Publications by authors named "Jean-Francois Desaphy"

75 Publications

Buprenorphine may be effective for treatment of paramyotonia congenita.

Muscle Nerve 2021 Apr 9. Epub 2021 Apr 9.

IRCCS Mondino Foundation, Pavia, Italy.

Introduction/aims: Paramyotonia congenita (PMC) is a skeletal muscle sodium channelopathy characterized by paradoxical myotonia, cold sensitivity, and exercise/cold-induced paralysis. Treatment with sodium-channel-blocking antiarrhythmic agents may expose patients to a risk of arrhythmia or may be poorly tolerated or ineffective. In this study we explored the effectiveness of non-antiarrhythmic sodium-channel blockers in two patients with PMC.

Methods: Earlier treatment with mexiletine was discontinued for gastrointestinal side effects in one of the patients and lack of clinical benefit in the other. One patient received lacosamide, ranolazine, and buprenorphine, and the other was given buprenorphine only. Drug efficacy was assessed by clinical scores, timed tests, and by long and short exercise tests.

Results: In both patients, buprenorphine improved pain scores by at least 50%, stiffness and weakness levels, and handgrip/eyelid-opening times. The fall in compound muscle action potential (CMAP) during short exercise normalized in both patients at baseline, and improved after cooling. During long exercise, one patient showed an earlier recovery of CMAP, and the other patient had a less severe decrease (<60%). With buprenorphine, the fall in CMAP induced by cooling normalized in one patient (from -72% to -4%) and improved (from -49% to -37%) in the other patient.

Discussion: Buprenorphine showed promising results for the treatment of exercise-induced paralysis and cold intolerance in the two patients assessed. The exercise test may be useful for quantitative assessment of treatment response. Further studies on a larger number of patients, under carefully controlled conditions, should be considered to address the effectiveness and long-term tolerability of this therapeutic option.
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http://dx.doi.org/10.1002/mus.27249DOI Listing
April 2021

Next-generation sequencing application to investigate skeletal muscle channelopathies in a large cohort of Italian patients.

Neuromuscul Disord 2021 Apr 14;31(4):336-347. Epub 2020 Dec 14.

Neurology IV Unit, Neuroimmunology and Neuromuscular Diseases, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

Non-dystrophic myotonias and periodic paralyses are a heterogeneous group of disabling diseases classified as skeletal muscle channelopathies. Their genetic characterization is essential for prognostic and therapeutic purposes; however, several genes are involved. Sanger-based sequencing of a single gene is time-consuming, often expensive; thus, we designed a next-generation sequencing panel of 56 putative candidate genes for skeletal muscle channelopathies, codifying for proteins involved in excitability, excitation-contraction coupling, and metabolism of muscle fibres. We analyzed a large cohort of 109 Italian patients with a suspect of NDM or PP by next-generation sequencing. We identified 24 patients mutated in CLCN1 gene, 15 in SCN4A, 3 in both CLCN1 and SCN4A, 1 in ATP2A1, 1 in KCNA1 and 1 in CASQ1. Eight were novel mutations: p.G395Cfs*32, p.L843P, p.V829M, p.E258E and c.1471+4delTCAAGAC in CLCN1, p.K1302R in SCN4A, p.L208P in ATP2A1 and c.280-1G>C in CASQ1 genes. This study demonstrated the utility of targeted next generation sequencing approach in molecular diagnosis of skeletal muscle channelopathies and the importance of the collaboration between clinicians and molecular geneticists and additional methods for unclear variants to make a conclusive diagnosis.
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http://dx.doi.org/10.1016/j.nmd.2020.12.003DOI Listing
April 2021

Emerging Roles for Ion Channels in Ovarian Cancer: Pathomechanisms and Pharmacological Treatment.

Cancers (Basel) 2021 Feb 7;13(4). Epub 2021 Feb 7.

Department of Biomedical Sciences and Human Oncology, School of Medicine, University of Bari Aldo Moro, 70124 Bari, Italy.

Ovarian cancer (OC) is the deadliest gynecologic cancer, due to late diagnosis, development of platinum resistance, and inadequate alternative therapy. It has been demonstrated that membrane ion channels play important roles in cancer processes, including cell proliferation, apoptosis, motility, and invasion. Here, we review the contribution of ion channels in the development and progression of OC, evaluating their potential in clinical management. Increased expression of voltage-gated and epithelial sodium channels has been detected in OC cells and tissues and shown to be involved in cancer proliferation and invasion. Potassium and calcium channels have been found to play a critical role in the control of cell cycle and in the resistance to apoptosis, promoting tumor growth and recurrence. Overexpression of chloride and transient receptor potential channels was found both in vitro and in vivo, supporting their contribution to OC. Furthermore, ion channels have been shown to influence the sensitivity of OC cells to neoplastic drugs, suggesting a critical role in chemotherapy resistance. The study of ion channels expression and function in OC can improve our understanding of pathophysiology and pave the way for identifying ion channels as potential targets for tumor diagnosis and treatment.
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http://dx.doi.org/10.3390/cancers13040668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7914442PMC
February 2021

Musculoskeletal Features without Ataxia Associated with a Novel de novo Mutation in Impairing the Voltage Sensitivity of Kv1.1 Channel.

Biomedicines 2021 Jan 14;9(1). Epub 2021 Jan 14.

Paediatric Neurology and Neuromuscular Disorders Unit, IRCCS Institute "G. Gaslini", 80131 Genoa, Italy.

