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Introduction: CACNA1S related congenital myopathy is an emerging recently described entity. In this report we describe 2 sisters with mutations in the CACNA1S gene and the novel phenotype of congenital myopathy and infantile onset episodic weakness. Clinical description: Both sisters had neonatal onset hypotonia, muscle weakness, and delayed walking. Episodic weakness started in infancy and continued thereafter, provoked mostly by cold exposure. Muscle imaging revealed fat replacement of gluteus maximus muscles. Next generation sequencing found themissense p. Cys944Tyr variant and the novel splicing variant c.3526-2A>G in CACNA1S. Minigene assay revealed the splicing variant caused skipping of exon 28 from the transcript, potentially affecting protein folding and/or voltage dependent activation. Conclusion: This novel phenotype supports the notion that there are age related diferences in the clinical expression of CACNA1S gene mutations. This expands our understanding of mutations located in regions of the CACNA1S outside the highly conserved S4 segment, where most mutations thus far have been identified. [ABSTRACT FROM AUTHOR]

Abstract Background The CACNA1S gene encodes the alpha 1 S-subunit of the voltage-gated calcium channel, which is primarily expressed in the skeletal muscle cells. Pathogenic variants of CACNA1S can cause hypokalemic periodic paralysis (HypoPP), malignant hyperthermia susceptibility, and congenital myopathy. We aimed to study the clinical and molecular features of a male child with a CACNA1S variant and depict the molecular sub-regional characteristics of different phenotypes associated with CACNA1S variants. Case presentation We presented a case of HypoPP with recurrent muscle weakness and hypokalemia. Genetic analyses of the family members revealed that the proband had a novel c.497 C > A (p.Ala166Asp) variant of CACNA1S, which was inherited from his father. The diagnosis of HypoPP was established in the proband as he met the consensus diagnostic criteria. The patient and his parents were informed to avoid the classical triggers of HypoPP. The attacks of the patient are prevented by lifestyle changes and nutritional counseling. We also showed the molecular sub-regional location of the variants of CACNA1S which was associated with different phenotypes. Conclusions Our results identified a new variant of CACNA1S and expanded the spectrum of variants associated with HypoPP. Early genetic diagnosis can help avoid diagnostic delays, perform genetic counseling, provide proper treatment, and reduce morbidity and mortality.

IntroductionCACNA1S related congenital myopathy is an emerging recently described entity. In this report we describe 2 sisters with mutations in the CACNA1S gene and the novel phenotype of congenital myopathy and infantile onset episodic weakness.Clinical descriptionBoth sisters had neonatal onset hypotonia, muscle weakness, and delayed walking. Episodic weakness started in infancy and continued thereafter, provoked mostly by cold exposure. Muscle imaging revealed fat replacement of gluteus maximus muscles. Next generation sequencing found the missense p.Cys944Tyr variant and the novel splicing variant c.3526-2A>G in CACNA1S. Minigene assay revealed the splicing variant caused skipping of exon 28 from the transcript, potentially affecting protein folding and/or voltage dependent activation.ConclusionThis novel phenotype supports the notion that there are age related differences in the clinical expression of CACNA1S gene mutations. This expands our understanding of mutations located in regions of the CACNA1S outside the highly conserved S4 segment, where most mutations thus far have been identified.

Background: The CACNA1S gene encodes the alpha 1 S-subunit of the voltage-gated calcium channel, which is primarily expressed in the skeletal muscle cells. Pathogenic variants of CACNA1S can cause hypokalemic periodic paralysis (HypoPP), malignant hyperthermia susceptibility, and congenital myopathy. We aimed to study the clinical and molecular features of a male child with a CACNA1S variant and depict the molecular sub-regional characteristics of different phenotypes associated with CACNA1S variants. Case presentation: We presented a case of HypoPP with recurrent muscle weakness and hypokalemia. Genetic analyses of the family members revealed that the proband had a novel c.497 C > A (p.Ala166Asp) variant of CACNA1S, which was inherited from his father. The diagnosis of HypoPP was established in the proband as he met the consensus diagnostic criteria. The patient and his parents were informed to avoid the classical triggers of HypoPP. The attacks of the patient are prevented by lifestyle changes and nutritional counseling. We also showed the molecular sub-regional location of the variants of CACNA1S which was associated with different phenotypes. Conclusions: Our results identified a new variant of CACNA1S and expanded the spectrum of variants associated with HypoPP. Early genetic diagnosis can help avoid diagnostic delays, perform genetic counseling, provide proper treatment, and reduce morbidity and mortality. [ABSTRACT FROM AUTHOR]

ABSTRACTThe CaV1.1 voltage-gated Ca2+ channel carries L-type Ca2+ current and is the voltage-sensor for excitation-contraction (EC) coupling in skeletal muscle. Significant breakthroughs in the EC coupling field have often been close on the heels of technological advancement. In particular, CaV1.1 was the first voltage-gated Ca2+ channel to be cloned, the first ion channel to have its gating current measured and the first ion channel to have an effectively null animal model. Though these innovations have provided invaluable information regarding how CaV1.1 detects changes in membrane potential and transmits intra- and inter-molecular signals which cause opening of the channel pore and support Ca2+ release from the sarcoplasmic reticulum remain elusive. Here, we review current perspectives on this topic including the recent application of functional site-directed fluorometry.

