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Bisphosphonates (BPs) are the most used bone-specific anti-resorptive agents, often chosen as first-line therapy in several bone diseases characterized by an imbalance between osteoblast-mediated bone production and osteoclast-mediated bone resorption. BPs target the farnesyl pyrophosphate synthase (FPPS) in osteoclasts, reducing bone resorption. Lately, there has been an increasing interest in BPs direct pro-survival/pro-mineralizing properties in osteoblasts and their pain-relieving effects. Even so, molecular targets involved in these effects appear now largely elusive. Ion channels are emerging players in bone homeostasis. Nevertheless, the effects of BPs on these proteins have been poorly described. Here we reviewed the actions of BPs on ion channels in musculoskeletal cells. In particular, the TRPV1 channel is essential for osteoblastogenesis. Since it is involved in bone pain sensation, TRPV1 is a possible alternative target of BPs. Ion channels are emerging targets and anti-target for bisphosphonates. Zoledronic acid can be the first selective musculoskeletal and vascular KATP channel blocker targeting with high affinity the inward rectifier channels Kir6.1-SUR2B and Kir6.2-SUR2A. The action of this drug against the overactive mutants of KCNJ9-ABCC9 genes observed in the Cantu’ Syndrome (CS) may improve the appropriate prescription in those CS patients affected by musculoskeletal disorders such as bone fracture and bone frailty.

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.

Background and Purpose Despite the fact that chloride channels are involved in several physiological processes and acquired diseases, the availability of compounds selectively targeting CLC proteins is rather limited. ClC-1 channels are responsible for sarcolemma repolarization after an action potential in skeletal muscle fibers and have been associated with myotonia congenita and myotonic dystrophy as well as with other muscular physiopathological conditions. To date only a few ClC-1 blockers have been discovered, such as anthracene-9-carboxylic acid (9-AC) and niflumic acid (NFA), whereas no useful activator exists. The absence of a ClC-1 structure and the limited information regarding the binding pockets in CLC channels hamper the identification of improved modulators. Experimental Approach The aim of this study was to provide an in-depth characterization of drug binding pockets in ClC-1 through an integrated in silico and experimental approach. We searched putative cavities in a homology model of ClC-1 built upon an eukaryotic CLC crystal structure, and validated in silico data by measuring the blocking ability of 9-AC and NFA on mutant ClC-1 channels expressed in HEK 293 cells. Key Results We identified four putative binding cavities in ClC-1. 9-AC appears to interact with K231, R421 and F484 within the channel pore that are pivotal for ClC-1 block. We also identified one preferential binding cavity for NFA and propose R421 and F484 as critical residues. Conclusion and Implications This study represents the first effort to delineate, through a computational-functional protocol, the binding sites of ClC-1. This information is fundamental to discover compounds useful in the treatment of ClC-1-associated dysfunctions and might represent a starting point for specifically targeting other CLC proteins.

Background and Purpose Human ClC-K chloride channels are highly attractive targets for drug discovery as they have a variety of important physiological functions and are associated with genetic disorders. These channels are crucial in the kidney as they control chloride reabsorption and water diuresis. In addition, loss-of-function mutations of CLCNKB and BSND genes cause Bartter's syndrome (BS), whereas CLCNKA and CLCNKB gain-of-function polymorphisms predispose to a rare form of salt sensitive hypertension. Both disorders lack a personalized therapy that is in most cases only symptomatic. The aim of this study was to identify novel ClC-K ligands from drugs already on the market, by exploiting the pharmacological side activity of drug molecules available from the FDA Adverse Effects Reporting System database. Experimental Approach We searched for drugs having a Bartter-like syndrome as a reported side effect, with the assumption that BS could be causatively related to the block of ClC-K channels. The ability of the selected BS-causing drugs to bind and block ClC-K channels was then validated through an integrated experimental and computational approach based on patch clamp electrophysiology in HEK293 cells and molecular docking simulations. Key Results Valsartan and olmesartan were able to block ClC-Ka channels and the molecular requirements for effective inhibition of these channels have been identified. Conclusion and Implications These results suggest additional mechanisms of action for these sartans further to their primary AT1 receptor antagonism and propose these compounds as leads for designing new potent ClC-K ligands.

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 hNav1.4 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 (IC50) 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 INa with an IC50 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 IC50 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 ED50 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.

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.

Mexiletine (Mex) has been recently appointed as an orphan-drug in myotonic-syndromes, being a potent use-dependent blocker of skeletal-muscle sodium channels (NaV1.4). Available evidences about a potential anti-oxidant effect of Mex and its tetramethyl-pyrroline-derivatives in vivo, suggest the possibility to further enlarge the therapeutic potential of Mex-like compounds in myopathies in which alteration of excitation-contraction coupling is paralleled by oxidative stress. In line with this and based on our previous structure-activity-relationship studies, we synthesized new compounds with a tetramethyl-pyrroline-ring on the amino-group of both Mex (VM11) and of its potent use-dependent isopropyl-derivative (CI16). The compounds were tested for their ability to block native NaV1.4 and to exert cyto-protective effects against oxidative-stress injury in myoblasts. Voltage-clamp-recordings on adult myofibers were performed to assess the tonic and use-dependent block of peak sodium-currents (INa) by VM11 and CI16, as well as Mex, VM11 and CI16 were 3 and 6-fold more potent than Mex in producing a tonic-block of peak sodium-currents (INa), respectively. Interestingly, CI16 showed a 40-fold increase of potency with respect to Mex during high-frequency stimulation (10-Hz), resulting the strongest use-dependent Mex-like compound so far. The derivatives also behaved as inactivated channel blockers, however the voltage dependent block was modest. The experimental data fitted with the molecular-modeling simulation based on previously proposed interaction of main pharmacophores with NaV1.4 binding-site. CI16 and VM11 were then compared to Mex and its isopropyl derivative (Me5) for the ability to protect C2C12-cells from H2O2-cytotoxicity in the concentration range effective on Nav1.4. Mex and Me5 showed a moderate cyto-protective effect in the presence of H2O2, Importantly, CI16 and VM11 showed a remarkable cyto-protection at concentrations effective for use-dependent block of NaV1.4. This effect was comparable to that of selected anti-oxidant drugs proved to exert protective effect in preclinical models of progressive myopathies such as muscular dystrophies. Then, the tetramethyl-pyrroline compounds have increased therapeutic profile as sodium channel blockers and an interesting cyto-protective activity. The overall profile enlarges therapeutic potential from channelopathies to myopathies in which alteration of excitation-contraction coupling is paralleled by oxidative-stress, i.e., muscular dystrophies.

Metaglidasen is a fibrate-like drug reported as a selective modulator of peroxisome proliferator-activated receptor γ (PPARγ), able to lower plasma glucose levels in the absence of the side effects typically observed with thiazolidinedione antidiabetic agents in current use. Herein we report an improved synthesis of metaglidasen's metabolically active form halofenic acid (R)-2 and that of its enantiomer (S)-2. The activity of the two stereoisomers was carefully examined on PPARα and PPARγ subtypes. As expected, both showed partial agonist activity toward PPARγ; the investigation of PPARα activity, however, led to unexpected results. In particular, (S)-2 was found to act as a partial agonist, whereas (R)-2 behaved as an antagonist. X-ray crystallographic studies with PPARγ were carried out to gain more insight on the molecular-level interactions and to propose a binding mode. Given the adverse effects provoked by fibrate drugs on skeletal muscle function, we also investigated the capacity of (R)-2 and (S)-2 to block conductance of the skeletal muscle membrane chloride channel. The results showed a more beneficial profile for (R)-2, the activity of which on skeletal muscle function, however, should not be overlooked in the ongoing clinical trials studying its long-term effects.

CLC-K chloride channels and their subunit, barttin, are crucial for renal NaCl reabsorption and for inner ear endolymph production. Mutations in CLC-Kb and barttin cause Bartter syndrome. Here, we identified two adjacent residues, F256 and N257, that when mutated hugely alter in Xenopus oocytes CLC-Ka's biphasic response to niflumic acid, a drug belonging to the fenamate class, with F256A being potentiated 37-fold and N257A being potently blocked with a KD ~ 1 μM. These residues are localized in the same extracellular I–J loop which harbors a regulatory Ca2 + binding site. This loop thus can represent an ideal and CLC-K specific target for extracellular ligands able to modulate channel activity. Furthermore, we demonstrated the involvement of the barttin subunit in the NFA potentiation. Indeed the F256A mutation confers onto CLC-K1 a transient potentiation induced by NFA which is found only when CLC-K1/F256A is co-expressed with barttin. Thus, in addition to the role of barttin in targeting and gating, the subunit participates in the pharmacological modulation of CLC-K channels and thus represents a further target for potential drugs., Graphical abstract, Highlights • CLC-K activators could be used for the treatment of Bartter syndrome. • NFA is the most potent activator of CLC-Ks. • Goal: knowledge of mechanisms and residues involved in CLC-K modulation by NFA. • I–J loop and barttin are promising pharmacological targets for extracellular ligands.