The gene encodes the α subunit of the voltage-gated Kv1.1 potassium channel that critically regulates neuronal excitability in the central and peripheral nervous systems. Mutations in have been classically associated with episodic ataxia type 1 (EA1), a movement disorder triggered by physical and emotional stress. Additional features variably reported in recent years include epilepsy, myokymia, migraine, paroxysmal dyskinesia, hyperthermia, hypomagnesemia, and cataplexy. Interestingly, a few individuals with neuromyotonia, either isolated or associated with skeletal deformities, have been reported carrying variants in the S2-S3 transmembrane segments of Kv1.1 channels in the absence of any other symptoms. Here, we have identified by whole-exome sequencing a novel de novo variant, T268K, in in a boy displaying recurrent episodes of neuromyotonia, muscle hypertrophy, and skeletal deformities. Through functional analysis in heterologous cells and structural modeling, we show that the mutation, located at the extracellular end of the S3 helix, causes deleterious effects, disrupting Kv1.1 function by altering the voltage dependence of activation and kinetics of deactivation, likely due to abnormal interactions with the voltage sensor in the S4 segment. Our study supports previous evidence suggesting that specific residues within the S2 and S3 segments of Kv1.1 result in a distinctive phenotype with predominant musculoskeletal presentation.
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http://dx.doi.org/10.3390/biomedicines9010075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829709PMC
January 2021

Editorial: New Insights in Skeletal Muscle Channelopathies - A Rapidly Expanding Field.

Front Neurol 2020 17;11:626772. Epub 2020 Dec 17.

Department of Biomedical Sciences and Human Oncology, School of Medicine, University of Bari Aldo Moro, Policlinico, Bari, Italy.

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http://dx.doi.org/10.3389/fneur.2020.626772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774510PMC
December 2020

Targeted Therapies for Skeletal Muscle Ion Channelopathies: Systematic Review and Steps Towards Precision Medicine.

J Neuromuscul Dis 2020 Dec 7. Epub 2020 Dec 7.

Sorbonne Université, INSERM, Assistance Publique Hôpitaux de Paris, Centre de Recherche en Myologie-UMR 974, Reference center in neuro-muscular channelopathies, Institute of Myology, Hôpital Universitaire Pitié-Salpêtrière, Paris, France.

Background: Skeletal muscle ion channelopathies include non-dystrophic myotonias (NDM), periodic paralyses (PP), congenital myasthenic syndrome, and recently identified congenital myopathies. The treatment of these diseases is mainly symptomatic, aimed at reducing muscle excitability in NDM or modifying triggers of attacks in PP.

Objective: This systematic review collected the evidences regarding effects of pharmacological treatment on muscle ion channelopathies, focusing on the possible link between treatments and genetic background.

Methods: We searched databases for randomized clinical trials (RCT) and other human studies reporting pharmacological treatments. Preclinical studies were considered to gain further information regarding mutation-dependent drug effects. All steps were performed by two independent investigators, while two others critically reviewed the entire process.

Results: For NMD, RCT showed therapeutic benefits of mexiletine and lamotrigine, while other human studies suggest some efficacy of various sodium channel blockers and of the carbonic anhydrase inhibitor (CAI) acetazolamide. Preclinical studies suggest that mutations may alter sensitivity of the channel to sodium channel blockers in vitro, which has been translated to humans in some cases. For hyperkalemic and hypokalemic PP, RCT showed efficacy of the CAI dichlorphenamide in preventing paralysis. However, hypokalemic PP patients carrying sodium channel mutations may have fewer benefits from CAI compared to those carrying calcium channel mutations. Few data are available for treatment of congenital myopathies.

Conclusions: These studies provided limited information about the response to treatments of individual mutations or groups of mutations. A major effort is needed to perform human studies for designing a mutation-driven precision medicine in muscle ion channelopathies.
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http://dx.doi.org/10.3233/JND-200582DOI Listing
December 2020

Synthesis and Evaluation of Voltage-Gated Sodium Channel Blocking Pyrroline Derivatives Endowed with Both Antiarrhythmic and Antioxidant Activities.

ChemMedChem 2021 Feb 28;16(3):578-588. Epub 2020 Oct 28.

Department of Pharmacy - Pharmaceutical Sciences, University of Bari Aldo Moro, via E. Orabona n. 4, 70126, Bari, Italy.

Under the hypothesis that cardioprotective agents might benefit from synergism between antiarrhythmic activity and antioxidant properties, a small series of mexiletine analogues were coupled with the 2,2,5,5-tetramethylpyrroline moiety, known for its antioxidant effect, in order to obtain dual-acting drugs potentially useful in the protection of the heart against post-ischemic reperfusion injury. The pyrroline derivatives reported herein were found to be more potent as antiarrhythmic agents than mexiletine and displayed antioxidant activity. The most interesting tetramethylpyrroline congener, a tert-butyl-substituted analogue, was at least 100 times more active as an antiarrhythmic than mexiletine.
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http://dx.doi.org/10.1002/cmdc.202000692DOI Listing
February 2021

Pathomechanisms of a Mutation Found in a Russian Family Suffering From Becker's Myotonia.

Front Neurol 2020 4;11:1019. Epub 2020 Sep 4.

Section of Pharmacology, Department of Biomedical Sciences and Human Oncology, School of Medicine, University of Bari Aldo Moro, Bari, Italy.

Myotonia congenita (MC) is a rare muscle disease characterized by sarcolemma over-excitability inducing skeletal muscle stiffness. It can be inherited either as an autosomal dominant (Thomsen's disease) or an autosomal recessive (Becker's disease) trait. Both types are caused by loss-of-function mutations in the gene, encoding for ClC-1 chloride channel. We found a ClC-1 mutation, p.G411C, identified in Russian patients who suffered from a severe form of Becker's disease. The purpose of this study was to provide a solid correlation between G411C dysfunction and clinical symptoms in the affected patient. We provide clinical and genetic information of the proband kindred. Functional studies include patch-clamp electrophysiology, biotinylation assay, western blot analysis, and confocal imaging of G411C and wild-type ClC-1 channels expressed in HEK293T cells. The G411C mutation dramatically abolished chloride currents in transfected HEK cells. Biochemical experiments revealed that the majority of G411C mutant channels did not reach the plasma membrane but remained trapped in the cytoplasm. Treatment with the proteasome inhibitor MG132 reduced the degradation rate of G411C mutant channels, leading to their expression at the plasma membrane. However, despite an increase in cell surface expression, no significant chloride current was recorded in the G411C-transfected cell treated with MG132, suggesting that this mutation produces non-functional ClC-1 chloride channels. These results suggest that the molecular pathophysiology of G411C is linked to a reduced plasma membrane expression and biophysical dysfunction of mutant channels, likely due to a misfolding defect. Chloride current abolition confirms that the mutation is responsible for the clinical phenotype.
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http://dx.doi.org/10.3389/fneur.2020.01019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500137PMC
September 2020

A guide to writing systematic reviews of rare disease treatments to generate FAIR-compliant datasets: building a Treatabolome.