S100A1, a calcium-binding protein, plays a crucial role in regulating Ca2+ signaling pathways in skeletal and cardiac myocytes via interactions with the ryanodine receptor (RyR) to affect Ca2+ release and contractile performance. Biophysical studies strongly suggest that S100A1 interacts with RyRs but have been inconclusive about both the nature of this interaction and its competition with another important calcium-binding protein, calmodulin (CaM). Thus, high-resolution cryo-EM studies of RyRs in the presence of S100A1, with or without additional CaM, were needed. The elegant work by Weninger et al. demonstrates the interaction between S100A1 and RyR1 through various experiments and confirms that S100A1 activates RyR1 at sub-micromolar Ca2+ concentrations, increasing the open probability of RyR1 channels. [Display omitted] [ABSTRACT FROM AUTHOR]

The skeletal muscle L-type Ca2+ channel (CaV1.1) works primarily as a voltage sensor for skeletal muscle action potential (AP)-evoked Ca2+ release. CaV1.1 contains four distinct voltage-sensing domains (VSDs), yet the contribution of each VSD to AP-evoked Ca2+ release remains unknown. To investigate the role of VSDs in excitation– contraction coupling (ECC), we encoded cysteine substitutions on each S4 voltage-sensing segment of CaV1.1, expressed each construct via in vivo gene transfer electroporation, and used in cellulo AP fluorometry to track the movement of each CaV1.1 VSD in skeletal muscle fibers. We first provide electrical measurements of CaV1.1 voltage sensor charge movement in response to an AP waveform. Then we characterize the fluorescently labeled channels’ VSD fluorescence signal responses to an AP and compare them with the waveforms of the electrically measured charge movement, the optically measured free myoplasmic Ca2+, and the calculated rate of Ca2+ release from the sarcoplasmic reticulum for an AP, the physiological signal for skeletal muscle fiber activation. A considerable fraction of the fluorescence signal for each VSD occurred after the time of peak Ca2+ release, and even more occurred after the earlier peak of electrically measured charge movement during an AP, and thus could not directly reflect activation of Ca2+ release or charge movement, respectively. However, a sizable fraction of the fluorometric signals for VSDs I, II, and IV, but not VSDIII, overlap the rising phase of charge moved, and even more for Ca2+ release, and thus could be involved in voltage sensor rearrangements or Ca2+ release activation. [ABSTRACT FROM AUTHOR]

Voltage-gated calcium channels control key functions of excitable cells, like synaptic transmission in neurons and the contraction of heart and skeletal muscles. To accomplish such diverse functions, different calcium channels activate at different voltages and with distinct kinetics. To identify the molecular mechanisms governing specific voltage sensing properties, we combined structure modeling, mutagenesis, and electrophysiology to analyze the structures, free energy, and transition kinetics of the activated and resting states of two functionally distinct voltage sensing domains (VSDs) of the eukaryotic calcium channel CaV1.1. Both VSDs displayed the typical features of the sliding helix model; however, they greatly differed in ion-pair formation of the outer gating charges. Specifically, stabilization of the activated state enhanced the voltage dependence of activation, while stabilization of resting states slowed the kinetics. This mechanism provides a mechanistic model explaining how specific ion-pair formation in separate VSDs can realize the characteristic gating properties of voltage-gated cation channels.

Abstract Background Human induced pluripotent stem cells-derived myogenic progenitors develop functional and ultrastructural features typical of skeletal muscle when differentiated in culture. Besides disease-modeling, such a system can be used to clarify basic aspects of human skeletal muscle development. In the present study, we focus on the development of the excitation-contraction (E-C) coupling, a process that is essential both in muscle physiology and as a tool to differentiate between the skeletal and cardiac muscle. The occurrence and maturation of E-C coupling structures (Sarcoplasmic Reticulum-Transverse Tubule (SR-TT) junctions), key molecular components, and Ca2+ signaling were examined, along with myofibrillogenesis. Methods Pax7+-myogenic progenitors were differentiated in culture, and developmental changes were examined from a few days up to several weeks. Ion channels directly involved in the skeletal muscle E-C coupling (RyR1 and Cav1.1 voltage-gated Ca2+ channels) were labeled using indirect immunofluorescence. Ultrastructural changes of differentiating cells were visualized by transmission electron microscopy. On the functional side, depolarization-induced intracellular Ca2+ transients mediating E-C coupling were recorded using Fura-2 ratiometric Ca2+ imaging, and myocyte contraction was captured by digital photomicrography. Results We show that the E-C coupling machinery occurs and operates within a few days post-differentiation, as soon as the myofilaments align. However, Ca2+ transients become effective in triggering myocyte contraction after 1 week of differentiation, when nascent myofibrils show alternate A-I bands. At later stages, myofibrils become fully organized into adult-like sarcomeres but SR-TT junctions do not reach their triadic structure and typical A-I location. This is mirrored by the absence of cross-striated distribution pattern of both RyR1 and Cav1.1 channels. Conclusions The E-C coupling machinery occurs and operates within the first week of muscle cells differentiation. However, while early development of SR-TT junctions is coordinated with that of nascent myofibrils, their respective maturation is not. Formation of typical triads requires other factors/conditions, and this should be taken into account when using in-vitro models to explore skeletal muscle diseases, especially those affecting E-C coupling.