Although the sodium channel blocker mexiletine is considered the first-line drug in myotonia, some patients experiment adverse effects, while others do not gain any benefit. Other antimyotonic drugs are thus needed to offer mexiletine alternatives. In the present study, we used a previously-validated rat model of myotonia congenita to compare six marketed sodium channel blockers to mexiletine. Myotonia was induced in the rat by injection of anthracen-9-carboxylic acid, a muscle chloride channel blocker. The drugs were given orally and myotonia was evaluated by measuring the time of righting reflex. The drugs were also tested on sodium currents recorded in a cell line transfected with the human skeletal muscle sodium channel hNav1.4 using patch-clamp technique. In vivo, carbamazepine and propafenone showed antimyotonic activity at doses similar to mexiletine (ED50 close to 5 mg/kg); flecainide and orphenadrine showed greater potency (ED50 near 1 mg/kg); lubeluzole and riluzole were the more potent (ED50 near 0.1 mg/kg). The antimyotonic activity of drugs in vivo was linearly correlated with their potency in blocking hNav1.4 channels in vitro. Deviation was observed for propafenone and carbamazepine, likely due to pharmacokinetics and multiple targets. The comparison of the antimyotonic dose calculated in rats with the current clinical dose in humans strongly suggests that all the tested drugs may be used safely for the treatment of human myotonia. Considering the limits of mexiletine tolerability and the occurrence of non-responders, this study proposes an arsenal of alternative drugs, which may prove useful to increase the quality of life of individuals suffering from non-dystrophic myotonia. Further clinical trials are warranted to confirm these results., Highlights • Seven sodium channel blockers show antimyotonic activity in a rat model of myotonia. • The ED50 value ranges from 0.1 (riluzole, lubeluzole) to 5 mg/kg (mexiletine, carbamazepine). • The drugs use-dependently block hNav1.4 channels in cells in myotonic-like conditions. • The IC50 values in vitro were well linearly correlated with the ED50 values in vivo. • The study discloses promising new therapeutic options for myotonic patients.

ATP-sensitive-K(+) (KATP) channels couple metabolism to the electrical activity of the cells. This channel is associated with glycolytic enzymes to form complexes regulating the channel activity in various tissues. The pyruvate-kinase (PK) enzyme is an antigen in the Paediatric Autoimmune Neuropsychiatric Disorders Associated Streptococcal infection known as PANDAS which is characterized by an abnormal production of auto-antibodies against PK. Here, the effects of the anti-pyruvate kinase antibody (anti-PK-ab) on the muscle and neuronal KATP channels were investigated in native rat skeletal muscle fibres and human neuroblastoma cell-line (SH-SY5Y), respectively. Furthermore, the interaction of PK with the inwardly rectifier potassium channel (Kir6.1/Kir6.2) subunits of the KATP channels was investigated by co-immunoprecipitation experiments in mouse brain using the anti-PK-ab. Patch-clamp experiments showed that the short-term incubation (1h) of the fibres with the anti-PK-ab at the dilutions of 1:500 and 1:300 enhanced the KATP current of 19.6% and 33.5%, respectively. As opposite, the long-term incubation (24h) of the fibres with the anti-PK-ab at the dilutions of 1:500 and 1:300 reduced the KATP current of 16% and 24%, respectively, reducing the diameter with atrophy. The direct application of the anti-PK-ab to the excised patches in the absence of intracellular ATP caused channel block, while in the presence of nucleotide channel opened. In neuronal cell line, in the short-term the anti-PK-ab potentiated KATP currents without affecting survival, while in the long-term the anti-PK-ab reduced KATP currents inducing neuronal death. Opening/blocking actions of the anti-PK antibodies on the KATP channels were observed, the blocking action causes fibre atrophy and neuronal death. We demonstrated that PK and Kir subunits are physically/functionally coupled in neurons. The KATP/PK complex can be proposed a novel target in the autoimmune diseases associated with anti-PK production as in PANDAS.

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.

OBJECTIVE Alterations in the handling of renal salt reabsorption may contribute to interindividual differences in blood pressure regulation and susceptibility to hypertension. CLC-K chloride channels and their accessory subunit barttin play a pivotal role in kidney by controlling chloride and water absorption. Compounds selective for CLC-Ks, such as the benzofuran derivative MT-189, may have a significant therapeutic potential. Here, we assessed the feasibility of using CLC-K blockers in hypertension and aimed at enhancing drug inhibitory affinity. METHODS AND RESULTS We demonstrated that acute in-vivo administration of MT-189 to spontaneously hypertensive rats (SHR) caused a reduction of blood pressure and defined the CLC-K/barttin gene expression pattern in kidney of SHR in comparison with normotensive Wistar-Kyoto rats. Based on MT-189, we designed and tested a new series of benzofuran derivatives on CLC-K chloride channels heterologously expressed in HEK293 cells. These studies enabled us to elucidate the causative molecular relationship for obtaining the most potent and selective inhibitor (SRA-36) described so far, with an IC50 of 6.6 ± 1 μmol/l. The biophysical and pharmacological characterization of A447T CLC-Ka and Y315F CLC-Ka, both polymorphisms associated with hypertension, showed that SRA-36 is an efficacious inhibitor of the chloride currents sustained by these polymorphisms. Molecular docking studies allowed hypothesizing an inhibition mechanism for the considered ligands, laying the foundations for the rational design of new and more effective CLC-K inhibitors. CONCLUSION The SRA-36 molecule represents a new potential therapeutic option for hypertension.

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.

Objective The human kidney-specific chloride channels ClC-Ka (rodent ClC-K1) and ClC-Kb (rodent ClC-K2) are important determinants of renal function, participating to urine concentration and blood pressure regulation mechanisms. Here we tested the hypothesis that these chloride channels could represent new drug targets for inducing diuretic and antihypertensive effects. Methods To this purpose, the CLC-K blockers benzofuran derivatives MT-189 and RT-93 (10, 50, 100 mg/kg), were acutely administered by gavage in Wistar rats, and pharmacodynamic and pharmacokinetic parameters determined by functional, bioanalytical, biochemical and molecular biology assays. Results Plasma concentration values for MT-189 and RT-93 were indicative of good bioavailability. Both MT-189 and RT-93 dose-dependently increased urine volume without affecting electrolyte balance. A comparable reduction of SBP was observed in rats after MT-189, RT-93 or furosemide administration. Benzofuran derivatives treatment did not affect kidney CLC-K mRNA level or inner medulla osmolality, whereas a significant vasopressin-independent down-regulation of aquaporin water channel type 2 was observed at protein and transcriptional levels. In rats treated with benzofuran derivatives, the observed polyuria was mainly water diuresis; this finding indirectly supports a cross-talk between chloride and water transport in nephron. Moreover, preliminary in-vitro evaluation of the drugs capability to cross the blood-inner ear barrier suggests that these compounds have a limited ability to induce potential auditory side effects. Conclusion CLC-K blockers may represent a new class of drugs for the treatment of conditions associated with expanded extracellular volume, with a hopeful high therapeutic potential for hypertensive patients carrying ClC-K gain-of-function polymorphisms.

Understanding the genetic background and the molecular mechanisms of inherited kidney channelopathies is essential to definition of targeted therapeutic approaches for affected patients. This article is protected by copyright. All rights reserved

New chiral mexiletine analogs were synthesized in their optically active forms and evaluated in vitro as use-dependent blockers of skeletal muscle sodium channels. Tests carried out on sodium currents of single muscle fibers of Rana esculenta demonstrated that all of them exerted a higher use-dependent block than mexiletine. The most potent analog, (S)-3-(2,6-dimethylphenoxy)-1-phenylpropan-1-amine (S)-(5), was six-fold more potent than (R)-Mex in producing a tonic block. As observed with mexiletine, the newly synthesized compounds exhibit modest enantioselective behavior, that is more evident in 3-(2,6-dimethylphenoxy)butan-1-amine (3). Chirality 22:299-307, 2010. V C 2009 Wiley-Liss, Inc.

The mechanism by which the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) induce skeletal muscle injury is still under debate. By using fura-2 cytofluorimetry on intact extensor digitorum longus muscle fibers, here we provided the first evidence that 2 months in vivo chronic treatment of rats with fluvastatin (5 and 20 mg kg-1) and atorvastatin (5 and 10 mg kg-1) caused an alteration of calcium homeostasis. All treated animals showed a significant increase of resting cytosolic calcium [Ca2+]i, up to 60% with the higher fluvastatin dose and up to 20% with the other treatments. The [Ca2+]i rise induced by statin administration was not due to an increase of sarcolemmal permeability to calcium. Furthermore, the treatments reduced caffeine responsiveness. In vitro application of fluvastatin caused changes of [Ca2+]i, resembling the effect obtained after the in vivo administration. Indeed, fluvastatin produced a shift of mechanical threshold for contraction toward negative potentials and an increase of resting [Ca2+]i. By using ruthenium red and cyclosporine A, we determined the sequence of the statin-induced Ca2+ release mechanism. Mitochondria appeared as the cellular structure responsible for the earlier event leading to a subsequent large sarcoplasmic reticulum Ca2+ release. In conclusion, we suggest that calcium homeostasis alteration may be a crucial event for myotoxicity induced by this widely used class of hypolipidemic drugs.