Orphanet J Rare Dis 2020 08 12;15(1):206. Epub 2020 Aug 12.

Children's Hospital of Eastern Ontario Research Institute, Ottawa, Canada.

Background: Rare diseases are individually rare but globally affect around 6% of the population, and in over 70% of cases are genetically determined. Their rarity translates into a delayed diagnosis, with 25% of patients waiting 5 to 30 years for one. It is essential to raise awareness of patients and clinicians of existing gene and variant-specific therapeutics at the time of diagnosis to avoid that treatment delays add up to the diagnostic odyssey of rare diseases' patients and their families.

Aims: This paper aims to provide guidance and give detailed instructions on how to write homogeneous systematic reviews of rare diseases' treatments in a manner that allows the capture of the results in a computer-accessible form. The published results need to comply with the FAIR guiding principles for scientific data management and stewardship to facilitate the extraction of datasets that are easily transposable into machine-actionable information. The ultimate purpose is the creation of a database of rare disease treatments ("Treatabolome") at gene and variant levels as part of the H2020 research project Solve-RD.

Results: Each systematic review follows a written protocol to address one or more rare diseases in which the authors are experts. The bibliographic search strategy requires detailed documentation to allow its replication. Data capture forms should be built to facilitate the filling of a data capture spreadsheet and to record the application of the inclusion and exclusion criteria to each search result. A PRISMA flowchart is required to provide an overview of the processes of search and selection of papers. A separate table condenses the data collected during the Systematic Review, appraised according to their level of evidence.

Conclusions: This paper provides a template that includes the instructions for writing FAIR-compliant systematic reviews of rare diseases' treatments that enables the assembly of a Treatabolome database that complement existing diagnostic and management support tools with treatment awareness data.
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http://dx.doi.org/10.1186/s13023-020-01493-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424983PMC
August 2020

From Riluzole to Dexpramipexole via Substituted-Benzothiazole Derivatives for Amyotrophic Lateral Sclerosis Disease Treatment: Case Studies.

Molecules 2020 Jul 22;25(15). Epub 2020 Jul 22.

Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari Aldo Moro, via Orabona 4, 70125 Bari, Italy.

The 1,3-benzothiazole (BTZ) ring may offer a valid option for scaffold-hopping from indole derivatives. Several BTZs have clinically relevant roles, mainly as CNS medicines and diagnostic agents, with riluzole being one of the most famous examples. Riluzole is currently the only approved drug to treat amyotrophic lateral sclerosis (ALS) but its efficacy is marginal. Several clinical studies have demonstrated only limited improvements in survival, without benefits to motor function in patients with ALS. Despite significant clinical trial efforts to understand the genetic, epigenetic, and molecular pathways linked to ALS pathophysiology, therapeutic translation has remained disappointingly slow, probably due to the complexity and the heterogeneity of this disease. Many other drugs to tackle ALS have been tested for 20 years without any success. Dexpramipexole is a BTZ structural analog of riluzole and was a great hope for the treatment of ALS. In this review, as an interesting case study in the development of a new medicine to treat ALS, we present the strategy of the development of dexpramipexole, which was one of the most promising drugs against ALS.
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http://dx.doi.org/10.3390/molecules25153320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435757PMC
July 2020

Long-Term Safety and Usefulness of Mexiletine in a Large Cohort of Patients Affected by Non-dystrophic Myotonias.

Front Neurol 2020 20;11:300. Epub 2020 May 20.

Department of Geriatric, Neurologic, Orthopedics and Head-Neck Science, Area of Neuroscience, Institute of Neurology, Fondazione Policlinico Universitario A. Gemelli, IRCCS, Rome, Italy.

The aim of our study was to evaluate the long-term efficacy and safety of mexiletine in 112 patients affected by genetically confirmed non-dystrophic myotonias. The study was performed at the Neurophysiologic Division of Fondazione Policlinico Universitario A. Gemelli Istituto di Ricerca e Cura a Carattere Scientifico (IRCCS), Rome and the Children's Hospital Bambino Gesù, Rome. The treatment was accepted by 59 patients according to clinical severity, individual needs, and concerns about a chronic medication. Forty-three patients were affected by recessive congenita myotonia, 11 by sodium channel myotonia, and five by dominant congenital myotonia. They underwent clinical examination before and after starting therapy, and Electromyography (EMG). A number of recessive myotonia patients underwent a protocol of repetitive nerve stimulations, for detecting and quantifying the transitory weakness, and a modified version of the Timed Up and Go test, to document and quantify the gait impairment. Treatment duration ranged from 1 month to 20 years and the daily dosages in adults ranged between 200 and 600 mg. No patient developed cardiac arrhythmias causing drug discontinuation. Mexiletine was suspended in 13 cases (22%); in three patients, affected by Sodium Channel myotonia, because flecainide showed better efficacy; in one patient because of a gastric cancer antecedent treatment; in four patients because of untreatable dyspepsia; and five patients considered the treatment not necessary. In our experience, mexiletine is very useful and not expensive. We did not observe any hazarding cardiac arrhythmias. Dyspepsia was the most frequent dose-limiting side effect.
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http://dx.doi.org/10.3389/fneur.2020.00300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326038PMC
May 2020

Privileged scaffold-based design to identify a novel drug-like 5-HT receptor-preferring agonist to target Fragile X syndrome.

Eur J Med Chem 2020 Aug 4;199:112395. Epub 2020 May 4.

Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari Aldo Moro, via Orabona 4, 70125, Bari, Italy. Electronic address:

Recent preclinical studies have shown that activation of the serotonin 5-HT receptor has the potential to treat neurodevelopmental disorders such as Fragile X syndrome, a rare disease characterized by autistic features. With the aim to provide the scientific community with diversified drug-like 5-HT receptor-preferring agonists, we designed a set of new long-chain arylpiperazines by exploiting structural fragments present in clinically approved drugs or in preclinical candidates (privileged scaffolds). The new compounds were synthesized, tested for their affinity at 5-HT and 5-HT receptors, and screened for their in vitro stability to microsomal degradation and toxicity. Selected compounds were characterized as 5-HT receptor-preferring ligands, endowed with high metabolic stability and low toxicity. Compound 7g emerged as a drug-like 5-HT receptor-preferring agonist capable to rescue synaptic plasticity and attenuate stereotyped behavior in a mouse model of Fragile X syndrome.
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http://dx.doi.org/10.1016/j.ejmech.2020.112395DOI Listing
August 2020

Skeletal muscle ClC-1 chloride channels in health and diseases.

Pflugers Arch 2020 07 2;472(7):961-975. Epub 2020 May 2.

Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", Bari, Italy.

In 1970, the study of the pathomechanisms underlying myotonia in muscle fibers isolated from myotonic goats highlighted the importance of chloride conductance for skeletal muscle function; 20 years later, the human ClC-1 chloride channel has been cloned; last year, the crystal structure of human protein has been solved. Over the years, the efforts of many researchers led to significant advances in acknowledging the role of ClC-1 in skeletal muscle physiology and the mechanisms through which ClC-1 dysfunctions lead to impaired muscle function. The wide spectrum of pathophysiological conditions associated with modification of ClC-1 activity, either as the primary cause, such as in myotonia congenita, or as a secondary adaptive mechanism in other neuromuscular diseases, supports the idea that ClC-1 is relevant to preserve not only for skeletal muscle excitability, but also for skeletal muscle adaptation to physiological or harmful events. Improving this understanding could open promising avenues toward the development of selective and safe drugs targeting ClC-1, with the aim to restore normal muscle function. This review summarizes the most relevant research on ClC-1 channel physiology, associated diseases, and pharmacology.
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http://dx.doi.org/10.1007/s00424-020-02376-3DOI Listing
July 2020

Safinamide's potential in treating nondystrophic myotonias: Inhibition of skeletal muscle voltage-gated sodium channels and skeletal muscle hyperexcitability in vitro and in vivo.

Exp Neurol 2020 06 20;328:113287. Epub 2020 Mar 20.

Department of Pharmacy & Drug Sciences, University of Bari Aldo Moro, Bari, Italy.

The antiarrhythmic sodium-channel blocker mexiletine is used to treat patients with myotonia. However, around 30% of patients do not benefit from mexiletine due to poor tolerability or suboptimal response. Safinamide is an add-on therapy to levodopa for Parkinson's disease. In addition to MAOB inhibition, safinamide inhibits neuronal sodium channels, conferring anticonvulsant activity in models of epilepsy. Here, we investigated the effects of safinamide on skeletal muscle hNa1.4 sodium channels and in models of myotonia, in-vitro and in-vivo. Using patch-clamp, we showed that safinamide reversibly inhibited sodium currents in HEK293T cells transfected with hNav1.4. At the holding potential (hp) of -120 mV, the half-maximum inhibitory concentrations (IC) were 160 and 33 μM at stimulation frequencies of 0.1 and 10 Hz, respectively. The calculated affinity constants of safinamide were dependent on channel state: 420 μM for closed channels and 9 μM for fast-inactivated channels. The p.F1586C mutation in hNav1.4 greatly impaired safinamide inhibition, suggesting that the drug binds to the local anesthetic receptor site in the channel pore. In a condition mimicking myotonia, i.e. hp. of -90 mV and 50-Hz stimulation, safinamide inhibited I with an IC of 6 μM, being two-fold more potent than mexiletine. Using the two-intracellular microelectrodes current-clamp method, action potential firing was recorded in vitro in rat skeletal muscle fibers in presence of the chloride channel blocker, 9-anthracene carboxylic acid (9-AC), to increase excitability. Safinamide counteracted muscle fiber hyperexcitability with an IC of 13 μM. In vivo, oral safinamide was tested in the rat model of myotonia. In this model, intraperitoneal injection of 9-AC greatly increased the time of righting reflex (TRR) due to development of muscle stiffness. Safinamide counteracted 9-AC induced TRR increase with an ED of 1.2 mg/kg, which is 7 times lower than that previously determined for mexiletine. In conclusion, safinamide is a potent voltage and frequency dependent blocker of skeletal muscle sodium channels. Accordingly, the drug was able to counteract abnormal muscle hyperexcitability induced by 9-AC, both in vitro and in vivo. Thus, this study suggests that safinamide may have potential in treating myotonia and warrants further preclinical and human studies to fully evaluate this possibility.
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http://dx.doi.org/10.1016/j.expneurol.2020.113287DOI Listing
June 2020

Characterization of Student Drinking Behaviors at the Beginning of the First Academic Year at One University in Southern Italy.

J Addict Nurs 2019 Jul/Sep;30(3):193-200

Maria A. De Salvia, PhD, Maria Tattoli, MD, PhD, Jean Francois Desaphy, PhD, and Arcangela Giustino, PhD, Department of Biomedical Sciences and Human Oncology, University of Bari "Aldo Moro," Bari, Italy. Angela Maria D'Uggento, PhD, Department of Economics and Finance, University of Bari "Aldo Moro," Bari, Italy. Giovanni Aquilino, PhD, Department of Humanities, University of Foggia, Foggia, Italy. Carmine Finelli, MD, Department of Internal Medicine, Hospital "Cavalier Raffaele Apicella"-ASL Naples, Naples, Italy. Paola Imbrici, PhD, Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro," Bari, Italy.

