II to the complex to drive GAL4-directed firefly luciferase expression. In a high throughput screen (HTS) of 27,705 chemicals, we identified several potential “hit” TR4 modulators that induced or inhibited TR4 LBD-directed GAL4UAS-directed luciferase expression by &amp;gt;3 -fold. Compounds from the HTS included ATP-sensitive K+ and calcium channel blockers, steroid hormones and modulators of cell proliferation and inflammation and several agonist and antagonist “hit” compounds induced TR4 &amp;gt;10 fold or inhibited TR4 by 90% respectively with minimal cell toxicity (&amp;lt;10%). In silico analysis of interactions between the TR4 LBD and these potential ligands indicated that the GLU-534 &amp; ARG-565 may be crucial residues involved in TR4 ligand binding. Dose-response curves to calculate EC50 of these potential TR4 hit compounds and mode-of-action characterization are in progress. Our studies have identified several potent TR4 agonistic and antagonistic compounds and lay the framework for the discovery of safe and efficacious lead TR4 ligands that can be further advanced as potential drug therapies for CD and other diseases. Presentation: Friday, June 16, 2023

Neuromuscular recovery from botulism involves multiple forms of compensatory plasticity.

Botulinum neurotoxin (BoNT) causes neuroparalytic disease and death by blocking neuromuscular transmission. There are no specific therapies for clinical botulism and the only treatment option is supportive care until neuromuscular function spontaneously recovers, which can take weeks or months after exposure. The highly specialized neuromuscular junction (NMJ) between phrenic motor neurons and diaphragm muscle fibers is the main clinical target of BoNT. Due to the difficulty in eliciting respiratory paralysis without a high mortality rate, few studies have characterized the neurophysiological mechanisms involved in diaphragm recovery from intoxication. Here, we develop a mouse model of botulism that involves partial paralysis of respiratory muscles with low mortality rates, allowing for longitudinal analysis of recovery. Mice challenged by systemic administration of 0.7 LD50 BoNT/A developed physiological signs of botulism, such as respiratory depression and reduced voluntary running activity, that persisted for an average of 8-12 d. Studies in isolated hemidiaphragm preparations from intoxicated mice revealed profound reductions in nerve-elicited, tetanic and twitch muscle contraction strengths that recovered to baseline 21 d after intoxication. Despite apparent functional recovery, neurophysiological parameters remained depressed for 28 d, including end plate potential (EPP) amplitude, EPP success rate, quantal content (QC), and miniature EPP (mEPP) frequency. However, QC recovered more quickly than mEPP frequency, which could explain the discrepancy between muscle function studies and neurophysiological recordings. Hypothesizing that differential modulation of voltage-gated calcium channels (VGCC) contributed to the uncoupling of QC from mEPP frequency, pharmacological inhibition studies were used to study the contributions of different VGCCs to neurophysiological function. We found that N-type VGCC and P/Q-type VGCC partially restored QC but not mEPP frequency during recovery from paralysis, potentially explaining the accelerated recovery of evoked release versus spontaneous release. We identified additional changes that presumably compensate for reduced acetylcholine release during recovery, including increased depolarization of muscle fiber resting membrane potential and increased quantal size. In addition to identifying multiple forms of compensatory plasticity that occur in response to reduced NMJ function, it is expected that insights into the molecular mechanisms involved in recovery from neuromuscular paralysis will support new host-targeted treatments for multiple neuromuscular diseases.

T-type calcium channel modulation by hydrogen sulfide in neuropathic pain conditions.

Neuropathic pain can appear as a direct or indirect nerve damage lesion or disease that affects the somatosensory nervous system. If the neurons are damaged or indirectly stimulated, immune cells contribute significantly to inflammatory and neuropathic pain. After nerve injury, peripheral macrophages/spinal microglia accumulate around damaged neurons, producing endogenous hydrogen sulfide (H2S) through the cystathionine-γ-lyase (CSE) enzyme. H2S has a pronociceptive modulation on the Cav3.2 subtype, the predominant Cav3 isoform involved in pain processes. The present review provides relevant information about H2S modulation on the Cav3.2 T-type channels in neuropathic pain conditions. We have discussed that the dual effect of H2S on T-type channels is concentration-dependent, that is, an inhibitory effect is seen at low concentrations of 10µM and an augmentation effect on T-current at 100µM. The modulation mechanism of the Cav3.2 channel by H2S involves the direct participation of the redox/Zn2+ affinity site located in the His191 in the extracellular loop of domain I of the channel, involving a group of extracellular cysteines, comprising C114, C123, C128, and C1333, that can modify the local redox environment. The indirect interaction pathways involve the regulation of the Cav3.2 channel through cytokines, kinases, and post-translational regulators of channel expression. The findings conclude that the CSE/H2S/Cav3.2 pathway could be a promising therapeutic target for neuropathic pain disorders.

Arrestin-3 Agonism at Dopamine D3 Receptors Defines a Subclass of Second-Generation Antipsychotics That Promotes Drug Tolerance

BackgroundSecond-generation antipsychotics (SGAs) are frontline treatments for serious mental illness. Often, individual patients benefit only from some SGAs and not others. The mechanisms underlying this unpredictability in treatment efficacy remain unclear. All SGAs bind the dopamine D3 receptor (D3R) and are traditionally considered antagonists for dopamine receptor signaling. MethodsHere, we used a combination of two-photon calcium imaging, in vitro signaling assays, and mouse behavior to assess signaling by SGAs at D3R. ResultsWe report that some clinically important SGAs function as arrestin-3 agonists at D3R, resulting in modulation of calcium channels localized to the site of action potential initiation in prefrontal cortex pyramidal neurons. We further show that chronic treatment with an arrestin-3 agonist SGA, but not an antagonist SGA, abolishes D3R function through postendocytic receptor degradation by GASP1 (G protein–coupled receptor–associated sorting protein-1). ConclusionsThese results implicate D3R–arrestin-3 signaling as a source of SGA variability, highlighting the importance of including arrestin-3 signaling in characterizations of drug action. Furthermore, they suggest that postendocytic receptor trafficking that occurs during chronic SGA treatment may contribute to treatment efficacy.