Taurine is a natural amino acid present as free form in many mammalian tissues and in particular in skeletal muscle. Taurine exerts many physiological functions, including membrane stabilization, osmoregulation and cytoprotective effects, antioxidant and anti-inflammatory actions as well as modulation of intracellular calcium concentration and ion channel function. In addition taurine may control muscle metabolism and gene expression, through yet unclear mechanisms. This review summarizes the effects of taurine on specific muscle targets and pathways as well as its therapeutic potential to restore skeletal muscle function and performance in various pathological conditions. Evidences support the link between alteration of intracellular taurine level in skeletal muscle and different pathophysiological conditions, such as disuse-induced muscle atrophy, muscular dystrophy and/or senescence, reinforcing the interest towards its exogenous supplementation. In addition, taurine treatment can be beneficial to reduce sarcolemmal hyper-excitability in myotonia-related syndromes. Although further studies are necessary to fill the gaps between animals and humans, the benefit of the amino acid appears to be due to its multiple actions on cellular functions while toxicity seems relatively low. Human clinical trials using taurine in various pathologies such as diabetes, cardiovascular and neurological disorders have been performed and may represent a guide-line for designing specific studies in patients of neuromuscular diseases.

Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7-1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously-expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogues, namely 20b, (R)-31b, and (S)-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S)-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S)-22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that use-dependent sodium channel blockade contributes to the analgesic activity of (R)-31b and (S)-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors.

CLC-K Cl(-) channels are selectively expressed in kidney and ear, where they are pivotal for salt homeostasis, and loss-of-function mutations of CLC-Kb produce Bartter's syndrome type III. The only ligand known for CLC-K channels is a derivative of the 2-p-chlorophenoxypropionic acid (CPP), 3-phenyl-CPP, which blocks CLC-Ka, but not CLC-Kb. Here we show that in addition to this blocking site, CLC-K channels bear an activating binding site that controls channel opening. Using the voltage-clamp technique on channels expressed in Xenopus laevis oocytes, we found that niflumic acid (NFA) increases CLC-Ka and CLC-Kb currents in the 10 to 1000 microM range. Flufenamic acid (FFA) derivatives or high doses of NFA produced instead an inhibitory effect on CLC-Ka, but not on CLC-Kb, and on blocker-insensitive CLC-Ka mutants, indicating that the activating binding site is distinct from the blocker site. Evaluation of the sensitivity of CLC-Ka to derivatives of NFA and FFA together with a modeling study of these ligands allow us to conclude that one major characteristic of activating compounds is the coplanarity of the two rings of the molecules, whereas block requires a noncoplanar configuration. These molecules provide a starting point for identification of diuretics or drugs useful in the treatment of Bartter's syndrome.

Flecainide, a class IC antiarrhythmic, was shown to improve myotonia caused by sodium channel mutations in situations where the class IB antiarrhythmic drug mexiletine was less efficient. Yet little is known about molecular interactions between flecainide and human skeletal muscle sodium (hNav1.4) channels. Whole-cell sodium currents (INa) were recorded in tsA201 cells expressing wild-type (WT) and mutant hNav1.4 channels (R1448C, paramyotonia congenita; G1306E, potassium-aggravated myotonia). At a holding potential (HP) of –120 mV, flecainide use-dependently blocked WT and G1306E INa equally but was more potent on R1448C channels. For WT, the extent of block depended on a holding voltage more negative than the activation threshold, being greater at –90 mV as compared to –120 and –180 mV. This behaviour was exacerbated by the R1448C mutation since block at –120 mV was greater than that at –180 mV. Thus flecainide can bind to inactivated sodium channels in the absence of channel opening. Nevertheless, all the channels showed the same closed-state affinity constant (KR∼480 μm) and the same inactivated-state affinity constant (KI∼18 μm). Simulations according to the modulated receptor hypothesis mimic the voltage-dependent block of WT and mutant channels by flecainide and mexiletine. All the results suggest similar blocking mechanisms for the two drugs. Yet, since flecainide exerts use-dependent block at lower frequency than mexiletine, it may exhibit greater benefit in all myotonic syndromes. Moreover, flecainide blocks hNav1.4 channel mutants with a rightward shift of availability voltage dependence more specifically than mexiletine, owing to a lower KR/KI ratio. This study offers a pharmacogenetic strategy to better address treatment in individual myotonic patients.

Both enantiomers of “para-hydroxymexiletine” (PHM), one of the main metabolites of mexiletine, were synthesized and fully characterized. Properties of (R)- and (S)-PHM, in terms of blocking potency and stereoselectivity on frog skeletal muscle Na+ channels, were evaluated. The presence of a hydroxy group on the aryloxy moiety in the 4-position, as in PHM, reduced potency with respect to mexiletine in reducing INa max. However, PHM showed clear use-dependent behavior similar to that of mexiletine and, in contrast with what is observed with the parent compound, maintained its stereoselectivity during the use-dependent block. Chirality 16:72–78, 2004. © 2004 Wiley-Liss, Inc.

The 2-(p-chlorophenoxy)propionic acid (CPP) modulates in a stereoselective manner the macroscopic chloride conductance (gCl), the electrical parameter sustained by the CLC-1 channel, of skeletal muscle. In order to determine the structural requirements for modulating native gCl and to identify high-affinity ligands, the effects of newly synthesised CPP analogues have been evaluated on gCl of rat EDL muscle fibres by means of the two-microelectrode current-clamp technique. Each type of the following independent modification of CPP structure led to a three- to 10-fold decrease or to a complete lack of gCl-blocking activity: replacement of the electron-attractive chlorine atom of the aromatic ring, substitution of the oxygen atom of the phenoxy group, modification at the chiral centre and substitution of the carboxylic function with a phosphonate one. The analogues bearing a second chlorophenoxy group on the asymmetric carbon atom showed a significant gCl-blocking activity. Similar to racemate CPP, the analogue with this group, spaced by an alkyl chain formed by three methylenic groups, blocked gCl by 45% at 100 μM. These latter derivatives were tested on heterelogously expressed CLC-1 performing inside-out patch-clamp recordings to further define how interaction between drug and channel protein could take place. Depending on the exact chemical nature of modification, these derivatives strongly blocked CLC-1 with KD values at −140 mV ranging from about 4 to 180 μM. In conclusion, we identified four molecular determinants pivotal for the interaction with the binding site on muscle CLC-1 channels: (a) the carboxylic group that confers the optimal acidity and the negative charge; (b) the chlorophenoxy moiety that might interact with a hydrophobic pocket; (c) the chiral centre that allows the proper spatial disposition of the molecule; (d) an additional phenoxy group that remarkably stabilises the binding by interacting with a second hydrophobic pocket. Keywords: Skeletal muscle, CLC-1 chloride channel, chloride conductance, clofibric acid analogues Introduction CLC-1 is a voltage-dependent Cl− channel selectively expressed in skeletal muscle, where it is responsible for the maintenance of the membrane potential at rest and consequently for the electrical stability of the sarcolemma. Genetic defects of CLC-1 lead to inheritable myotonia, a disease characterised by abnormal membrane hyperexcitability and muscle stiffness, in mice, goats and humans (Steinmeyer et al., 1991; Koch et al., 1992). Both the small single-channel conductance (Pusch et al., 1994) and the probable localisation of the channel in the transverse tubules (Chua & Betz, 1991) do not allow the study of this muscle Cl− channel by direct inspection with the patch-clamp technique on native fibres. The measurement of the macroscopic Cl− conductance (gCl), the electrical parameter sustained by CLC-1, allows to gain insight into the activity and pharmacology of the Cl− channel in its native environment. The 2-(p-chlorophenoxy) propionic acid (CPP) and the 9-anthracenecarboxylic acid (9-AC) are two specific ligands of the CLC-1 channel (Jentsch et al., 2002; Pusch et al., 2002). Pioneering studies conducted in our laboratory on native skeletal muscle have demonstrated that CPP, having an asymmetric carbon atom α to the carboxylic group, is capable of stereoselectively modulating the native gCl (Conte Camerino et al., 1988; De Luca et al., 1992b). In particular, S(−)CPP blocks gCl in a concentration-dependent manner with a half-maximal concentration in the range of 10–20 μM, whereas the R(+) enantiomer produces a typical biphasic effect, enhancing gCl slightly at low concentrations (1–10 μM) and decreasing it at higher concentrations (>10 μM). The opposite effects produced by the individual enantiomers allowed us to hypothesise the presence of two different receptors or binding sites able, respectively, to enhance and decrease channel activity. The mechanism of the inhibition mediated by CPP has been studied in great detail using cellular lines or Xenopus oocytes as heterologous expression systems (Aromataris et al., 1999; Pusch et al., 2000). S(−)CPP blocks CLC-1 current in an apparently complex voltage-dependent manner and interferes with channel gating acting from the intracellular side. In particular, as demonstrated by single-channel recording of homologous CLC-0 chloride currents, S(−)CPP inhibits the individual protopores of the double-barrelled channel by binding to the pore preferentially when the channel is closed (Pusch et al., 2001). Furthermore, CPP is a specific ligand for the muscle CLC-1 channel, being much less effective on the ubiquitous CLC-2 channel and completely ineffective on renal CLC-5 and CLC-K channels from the extracellular side (Pusch et al., 2000; Liantonio et al., 2002). The phosphorylation of the CLC-1 protein by Ca-dependent protein kinase C (PKC) modulates channel activity in native skeletal muscle and in heterologous expression systems (De Luca et al., 1998; Rosenbohm et al., 1999). Furthermore, the sensitivity of CLC-1 to CPP enantiomers changes with the PKC-dependent phosphorylation state of the channel protein (De Luca et al., 1994; 1992a). These findings corroborate the idea that the measurement of gCl of native skeletal muscle fibres represents a useful system to obtain information about the modulation of CLC-1 activity, preserving the mechanisms of biochemical constituents that are fundamental for the channel physiology. On the other hand, taking into account the intracellular localisation of the hypothetical binding site of CPP (Pusch et al., 2000; 2001), inside-out patch-clamp recordings on heterologously expressed CLC-1 channel allow to study the interaction between the drugs and the amino-acid residues involved in the binding site independently from the capability of the molecule to cross the plasma membrane. The stereoselective effect of CPP and the reduced potency of clofibric acid and p-chlorophenoxyacetic acid (CPA), two achiral CPP-like compounds, in blocking native gCl (Conte Camerino et al., 1988; Loiodice et al., 1993), suggest that the asymmetric carbon atom of the drug molecule represents an important pharmacophore moiety. In order to determine the molecular requisites for the modulation of the native gCl and consequently the activity of CLC-1 channel, and with the goal of identifying high-affinity ligands, we performed a structure–activity relationship study by synthesising and evaluating the effects of a series of CPP derivatives (Figure 1). We initially screened the CPP-like compounds on the native chloride conductance of rat skeletal muscle using the two-microelectrode current-clamp technique. All tested derivatives resulted less potent with respect to CPP. Taking into account that membrane permeability can be a limiting step for the effect on gCl, the derivatives having physical–chemical properties different from that of CPP, as the phosphonate derivatives and bis-phenoxy derivatives (N.23 and N.25–33), were also tested on heterologously expressed CLC-1 performing inside-out patch-clamp recordings. This approach allowed to better define the interaction between drug and channel protein, corroborating a high-affinity interaction of the bis-phenoxy derivatives, in spite of their limited permeability. Figure 1 Chemical structures of different analogues of CPP. Methods Experimental procedure of muscle preparation Adult male Wistar rats of 350–400 g were used for the experiments. The electrophysiological experiments were made on isolated extensor digitorum longus (EDL) muscle. The muscle was removed under urethane anaesthesia (1.2 g kg−1, i.p.). Soon after the biopsy, the EDL muscle was stretched to about 1.5 times its resting length on a 3 mm plastic rod in a temperature-controlled muscle chamber at 30°C and perfused with a physiological solution (see below) in the absence and in the presence of tested compounds (De Luca et al., 1992b).