It is well recognized that both college and noncollege students are at-risk age groups for alcohol consumption. We investigated the alcohol consumption habits of undergraduate students with an emphasis on binge drinking. Participants (N = 809, 61.2% female) were freshmen attending courses at one of the main universities of southern Italy. They were asked to fill out a paper-and-pencil questionnaire that was administered between October 2017 and January 2018. Nearly 90% of the questioned students reported drinking alcohol during the 12 months before the survey. Among them, 31.4% of female students and 41.5% of male students engaged in binge drinking, mainly once a month; binge drinkers preferred highly alcoholic beverages during parties, underestimated the alcoholic content of their drinks, started drinking alcohol at a younger age than nonbinge drinkers, and drank weekly and between meals. Binge drinkers started smoking earlier than their peers, and a great number of them consumed illicit drugs. Moreover, 30.3% of female and 34.8% of male nonbinge drinkers declared that they consumed 6 or more units of alcohol in one occasion, making them unaware binge drinkers. Furthermore, approximately 50% of students recognized that alcohol consumption has effects similar to those induced by illicit drugs but only considered their peers' drinking behavior to be risky.This study highlights that most students involved in this survey expose themselves to a risky lifestyle by heavy drinking and, most alarmingly, that some of them are not even aware of that.
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http://dx.doi.org/10.1097/JAN.0000000000000288DOI Listing
February 2020

Elucidating the Contribution of Skeletal Muscle Ion Channels to Amyotrophic Lateral Sclerosis in search of new therapeutic options.

Sci Rep 2019 02 28;9(1):3185. Epub 2019 Feb 28.

Department of Pharmacy-Drug Sciences, University of Bari Aldo Moro, 70125, Bari, Italy.

The discovery of pathogenetic mechanisms is essential to identify new therapeutic approaches in Amyotrophic Lateral Sclerosis (ALS). Here we investigated the role of the most important ion channels in skeletal muscle of an ALS animal model (MLC/SOD1) carrying a mutated SOD1 exclusively in this tissue, avoiding motor-neuron involvement. Ion channels are fundamental proteins for muscle function, and also to sustain neuromuscular junction and nerve integrity. By a multivariate statistical analysis, using machine learning algorithms, we identified the discriminant genes in MLC/SOD1 mice. Surprisingly, the expression of ClC-1 chloride channel, present only in skeletal muscle, was reduced. Also, the expression of Protein Kinase-C, known to control ClC-1 activity, was increased, causing its inhibition. The functional characterization confirmed the reduction of ClC-1 activity, leading to hyperexcitability and impaired relaxation. The increased expression of ion channel coupled AMPA-receptor may contribute to sustained depolarization and functional impairment. Also, the decreased expression of irisin, a muscle-secreted peptide protecting brain function, may disturb muscle-nerve connection. Interestingly, the in-vitro application of chelerythrine or acetazolamide, restored ClC-1 activity and sarcolemma hyperexcitability in these mice. These findings show that ion channel function impairment in skeletal muscle may lead to motor-neuron increased vulnerability, and opens the possibility to investigate on new compounds as promising therapy.
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http://dx.doi.org/10.1038/s41598-019-39676-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395744PMC
February 2019

Pharmacogenetics of myotonic hNav1.4 sodium channel variants situated near the fast inactivation gate.

Pharmacol Res 2019 03 3;141:224-235. Epub 2019 Jan 3.

Dept. of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro, Bari, Italy. Electronic address:

Sodium channel myotonia and paramyotonia congenita are caused by gain-of-function mutations in the skeletal muscle voltage-gated sodium channel hNav1.4. The first-line drug is the sodium channel blocker mexiletine; however, some patients show side effects or limited responses. We previously showed that two hNav1.4 mutations, p.G1306E and p.P1158L, reduce mexiletine potency in vitro, whereas another sodium channel blocker, flecainide, is less sensitive to mutation-induced gating defects. This observation was successfully translated to p.G1306E and p.P1158L carriers. Thus, the aim of this study was to perform a pharmacological characterization of myotonic Nav1.4 mutations clustered near the fast inactivation gate of the channel. We chose seven mutations (p.V1293I, p.N1297S, p.N1297K, p.F1298C, p.G1306E, p.I1310N, and p.T1313M) from the database of Italian and French networks for muscle channelopathies. Recombinant hNav1.4 mutants were expressed in HEK293T cells for functional and pharmacological characterization using the patch-clamp technique. All the studied mutations impair the kinetics and/or voltage dependence of fast inactivation, which is likely the main mechanism responsible for myotonia. The severity of myotonia is well-correlated to the enhancement of window currents generated by the intersection of the activation and fast inactivation voltage dependence. Five of the six mutants displaying a significant positive shift of fast inactivation voltage dependence reduced mexiletine inhibition in an experimental condition mimicking myotonia. In contrast, none of the mutations impairs flecainide block nor does p.T1313M impair propafenone block, indicating that class Ic antiarrhythmics may constitute a valuable alternative. Our study suggests that mutation-driven therapy would be beneficial to myotonic patients, greatly improving their quality of life.
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http://dx.doi.org/10.1016/j.phrs.2019.01.004DOI Listing
March 2019

The analysis of myotonia congenita mutations discloses functional clusters of amino acids within the CBS2 domain and the C-terminal peptide of the ClC-1 channel.

Hum Mutat 2018 09 4;39(9):1273-1283. Epub 2018 Jul 4.

Department of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro, Polyclinic, Bari, Italy.

Myotonia congenita (MC) is a skeletal-muscle hyperexcitability disorder caused by loss-of-function mutations in the ClC-1 chloride channel. Mutations are scattered over the entire sequence of the channel protein, with more than 30 mutations located in the poorly characterized cytosolic C-terminal domain. In this study, we characterized, through patch clamp, seven ClC-1 mutations identified in patients affected by MC of various severities and located in the C-terminal region. The p.Val829Met, p.Thr832Ile, p.Val851Met, p.Gly859Val, and p.Leu861Pro mutations reside in the CBS2 domain, while p.Pro883Thr and p.Val947Glu are in the C-terminal peptide. We showed that the functional properties of mutant channels correlated with the clinical phenotypes of affected individuals. In addition, we defined clusters of ClC-1 mutations within CBS2 and C-terminal peptide subdomains that share the same functional defect: mutations between 829 and 835 residues and in residue 883 induced an alteration of voltage dependence, mutations between 851 and 859 residues, and in residue 947 induced a reduction of chloride currents, whereas mutations on 861 residue showed no obvious change in ClC-1 function. This study improves our understanding of the mechanisms underlying MC, sheds light on the role of the C-terminal region in ClC-1 function, and provides information to develop new antimyotonic drugs.
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http://dx.doi.org/10.1002/humu.23581DOI Listing
September 2018

Coexistence of CLCN1 and SCN4A mutations in one family suffering from myotonia.