Loss of total muscle force during aging has both atrophic and non-atrophic components. The former deficit is a direct consequence of reduced muscle mass while the latter has been attributed to a depression of excitation-contraction (EC) coupling. It is well established that age-onset reductions in sex hormone production regulate the atrophic component in both males and females. However, it is unknown whether the non-atrophic component is influenced by sex hormones. Since the non-atrophic component has been linked mechanistically to reduced expression of the skeletal muscle L-type Ca2+ channel (Ca V 1.1), we recorded L-type Ca2+ currents, gating charge movements and depolarization-induced changes in myoplasmic Ca2+ from flexor digitorum brevis (FDB) fibers of naïve and gonadectomized mice of both sexes. Our first set of experiments sought to identify any basal differences in EC coupling or L-type Ca2+ flux between the sexes; no detectable differences in any of the aforementioned parameters were observed between FDB harvested from either naïve males or females. In the latter segments of the study, ovariectomy (OVX) and orchiectomy (ORX) models were used to assess the possible influence of sex hormones on EC coupling and/or L-type Ca2+ flux. In these experiments, FDB fibers harvested from OVX and ORX mice both showed no differences in L-type Ca2+ current, gating charge movement or depolarization-induced changes in Ca2+ release from the sarcoplasmic reticulum. Taken together, our results indicate L-type Ca2+ channel function and EC coupling are: 1) equivalent between the sexes, and 2) not significantly regulated by sex hormones. Since recent NIH review guidelines mandate the consideration of sex differences as a criterion for review, our work indicates the suitability of either sex for the study of the fundamental mechanisms of EC coupling. Thus, our findings may accelerate the research process by conserving animals, labor and financial resources. • Sex differences in skeletal excitation-contraction coupling were investigated. • No differences were observed in L-current or Ca2+ release within males and females. • L-channel function and EC coupling were unaffected by ovariectomy or orchiectomy. • Our results indicate the suitability of either sex for the study of EC coupling. • Our findings may accelerate research by conserving animals and other resources. [ABSTRACT FROM AUTHOR]

Excitation-contraction coupling (ECC) is a physiological process that links excitation of muscles by the nervous system to their mechanical contraction. In skeletal muscle, ECC is initiated with an action potential, generated by the somatic nervous system, which causes a depolarisation of the muscle fibre membrane (sarcolemma). This leads to a rapid change in the transmembrane potential, which is detected by the voltage-gated Ca2+ channel dihydropyridine receptor (DHPR) embedded in the sarcolemma. DHPR transmits the contractile signal to another Ca2+ channel, ryanodine receptor (RyR1), embedded in the membrane of the sarcoplasmic reticulum (SR), which releases a large amount of Ca2+ ions from the SR that initiate muscle contraction. Despite the fundamental role of ECC in skeletal muscle function of all vertebrate species, the molecular mechanism underpinning the communication between the two key proteins involved in the process (DHPR and RyR1) is still largely unknown. The goal of this work is to review the recent progress in our understanding of ECC in skeletal muscle from the point of view of the structure and interactions of proteins involved in the process, and to highlight the unanswered questions in the field. [ABSTRACT FROM AUTHOR]

CACNA1S-related myopathy, due to pathogenic variants in the CACNA1S gene, is a recently described congenital muscle disease. Disease associated variants result in loss of gene expression and/or reduction of Cav1.1 protein stability. There is an incomplete understanding of the underlying disease pathomechanisms and no effective therapies are currently available. A barrier to the study of this myopathy is the lack of a suitable animal model that phenocopies key aspects of the disease. To address this barrier, we generated knockouts of the two zebrafish CACNA1S paralogs, cacna1sa and cacna1sb. Double knockout fish exhibit severe weakness and early death, and are characterized by the absence of Cav1.1 α1 subunit expression, abnormal triad structure, and impaired excitation-contraction coupling, thus mirroring the severe form of human CACNA1S-related myopathy. A double mutant (cacna1sa homozygous, cacna1sb heterozygote) exhibits normal development, but displays reduced body size, abnormal facial structure, and cores on muscle pathologic examination, thus phenocopying the mild form of human CACNA1S-related myopathy. In summary, we generated and characterized the first cacna1s zebrafish loss-of-function mutants, and show them to be faithful models of severe and mild forms of human CACNA1S-related myopathy suitable for future mechanistic studies and therapy development., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Abstract Excitation-contraction coupling is the signaling process by which action potentials control calcium release and consequently the force of muscle contraction. Until recently, three triad proteins were known to be essential for skeletal muscle EC coupling: the voltage-gated calcium channel Ca V 1.1 acting as voltage sensor, the SR calcium release channel RyR1 representing the only relevant calcium source, and the auxiliary Ca V β 1a subunit. Whether Ca V 1.1 and RyR1 are directly coupled or whether their interaction is mediated by another triad protein is still unknown. The recent identification of the adaptor protein STAC3 as fourth essential component of skeletal muscle EC coupling prompted vigorous research to reveal its role in this signaling process. Accumulating evidence supports its possible involvement in linking Ca V 1.1 and RyR1 in skeletal muscle EC coupling, but also indicates a second, much broader role of STAC proteins in the regulation of calcium/calmodulin-dependent feedback regulation of L-type calcium channels. Graphical abstract Unlabelled Image Highlights • Skeletal muscle EC coupling converts an electrical signal into contraction. • The classical essential EC coupling proteins are Ca V 1.1, β 1a , and RyR1. • Recently STAC3 has been identified as fourth essential EC coupling protein. • In skeletal muscle STAC3 may couple Ca V 1.1 with RyR1 calcium release. • In other tissues STAC proteins modulate L-type calcium channel inactivation. [ABSTRACT FROM AUTHOR]

The Ca V 1.1 voltage-gated Ca 2+ channel carries L-type Ca 2+ current and is the voltage-sensor for excitation-contraction (EC) coupling in skeletal muscle. Significant breakthroughs in the EC coupling field have often been close on the heels of technological advancement. In particular, Ca V 1.1 was the first voltage-gated Ca 2+ channel to be cloned, the first ion channel to have its gating current measured and the first ion channel to have an effectively null animal model. Though these innovations have provided invaluable information regarding how Ca V 1.1 detects changes in membrane potential and transmits intra- and inter-molecular signals which cause opening of the channel pore and support Ca 2+ release from the sarcoplasmic reticulum remain elusive. Here, we review current perspectives on this topic including the recent application of functional site-directed fluorometry.