(1) Growth hormone secretagogues (GHS) exhibit potent growth hormone (GH)-releasing activity through the activation of a pituitary receptor. Here, we consider the possibility that GHS can target a specific receptor in rat skeletal muscle and have a role in the control of muscle function. (2) By means of the intracellular microelectrode technique, we found that in vitro application of hexarelin and L-163,255 dose dependently reduced resting chloride (gCl) and potassium (gK) conductances in rat skeletal muscle. These effects were prevented by the GHS-receptor antagonist [D-Lys-3]-GHRP-6, and by either phospholipase C or protein kinase C (PKC) inhibitors. Ghrelin, a natural ligand of GHS receptors, also induced a reduction of muscle gCl and gK, which was antagonised by [D-Lys-3]-GHRP-6. (3) Both GHS shifted the mechanical threshold for the contraction of muscle fibres towards more negative voltages. Accordingly, by means of FURA-2 fluorescent measurements, we demonstrated that L-163,255 induced a resting [Ca(2+)](i) increase, which was reversible and not blocked by nifedipine or removal of external Ca(2+). (4) Ageing is a condition characterised by a deficit of GH secretion, which in turn modifies the electrical and contractile properties of skeletal muscle. In contrast to GH, chronic treatment of aged rats with hexarelin or L-163,255 failed to restore the electrical and contractile muscle properties. Moreover, the two GHS applied in vitro were able to antagonise the beneficial effect on gCl and gK obtained through chronic treatment of aged animals with GH. (5) Thus, skeletal muscle expresses a specific GHS receptor able to decrease gCl and gK through a PKC-mediated intracellular pathway. This peripheral action may account for the lack of restoration of skeletal muscle function in long-term GHS-treated aged animals.

New 2H-1,4-benzoxazine derivatives were synthesized and tested for their agonist properties on the ATP-sensitive K+ channels (KATP) of native rat skeletal muscle fibres by using the patch-clamp technique. The novel modifications involved the introduction at position 2 of the benzoxazine ring of alkyl substituents such as methyl (–CH3), ethyl (–C2H5) or propyl (–C3H7) groups, while maintaining pharmacophore groups critical for conferring agonist properties. The effects of these molecules were compared with those of cromakalim in the presence or absence of internal ATP (10−4M). In the presence of internal ATP, all the compounds increased the macropatch KATP currents. The order of potency of the molecules as agonists was −C3H7 (DE50=1.63 × 10−8M) >−C2H5 (DE50=1.11 × 10−7M)>–CH3 (DE50=2.81 × 10−7M)>cromak-slim (DE50= 1.42 × 10−5M). Bell-shaped dose–response curves were observed for these compounds and cromakalim indicating a downturn in response when a certain dose was exceeded. In contrast, in the absence of internal ATP, all molecules including cromakalim inhibited the KATP currents. The order of increasing potency as antagonists was cromakalim (IC50=1.15 × 10−8M)–CH3 (IC50=2.6 × 10−8M)>–C2H5 (IC50=4.4 × 10−8M)>–C3H7 (IC50=1.68 × 10−7M) derivatives. These results suggest that the newly synthesized molecules and cromakalim act on muscle KATP channel by binding on two receptor sites that have opposite actions. Alternatively, a more simple explanation is to consider the existence of a single site for potassium channel openers regulated by ATP which favours the transduction of the channel opening. The alkyl chains at position 2 of the 2H-1,4-benzoxazine nucleus is pivotal in determining the potency of benzoxazine derivatives as agonists or antagonists. British Journal of Pharmacology (2003) 139, 255–262. doi:10.1038/sj.bjp.0705233

Activation of muscle beta(2)-adrenergic receptors successfully counteracted sarcolemma inexcitability in patients suffering from hyperkalemic periodic paralysis (HPP), a hereditary disease caused by mutations in the gene encoding the skeletal muscle sodium channel. Looking for potential modulation of these channels by beta(2)-adrenergic pathway using patch-clamp technique, we found that clenbuterol blocked sodium currents (I(Na)) in rat skeletal muscle fibers and in tsA201 cells transfected with the human channel isoform, whereas salbutamol did not. The effects of clenbuterol were independent of beta-adrenoceptor stimulation. Instead, clenbuterol structure and physicochemical characteristics as well as I(Na) blocking properties resembled those of local anesthetics, suggesting direct binding to the channels. Similar experiments with the chemically similar beta-antagonists propranolol and nadolol, suggested the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. Importantly, clenbuterol use-dependently inhibited action potential firing in rat skeletal muscle fibers, owing to beta-adrenoceptor-independent I(Na) block. From a clinical point of view, our study defines the rationale for the safe use of salbutamol in HPP patients, whereas clenbuterol may be more indicated in patients suffering from myotonic syndromes, a condition characterized by sarcolemmal overexcitability, because use-dependent I(Na) block can inhibit abnormal runs of action potentials.

