Ion channels as biomarkers of altered myogenesis in myofiber precursors of Duchenne muscular dystrophy.

Myogenesis is essential for skeletal muscle formation, growth, and regeneration and can be altered in Duchenne muscular dystrophy (DMD), an X-linked disorder due to the absence of the cytoskeletal protein dystrophin. Ion channels play a pivotal role in muscle differentiation and interact with the dystrophin complex. To investigate ion channel involvement in myogenesis in dystrophic settings, we performed electrophysiological characterization of two immortalized mouse cell lines, wild-type (WT) H2K-2B4 and the dystrophic (DYS) H2K-SF1, and measured gene expression of differentiation markers and ion channels. Inward and outward currents/density increased as differentiation progressed in both WT and DYS cells. However, day-11 DYS cells showed higher (27%) inward current density with an increased expression ratio of Scn5a/Scn4a and decreased (48%) barium-sensitive outward current compared to WT. Furthermore, day-11 DYS cells showed more positive resting membrane potential (+10 mV) and lower membrane capacitance (50%) compared to WT. DYS cells also had reduced Myog and Myf5 expression at days 6 and 11. Overall, ion channel profile and myogenesis appeared altered in DYS cells. These results are a first step in validating ion channels as potential drug targets to ameliorate muscle degeneration in DMD settings and as differentiation biomarkers in innovative platforms.

TISBE: A Public Web Platform for the Consensus-Based Explainable Prediction of Developmental Toxicity.

Despite being extremely relevant for the protection of prenatal and neonatal health, the developmental toxicity (Dev Tox) is a highly complex endpoint whose molecular rationale is still largely unknown. The lack of availability of high-quality data as well as robust nontesting methods makes its understanding even more difficult. Thus, the application of new explainable alternative methods is of utmost importance, with Dev Tox being one of the most animal-intensive research themes of regulatory toxicology. Descending from TIRESIA (Toxicology Intelligence and Regulatory Evaluations for Scientific and Industry Applications), the present work describes TISBE (TIRESIA Improved on Structure-Based Explainability), a new public web platform implementing four fundamental advancements for in silico analyses: a three times larger dataset, a transparent XAI (explainable artificial intelligence) framework employing a fragment-based fingerprint coding, a novel consensus classifier based on five independent machine learning models, and a new applicability domain (AD) method based on a double top-down approach for better estimating the prediction reliability. The training set (TS) includes as many as 1008 chemicals annotated with experimental toxicity values. Based on a 5-fold cross-validation, a median value of 0.410 for the Matthews correlation coefficient was calculated; TISBE was very effective, with a median value of sensitivity and specificity equal to 0.984 and 0.274, respectively. TISBE was applied on two external pools made of 1484 bioactive compounds and 85 pediatric drugs taken from ChEMBL (Chemical European Molecular Biology Laboratory) and TEDDY (Task-Force in Europe for Drug Development in the Young) repositories, respectively. Notably, TISBE gives users the option to clearly spot the molecular fragments responsible for the toxicity or the safety of a given chemical query and is available for free at https://prometheus.farmacia.uniba.it/tisbe.

Meta-regression analysis on the impact of medical therapy on long-term outcome in spontaneous coronary artery dissection.

Background: Spontaneous coronary artery dissection (SCAD) is a relatively rare condition affecting predominantly young adults, with a prevalence of female sex. The best management of SCAD is still unclear and not adequately evidence-based both in the acute phase but especially over the long-term. We therefore aimed to evaluate the impact of medical therapy usually adopted for coronary artery disease on long-term outcome in SCAD patients. Methods: We performed a meta-regression analysis including all the studies evaluating the long-term outcome of patients affected by SCAD. We used long-term mortality, recurrent SCAD, admission for angina and major adverse cardio-vascular events (MACE) as dependent variables and the rates of discharge drug rates (beta-blockers, statins, renin-angiotensin-aldosterone system inhibitors, aspirin, dual antiplatelet therapy (DAPT)) as independent variables. Results: Fourteen observational studies were included with a long-term follow-up of 3.5 ± 1.7 years. No statistically significant correlations between drug therapy (beta-blockers, statins, calcium channel blockers, nitrates, renin-angiotensin-aldosterone inhibitors) and mortality, MACE, admission for angina, and SCAD recurrence were found. Higher aspirin use rates were significantly correlated with lower admission rates for angina (p < 0.05); DAPT, however, showed a borderline correlation with higher rates of SCAD recurrence (p = 0.068). Conclusions: In a meta-regression analysis including observational studies aspirin use rates correlated with lower long-term rates of admission for angina, while a borderline correlation between DAPT and rates of SCAD recurrence was found. Other drugs usually used for the treatment of coronary artery disease do not seem to impact long-term outcome of SCAD patients.