Background: Human induced pluripotent stem cells-derived myogenic progenitors develop functional and ultrastructural features typical of skeletal muscle when differentiated in culture. Besides disease-modeling, such a system can be used to clarify basic aspects of human skeletal muscle development. In the present study, we focus on the development of the excitation-contraction (E-C) coupling, a process that is essential both in muscle physiology and as a tool to differentiate between the skeletal and cardiac muscle. The occurrence and maturation of E-C coupling structures (Sarcoplasmic Reticulum-Transverse Tubule (SR-TT) junctions), key molecular components, and Ca2+ signaling were examined, along with myofibrillogenesis. Methods: Pax7+-myogenic progenitors were differentiated in culture, and developmental changes were examined from a few days up to several weeks. Ion channels directly involved in the skeletal muscle E-C coupling (RyR1 and Cav1.1 voltage-gated Ca2+ channels) were labeled using indirect immunofluorescence. Ultrastructural changes of differentiating cells were visualized by transmission electron microscopy. On the functional side, depolarization-induced intracellular Ca2+ transients mediating E-C coupling were recorded using Fura-2 ratiometric Ca2+ imaging, and myocyte contraction was captured by digital photomicrography. Results: We show that the E-C coupling machinery occurs and operates within a few days postdifferentiation, as soon as the myofilaments align. However, Ca2+ transients become effective in triggering myocyte contraction after 1 week of differentiation, when nascent myofibrils show alternate A-I bands. At later stages, myofibrils become fully organized into adult-like sarcomeres but SR-TT junctions do not reach their triadic structure and typical A-I location. This is mirrored by the absence of cross-striated distribution pattern of both RyR1 and Cav1.1 channels. Conclusions: The E-C coupling machinery occurs and operates within the first week of muscle cells differentiation. However, while early development of SR-TT junctions is coordinated with that of nascent myofibrils, their respective maturation is not. Formation of typical triads requires other factors/conditions, and this should be taken into account when using in-vitro models to explore skeletal muscle diseases, especially those affecting E-C coupling. [ABSTRACT FROM AUTHOR]

Skeletal muscle contraction is triggered by Ca2+ release from the sarcoplasmic reticulum (SR) in response to plasma membrane (PM) excitation. In vertebrates, this depends on activation of the RyR1 Ca2+ pore in the SR, under control of conformational changes of Cav1.1, located ~12 nm away in the PM. Over the last ~30 y, gene knockouts have revealed that Cav1.1/RyR1 coupling requires additional proteins, but leave open the possibility that currently untested proteins are also necessary. Here, we demonstrate the reconstitution of conformational coupling in tsA201 cells by expression of Cav1.1, β1a, Stac3, RyR1, and junctophilin2. As in muscle, depolarization evokes Ca2+ transients independent of external Ca2+ entry and having amplitude with a saturating dependence on voltage. Moreover, freeze-fracture electron microscopy indicates that the five identified proteins are sufficient to establish physical links between Cav1.1 and RyR1. Thus, these proteins constitute the key elements essential for excitation--contraction coupling in skeletal muscle. [ABSTRACT FROM AUTHOR]

Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation-contraction coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca2+ transients without affecting membrane integrity or causing sarcoplasmic reticulum calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not sarcoplasmic reticulum membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na+/K+-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX) activity and protein content of NOX2 subunits (p47phox and gp91phox), implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs excitation-contraction coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged animals.

Background: Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within 3-5 years of diagnosis. While motoneuron death is the defining characteristic of ALS, the events that underlie its pathology are not restricted to the nervous system. In this regard, ALS muscle atrophies and weakens significantly before presentation of neurological symptoms. Since the skeletal muscle L-type Ca2+ channel (CaV1.1) is a key regulator of both mass and force, we investigated whether CaV1.1 function is impaired in the muscle of two distinct mouse models carrying an ALS-linked mutation. Methods: We recorded L-type currents, charge movements, and myoplasmic Ca2+ transients from dissociated flexor digitorum brevis (FDB) fibers to assess CaV1.1 function in two mouse models expressing a type 1 Cu/Zn superoxide dismutase mutant (SOD1G93A). Results: In FDB fibers obtained from "symptomatic" global SOD1G93A mice, we observed a substantial reduction of SR Ca2+ release in response to depolarization relative to fibers harvested from age-matched control mice. L-type current and charge movement were both reduced by ~40 % in symptomatic SOD1G93A fibers when compared to control fibers. Ca2+ transients were not significantly reduced in similar experiments performed with FDB fibers obtained from "early-symptomatic" SOD1G93A mice, but L-type current and charge movement were decreased (~30 and ~20 %, respectively). Reductions in SR Ca2+ release (~35 %), L-type current (~20 %), and charge movement (~15 %) were also observed in fibers obtained from another model where SOD1G93A expression was restricted to skeletal muscle. Conclusions: We report reductions in EC coupling, L-type current density, and charge movement in FDB fibers obtained from symptomatic global SOD1G93A mice. Experiments performed with FDB fibers obtained from early-symptomatic SOD1G93A and skeletal muscle autonomous MLC/ SOD1G93A mice support the idea that events occurring locally in the skeletal muscle contribute to the impairment of CaV1.1 function in ALS muscle independently of innervation status. [ABSTRACT FROM AUTHOR]