Neurogenetics 2017 Dec 9;18(4):219-225. Epub 2017 Oct 9.

Center for Neuromuscular Diseases and Neuropathies, Unit of Neurology ASST "Spedali Civili", University of Brescia, Brescia, Italy.

Non-dystrophic myotonias are characterized by clinical overlap making it challenging to establish genotype-phenotype correlations. We report clinical and electrophysiological findings in a girl and her father concomitantly harbouring single heterozygous mutations in SCN4A and CLCN1 genes. Functional characterization of N1297S hNav1.4 mutant was performed by patch clamp. The patients displayed a mild phenotype, mostly resembling a sodium channel myotonia. The CLCN1 c.501C>G (p.F167L) mutation has been already described in recessive pedigrees, whereas the SCN4A c.3890A>G (p.N1297S) variation is novel. Patch clamp experiments showed impairment of fast and slow inactivation of the mutated Nav1.4 sodium channel. The present findings suggest that analysis of both SCN4A and CLCN1 genes should be considered in myotonic patients with atypical clinical and neurophysiological features.
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http://dx.doi.org/10.1007/s10048-017-0525-5DOI Listing
December 2017

Effects of Benzothiazolamines on Voltage-Gated Sodium Channels.

Handb Exp Pharmacol 2018 ;246:233-250

Section of Pharmacology, Department of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro, Policlinico, Piazza G. Cesare 11, 70124, Bari, Italy.

Benzothiazole is a versatile fused heterocycle that aroused much interest in drug discovery as anticonvulsant, neuroprotective, analgesic, anti-inflammatory, antimicrobial, and anticancer. Two benzothiazolamines, riluzole and lubeluzole, are known blockers of voltage-gated sodium (Na) channels. Riluzole is clinically used as a neuroprotectant in amyotrophic lateral sclerosis. Inhibition of Na channels by riluzole is voltage-dependent due to preferential binding to inactivated sodium channels. Yet the drug exerts little use-dependent block, probably because it lacks protonable amine. One important property is riluzole ability to inhibit persistent Na currents, which likely contributes to its neuroprotective activity. Lubeluzole showed promising neuroprotective effects in animal stroke models, but failed to show benefits in acute ischemic stroke in humans. One important concern is its propensity to prolong the cardiac QT interval, due to hERG K channel block. Lubeluzole very potently inhibits Na channels in a voltage- and use-dependent manner, due to its great preferential affinity for inactivated channels and the presence of a protonable amine group. Patch-clamp experiments suggest that the binding sites of both drugs overlap the local anesthetic receptor within the ion-conducting pathway. Riluzole and lubeluzole displayed very potent antimyotonic activity in a rat model of myotonia, a pathological skeletal muscle condition characterized by high-frequency runs of action potentials. Such results well support the repurposing of riluzole as an antimyotonic drug, allowing the launch of a pilot study in myotonic patients. Riluzole, lubeluzole, and new Na channel blockers built on the benzothiazolamine scaffold will certainly continue to be investigated for possible clinical applications.
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http://dx.doi.org/10.1007/164_2017_46DOI Listing
September 2018

Risk of Myopathy in Patients in Therapy with Statins: Identification of Biological Markers in a Pilot Study.

Front Pharmacol 2017 27;8:500. Epub 2017 Jul 27.

Section of Pharmacology, Department of Pharmacy and Drug Sciences, University of Bari Aldo MoroBari, Italy.

Statin therapy may induce skeletal muscle damage ranging from myalgia to severe rhabdomyolysis. Our previous preclinical studies showed that statin treatment in rats involves the reduction of skeletal muscle ClC-1 chloride channel expression and related chloride conductance (gCl). An increase of the activity of protein kinase C theta (PKC theta) isoform, able to inactivate ClC-1, may contribute to destabilize sarcolemma excitability. These effects can be detrimental for muscle function leading to drug-induced myopathy. Our goal is to study the causes of statin-induced muscle side effects in patients at the aim to identify biological markers useful to prevent and counteract statin-induced muscle damage. We examined 10 patients, who experienced myalgia and hyper-CK-emia after starting statin therapy compared to 9 non-myopathic subjects not using lipid-lowering drugs. Western Blot (WB) analysis showed a 40% reduction of ClC-1 protein and increased expression of phosphorylated PKC in muscle biopsies of statin-treated patients with respect to untreated subjects, independently from their age and statin type. Real-time PCR analysis showed that despite reduction of the protein, the ClC-1 mRNA was not significantly changed, suggesting post-transcriptional modification. The mRNA expression of a series of genes was also evaluated. MuRF-1 was increased in accord with muscle atrophy, MEF-2, calcineurin (CN) and GLUT-4 transporter were reduced, suggesting altered transcription, alteration of glucose homeostasis and energy deficit. Accordingly, the phosphorylated form of AMPK, measured by WB, was increased, suggesting cytoprotective process activation. In parallel, mRNA expression of Notch-1, involved in muscle cell proliferation, was highly expressed in statin-treated patients, indicating active regeneration. Also, PGC-1-alpha and isocitrate-dehydrogenase increased expression together with increased activity of mitochondrial citrate-synthase, measured by spectrophotometric assay, suggests mitochondrial biogenesis. Thus, the reduction of ClC-1 protein and consequent sarcolemma hyperexcitability together with energy deficiency appear to be among the most important alterations to be associated with statin-related risk of myopathy in humans. Thus, it may be important to avoid statin treatment in pathologies characterized by energy deficit and chloride channel malfunction. This study validates the measure of ClC-1 expression as a reliable clinical test for assessing statin-dependent risk of myopathy.
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http://dx.doi.org/10.3389/fphar.2017.00500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529355PMC
July 2017

A novel KCNA1 mutation in a patient with paroxysmal ataxia, myokymia, painful contractures and metabolic dysfunctions.

Mol Cell Neurosci 2017 09 28;83:6-12. Epub 2017 Jun 28.

Department of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro, Bari, Italy.