SCN5A mutation is associated with a higher Shanghai Score in patients with type 1 Brugada ECG pattern.

AIMS: Brugada syndrome (BrS) is an inherited arrhythmic disease characterized by a coved ST-segment elevation in the right precordial electrocardiogram leads (type 1 ECG pattern) and is associated with a risk of malignant ventricular arrhythmias and sudden cardiac death. In order to assess the predictive value of the Shanghai Score System for the presence of a SCN5A mutation in clinical practice, we studied a cohort of 125 patients with spontaneous or fever/drug-induced BrS type 1 ECG pattern, variably associated with symptoms and a positive family history. METHODS: The Shanghai Score System items were collected for each patient and PR and QRS complex intervals were measured. Patients were genotyped through a next-generation sequencing (NGS) custom panel for the presence of SCN5A mutations and the common SCN5A polymorphism (H558R). RESULTS: The total Shanghai Score was higher in SCN5A+ patients than in SCN5A- patients. The 81% of SCN5A+ patients and the 100% of patients with a SCN5A truncating variant exhibit a spontaneous type 1 ECG pattern. A significant increase in PR (P = 0.006) and QRS (P = 0.02) was detected in the SCN5A+ group. The presence of the common H558R polymorphism did not significantly correlate with any of the items of the Shanghai Score, nor with the total score of the system. CONCLUSION: Data from our study suggest the usefulness of Shanghai Score collection in clinical practice in order to maximize genetic test appropriateness. Our data further highlight SCN5A mutations as a cause of conduction impairment in BrS patients.

Brugada Syndrome: More than a Monogenic Channelopathy.

Brugada syndrome (BrS) is an inherited cardiac channelopathy first diagnosed in 1992 but still considered a challenging disease in terms of diagnosis, arrhythmia risk prediction, pathophysiology and management. Despite about 20% of individuals carrying pathogenic variants in the SCN5A gene, the identification of a polygenic origin for BrS and the potential role of common genetic variants provide the basis for applying polygenic risk scores for individual risk prediction. The pathophysiological mechanisms are still unclear, and the initial thinking of this syndrome as a primary electrical disease is evolving towards a partly structural disease. This review focuses on the main scientific advancements in the identification of biomarkers for diagnosis, risk stratification, pathophysiology and therapy of BrS. A comprehensive model that integrates clinical and genetic factors, comorbidities, age and gender, and perhaps environmental influences may provide the opportunity to enhance patients' quality of life and improve the therapeutic approach.

An activator of voltage-gated K+ channels Kv1.1 as a therapeutic candidate for episodic ataxia type 1.

Loss-of-function mutations in the KCNA1(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. Here, we uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. We show that through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies (Sh5). By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery.

Biophysical Aspects of Neurodegenerative and Neurodevelopmental Disorders Involving Endo-/Lysosomal CLC Cl-/H+ Antiporters.

Endosomes and lysosomes are intracellular vesicular organelles with important roles in cell functions such as protein homeostasis, clearance of extracellular material, and autophagy. Endolysosomes are characterized by an acidic luminal pH that is critical for proper function. Five members of the gene family of voltage-gated ChLoride Channels (CLC proteins) are localized to endolysosomal membranes, carrying out anion/proton exchange activity and thereby regulating pH and chloride concentration. Mutations in these vesicular CLCs cause global developmental delay, intellectual disability, various psychiatric conditions, lysosomal storage diseases, and neurodegeneration, resulting in severe pathologies or even death. Currently, there is no cure for any of these diseases. Here, we review the various diseases in which these proteins are involved and discuss the peculiar biophysical properties of the WT transporter and how these properties are altered in specific neurodegenerative and neurodevelopmental disorders.

Short-term anti-remodeling effects of gliflozins in diabetic patients with heart failure and reduced ejection fraction: an explainable artificial intelligence approach.