Sodium channel myotonias are inherited muscle diseases linked to mutations in the voltage-gated sodium (Nav1.4) channel [1]. Today, mexiletine is empirically considered as a first choice drug in non-dystrophic myotonias. Mexiletine likely acts by reducing muscle cell excitability through use-dependent Nav1.4 channel block. Nevertheless, not all patients benefit from mexiletine because of side effects (gastrointestinal discomfort, dizziness) or lack of efficacy. Here we report the case of a mother and her son, both carrying the G1306E sodium channel mutation associated with a severe phenotype of myotonia permanens and little improvement with mexiletine. Since the G1306E mutant was more sensitive to flecainide than mexiletine in vitro [2], we decided to shift treatment to flecainide. The proband came to our observation when she was 20 years old, complaining since early childhood of severe and painful generalized muscle stiffness, exacerbated after exercise, in wet weather and during febrile episodes. Generalized muscle hypertrophy was present. The lid-lag phenomenon lasted for several seconds, while relaxation time of the grip was lengthened to 30–40 s. Long exercise and cooling tests did not induce weakness. Electromyography showed normal nerve conduction but continuous myotonic running at rest. Serum creatine kinase was elevated to 400–900 IU/L. A biopsy of the left biceps muscle showed abnormal central nuclei and fiber size. The G1306E mutation was found in the SCN4A gene [3]. Previous electrophysiological studies described the alteration of mutant sodium channel gating and demonstrated its responsibility for the phenotype [3, 4]. During her life, the patient was given empiric trials of carbamazepine (3 × 200 mg/day), hydroquinidine (3 × 200 mg/day), tocainide (3 × 400 mg/day), and mexiletine (4 × 200 mg/day) with limited benefit. During pregnancy (age 36 and 39 years), the proband experienced a striking worsening of myotonia. One son is affected, carrying the same G1306E mutation. He shows a very severe phenotype, including facial and ocular muscle myotonia since his birth, generalized painful muscle myotonia, and muscle hypertrophy. His general motor and mental development is normal. Since early childhood he has taken mexiletine with very poor benefit. Examination at age 5 years revealed forced internal rotation of the gleno-humeral joint and elevation of the shoulders, probably due to shoulder muscle stiffness and hypertrophy. Prolonged lid-lag phenomenon was found associated with severe orbicular myotonia, extraocular muscles and tongue myotonia. On the basis of in vitro experiments [2], we decided to initiate treatment with flecainide. In the proband (age 43 years, body weight 65 kg), mexiletine (4 × 200 mg/day) was suspended in the afternoon and flecainide (100 mg/day) assumed next morning. Ambulatory cardiac function was evaluated. After 2 days of malaise, the patient started to improve. Four days later, flecainide dose was increased to 100 mg twice a day. The patient noticed an enhanced capability to move spontaneously after rest and a dramatic improvement of her periorbital and extraocular myotonia. One month later, the proband child (age 7 years, body weight 25 kg) was changed from mexiletine to 35 mg flecainide twice a day. After 10 days, muscle stiffness decreased so that the internal rotation of the gleno-humeral joint and elevation of the shoulders were significantly reduced. The mother reported that he became able to have a meal and dress himself without any help. At last follow-up the child was taking 60 mg flecainide twice a day. A functional chair test [5] showed a remarkable improvement after flecainide introduction and a further improvement 9 months later (Fig. 1). The quality of life of the proband was evaluated with the 36-Item Short-Form Health Survey [6]. The physical component scale increased from 29 during mexiletine therapy to 38 after flecainide introduction. Improvement included physical functioning, role limitations due to physical problems, body pain, and general health perceptions. After almost 2 years of treatment no side effects have been reported. Fig. 1 Chair test. The functional chair test was performed at the same time of day, in a comfortable room with an ambient temperature of 25 °C. Both patients were asked to rise from a chair after a 5 min rest and perform three turns around the ... There are only a few reports on myotonia permanens associated with G1306E mutation, including a familial case with autosomal dominant inheritance [7]. In this study, the proband did not have any symptom relief with mexiletine, oxcarbazepine, or thiazide diuretics. Although flecainide has been shown to be an effective hNav1.4 channel blocker in vitro [2, 8, 9], its clinical use as an antimyotonic agent is rarely reported [10]. The improvement of myotonia by flecainide obtained here suggests that there may be a relationship between the G1306E mutation and the response to flecainide therapy. Indeed we previously demonstrated that flecainide was more efficient than mexiletine in blocking functionally expressed G1306E channels, probably due to the positive shift of channel availability voltage-dependence by the mutation [2]. On the same basis, we may hypothesize that at least 15 out of 33 identified SCN4A mutations, which are known to induce a positive shift of voltage-dependence, may be more sensitive to flecainide than mexiletine, and patients carrying these mutations might have more benefit from flecainide. Such hypothesis merits further investigation. The present study provides a paradigm of a bench-to-bedside pharmacogenetics strategy and indicates the relevance of pursuing the pharmacological characterization of sodium channel mutants in vitro in order to define a drug-genotype relationship. A clinical trial would be then warranted to develop a pharmacogenetic strategy to better address treatment in individual myotonic patients. Such an approach may be also of great interest in other familial diseases, including cardiac arrhythmias, epilepsies, and neuropathic pain, all characterized by mutations in sodium channel genes and by possible treatment with sodium channel blockers.

We investigated on the role of the genes encoding for the ATP-sensitive K(+)-channel (KATP) subunits (SUR1-2A/B, Kir6.2) in the atrophy induced "in vitro" by staurosporine (STS) in different skeletal muscle phenotypes of mouse. Patch-clamp and gene expression experiments showed that the expression/activity of the sarcolemma KATP channel subunits was higher in the fast-twitch than in the slow-twitch fibers. After 1 to 3h of incubation time, the STS (2.14×10(-6)M) treatment enhanced the expression/activity of the SUR2B, SUR1 and Kir6.2 subunit genes, but not SUR2A, in the slow-twitch muscle fibers, induced the caspase-3-9, Atrogin-1 and Murf-1 gene expression without affecting protein content. After 3 to 6h, the STS-related atrophy markedly down-regulated the SUR2B, SUR1 and Kir6.2 genes reducing the KATP currents and reduced the protein content/muscle weight ratio of the slow-twitch muscle by -36.4±6% (p

The ATP-sensitive K(+) (KATP) channel is an emerging pathway in the skeletal muscle atrophy which is a comorbidity condition in diabetes. The "in vitro" effects of the sulfonylureas and glinides were evaluated on the protein content/muscle weight, fibers viability, mitochondrial succinic dehydrogenases (SDH) activity, and channel currents in oxidative soleus (SOL), glycolitic/oxidative flexor digitorum brevis (FDB), and glycolitic extensor digitorum longus (EDL) muscle fibers of mice using biochemical and cell-counting Kit-8 assay, image analysis, and patch-clamp techniques. The sulfonylureas were: tolbutamide, glibenclamide, and glimepiride; the glinides were: repaglinide and nateglinide. Food and Drug Administration-Adverse Effects Reporting System (FDA-AERS) database searching of atrophy-related signals associated with the use of these drugs in humans has been performed. The drugs after 24 h of incubation time reduced the protein content/muscle weight and fibers viability more effectively in FDB and SOL than in the EDL. The order of efficacy of the drugs in reducing the protein content in FDB was: repaglinide (EC50 = 5.21 × 10(-6)) ≥ glibenclamide(EC50 = 8.84 × 10(-6))glimepiride(EC50 = 2.93 × 10(-5))tolbutamide(EC50 = 1.07 × 10(-4))nateglinide(EC50 = 1.61 × 10(-4)) and it was: repaglinide(7.15 × 10(-5)) ≥ glibenclamide(EC50 = 9.10 × 10(-5))nateglinide(EC50 = 1.80 × 10(-4)) ≥ tolbutamide(EC50 = 2.19 × 10(-4))glimepiride(EC50=-) in SOL. The drug-induced atrophy can be explained by the KATP channel block and by the enhancement of the mitochondrial SDH activity. In an 8-month period, muscle atrophy was found in 0.27% of the glibenclamide reports in humans and in 0.022% of the other not sulfonylureas and glinides drugs. No reports of atrophy were found for the other sulfonylureas and glinides in the FDA-AERS. Glibenclamide induces atrophy in animal experiments and in human patients. Glimepiride shows less potential for inducing atrophy.

