113 research outputs found
Ion Channels Involvement in Neurodevelopmental Disorders
Inherited and sporadic mutations in genes encoding for brain ion channels, affecting membrane expression or biophysical properties, have been associated with neurodevelopmental disorders characterized by epilepsy, cognitive and behavioral deficits with significant phenotypic and genetic heterogeneity. Over the years, the screening of a growing number of patients and the functional characterization of newly identified mutations in ion channels genes allowed to recognize new phenotypes and to widen the clinical spectrum of known diseases. Furthermore, advancements in understanding disease pathogenesis at atomic level or using patient-derived iPSCs and animal models have been pivotal to orient therapeutic intervention and to put the basis for the development of novel pharmacological options for drug-resistant disorders. In this review we will discuss major improvements and critical issues concerning neurodevelopmental disorders caused by dysfunctions in brain sodium, potassium, calcium, chloride and ligand-gated ion channels
I-J loop involvement in the pharmacological profile of CLC-K channels expressed in Xenopus oocytes
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~1μM. These residues are localized in the same extracellular I-J loop which harbors a regulatory Ca(2+) 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
Fluvastatin and atorvastatin affect calcium homeostasis of rat skeletal muscle fibers in vivo and in vitro by impairing the sarcoplasmic reticulum/mitochondria Ca2+-release system
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 drug
Calcium homeostasis is altered in skeletal muscle of spontaneously hypertensive rats cytofluorimetric and gene expression analysis
Hypertension is often associated with skeletal muscle pathological conditions related to function and metabolism. The mechanisms underlying the development of these pathological conditions remain undefined. Because calcium homeostasis is a biomarker of muscle function, we assessed whether it is altered in hypertensive muscles. We measured resting intracellular calcium and store-operated calcium entry (SOCE) in fast- and slow-twitch muscle fibers from normotensive Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs) by cytofluorimetric technique and determined the expression of SOCE gene machinery by real-time PCR. Hypertension caused a phenotype-dependent dysregulation of calcium homeostasis; the resting intracellular calcium of extensor digitorum longus and soleus muscles of SHRs were differently altered with respect to the related muscle of normotensive animals. In addition, soleus muscles of SHR showed reduced activity of the sarcoplasmic reticulum and decreased sarcolemmal calcium permeability at rest and after SOCE activation. Accordingly, we found an alteration of the expression levels of some SOCE components, such as stromal interaction molecule 1, calcium release-activated calcium modulator 1, and transient receptor potential canonical 1. The hypertension-induced alterations of calcium homeostasis in the soleus muscle of SHRs occurred with changes of some functional outcomes as excitability and resting chloride conductance. We provide suitable targets for therapeutic interventions aimed at counterbalancing muscle performance decline in hypertension, and propose the reported calcium-dependent parameters as indexes to predict how the antihypertensive drugs could influence muscle function
Growth hormone secretagogues exert differential effects on skeletal muscle calcium homeostasis in male rats depending on the peptidyl/non-peptidyl structure
TheorexigenicandanaboliceffectsinducedbyghrelinandthesyntheticGHsecretagogues(GHSs)
are thought to positively contribute to therapeutic approaches and the adjunct treatment of a
number of diseases associated with muscle wasting such as cachexia and sarcopenia. However,
manyquestionsaboutthepotentialutilityandsafetyofGHSsinboththerapyandskeletalmuscle
functionremainunanswered.Byusingfura-2cytofluorimetrictechnique,wedeterminedtheacute
effectsofghrelin,aswellasofpeptidylandnonpeptidylsyntheticGHSsoncalciumhomeostasis,
a critical biomarker of muscle function, in isolated tendon-to-tendon male rat skeletal muscle
fibers.ThesyntheticnonpeptidylGHSs,butnotpeptidylghrelinandhexarelin,wereabletosignificantlyincreaserestingcytosoliccalcium[Ca2]i.ThenonpeptidylGHS-induced[Ca2]
iincrease
was independent of GHS-receptor 1a but was antagonized by both thapsigargin/caffeine and
cyclosporineA,indicatingtheinvolvementofthesarcoplasmicreticulumandmitochondria.EvaluationoftheeffectsofapseudopeptidylGHSandanonpeptidylantagonistoftheGHS-receptor
1a together with a drug-modeling study suggest the conclusion that the lipophilic nonpeptidyl
structureofthetestedcompoundsisthekeychemicalfeaturecrucialfortheGHS-inducedcalcium
alterationsintheskeletalmuscle.Thus,syntheticGHSscanhavedifferenteffectsonskeletalmuscle
fibersdependingontheirmolecularstructures.Thecalciumhomeostasisdysregulationspecifically
induced by the nonpeptidyl GHSs used in this study could potentially counteract the beneficial
effects associated with these drugs in the treatment of muscle wasting of cachexia- or other
age-related disorders
ATP Sensitive Potassium Channels in the Skeletal Muscle Function: Involvement of the KCNJ11(Kir6.2) Gene in the Determination of Mechanical Warner Bratzer Shear Force
The ATP-sensitive K-channels (KATP) are distributed in the tissues coupling metabolism with K ions efflux. KATP subunits are encoded by KCNJ8 (Kir6.1), KCNJ11 (Kir6.2), ABCC8 (SUR1), and ABCC9 (SUR2) genes, alternative RNA splicing give rise to SUR variants that confer distinct physiological properties on the channel. An high expression/activity of the sarco-KATP channel is observed in various rat fast-twitch muscles, characterized by elevated muscle strength, while a low expression/activity is observed in the slow twitch muscles characterized by reduced strength and frailty. Down-regulation of the KATP subunits of fast-twitch fibers is found in conditions characterized by weakness and frailty. KCNJ11 gene knockout mice have reduced glycogen, lean phenotype, lower body fat, and weakness. KATP channel is also a sensor of muscle atrophy. The KCNJ11 gene is located on BTA15, close to a QTL for meat tenderness, it has also a role in glycogen storage, a key mechanism of the postmortem transformation of muscle into meat. The role of KCNJ11 gene in muscle function may underlie an effect of KCNJ11 genotypes on meat tenderness, as recently reported. The fiber phenotype and genotype are important in livestock production science. Quantitative traits including meat production and quality are influenced both by environment and genes. Molecular markers can play an important role in the genetic improvement of animals through breeding strategies. Many factors influence the muscle Warner-Bratzler shear force including breed, age, feeding, the biochemical, and functional parameters. The role of KCNJ11gene and related genes on muscle tenderness will be discussed in the present review
Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ
In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process
A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels
The two human CLC Cl− channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel β subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca2+ and H+ on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca2+]ext without full saturation up to 50 mM. However, in the absence of Ca2+, ClC-Ka currents were still 20% of currents in 10 mM [Ca2+]ext, demonstrating that Ca2+ is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H+]ext with a practically complete block at pH 6. Ca2+ and H+ act as gating modifiers without changing the single-channel conductance. Dose–response analysis suggested that two protons are necessary to induce block with an apparent pK of ∼7.1. A simple four-state allosteric model described the modulation by Ca2+ assuming a 13-fold higher Ca2+ affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca2+ and H+
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