Introduction: Sodium-glucose cotransporter type 2 inhibitors (SGLT2i), gliflozins, play an emerging role for the treatment of heart failure with reduced left ventricular ejection fraction (HFrEF). Nevertheless, the effects of SGLT2i on ventricular remodeling and function have not been completely understood yet. Explainable artificial intelligence represents an unprecedented explorative option to clinical research in this field. Based on echocardiographic evaluations, we identified some key clinical responses to gliflozins by employing a machine learning approach. Methods: Seventy-eight consecutive diabetic outpatients followed for HFrEF were enrolled in the study. Using a random forests classification, a single subject analysis was performed to define the profile of patients treated with gliflozins. An explainability analysis using Shapley values was used to outline clinical parameters that mostly improved after gliflozin therapy and machine learning runs highlighted specific variables predictive of gliflozin response. Results: The five-fold cross-validation analyses showed that gliflozins patients can be identified with a 0.70 ± 0.03% accuracy. The most relevant parameters distinguishing gliflozins patients were Right Ventricular S'-Velocity, Left Ventricular End Systolic Diameter and E/e' ratio. In addition, low Tricuspid Annular Plane Systolic Excursion values along with high Left Ventricular End Systolic Diameter and End Diastolic Volume values were associated to lower gliflozin efficacy in terms of anti-remodeling effects. Discussion: In conclusion, a machine learning analysis on a population of diabetic patients with HFrEF showed that SGLT2i treatment improved left ventricular remodeling, left ventricular diastolic and biventricular systolic function. This cardiovascular response may be predicted by routine echocardiographic parameters, with an explainable artificial intelligence approach, suggesting a lower efficacy in case of advanced stages of cardiac remodeling.

Small Molecules Targeting Kidney ClC-K Chloride Channels: Applications in Rare Tubulopathies and Common Cardiovascular Diseases.

Given the key role played by ClC-K chloride channels in kidney and inner ear physiology and pathology, they can be considered important targets for drug discovery. Indeed, ClC-Ka and ClC-Kb inhibition would interfere with the urine countercurrent concentration mechanism in Henle's loop, which is responsible for the reabsorption of water and electrolytes from the collecting duct, producing a diuretic and antihypertensive effect. On the other hand, ClC-K/barttin channel dysfunctions in Bartter Syndrome with or without deafness will require the pharmacological recovery of channel expression and/or activity. In these cases, a channel activator or chaperone would be appealing. Starting from a brief description of the physio-pathological role of ClC-K channels in renal function, this review aims to provide an overview of the recent progress in the discovery of ClC-K channel modulators.

Sarcoplasmic Reticulum Ca2+ Buffer Proteins: A Focus on the Yet-To-Be-Explored Role of Sarcalumenin in Skeletal Muscle Health and Disease.

Sarcalumenin (SAR) is a luminal Ca2+ buffer protein with high capacity but low affinity for calcium binding found predominantly in the longitudinal sarcoplasmic reticulum (SR) of fast- and slow-twitch skeletal muscles and the heart. Together with other luminal Ca2+ buffer proteins, SAR plays a critical role in modulation of Ca2+ uptake and Ca2+ release during excitation-contraction coupling in muscle fibers. SAR appears to be important in a wide range of other physiological functions, such as Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) stabilization, Store-Operated-Calcium-Entry (SOCE) mechanisms, muscle fatigue resistance and muscle development. The function and structural features of SAR are very similar to those of calsequestrin (CSQ), the most abundant and well-characterized Ca2+ buffer protein of junctional SR. Despite the structural and functional similarity, very few targeted studies are available in the literature. The present review provides an overview of the role of SAR in skeletal muscle physiology, as well as of its possible involvement and dysfunction in muscle wasting disorders, in order to summarize the current knowledge on SAR and drive attention to this important but still underinvestigated/neglected protein.

Blockers of Skeletal Muscle Nav1.4 Channels: From Therapy of Myotonic Syndrome to Molecular Determinants of Pharmacological Action and Back.

The voltage-gated sodium channels represent an important target for drug discovery since a large number of physiological processes are regulated by these channels. In several excitability disorders, including epilepsy, cardiac arrhythmias, chronic pain, and non-dystrophic myotonia, blockers of voltage-gated sodium channels are clinically used. Myotonia is a skeletal muscle condition characterized by the over-excitability of the sarcolemma, resulting in delayed relaxation after contraction and muscle stiffness. The therapeutic management of this disorder relies on mexiletine and other sodium channel blockers, which are not selective for the Nav1.4 skeletal muscle sodium channel isoform. Hence, the importance of deepening the knowledge of molecular requirements for developing more potent and use-dependent drugs acting on Nav1.4. Here, we review the available treatment options for non-dystrophic myotonia and the structure-activity relationship studies performed in our laboratory with a focus on new compounds with potential antimyotonic activity.