Searching for the structural requirements improving the potency and the stereoselectivity of Na+ channel blockers as antimyotonic agents, new derivatives of tocainide, in which the chiral carbon atom is constrained in a rigid α-proline or pyrrolo-imidazolic cycle, were synthesized as pure enantiomers. Their ability to block Na+ currents, elicited from −100 to −20 mV at 0.3 Hz (tonic block) and 2 – 10 Hz (use-dependent block) frequencies, was investigated in vitro on single fibres of frog semitendinosus muscle using the vaseline-gap voltage-clamp method. The α-proline derivative, To5, was 5 and 21 fold more potent than tocainide in producing tonic and 10 Hz-use-dependent block, respectively. Compared to To5, the presence of one methyl group on the aminic (To6) or amidic (To7) nitrogen atom decreased use-dependence by 2- and 6-times, respectively. When methylene moieties were present on both nitrogen atoms (To8), both tonic and use-dependent block were reduced. Contrarily to tocainide, all proline derivatives were stereoselective in relation to an increased rigidity. A further increase in the molecular rigidity as in pyrrolo-imidazolic derivatives markedly decreased the drug potency with respect to tocainide. Antimyotonic activity, evaluated as the shortening of the time of righting reflexes of myotonic adr/adr mice upon acute drug in vivo administration was 3 fold more effective for R-To5 than for R-Tocainide. Thus, constraining the chiral centre of tocainide in α-proline cycle leads to more potent and stereoselective use-dependent Na+ channel blockers with improved therapeutic potential. Keywords: Na+ channel, native frog skeletal muscle fibres, vaseline-gap voltage-clamp, tocainide derivatives, use-dependent block, stereoselectivity, antimyotonic activity, adr/adr mice Introduction Tocainide is an orally effective lidocaine derivative, able to block the voltage-gated Na+ channels in a use-dependent manner, a feature that enhances its potency in situations of prolonged depolarization and/or excessive firing of action potentials with respect to a physiological excitability (Catterall, 1987; de luca et al., 1997b; Conte Camerino et al., 2000). Thus, tocainide is actually among the few drugs clinically used for the symptomatic treatment of hyperexcitability of myotonic syndromes, hereditary disorders of skeletal muscle due to genetic mutations on either sodium or chloride channel genes (Streib, 1986; Kwiecinski et al., 1992, Rudel et al., 1994; Lehmann-Horn & Rudel, 1996; Cannon, 1997; Ptacek, 1998). However, its clinical use presents some disadvantages, due to the high doses required for its antimyotonic effectiveness with the risk of a greater incidence of serious side effects, especially at the cardiac, haematopoietic and central nervous system levels (Roden & Woosley, 1986; Puniani & Bertorini, 1991; Rudel et al., 1994). The use-dependent inhibition of Na+ channels by tocainide and other local anaesthetic (LA) drugs relies on the higher affinity of these drugs for their receptor site on the α-subunit of the channel when this latter is open and/or inactivated, and to a slow recovery from inactivation of the drug-bound channels during membrane repolarization (Catterall, 1987; de luca et al., 1997a). At the resting state, the LA interacts with its presumed receptor through hydrophobic interactions with two aromatic amino acid residues at the D4 – S6 segment (Ragsdale et al., 1994; Wright et al., 1998). Gating movements due to the channel opening may then allow further drug interactions with other residues in the pore that could stabilize the drug-bound channel in an inactivated state (Wang et al., 2000; Yarov-Yarovoy et al., 2001). Furthermore, a site in D1 – S6 segment would also be involved in determining the drug stereoselectivity (Nau et al., 1999). This feature is important because tocainide and mexiletine, another LA-like compound, have a chiral carbon atom that contributes to the potency and the stereoselectivity of the drug (Tricarico et al., 1991; de luca et al., 1997a; 2000; Desaphy et al., 1999). In particular, we found that the introduction of an aromatic lipophilic group on the chiral centre of mexiletine highly increases the drug potency suggesting the establishment of a strong two-points interaction with the two hydrophobic pockets of the receptor in D4 – S6 segment (Wright et al., 1998; de luca et al., 2000). Furthermore, the nature of the group on the stereogenic centre is important for the drug stereoselectivity because the introduction of an unbending lipophilic group such as the isopropyl one enhanced the stereoselectivity, likely due to the occurrence of a three-points interaction of this more rigid structure with the receptor (de luca et al., 1997a; 2000). In agreement with previous data, we confirmed that a higher basicity in the molecule determines a greater use-dependent behaviour (de luca et al., 1997a). To go further in the study of the structure-activity relationship and to better evaluate the role of the chiral centre in the potency and/or stereoselectivity of drug for blocking Na+ channels, the effects of the pure enantiomers of a new derivative of tocainide, in which the asymmetric carbon atom is constrained in a rigid α-proline cycle (To5, Figure 1) were evaluated on Na+ currents of native frog skeletal muscle fibres. In addition, by introducing methyl groups on one or both of the nitrogen atoms of the proline derivative (as in To6, To7 and To8, Figure 1) or by constraining the chiral centre in a pyrrolo-imidazolic cycle (To3 and To4, Figure 1), we have investigated whether a further decrease in the ability of the molecule to transit through various conformations could modify the drug potency and/or stereoselectivity, and consequently the interaction with the receptor. The results indicate that constraining the stereogenic centre in a α-proline cycle, as in To5, highly improves potency, stereoselectivity and use-dependent behaviour of Na+ channel blockers. Accordingly, an acute in vivo administration of tocainide and To5 in myotonic adr/adr mice showed that To5 was 3 fold more effective than its parent compound in decreasing the time of righting reflex of myotonic mice. On the basis of these data, we can conclude that the newly synthesized proline derivative of tocainide, To5, may be considered as a potential therapeutic agent in the treatment of myotonic syndromes. Figure 1 Chemical structure of tocainide and its newly synthesized α-proline and pyrrolo-imidazolic analogues. Methods Fibre preparation and voltage clamp apparatus Segments of undamaged single muscle fibres (about 1 cm in length) were obtained by microsurgery (plucking procedure) from the ventral branch of the semitendinosus muscle of Rana Esculenta bathed in normal physiological solution at room temperature. The cut-end fibre was then superfused with an internal solution and mounted across three chamber partitions, which delineated the four pools. Three strips of vaseline were applied over the fibre and carefully sealed to the fibre to reduce leakage. The width of the gaps of the central pools (A and B) had been previously set to 70 – 100 μM and 200 μM, respectively. Four KCl/agar bridges electrodes connected the recording chamber to the voltage clamp amplifier based on methods described by Hille & Campbell (1976) and detailed elsewhere (de luca et al., 1995; 1997a). When the solution level was lowered below the vaseline strips, the four pools were physically and electrically independent from each other: this was confirmed by verifying that no leak current was flowing upon increasing the amplifier gain. Then the solution in the pool A was replaced with the external solution and after about 10 min of equilibration the recordings were performed at 10°C. The usual holding potential (h.p.) was −100 mV. Sodium currents were recorded using an amplifier connected via a A/D and D/A Digidata 1200 Interface (Axon Instruments, Foster City, CA., U.S.A.) to a 486 DX2/66 personal computer and stored on the hard disk. The stimulation protocols and data acquisition were driven by the Clampex program (pClamp6 software package; Axon Instruments). The currents flowing in response to depolarizing command voltages were low pass filtered at 10 kHz (Frequency Devices, U.S.A.), visualized on an oscilloscope and sampled at 20 kHz. When necessary, leak and capacities currents were subtracted by P/4 method. The acquired traces were analysed later using Clampfit program (pClamp6 software package; Axon Instruments).

No clear data is available about functional alterations in the calcium-dependent excitation-contraction (e-c) coupling mechanism of dystrophin-deficient muscle of mdx mice. By means of the intracellular microelectrode ‘point’ voltage clamp method, we measured the voltage threshold for contraction (mechanical threshold; MT) in intact extensor digitorum longus (EDL) muscle fibres of dystrophic mdx mouse of two different ages: 8–12 weeks, during the active regeneration of hind limb muscles, and 6–8 months, when regeneration is complete. The EDL muscle fibres of 8–12-week-old wildtype animals had a more negative rheobase voltage (potential of equilibrium for contraction- and relaxation-related calcium movements) with respect to control mice of 6–8 months. However, at both ages, the EDL muscle fibres of mdx mice contracted at more negative potentials with respect to age-matched controls and had markedly slower time constants to reach the rheobase. The in vitro application of 60 mM taurine, whose normally high intracellular muscle levels play a role in e-c coupling, was without effect on 6–8-month-old wildtype EDL muscle, while it significantly ameliorated the MT of mdx mouse. HPLC determination of taurine content at 6–8 months showed a significant 140% rise of plasma taurine levels and a clear trend toward a decrease in amino acid levels in hind limb muscles, brain and heart, suggesting a tissue difficulty in retaining appropriate levels of the amino acid. The data is consistent with a permanent alteration of e-c coupling in mdx EDL muscle fibres. The alteration could be related to the proposed increase in intracellular calcium, and can be ameliorated by taurine, suggesting a potential therapeutic role of the amino acid. British Journal of Pharmacology (2001) 132, 1047–1054; doi:10.1038/sj.bjp.0703907

Taurine is a sulphonic aminoacid present in high amounts in various tissues including cardiac and skeletal muscles showing different properties such as antioxidative, antimyotonic and anti-schaemic effects. The cellular mechanism of action of taurine is under investigation and appears to involve the interaction of the sulphonic aminoacid with several ion channels. Using the patch-clamp technique we studied the effects of taurine in rat skeletal muscle fibres on ATP-sensitive K(+) channel (K(ATP)) immediately after excision and on channels that underwent rundown. The cytoplasmic application of 20 mM of taurine reduced the K(ATP) current; this effect was reverted by washout of the drug solution. In this experimental condition the IC(50) was 20.1 mM. After rundown, taurine inhibited the K(ATP) current with similar efficacy. Competition experiments showed that taurine shifted the dose-response inhibition curve of glybenclamide to the left on the log-dose axis without significantly affecting those of ATP or Ca(2+) ion. Single channel recording revealed that taurine affects the close state of the channel prolonging it and reducing the bursts duration. Our data indicate that taurine inhibits the muscular K(ATP) channel interfering with the glybenclamide site on the sulphonylurea receptor of the channel or on the site allosterically coupled to it. During ischaemia and hypoxia, the skeletal and heart muscles undergo several changes; for example, the activation of K(ATP) channels and loss of the intracellular taurine content. The depletion of taurine during ischaemia would contribute to the early activation of K(ATP) channels and salvage the intracellular ATP content.