Episodic ataxia type 1 (EA1) is a human dominant neurological syndrome characterized by continuous myokymia, episodic attacks of ataxic gait and spastic contractions of skeletal muscles that can be triggered by emotional stress and fatigue. This rare disease is caused by missense mutations in the KCNA1 gene coding for the neuronal voltage gated potassium channel Kv1.1, which contributes to nerve cell excitability in the cerebellum, hippocampus, cortex and peripheral nervous system. We identified a novel KCNA1 mutation, E283K, in an Italian proband presenting with paroxysmal ataxia and myokymia aggravated by painful contractures and metabolic dysfunctions. The E283K mutation is located in the S3-S4 extracellular linker belonging to the voltage sensor domain of Kv channels. In order to test whether the E283K mutation affects Kv1.1 biophysical properties we transfected HEK293 cells with WT or mutant cDNAs alone or in a 1:1 combination, and recorded relative potassium currents in the whole-cell configuration of patch-clamp. Mutant E283K channels display voltage-dependent activation shifted by 10mV toward positive potentials and kinetics of activation slowed by ~2 fold compared to WT channels. Potassium currents resulting from heteromeric WT/E283K channels show voltage-dependent gating and kinetics of activation intermediate between WT and mutant homomeric channels. Based on homology modeling studies of the mutant E283K, we propose a molecular explanation for the reduced voltage sensitivity and slow channel opening. Overall, our results suggest that the replacement of a negatively charged residue with a positively charged lysine at position 283 in Kv1.1 causes a drop of potassium current that likely accounts for EA-1 symptoms in the heterozygous carrier.
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http://dx.doi.org/10.1016/j.mcn.2017.06.006DOI Listing
September 2017

Toward precision medicine in myotonic syndromes.

Oncotarget 2017 Feb;8(9):14279-14280

Department of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro, Bari, Italy.

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http://dx.doi.org/10.18632/oncotarget.15263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362403PMC
February 2017

Increased sodium channel use-dependent inhibition by a new potent analogue of tocainide greatly enhances in vivo antimyotonic activity.

Neuropharmacology 2017 02 13;113(Pt A):206-216. Epub 2016 Oct 13.

Section of Pharmacology, Department of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro, 70126 Bari, Italy. Electronic address:

Although the sodium channel blocker, mexiletine, is the first choice drug in myotonia, some myotonic patients remain unsatisfied due to contraindications, lack of tolerability, or incomplete response. More therapeutic options are thus needed for myotonic patients, which require clinical trials based on solid preclinical data. In previous structure-activity relationship studies, we identified two newly-synthesized derivatives of tocainide, To040 and To042, with greatly enhanced potency and use-dependent behavior in inhibiting sodium currents in frog skeletal muscle fibers. The current study was performed to verify their potential as antimyotonic agents. Patch-clamp experiments show that both compounds, especially To042, are greatly more potent and use-dependent blockers of human skeletal muscle hNav1.4 channels compared to tocainide and mexiletine. Reduced effects on F1586C hNav1.4 mutant suggest that the compounds bind to the local anesthetic receptor, but that the increased hindrance and lipophilia of the N-substituent may further strengthen drug-receptor interaction and use-dependence. Compared to mexiletine, To042 was 120 times more potent to block hNav1.4 channels in a myotonia-like cellular condition and 100 times more potent to improve muscle stiffness in vivo in a previously-validated rat model of myotonia. To explore toxicological profile, To042 was tested on hERG potassium currents, motor coordination using rotarod, and C2C12 cell line for cytotoxicity. All these experiments suggest a satisfactory therapeutic index for To042. This study shows that, owing to a huge use-dependent block of sodium channels, To042 is a promising candidate drug for myotonia and possibly other membrane excitability disorders, warranting further preclinical and human studies.
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http://dx.doi.org/10.1016/j.neuropharm.2016.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154332PMC
February 2017

Statin-induced myotoxicity is exacerbated by aging: A biophysical and molecular biology study in rats treated with atorvastatin.

Toxicol Appl Pharmacol 2016 09 1;306:36-46. Epub 2016 Jul 1.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari - Aldo Moro, Bari, Italy. Electronic address:

Statin-induced skeletal muscle damage in rats is associated to the reduction of the resting sarcolemmal chloride conductance (gCl) and ClC-1 chloride channel expression. These drugs also affect the ClC-1 regulation by increasing protein kinase C (PKC) activity, which phosphorylate and close the channel. Also the intracellular resting calcium (restCa) level is increased. Similar alterations are observed in skeletal muscles of aged rats, suggesting a higher risk of statin myotoxicity. To verify this hypothesis, we performed a 4-5-weeks atorvastatin treatment of 24-months-old rats to evaluate the ClC-1 channel function by the two-intracellular microelectrodes technique as well as transcript and protein expression of different genes sensitive to statins by quantitative real-time-PCR and western blot analysis. The restCa was measured using FURA-2 imaging, and histological analysis of muscle sections was performed. The results show a marked reduction of resting gCl, in agreement with the reduced ClC-1 mRNA and protein expression in atorvastatin-treated aged rats, with respect to treated adult animals. The observed changes in myocyte-enhancer factor-2 (MEF2) expression may be involved in ClC-1 expression changes. The activity of PKC was also increased and further modulate the gCl in treated aged rats. In parallel, a marked reduction of the expression of glycolytic and mitochondrial enzymes demonstrates an impairment of muscle metabolism. No worsening of restCa or histological features was found in statin-treated aged animals. These findings suggest that a strong reduction of gCl and alteration of muscle metabolism coupled to muscle atrophy may contribute to the increased risk of statin-induced myopathy in the elderly.
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http://dx.doi.org/10.1016/j.taap.2016.06.032DOI Listing
September 2016

Multidisciplinary study of a new ClC-1 mutation causing myotonia congenita: a paradigm to understand and treat ion channelopathies.

FASEB J 2016 10 20;30(10):3285-3295. Epub 2016 Jun 20.

Department of Pharmacy, Drug Sciences, University of Bari "Aldo Moro," Bari, Italy.