Dapagliflozin protects the kidney in a non-diabetic model of cardiorenal syndrome.

Cardiorenal syndrome encompasses a spectrum of disorders involving heart and kidney dysfunction, and sharing common risk factors, such as hypertension and diabetes. Clinical studies have shown that patients with and without diabetes may benefit from using sodium-glucose cotransporter 2 inhibitors to reduce the risk of heart failure and ameliorate renal endpoints. Because the underlying mechanisms remain elusive, we investigated the effects of dapagliflozin on the progression of renal damage, using a model of non-diabetic cardiorenal disease. Dahl salt-sensitive rats were fed a high-salt diet for five weeks and then randomized to dapagliflozin or vehicle for the following six weeks. After treatment with dapagliflozin, renal function resulted ameliorated as shown by decrease of albuminuria and urine albumin-to-creatinine ratio. Functional benefit was accompanied by a decreased accumulation of extracellular matrix and a reduced number of sclerotic glomeruli. Dapagliflozin significantly reduced expression of inflammatory and endothelial activation markers such as NF-κB and e-selectin. Upregulation of pro-oxidant-releasing NADPH oxidases 2 and 4 as well as downregulation of antioxidant enzymes were also counteracted by drug treatment. Our findings also evidenced the modulation of both classic and non-classic renin-angiotensin-aldosterone system (RAAS), and effects of dapagliflozin on gene expression of ion channels/transporters involved in renal homeostasis. Thus, in a non-diabetic model of cardiorenal syndrome, dapagliflozin provides renal protection by modulating inflammatory response, endothelial activation, fibrosis, oxidative stress, local RAAS and ion channels.

Coexistence of SCN4A and CLCN1 mutations in a family with atypical myotonic features: A clinical and functional study.

Non-dystrophic myotonias include several entities with possible clinical overlap, i.e. myotonia congenita caused by CLCN1 gene mutations, as well as paramyotonia congenita and sodium channel myotonia caused by SCN4A gene mutations. Herein, we describe the clinical features of five relatives affected by clinical and neurophysiological myotonia, with an aspecific and mixed phenotype. Next-generation sequencing identified the novel p.K1302R variant in SCN4A and the p.H838P variant in CLCN1. Segregation of the two mutations with the disease was confirmed by genotyping affected and non-affected family members. Patch-clamp experiments showed that sodium currents generated by p.K1302R and WT hNav1.4 were very similar. Mutant channel showed a small negative shift (5 mV) in the voltage-dependence of activation, which increased the likelihood of the channel to open at more negative voltages. The p.H838P mutation caused a reduction in chloride current density and a small voltage-dependence shift towards less negative potentials, in agreement with its position into the CBS2 domain of the C-terminus. Our results demonstrated that the mild functional alterations induced by p.K1302R and p.H838P in combination may be responsible for the mixed myotonic phenotypes. The K1302R mutant was sensitive to mexiletine and lamotrigine, suggesting that both drugs might be useful for the K1302R carriers.

How brachial access compares to femoral access for invasive cardiac angiography when radial access is not feasible: A meta-analysis.

BACKGROUND: Trans-radial access is considered the best approach for cardiac catheterization. The choice of an alternative access route may be complex and trans-femoral access (TFA) is generally preferred. However, trans-brachial approach (TBA) may represent another feasible alternative. We therefore aimed to compare TBA and TFA in terms of access site bleeding and complications in a meta-analysis study. METHODS: We systematically searched principal databases for studies comparing femoral and brachial approach in terms of in-hospital vascular complications in patients undergoing cardiac catheterization (coronary angiography or percutaneous coronary intervention). RESULTS: Five retrospective studies and one randomized study were identified for the meta-analysis; 2756 patients undergoing a TBA and 331.208 patients undergoing a TFA for cardiac catheterization were included in the final study. No significant differences between access routes were found in terms of risk of any vascular complications (relative risk 1.18; 95% CI: 0.91-1.53; p n.s.). Brachial access was associated with a significantly lower risk of access site bleeding (relative risk 0.46; 95% CI 0.24-0.88, p = 0.02). CONCLUSIONS: TBA for cardiac catheterization was associated with a lower risk of access site bleeding and a comparable risk of any vascular complications compared with TFA. TBA may be considered a reasonable alternative access route for cardiac catheterization, at least as femoral approach.