In skeletal muscle, excitation-contraction (EC) coupling relies on the transmission of an intermolecular signal from the voltage-sensing regions of the L-type Ca2+ channel (CaV1.1) in the plasma membrane to the channel pore of the type 1 ryanodine receptor (RyR1) nearly 10 nm away in the membrane of the sarcoplasmic reticulum (SR). Even though the roles of CaV1.1 and RyR1 as voltage sensor and SR Ca2+ release channel, respectively, have been established for nearly 25 years, the mechanism underlying communication between these two channels remains undefined. In the course of this article, I will review current viewpoints on this topic with particular emphasis on recent studies. [ABSTRACT FROM AUTHOR]

Background and purpose: CACNA1S encodes Cav1.1, a voltage sensor for muscle excitation−contraction coupling, which activates the ryanodine receptor 1 (RYR1) leading to calcium release from the sarcoplasmic reticulum. CACNA1S mutations cause hypokalemic periodic paralysis, malignant hyperthermia and congenital myopathy. RYR1 mutations result in congenital myopathy, malignant hyperthermia and rhabdomyolysis. Methods: The aim was to describe a novel phenotype associated with a CACNA1S variant at a site previously linked to hypokalemic periodic paralysis. Results: The patient presented with fluctuating asymptomatic creatine kinase elevation after an episode of rhabdomyolysis but has no history of periodic paralysis. His muscle biopsy showed core‐like structures occurring mainly in type 2 fibers. He carries a novel Cav1.1 variant (p.Arg528Leu) affecting a highly conserved amino acid. Different mutations at the same location cause hypokalemic periodic paralysis. Conclusion: This case underscores the similarity between the phenotypes caused by mutations in two functionally linked proteins, RYR1 and Cav1.1. [ABSTRACT FROM AUTHOR]

Drug-drug interaction of the antiviral sofosbuvir and the antiarrhythmics amiodarone has been reported to cause fatal heartbeat slowing. Sofosbuvir and its analog, MNI-1, were reported to potentiate the inhibition of cardiomyocyte calcium handling by amiodarone, which functions as a multi-channel antagonist, and implicate its inhibitory effect on L-type Ca v channels, but the molecular mechanism has remained unclear. Here we present systematic cryo-EM structural analysis of Ca v 1.1 and Ca v 1.3 treated with amiodarone or sofosbuvir alone, or sofosbuvir/MNI-1 combined with amiodarone. Whereas amiodarone alone occupies the dihydropyridine binding site, sofosbuvir is not found in the channel when applied on its own. In the presence of amiodarone, sofosbuvir/MNI-1 is anchored in the central cavity of the pore domain through specific interaction with amiodarone and directly obstructs the ion permeation path. Our study reveals the molecular basis for the physical, pharmacodynamic interaction of two drugs on the scaffold of Ca v channels. [Display omitted] • Antiarrhythmic agent amiodarone (AMIO) binds to the III-IV fenestration of LTCCs • Sofosbuvir (SOF), applied alone, is not observed in the EM structure of Ca v 1.3 • Synergistic inhibition of LTCCs by AMIO and SOF (or its analogue MNI-1) • AMIO anchors SOF/MNI-1 to the central cavity of LTCCs to block ion conduction Co-administration of sofosbuvir and amiodarone can lead to fatal heartbeat slowing, yet the underlying mechanism has not been clear. Analyzing six cryo-EM structures of Ca v 1.1 and Ca v 1.3 in the presence of these drugs reveals their direct interaction within the channels and explains this clinical observation. [ABSTRACT FROM AUTHOR]

Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation-contraction (E-C) coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-b-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca2+ transients without affecting membrane integrity or causing sarcoplasmic reticulum (SR) calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain 10% attached transverse tubules, but not SR membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na+/K+-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX) activity and protein content of NOX2 subunits (p47phox and gp91phox), implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs E-C coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged animals. [ABSTRACT FROM AUTHOR]

Background: Ca2+ influx through CaV1.1 is not required for skeletal muscle excitation-contraction coupling, but whether Ca2+ permeation through CaV1.1 during sustained muscle activity plays a functional role in mammalian skeletal muscle has not been assessed. Methods: We generated a mouse with a Ca2+ binding and/or permeation defect in the voltage-dependent Ca2+ channel, CaV1.1, and used Ca2+ imaging, western blotting, immunohistochemistry, proximity ligation assays, SUnSET analysis of protein synthesis, and Ca2+ imaging techniques to define pathways modulated by Ca2+ binding and/or permeation of CaV1.1. We also assessed fiber type distributions, cross-sectional area, and force frequency and fatigue in isolated muscles. Results: Using mice with a pore mutation in CaV1.1 required for Ca2+ binding and/or permeation (E1014K, EK), we demonstrate that CaV1.1 opening is coupled to CaMKII activation and refilling of sarcoplasmic reticulum Ca2+ stores during sustained activity. Decreases in these Ca2+-dependent enzyme activities alter downstream signaling pathways (Ras/Erk/mTORC1) that lead to decreased muscle protein synthesis. The physiological consequences of the permeation and/or Ca2+ binding defect in CaV1.1 are increased fatigue, decreased fiber size, and increased Type IIb fibers. Conclusions: While not essential for excitation-contraction coupling, Ca2+ binding and/or permeation via the CaV1.1 pore plays an important modulatory role in muscle performance. [ABSTRACT FROM AUTHOR]