Myotonia congenita is an inherited disease that is characterized by impaired muscle relaxation after contraction caused by loss-of-function mutations in the skeletal muscle ClC-1 channel. We report a novel ClC-1 mutation, T335N, that is associated with a mild phenotype in 1 patient, located in the extracellular I-J loop. The purpose of this study was to provide a solid correlation between T335N dysfunction and clinical symptoms in the affected patient as well as to offer hints for drug development. Our multidisciplinary approach includes patch-clamp electrophysiology on T335N and ClC-1 wild-type channels expressed in tsA201 cells, Western blot and quantitative PCR analyses on muscle biopsies from patient and unaffected individuals, and molecular dynamics simulations using a homology model of the ClC-1 dimer. T335N channels display reduced chloride currents as a result of gating alterations rather than altered surface expression. Molecular dynamics simulations suggest that the I-J loop might be involved in conformational changes that occur at the dimer interface, thus affecting gating. Finally, the gene expression profile of T335N carrier showed a diverse expression of K channel genes, compared with control individuals, as potentially contributing to the phenotype. This experimental paradigm satisfactorily explained myotonia in the patient. Furthermore, it could be relevant to the study and therapy of any channelopathy.-Imbrici, P., Altamura, C., Camerino, G. M., Mangiatordi, G. F., Conte, E., Maggi, L., Brugnoni, R., Musaraj, K., Caloiero, R., Alberga, D., Marsano, R. M., Ricci, G., Siciliano, G., Nicolotti, O., Mora, M., Bernasconi, P., Desaphy, J.-F., Mantegazza, R., Camerino, D. C. Multidisciplinary study of a new ClC-1 mutation causing myotonia congenita: a paradigm to understand and treat ion channelopathies.
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http://dx.doi.org/10.1096/fj.201500079RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024700PMC
October 2016

Therapeutic Approaches to Genetic Ion Channelopathies and Perspectives in Drug Discovery.

Front Pharmacol 2016 10;7:121. Epub 2016 May 10.

Department of Pharmacy - Drug Sciences, University of Bari "Aldo Moro" Bari, Italy.

In the human genome more than 400 genes encode ion channels, which are transmembrane proteins mediating ion fluxes across membranes. Being expressed in all cell types, they are involved in almost all physiological processes, including sense perception, neurotransmission, muscle contraction, secretion, immune response, cell proliferation, and differentiation. Due to the widespread tissue distribution of ion channels and their physiological functions, mutations in genes encoding ion channel subunits, or their interacting proteins, are responsible for inherited ion channelopathies. These diseases can range from common to very rare disorders and their severity can be mild, disabling, or life-threatening. In spite of this, ion channels are the primary target of only about 5% of the marketed drugs suggesting their potential in drug discovery. The current review summarizes the therapeutic management of the principal ion channelopathies of central and peripheral nervous system, heart, kidney, bone, skeletal muscle and pancreas, resulting from mutations in calcium, sodium, potassium, and chloride ion channels. For most channelopathies the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a significant number of patients. Other channelopathies can exploit ion channel targeted drugs, such as marketed sodium channel blockers. Developing new and more specific therapeutic approaches is therefore required. To this aim, a major advancement in the pharmacotherapy of channelopathies has been the discovery that ion channel mutations lead to change in biophysics that can in turn specifically modify the sensitivity to drugs: this opens the way to a pharmacogenetics strategy, allowing the development of a personalized therapy with increased efficacy and reduced side effects. In addition, the identification of disease modifiers in ion channelopathies appears an alternative strategy to discover novel druggable targets.
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http://dx.doi.org/10.3389/fphar.2016.00121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861771PMC
May 2016

Translational approach to address therapy in myotonia permanens due to a new SCN4A mutation.

Neurology 2016 May 29;86(22):2100-8. Epub 2016 Apr 29.

From the Departments of Biomedical Sciences and Human Oncology (J.-F.D.) and Pharmacy & Drug Sciences (R.C., J.R., P.I., D.C.C.), University of Bari Aldo Moro, Bari; Unit of Neuromuscular and Neurodegenerative Disorders (A.D.), Bambino Gesù Children's Hospital, Rome; Departments of Geriatrics, Neurosciences, and Orthopedics (A.M., M.L.M.), Institute of Neurology, Catholic University of the Sacred Heart, Rome; Dino Ferrari Centre (S.P., S.L.), Neuroscience Section, Department of Pathophysiology and Transplantation (DEPT), University of Milan; and Neurology Unit (S.P., S.L.), IRCCS Foundation Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy.

Objective: We performed a clinical, functional, and pharmacologic characterization of the novel p.P1158L Nav1.4 mutation identified in a young girl presenting a severe myotonic phenotype.

Methods: Wild-type hNav1.4 channel and P1158L mutant were expressed in tsA201 cells for functional and pharmacologic studies using patch-clamp.

Results: The patient shows pronounced myotonia, slowness of movements, and generalized muscle hypertrophy. Because of general discomfort with mexiletine, she was given flecainide with satisfactory response. In vitro, mutant channels show a slower current decay and a rightward shift of the voltage dependence of fast inactivation. The voltage dependence of activation and slow inactivation were not altered. Mutant channels were less sensitive to mexiletine, whereas sensitivity to flecainide was not altered. The reduced inhibition of mutant channels by mexiletine was also observed using clinically relevant drug concentrations in a myotonic-like condition.

Conclusions: Clinical phenotype and functional alterations of P1158L support the diagnosis of myotonia permanens. Impairment of fast inactivation is consistent with the possible role of the channel domain III S4-S5 loop in the inactivation gate docking site. The reduced sensitivity of P1158L to mexiletine may have contributed to the unsatisfactory response of the patient. The success of flecainide therapy underscores the usefulness of in vitro functional studies to help in the choice of the best drug for each individual.
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http://dx.doi.org/10.1212/WNL.0000000000002721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891212PMC
May 2016

Editorial: Recent Advances in Voltage-Gated Sodium Channels, their Pharmacology and Related Diseases.

Front Pharmacol 2016 8;7:20. Epub 2016 Feb 8.

Section of Pharmacology, Department of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro Bari, Italy.

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http://dx.doi.org/10.3389/fphar.2016.00020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744839PMC
February 2016