Locus Coeruleus Neurons' Firing Pattern Is Regulated by ERG Voltage-Gated K+ Channels.

Locus coeruleus (LC) neurons, with their extensive innervations throughout the brain, control a broad range of physiological processes. Several ion channels have been characterized in LC neurons that control intrinsic membrane properties and excitability. However, ERG (ether-à-go-go-related gene) K+ channels that are particularly important in setting neuronal firing rhythms and automaticity have not as yet been discovered in the LC. Moreover, the neurophysiological and pathophysiological roles of ERG channels in the brain remain unclear despite their expression in several structures. By performing immunohistochemical investigations, we found that ERG-1A, ERG-1B, ERG-2 and ERG-3 are highly expressed in the LC neurons of mice. To examine the functional role of ERG channels, current-clamp recordings were performed on mouse LC neurons in brain slices under visual control. ERG channel blockade by WAY-123,398, a class III anti-arrhythmic agent, increased the spontaneous firing activity and discharge irregularity of LC neurons. Here, we have shown the presence of distinct ERG channel subunits in the LC which play an imperative role in modulating neuronal discharge patterns. Thus, we propose that ERG channels are important players behind the changes in, and/or maintenance of, LC firing patterns that are implicated in the generation of different behaviors and in several disorders.

Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita.

Myotonia congenita (MC) is an inherited rare disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. It is caused by loss-of-function mutations in the skeletal muscle chloride channel ClC-1, important for the stabilization of resting membrane potential and for the repolarization phase of action potentials. Thanks to in vitro functional studies, the molecular mechanisms by which ClC-1 mutations alter chloride ion influx into the cell have been in part clarified, classifying them in "gating-defective" or "expression-defective" mutations. To date, the treatment of MC is only palliative because no direct ClC-1 activator is available. An ideal drug should be one which is able to correct biophysical defects of ClC-1 in the case of gating-defective mutations or a drug capable to recover ClC-1 protein expression on the plasma membrane for trafficking-defective ones. In this study, we tested the ability of niflumic acid (NFA), a commercial nonsteroidal anti-inflammatory drug, to act as a pharmacological chaperone on trafficking-defective MC mutants (A531V, V947E). Wild-type (WT) or MC mutant ClC-1 channels were expressed in HEK293 cells and whole-cell chloride currents were recorded with the patch-clamp technique before and after NFA incubation. Membrane biotinylation assays and western blot were performed to support electrophysiological results. A531V and V947E mutations caused a decrease in chloride current density due to a reduction of ClC-1 total protein level and channel expression on the plasma membrane. The treatment of A531V and V947E-transfected cells with 50 µM NFA restored chloride currents, reaching levels similar to those of WT. Furthermore, no significant difference was observed in voltage dependence, suggesting that NFA increased protein membrane expression without altering the function of ClC-1. Indeed, biochemical experiments confirmed that V947E total protein expression and its plasma membrane distribution were recovered after NFA incubation, reaching protein levels similar to WT. Thus, the use of NFA as a pharmacological chaperone in trafficking defective ClC-1 channel mutations could represent a good strategy in the treatment of MC. Because of the favorable safety profile of this drug, our study may easily open the way for confirmatory human pilot studies aimed at verifying the antimyotonic activity of NFA in selected patients carrying specific ClC-1 channel mutations.

Clinical and Functional Study of a De Novo Variant in the PVP Motif of Kv1.1 Channel Associated with Epilepsy, Developmental Delay and Ataxia.

