Structure of the voltage-gated calcium channel Cav1.1 at 3.6 Å resolution

Voltage-gated ion channels (VGICs) mediate electrical signaling within the nervous system and regulate a wide range of physiological processes. Voltage-gated sodium (Nav) channels are responsible for initiating action potentials and their rapid activation, sodium selectivity, and drug sensitivity are unique among VGICs. Nav channels are the molecular targets of drugs used in local anaesthesia and in the treatment of genetic and sporadic Nav channelopathies including inherited epilepsy, migraine, periodic paralysis, cardiac arrhythmia, and chronic pain syndromes. Recent crystal structures of a Nav channel from the bacterium Arcobacter butzleri (NavAb) have revealed surprising insights into the structural basis for voltage-dependent activation, sodium selectivity, drug block, and slow inactivation (1,2). The available structures of NavAb will be described alongside complementary functional and molecular dynamic studies. Distinct from Nav channels, the closely related voltage-gated calcium (Cav) channels initiate processes such as synaptic transmission, muscle contraction, and hormone secretion in response to membrane depolarization. Cav channels catalyze the rapid and highly selective influx of calcium ions into cells despite a 70-fold higher extracellular concentration of sodium. By grafting a Cav channel selectivity filter onto NavAb, crystallographic and functional analyses of the resulting CavAb channel will be described that have revealed a multi-ion selectivity filter which establishes a structural framework for understanding the mechanisms of ion selectivity and conductance in vertebrate Cav channels (3).

Voltage-gated calcium (Cav) channels are miniature membrane transistors that convert membrane electrical signals to intracellular Ca2+ transients that trigger many physiological events. In mammals, there are ten subtypes of Cav channel, among which Cav1.1 is the first Cavα1 to be cloned. Cav1.1 is specified for the excitation-contraction coupling of skeletal muscles, and has been a prototype in the structural investigations of Cav channels. This article summarized the recent advances in the structural elucidation of Cav1.1 and the mechanistic insights derived from the 3.6Å structure obtained using single-particle, electron cryomicroscopy. The structure of the Cav1.1 complex established the framework for mechanistic understanding of excitation-contraction coupling and provides the template for molecular interpretations of the functions and disease mechanisms of Cav and Nav channels.

Excitation-Contraction Coupling in Skeletal, Cardiac, and Smooth Muscle

A versatile residue numbering scheme for Nav and Cav channels

Voltage-gated calcium (Cav) channels serve dual roles in the cell, where they can both depolarize the membrane potential for electrical excitability, and activate transient cytoplasmic Ca2+ signals. In animals, Cav channels play crucial roles including driving muscle contraction (excitation-contraction coupling), gene expression (excitation-transcription coupling), pre-synaptic and neuroendocrine exocytosis (excitation-secretion coupling), regulation of flagellar/ciliary beating, and regulation of cellular excitability, either directly or through modulation of other Ca2+-sensitive ion channels. In recent years, genome sequencing has provided significant insights into the molecular evolution of Cav channels. Furthermore, expanded gene datasets have permitted improved inference of the species phylogeny at the base of Metazoa, providing clearer insights into the evolution of complex animal traits which involve Cav channels, including the nervous system. For the various types of metazoan Cav channels, key properties that determine their cellular contribution include: Ion selectivity, pore gating, and, importantly, cytoplasmic protein-protein interactions that direct sub-cellular localization and functional complexing. It is unclear when these defining features, many of which are essential for nervous system function, evolved. In this review, we highlight some experimental observations that implicate Cav channels in the physiology and behavior of the most early-diverging animals from the phyla Cnidaria, Placozoa, Porifera, and Ctenophora. Given our limited understanding of the molecular biology of Cav channels in these basal animal lineages, we infer insights from better-studied vertebrate and invertebrate animals. We also highlight some apparently conserved cellular functions of Cav channels, which might have emerged very early on during metazoan evolution, or perhaps predated it.