Aims The pathogenesis of myotonic dystrophy type 1 ( DM1) and type 2 ( DM2) has been related to the aberrant splicing of several genes, including those encoding for ryanodine receptor 1 ( RYR1), sarcoplasmatic/endoplasmatic Ca2+- ATPase ( SERCA) and α1S subunit of voltage-gated Ca2+ channels ( Cav1.1). The aim of this study is to determine whether alterations of these genes are associated with changes in the regulation of intracellular Ca2+ homeostasis and signalling. Methods We analysed the expression of RYR1, SERCA and Cav1.1 and the intracellular Ca2+ handling in cultured myotubes isolated from DM1, DM2 and control muscle biopsies by semiquantitative RT-PCR and confocal Ca2+ imaging respectively. Results (i) The alternative splicing of RYR1, SERCA and Cav1.1 was more severely affected in DM1 than in DM2 myotubes; (ii) DM1 myotubes exhibited higher resting intracellular Ca2+ levels than DM2; (iii) the amplitude of intracellular Ca2+ transients induced by sustained membrane depolarization was higher in DM1 myotubes than in controls, whereas DM2 showed opposite behaviour; and (iv) in both DM myotubes, Ca2+ release from sarcoplasmic reticulum through RYR1 was lower than in controls. Conclusion The aberrant splicing of RYR1, SERCA1 and Cav1.1 may alter intracellular Ca2+ signalling in DM1 and DM2 myotubes. The differing dysregulation of intracellular Ca2+ handling in DM1 and DM2 may explain their distinct sarcolemmal hyperexcitabilities. [ABSTRACT FROM AUTHOR]

Hyperkalemic periodic paralysis (HyperKPP) is characterized by myotonic discharges that occur between episodic attacks of paralysis. Individuals with HyperKPP rarely suffer respiratory distress even though diaphragm muscle expresses the same defective Na+ channel isoform (NaV1.4) that causes symptoms in limb muscles. We tested the hypothesis that the extent of the HyperKPP phenotype (low force generation and shift toward oxidative type I and IIA fibers) in muscle is a function of 1) the NaV1.4 channel content and 2) the Na+ influx through the defective channels [i.e., the tetrodotoxin (TTX)-sensitive Na+ influx]. We measured NaV1.4 channel protein content, TTXsensitive Na+ influx, force generation, and myosin isoform expression in four muscles from knock-in mice expressing a NaV1.4 isoform corresponding to the human M1592V mutant. The HyperKPP flexor digitorum brevis muscle showed no contractile abnormalities, which correlated well with its low NaV1.4 protein content and by far the lowest TTX-sensitive Na+ influx. In contrast, diaphragm muscle expressing the HyperKPP mutant contained high levels of NaV1.4 protein and exhibited a TTX-sensitive Na+ influx that was 22% higher compared with affected extensor digitorum longus (EDL) and soleus muscles. Surprisingly, despite this high burden of Na+ influx, the contractility phenotype was very mild in mutant diaphragm compared with the robust abnormalities observed in EDL and soleus. This study provides evidence that HyperKPP phenotype does not depend solely on the NaV1.4 content or Na+ influx and that the diaphragm does not depend solely on Na+-K+ pumps to ameliorate the phenotype. [ABSTRACT FROM AUTHOR]

Abstract: CaV1.1 is the prototype for the other nine known CaV channel isoforms, yet it has functional properties that make it truly atypical of this group. Specifically, CaV1.1 is expressed solely in skeletal muscle where it serves multiple purposes; it is the voltage sensor for excitation–contraction coupling and it is an L-type Ca2+ channel which contributes to a form of activity-dependent Ca2+ entry that has been termed Excitation-coupled Ca2+ entry. The ability of CaV1.1 to serve as voltage-sensor for excitation–contraction coupling appears to be unique among CaV channels, whereas the physiological role of its more conventional function as a Ca2+ channel has been a matter of uncertainty for nearly 50years. In this chapter, we discuss how CaV1.1 supports excitation–contraction coupling, the possible relevance of Ca2+ entry through CaV1.1 and how alterations of CaV1.1 function can have pathophysiological consequences. This article is part of a Special Issue entitled: Calcium channels. [Copyright &y& Elsevier]

Voltage-gated Ca(2+) channels are multi-subunit membrane proteins that transduce depolarization into cellular functions such as excitation-contraction coupling in muscle or neurotransmitter release in neurons. The auxiliary β subunits function in membrane targeting of the channel and modulation of its gating properties. However, whether β subunits can reversibly interact with, and thus differentially modulate, channels in the membrane is still unresolved. In the present study we applied fluorescence recovery after photobleaching (FRAP) of GFP-tagged α1 and β subunits expressed in dysgenic myotubes to study the relative dynamics of these Ca(2+) channel subunits for the first time in a native functional signaling complex. Identical fluorescence recovery rates of both subunits indicate stable interactions, distinct recovery rates indicate dynamic interactions. Whereas the skeletal muscle β1a isoform formed stable complexes with CaV1.1 and CaV1.2, the non-skeletal muscle β2a and β4b isoforms dynamically interacted with both α1 subunits. Neither replacing the I-II loop of CaV1.1 with that of CaV2.1, nor deletions in the proximal I-II loop, known to change the orientation of β relative to the α1 subunit, altered the specific dynamic properties of the β subunits. In contrast, a single residue substitution in the α interaction pocket of β1aM293A increased the FRAP rate threefold. Taken together, these findings indicate that in skeletal muscle triads the homologous β1a subunit forms a stable complex, whereas the heterologous β2a and β4b subunits form dynamic complexes with the Ca(2+) channel. The distinct binding properties are not determined by differences in the I-II loop sequences of the α1 subunits, but are intrinsic properties of the β subunit isoforms. [ABSTRACT FROM AUTHOR]