Mutations in the KCNA1 gene, encoding the voltage-gated potassium channel Kv1.1, have been associated with a spectrum of neurological phenotypes, including episodic ataxia type 1 and developmental and epileptic encephalopathy. We have recently identified a de novo variant in KCNA1 in the highly conserved Pro-Val-Pro motif within the pore of the Kv1.1 channel in a girl affected by early onset epilepsy, ataxia and developmental delay. Other mutations causing severe epilepsy are located in Kv1.1 pore domain. The patient was initially treated with a combination of antiepileptic drugs with limited benefit. Finally, seizures and ataxia control were achieved with lacosamide and acetazolamide. The aim of this study was to functionally characterize Kv1.1 mutant channel to provide a genotype-phenotype correlation and discuss therapeutic options for KCNA1-related epilepsy. To this aim, we transfected HEK 293 cells with Kv1.1 or P403A cDNAs and recorded potassium currents through whole-cell patch-clamp. P403A channels showed smaller potassium currents, voltage-dependent activation shifted by +30 mV towards positive potentials and slower kinetics of activation compared with Kv1.1 wild-type. Heteromeric Kv1.1+P403A channels, resembling the condition of the heterozygous patient, confirmed a loss-of-function biophysical phenotype. Overall, the functional characterization of P403A channels correlates with the clinical symptoms of the patient and supports the observation that mutations associated with severe epileptic phenotype cluster in a highly conserved stretch of residues in Kv1.1 pore domain. This study also strengthens the beneficial effect of acetazolamide and sodium channel blockers in KCNA1 channelopathies.

Alteration of STIM1/Orai1-Mediated SOCE in Skeletal Muscle: Impact in Genetic Muscle Diseases and Beyond.

Intracellular Ca2+ ions represent a signaling mediator that plays a critical role in regulating different muscular cellular processes. Ca2+ homeostasis preservation is essential for maintaining skeletal muscle structure and function. Store-operated Ca2+ entry (SOCE), a Ca2+-entry process activated by depletion of intracellular stores contributing to the regulation of various function in many cell types, is pivotal to ensure a proper Ca2+ homeostasis in muscle fibers. It is coordinated by STIM1, the main Ca2+ sensor located in the sarcoplasmic reticulum, and ORAI1 protein, a Ca2+-permeable channel located on transverse tubules. It is commonly accepted that Ca2+ entry via SOCE has the crucial role in short- and long-term muscle function, regulating and adapting many cellular processes including muscle contractility, postnatal development, myofiber phenotype and plasticity. Lack or mutations of STIM1 and/or Orai1 and the consequent SOCE alteration have been associated with serious consequences for muscle function. Importantly, evidence suggests that SOCE alteration can trigger a change of intracellular Ca2+ signaling in skeletal muscle, participating in the pathogenesis of different progressive muscle diseases such as tubular aggregate myopathy, muscular dystrophy, cachexia, and sarcopenia. This review provides a brief overview of the molecular mechanisms underlying STIM1/Orai1-dependent SOCE in skeletal muscle, focusing on how SOCE alteration could contribute to skeletal muscle wasting disorders and on how SOCE components could represent pharmacological targets with high therapeutic potential.

A Novel KCNA2 Variant in a Patient with Non-Progressive Congenital Ataxia and Epilepsy: Functional Characterization and Sensitivity to 4-Aminopyridine.

Kv1.2 channels, encoded by the KCNA2 gene, are localized in the central and peripheral nervous system, where they regulate neuronal excitability. Recently, heterozygous mutations in KCNA2 have been associated with a spectrum of symptoms extending from epileptic encephalopathy, intellectual disability, and cerebellar ataxia. Patients are treated with a combination of antiepileptic drugs and 4-aminopyridine (4-AP) has been recently trialed in specific cases. We identified a novel variant in KCNA2, E236K, in a Serbian proband with non-progressive congenital ataxia and early onset epilepsy, treated with sodium valproate. To ascertain the pathogenicity of E236K mutation and to verify its sensitivity to 4-AP, we transfected HEK 293 cells with Kv1.2 WT or E236K cDNAs and recorded potassium currents through the whole-cell patch-clamp. In silico analysis supported the electrophysiological data. E236K channels showed voltage-dependent activation shifted towards negative potentials and slower kinetics of deactivation and activation compared with Kv1.2 WT. Heteromeric Kv1.2 WT+E236K channels, resembling the condition of the heterozygous patient, confirmed a mixed gain- and loss-of-function (GoF/LoF) biophysical phenotype. 4-AP inhibited both Kv1.2 and E236K channels with similar potency. Homology modeling studies of mutant channels suggested a reduced interaction between the residue K236 in the S2 segment and the gating charges at S4. Overall, the biophysical phenotype of E236K channels correlates with the mild end of the clinical spectrum reported in patients with GoF/LoF defects. The response to 4-AP corroborates existing evidence that KCNA2-disorders could benefit from variant-tailored therapeutic approaches, based on functional studies.