Tocainide is effective in the symptomatic treatment of myotonic syndromes for its ability to reduce the high frequency discharges of action potentials typical of the disease, by blocking voltage-gated sodium channels. However, its use is restricted by serious side effects. In spite of its chiral structure, tocainide is clinically used as a racemic mixture. Since the optical isomers may differ in their efficacy and toxicity, the present study was aimed at evaluating the antimyotonic activity of the pure R (−) and S (+) enantiomers of tocainide, on the abnormal membrane hyperexcitability of external intercostal muscle fibers of congenitally myotonic goats. The excitability parameters were recorded in vitro by means of the standard two-microelectrode current-clamp technique before and after the addition of the compounds. The R (−) enantiomer of tocainide at concentrations as low as 10 μM potently counteracted the abnormal excitability of myotonic fibers, by increasing the threshold current, and decreasing the latency of the action potential and firing capability. Also, this concentration of R −(−) tocainide almost completely abolished the abnormal spontaneous electrical activity occurring in about 70–80% of the myotonic fiber. The S (+) enantiomer was remarkably less potent since up to 100 μM did not restore the normal excitability pattern. The results show that most of the antimyotonic activity of tocainide resides in the R (−) enantiomer suggesting that its clinical use may allow a significant reduction of the doses and possibly of the side effects.

1 The antiarrhythmic drug mexiletine (Mex) is also used against myotonia. Searching for a more eAcient drug, a new compound (Me5) was synthesized substituting the methyl group on the chiral carbon atom of Mex by an isopropyl group. EAects of Me5 on Na + channels were compared to those of Mex in rat skeletal muscle fibres using the cell-attached patch clamp method. 2 Me5 (10 mM) reduced the maximal sodium current (INa) by 29.7+4.4 % (n=6) at a frequency of stimulation of 0.3 Hz and 65.7+4.4 % (n=6) at 1 Hz. At same concentration (10 mM), Mex was incapable of producing any eAect (n=3). Me5 also shifted the steady-state inactivation curves by 77.9+0.9 mV (n=6) at 0.3 Hz and 712.2+1.0 mV (n=6) at 1 Hz. 3 In the presence of sea anemone toxin II (ATX; 5 mM), INa decayed more slowly and no longer to zero, providing a model of sodium channel myotonia. The eAects of Me5 on peak INa were similar whatever ATX was present or not. Interestingly, Me5 did not modify the INa decay time constant nor the steady-state INa to peak INa ratio. 4 Analysis of ATX-induced late Na + channel activity shows that Me5 did not aAect mean open times and single-channel conductance, thus excluding open channel block property. 5 These results indicate that increasing hindrance on the chiral atom of Mex increases drug potency on wild-type and ATX-induced noninactivating INa and that Me5 might improve the prophylaxis of myotonia.

The human chloride channels CLC-Ka and CLC-Kb play a pivotal role in kidney by controlling chloride and water absorption. Both channels require barttin as an accessory subunit for full activity. Mutations in CLC-Kb and barttin genes lead to severe renal salt loss while CLC-K gain of function polymorphisms could predispose to hypertension. Thus, compounds that selectively bind to CLC-Ka and/or CLC-Kb channels may have a significant therapeutic potential. Recently, we explored the pharmacological profile of CLC-K/barttin expressed in mammalian HEK-293 cells and demonstrated that HEK cells represent a valid biological system to screen CLC-K high affinity blockers (Imbrici et al., Biochim Biophys Acta, 2014). Here, by using molecular modeling and patch-clamp technique, we developed a new series of benzofuran derivatives and evaluated their efficacy on CLC-K channels expressed in HEK 293 cells. Chemical modifications regarding the hydrophobic group at C-5 and C-3 position of the benzofuran nucleus of the lead compounds RT-93 and JBL-44 (IC50 within 10-30 μM range), allowed us to define the structural requirements to ensure an efficacious CLC-Ka block, finally identifying SRA-36 the most potent compound so far described, with an IC50 of 2.6 ± 1 μM. Interestingly, besides capable of inhibiting CLC-Kb and CLC-K1 isoforms, this compound was also efficacious in blocking A447T CLC-Ka, a polymorphism associated with hypertension. Thus, the SRA-36 molecule could represent a useful probe for exploring CLC-K molecular mechanisms of gating as well as a new potential therapeutic option for hypertensive patients carrying CLC-K gene polymorphisms (Telethon GGP14096).

1. The effects of mexiletine were evaluated on the ATP-sensitive K+ channel (K(ATP)) of rat skeletal muscle fibres using patch clamp techniques. The effects of mexiletine were studied on macropatch currents 20 s (maximally activated), 8 min (early stage of rundown) and 15 min (late stage of rundown) after excision in the absence or in the presence of internal ADP (50-100 microM) or UDP (500 microM). In addition, the effects of mexiletine were tested on single channel. 2. In the absence of ADP and UDP, mexiletine inhibited the current through maximally activated channels with an IC50 of -5.58+/-0.3 M. Nucleoside diphosphates shifted the current versus mexiletine concentration relationship to the right on the log concentration axis. UDP (500 microM) was more efficacious than ADP (50-100 microM) in this effect. 3. At the early stage of rundown, the sensitivity of the channel to mexiletine was reduced and nucleoside diphosphates, particularly UDP, antagonized the effect of mexiletine. At the late stage of rundown, mexiletine did not affect the currents. 4. At the single channel level, 1 microM mexiletine reduced the mean burst duration by 63% and prolonged the arithmetic mean closed time intervals between the bursts of openings without altering the open time and closed time distributions. Mexiletine did not affect the single channel conductance. 5. These results show that in skeletal muscle, mexiletine is a state-dependent K(ATP) channel inhibitor which either acts through the nucleotide binding site or a site allosterically coupled to it.

1 In the present study we tested the hypothesis that insulin-like growth factor-1 (IGF-1) modulates resting chloride conductance (GCl) of rat skeletal muscle by activating a phosphatase and that the chloride channel, based on the activity of phosphorylating-dephosphorylating pathways, has different sensitivity to specific ligands, such as the enantiomers of 2-(p-chlorophenoxy) propionic acid (CPP). 2 For this purpose GCl in EDL muscle isolated from adult rat was first lowered by treatment with 5 nM 4-β-phorbol 12,13 dibutyrate (4-β-PDB), presumably activating protein kinase C (PKC). The effects of IGF-1 and of the enantiomers of CPP on GCl were then tested. 3 IGF-1 (3.3 nM) had no effect of GCl on EDL muscle fibres in normal physiological solution, whereas it completely counteracted the 30% decrease of GCl induced by 4-β-PDB. No effects of IGF-1 were observed on GCl lowered by the phosphatase inhibitor okadaic acid (0.25 μM). 4 Ceramide, reported to activate on okadaic acid-sensitive phosphatase, mimicked the effects of IGF-1. In fact, N-acetyl-sphingosine (2.5–5 μM), not very effective in control conditions, increased the GCl lowered by the phorbol ester, but not the GCl lowered by okadaic acid. 5 In the presence of 4-β-PDB, GCl was differently affected by the enantiomers of CPP. The S(−)-CPP was remarkably less potent in producing the concentration-dependent reduction of GCl, whereas the R(+)-CPP caused an increase of GCl at all the concentrations tested. 6 In conclusion, the PKC-induced lowering of GCl is counteracted by IGF-1 through an okadaic acid sensitive phosphatase, and this effect can have therapeutic relevance in situations characterized by excessive channel phosphorylation. In turn the phosphorylation state of the channel can modulate the effects and the therapeutic potential of direct channel ligands. British Journal of Pharmacology (1998) 125, 477–482; doi:10.1038/sj.bjp.0702107

We evaluated the involvement of the sarcolemmal ATP-sensitive K+ channel in the depolarization of skeletal muscle fibers occurring in an animal model of human hypokalemic periodic paralysis, the K+-depleted rat. After 23-36 days of treatment with a K+-free diet, an hypokalemia was observed in the rats. No difference in the fasting insulinemia and glycemia was found between normokalemic and hypokalemic rats. The fibers of the hypokalemic rats were depolarized. In these fibers, the current of sarcolemmal ATP-sensitive K+ channels measured by the patch-clamp technique was abnormally reduced. Cromakalim, a K+ channel opener, enhanced the current and repolarized the fibers. At channel level, two open conductance states blocked by ATP and stimulated by cromakalim were found in the hypokalemic rats. The two states could be distinguished on the basis of their slope conductance and open probability and were never detected on muscle fibers of normokalemic rats. It is known that insulin in humans affected by hypokalemic periodic paralysis leads to fiber depolarization and provokes paralysis. We therefore examined the effects of insulin at macroscopic and single-channel level on hypokalemic rats. In normokalemic animals, insulin applied in vitro to the muscles induced a glybenclamide-sensitive hyperpolarization of the fibers and also stimulated the sarcolemmal ATP-sensitive K+ channels. In contrast, in hypokalemic rats, insulin caused a pronounced fiber depolarization and reduced the residual currents. Our data indicated that in hypokalemic rats, an abnormally low activity of ATP-sensitive K+ channel is responsible for the fiber depolarization that is aggravated by insulin.