Abstract: Caveolin-3 is the striated muscle specific isoform of the scaffolding protein family of caveolins and has been shown to interact with a variety of proteins, including ion channels. Mutations in the human CAV3 gene have been associated with several muscle disorders called caveolinopathies and among these, the P104L mutation (Cav-3P104L) leads to limb girdle muscular dystrophy of type 1C characterized by the loss of sarcolemmal caveolin. There is still no clear-cut explanation as to specifically how caveolin-3 mutations lead to skeletal muscle wasting. Previous results argued in favor of a role for caveolin-3 in dihydropyridine receptor (DHPR) functional regulation and/or T-tubular membrane localization. It appeared worth closely examining such a functional link and investigating if it could result from the direct physical interaction of the two proteins. Transient expression of Cav-3P104L or caveolin-3 specific siRNAs in C2C12 myotubes both led to a significant decrease of the L-type Ca2+ channel maximal conductance. Immunolabeling analysis of adult skeletal muscle fibers revealed the colocalization of a pool of caveolin-3 with the DHPR within the T-tubular membrane. Caveolin-3 was also shown to be present in DHPR-containing triadic membrane preparations from which both proteins co-immunoprecipitated. Using GST-fusion proteins, the I–II loop of Cav1.1 was identified as the domain interacting with caveolin-3, with an apparent affinity of 60nM. The present study thus revealed a direct molecular interaction between caveolin-3 and the DHPR which is likely to underlie their functional link and whose loss might therefore be involved in pathophysiological mechanisms associated to muscle caveolinopathies. [Copyright &y& Elsevier]

The subunit structure of low voltage activated T-type Ca2+ channels is still unknown. Co-expression of dihydropyridine receptor (DHPR) auxiliary subunits with T-type α1 subunits in heterologous systems has produced conflicting results. In developing foetal skeletal muscle fibres which abundantly express DHPR subunits, Cav3.2 (α1H) subunits are believed to underlie T-type calcium currents which disappear 2 to 3 weeks after birth. Therefore, a possible regulation of foetal skeletal muscle T-type Ca2+ channels by DHPR subunits was investigated in freshly isolated foetal skeletal muscle using knockout mice, which provide a powerful tool to address this question. The possible involvement of α1S (Cav1.1), β1 and γ1 DHPR subunits was tested using dysgenic (α1S-null), β1a and γ1 knockout mice. The results show that the absence of α1S, β1 or γ1 DHPR subunits does not significantly affect the electrophysiological properties of T-type Ca2+ currents in skeletal muscle, suggesting that (1) native Cav3.2 is not regulated by β1 or γ1 DHPR subunits; (2) T-type and L-type currents have distinct and not interchangeable roles. [ABSTRACT FROM AUTHOR]

Abstract: The dihydropyridine receptor (DHPR) α1S II–III loop has been shown to be critical for excitation–contraction (EC) coupling in skeletal muscle, but the importance of other cytoplasmic regions, especially the N-terminus (residues 1–51), remains unclear. In this study, we found that deletion of α1S residues 2–37 (weakly conserved with N-termini of other L-type Ca2+ channels) had little effect on the ability of α1S to serve as a Ca2+ channel or voltage sensor for EC coupling. Strikingly, deletion of 10 additional residues, which are conserved in L-type channels, resulted in ablation of DHPR function. Specifically, confocal microscopy and measurement of charge movement showed that removal of residues 2–47 resulted in a failure of sarcolemmal insertion. Our results indicate that the weakly conserved, distal α1S N-terminus is not critical for EC coupling or function as a Ca2+ channel. However, integrity of the proximal α1S N-terminus is necessary for sarcolemmal expression of the DHPR. [Copyright &y& Elsevier]

We study the CaM-peptide interactions for four CaM-related peptides with different calcium equivalents, using the h CaM-M124C- mBBr biosensor and Molecular Dynamics (MD). Due to the high sensitivity of the biosensor, we were able to calculate five K based on the number of calcium equivalents for each peptide, showing a directly proportional relationship between the degree of calcium saturation and the increased affinity for the Calspermin, nNOS, and skMLSK peptides; while the CaV1.1 peptide has a degree of affinity independent of the number of calcium equivalent. On the other hand, the MD studies were designed based on the experimental results; I) visualizing the effect of the gradual elimination of calcium in Holo-CaM and II) analyzing the CaM-Peptide complexes with and without calcium. We observe that the gradual addition of calcium increases the flexibility of Holo-CaM. Concerning CaM-Peptide complexes, it presents differences in both the ΔG ds based on the number of calcium equivalents for each peptide, showing a directly proportional relationship between the degree of calcium saturation and the increased affinity for the Calspermin, nNOS, and skMLSK peptides; while the CaV1.1 peptide has a degree of affinity independent of the number of calcium equivalent. On the other hand, the MD studies were designed based on the experimental results; I) visualizing the effect of the gradual elimination of calcium in Holo-CaM and II) analyzing the CaM-Peptide complexes with and without calcium. We observe that the gradual addition of calcium increases the flexibility of Holo-CaM. Concerning CaM-Peptide complexes, it presents differences in both the ΔG T and the RMSD. These results demonstrate the importance of the use of biosensors and the power of MD to make inferences in systems such as CaM-peptide complexes.