The effects of a 4-month daily treatment with recombinant human growth hormone (GH) (150 μg kg−1) to aged rats were evaluated on the passive and active membrane electrical properties of extensor digitorum longus (EDL) muscle fibres in vitro by means of a two intracellular microelectrode technique. Chronic GH treatment completely restored the diameter and the membrane capacitance of aged EDL muscle fibres and significantly lowered the membrane resistance towards the adult value. There was also an increase of the threshold current, a shortening of the latency and an increase of the amplitude of the action potential and a significant amelioration of the membrane firing capability. The effects were almost fully attributable to a significant 50% increase of resting conductance to chloride ions (GCl), although an observed restoration of potassium conductance and a possible effect on voltage-activated sodium channels could contribute to the effects. EDL muscle fibres of untreated aged rats showed a different pharmacological response to 2-(p-chlorophenoxy) propionic acid (CPP) enantiomers from that seen in adult rats; the S-(−) isomer was less potent in blocking GCl and the R-(+) isomer always increased GCl instead of producing the typical biphasic effect observed in adult fibres (an increase of GCl at 1–10 μM and a decrease at higher concentrations). The 4-month-GH-treated aged rats showed a pharmacological sensitivity to CPP enantiomers similar to that of adults. The in vitro application of insulin-like growth factor I (IGF-I), the peripheral mediator of GH, produced a significant and irreversible increase of GCl of EDL muscle of untreated aged rats, an effect not observed in adults. This effect was completely inhibited by preincubation with 0.5 μM okadaic acid, suggesting that the IGF-I receptor transduction pathway can act on the phosphorylation state of the chloride channel through a serine-threonine protein phosphatase. The results show that the skeletal muscle chloride channel is a target of the impairment of GH/IGF-I axis occurring in aged subjects. The acute and chronic effects observed on GCl of aged muscle fibres suggest that the GH/IGF-I stimuli act through a modulation of channel phosphorylation state and through the synthesis of ‘adult’-like type chloride channels.

Emerging evidences suggest that Ca(2+)activated-K(+)-(BK) channel is involved in the regulation of cell viability. The changes of the cell viability observed under hyperkalemia (15 mEq/L) or hypokalemia (0.55 mEq/L) conditions were investigated in HEK293 cells expressing the hslo subunit (hslo-HEK293) in the presence or absence of BK channel modulators. The BK channel openers(10(-11)-10(-3)M) were: acetazolamide(ACTZ), Dichlorphenamide(DCP), methazolamide(MTZ), bendroflumethiazide(BFT), ethoxzolamide(ETX), hydrochlorthiazide(HCT), quercetin(QUERC), resveratrol(RESV) and NS1619; and the BK channel blockers(2 x 10(-7)M-5 x 10(-3)M) were: tetraethylammonium(TEA), iberiotoxin(IbTx) and charybdotoxin(ChTX). Experiments on cell viability and channel currents were performed using cell counting kit-8 and patch-clamp techniques, respectively. Hslo whole-cell current was potentiated by BK channel openers with different potency and efficacy in hslo-HEK293. The efficacy ranking of the openers at -60 mV(Vm) was BFT> ACTZ >DCP ≥RESV≥ ETX> NS1619> MTZ≥ QUERC; HCT was not effective. Cell viability after 24 h of incubation under hyperkalemia was enhanced by 82+6% and 33+7% in hslo-HEK293 cells and HEK293 cells, respectively. IbTx, ChTX and TEA enhanced cell viability in hslo-HEK293. BK openers prevented the enhancement of the cell viability induced by hyperkalemia or IbTx in hslo-HEK293 showing an efficacy which was comparable with that observed as BK openers. BK channel modulators failed to affect cell currents and viability under hyperkalemia conditions in the absence of hslo subunit. In contrast, under hypokalemia cell viability was reduced by -22+4% and -23+6% in hslo-HEK293 and HEK293 cells, respectively; the BK channel modulators failed to affect this parameter in these cells. In conclusion, BK channel regulates cell viability under hyperkalemia but not hypokalemia conditions. BFT and ACTZ were the most potent drugs either in activating the BK current and in preventing the cell proliferation induced by hyperkalemia. These findings may have relevance in disorders associated with abnormal K(+) ion homeostasis including periodic paralysis and myotonia.

Voltage-gated ion channels are important mediators of physiological functions in the central nervous system. The cyclic activation of these channels influences neurotransmitter release, neuron excitability, gene transcription and plasticity, providing distinct brain areas with unique physiological and pharmacological response. A growing body of data has implicated ion channels in the susceptibility or pathogenesis of psychiatric diseases. Indeed, population studies support the association of polymorphisms in calcium and potassium channels with the genetic risk for bipolar disorders or schizophrenia. Moreover, point mutations in calcium, sodium and potassium channel genes have been identified in some childhood developmental disorders. Finally, antibodies against potassium channel complexes occur in a series of autoimmune psychiatric diseases. Here we report recent studies assessing the role of calcium, sodium and potassium channels in bipolar disorder, schizophrenia and autism spectrum disorders, and briefly summarize promising pharmacological strategies targeted on ion channels for the therapy of mental illness and related genetic tests.

The pharmacological action of taurine on skeletal muscle is to stabilize sarcolemma by increasing macroscopic conductance to Cl- (GCl), whereas a proposed physiological role for the amino acid is to modulate excitation-contraction coupling mechanism via Ca2+ availability. To get insight in the physiological role of taurine in skeletal muscle, the effects of its depletion were evaluated on voltage threshold for mechanical activation and GCl with the two intracellular microelectrode method in 'point' voltage clamp mode and current clamp mode, respectively. The experiments were performed on extensor digitorum longus muscle fibers from rats depleted of taurine by a chronic 4 week treatment with guanidinoethane sulfonate, a known inhibitor of taurine transporter. The treatment significantly modified the mechanical threshold of striated fibers; i.e. at each pulse duration they needed significantly less depolarization to contract and the fitted rheobase voltage was more negative by 10 mV with respect to untreated muscle fibers. In parallel, the treatment with guanidinoethane sulfonate produced a significant 40% lowering of GCl. In vitro application of 60 mM of taurine to such depleted muscles almost completely restored the mechanical threshold and increased GCl even above the value of untreated control. However, in vitro application of 60 mM of either taurine or guanidinoethane sulfonate to untreated control muscles did not cause any change of the mechanical threshold but increased GCl by 40% and 21%, respectively. Furthermore, 100 microM of the S-(-) enantiomer of 2-(p-chlorophenoxy)propionic acid almost fully blocked GCl but did not produce any change in the mechanical threshold of normal muscle fibers. The present results show that the large amount of intracellular taurine plays a role in the excitation-contraction coupling mechanism of striated muscle fibers. This action is independent from any effect involving muscle Cl- channels, but it is likely mediated by the proposed ability of taurine to modulate Ca2+ availability through the interaction with the Ca2+ transporters present on sarcoplasmic reticulum.

Lubeluzole, which acts on various targets in vitro, including voltage-gated sodium channels, was initially proposed as a neuroprotectant. The lubeluzole structure contains a benzothiazole moiety [N-methyl-1,3-benzothiazole-2-amine (R-like)] related to riluzole and a phenoxy-propranol-amine moiety [(RS)-1-(3,4-difluorophenoxy)-3-(piperidin-1-yl)propan-2-ol (A-core)] recalling propranolol. Both riluzole and propranolol are efficient sodium channel blockers. We studied in detail the effects of lubeluzole (racemic mixture and single isomers), the aforementioned lubeluzole moieties, and riluzole on sodium channels to increase our knowledge of drug-channel molecular interactions. Compounds were tested on hNav1.4 sodium channels, and on F1586C or Y1593C mutants functionally expressed in human embryonic kidney 293 cells, using the patch-clamp technique. Lubeluzole blocked sodium channels with a remarkable effectiveness. No stereoselectivity was found. Compared with mexiletine, the dissociation constant for inactivated channels was ~600 times lower (~11 nM), conferring to lubeluzole a huge use-dependence of great therapeutic value. The F1586C mutation only partially impaired the use-dependent block, suggesting that additional amino acids are critically involved in high-affinity binding. Lubeluzole moieties were modest sodium channel blockers. Riluzole blocked sodium channels efficiently but lacked use dependence, similar to R-like. F1586C fully abolished A-core use dependence, suggesting that A-core binds to the local anesthetic receptor. Thus, lubeluzole likely binds to the local anesthetic receptor through its phenoxy-propranol-amine moiety, with consequent use-dependent behavior. Nevertheless, compared with other known sodium channel blockers, lubeluzole adds a third pharmacophoric point through its benzothiazole moiety, which greatly enhances high-affinity binding and use-dependent block. If sufficient isoform specificity can be attained, the huge use-dependent block may help in the development of new sodium channel inhibitors to provide pharmacotherapy for membrane excitability disorders, such as myotonia, epilepsy, or chronic pain.