• Big gains have been made in understanding the impact of RGK proteins in the heart. • Relatively little is known regarding the roles of RGK proteins in skeletal muscle. • The RGK proteins Rad and Rem use different mechanisms to inhibit Ca V 1.1. • Elevated RGK protein expression is associated with skeletal muscle atrophy. • Rad expression is considerably elevated in stressed and/or diseased muscle. The RGK (Rad, Rem, Rem2 and Gem/Kir) family of small GTPases are potent endogenous inhibitors of voltage-gated Ca2+ channels (VGCCs). While the impact of RGK proteins on cardiac physiology has been investigated extensively, much less is known regarding their influence on skeletal muscle biology. Thus, the purpose of this article is to establish a basis for future investigation into the role of RGK proteins in regulating the skeletal muscle excitation-contraction (EC) coupling complex via modulation of the L-type Ca V 1.1 VGCC. The pathological consequences of elevated muscle RGK protein expression in Type II Diabetes, Amyotrophic Lateral Sclerosis (ALS), Duchenne's Muscular Dystrophy and traumatic nerve injury are also discussed. [Display omitted] [ABSTRACT FROM AUTHOR]

The skeletal muscle L-type Ca 2+ channel (Ca V 1.1) works primarily as a voltage sensor for skeletal muscle action potential (AP)-evoked Ca 2+ release. Ca V 1.1 contains four distinct voltage-sensing domains (VSDs), yet the contribution of each VSD to AP-evoked Ca 2+ release remains unknown. To investigate the role of VSDs in excitation-contraction coupling (ECC), we encoded cysteine substitutions on each S4 voltage-sensing segment of Ca V 1.1, expressed each construct via in vivo gene transfer electroporation, and used in cellulo AP fluorometry to track the movement of each Ca V 1.1 VSD in skeletal muscle fibers. We first provide electrical measurements of Ca V 1.1 voltage sensor charge movement in response to an AP waveform. Then we characterize the fluorescently labeled channels' VSD fluorescence signal responses to an AP and compare them with the waveforms of the electrically measured charge movement, the optically measured free myoplasmic Ca 2+ , and the calculated rate of Ca 2+ release from the sarcoplasmic reticulum for an AP, the physiological signal for skeletal muscle fiber activation. A considerable fraction of the fluorescence signal for each VSD occurred after the time of peak Ca 2+ release, and even more occurred after the earlier peak of electrically measured charge movement during an AP, and thus could not directly reflect activation of Ca 2+ release or charge movement, respectively. However, a sizable fraction of the fluorometric signals for VSDs I, II, and IV, but not VSDIII, overlap the rising phase of charge moved, and even more for Ca 2+ release, and thus could be involved in voltage sensor rearrangements or Ca 2+ release activation., Competing Interests: The authors declare no competing interest.

• Skeletal muscle dihydropyridine receptor functions as a voltage-gated Ca2+ channel. • N617D mutation in the DHPRα 1S subunit abolishes Ca2+ permeation through the channel. • Ba2+ and Mn2+ ions are found to permeate the N617D mutant channel. • External Ca2+ strongly blocks Ba2+ currents through the mutant channel. • N617D mutation located outside the selectivity filter influences channel permeation. In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca2+ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca2+ channel that conducts L-type Ca2+ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca2+ influx in mammalian muscle, a knock-in (ki) mouse model (nc DHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα 1S subunit abolishing Ca2+ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn2+ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca2+ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca2+ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca2+, Mn2+ entry and subsequent quenching did occur because Mn2+ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba2+ and Ba2+ currents were strongly blocked by external Ca2+. Ba2+ permeation was smaller, current kinetics slower and Ca2+ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca2+ impeding in this way permeation. Because Ba2+ binds with lower affinity to D, Ba2+ currents occur, but with reduced amplitudes as compared to Ba2+ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment. [ABSTRACT FROM AUTHOR]

Voltage-gated calcium channels control key functions of excitable cells, like synaptic transmission in neurons and the contraction of heart and skeletal muscles. To accomplish such diverse functions, different calcium channels activate at different voltages and with distinct kinetics. To identify the molecular mechanisms governing specific voltage sensing properties, we combined structure modeling, mutagenesis, and electrophysiology to analyze the structures, free energy, and transition kinetics of the activated and resting states of two functionally distinct voltage sensing domains (VSDs) of the eukaryotic calcium channel Ca V 1.1. Both VSDs displayed the typical features of the sliding helix model; however, they greatly differed in ion-pair formation of the outer gating charges. Specifically, stabilization of the activated state enhanced the voltage dependence of activation, while stabilization of resting states slowed the kinetics. This mechanism provides a mechanistic model explaining how specific ion-pair formation in separate VSDs can realize the characteristic gating properties of voltage-gated cation channels., Competing Interests: MF, YE, PT, MC, KL, BF No competing interests declared, (© 2021, Fernández-Quintero et al.)