Comparative Mechanisms for Contraction of Cardiac and Skeletal Muscle

<p>Voltage-gated Ca2+ (Cav) channels are activated upon depolarization. They specifically allow Ca2+ ions to come into the cell. These Ca2+ ions are bi-functional because they not only control cell excitability but also couple electrical activity to complex downstream signaling events, such as excitation-contraction coupling in muscles and neurotransmitter release in neurons. In the brain, Cav channels are expressed in the pre- or post-synaptic membrane of most excitable cells, neurons. In the past few years, their expression and function have also been characterized in many nonexcitable cells such as astrocytes. This dissertation focuses on the regulation of one subtype of postsynaptic Cav channels, Cav1.2, in neurons. In the first part of chapter I, I provide a literature overview of Cav channels in terms of their subtypes, localizations, physiological functions, and biophysical properties.</p> <p>For years, Cav channels were studied as single entities. But now, based on multiple proteomic studies, we know that these channels actually do not live alone. They interact with numerous proteins depending on the physiological conditions. Such interactions can anchor the channels to optimal sites of action, and tether Cav channels to their modulatory molecules. Therefore, it is crucial to understand how Cav channels are regulated by their macromolecular assembly. Among these protein partners, our lab studied the regulation of Cav channels by a subset of PDZ-domain containing proteins. Because these proteins play an important role in scaffolding and they colocalize with both pre- and post-synaptic Cav channels. Indeed, previous studies from our lab and other groups have revealed that PDZ proteins participate in a multitude of Cav regulation. The second part of chapter I introduces the diverse modulation of neuronal Cav channels by numerous PDZ proteins.</p> <p>In neurons, Cav1.2 channels regulate neuronal excitability and synaptic plasticity. Their functions have been implicated in learning, memory, and mood regulation. A study published in the journal Lancet showed that the gene encoding Cav1.2 is a common risk factor for five major psychiatric disorders. A PDZ protein, densin-180 (densin) is an excitatory synapse protein that promotes Ca2+-dependent facilitation of voltage-gated Cav1.3 Ca2+ channels in transfected cells. Mice lacking densin exhibit similar behavioral phenotypes that closely match those in mice lacking Cav1.2. In chapter II and III, we investigated the functional impact of densin on Cav1.2 channels and their auxiliary subunit β2a.</p> <p>Besides the regulation of Cav channels by their interactome, we have also known for a long time that Ca2+ currents undergo a negative feedback regulation. This regulation is called Ca2+-dependent inactivation (CDI) and it is mediated by Ca2+ that directly traverses the pore. CDI has been described for Cav channels in multiple cell types. In the heart, CDI prevents excessively long cardiac action potentials, which in turn can prevent activity-dependent arrhythmia. In neurons, CDI may be neuroprotective by preventing excitotoxic Ca2+ overloads. In the last 18 years, two essential components have been revealed in the mechanism of CDI. One is the protein calmodulin (CaM). CaM interacts directly with sites on the C-terminus of Cav channels. It binds to the incoming Ca2+ ions and produces a mysterious conformational change that determines the conductance of the channel. The other molecular player is Cavβ protein family. Cavβ comprises four subfamilies β1 through β4, which generally enhance the channel inactivation, except β2a. In chapter IV, Xiaohan Wang from Roger Colbran’s lab in Vanderbilt University, and I identified a new molecular determinant for Cav1.2 CDI.</p> <p>The α2δ subunit is an extracellular component of the Cav channel complex. Similar to Cavβ subunits, α2δ subunits are essential for the biophysical properties, surface level, and trafficking of Cavα1 subunits. There are four isoforms of α2δ subunits (α2δ1 to α2δ4). They display distinct tissue distributions. Although the roles of α2δ subunits in Cav channel regulation were studied extensively, studies have proposed that the function of α2δ subunits may be in part or entirely independent of Cav channel complex, such as synaptogenesis. Considering the important role of α2δ in physiology and pathology, it is imperative to identify the factors that regulate the properties of α2δ. In chapter V, I explored the trafficking dynamics of α2δ1 and revealed a potential regulator of α2δ1 for its protein stability and localization.</p> <p>One beauty of doing research is that it always motivates us to think and ask more questions on our journey of demystifying nature. While looking at the evidence that I find, I realize how much more we could do in the future. In chapter VI, I conclude the findings of each chapter and share my perspectives on the future direction for these research projects.</p>

How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4–12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel.

The Tumor Suppressor eIF3e Mediates Calcium-Dependent Internalization of the L-Type Calcium Channel CaV1.2

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Skeletal muscles are a highly structured tissue responsible for movement and metabolic regulation, which can be broadly subdivided into fast and slow twitch muscles with each type expressing common as well as specific sets of proteins. Congenital myopathies are a group of muscle diseases leading to a weak muscle phenotype caused by mutations in a number of genes including RYR1. Patients carrying recessive RYR1 mutations usually present from birth and are generally more severely affected, showing preferential involvement of fast twitch muscles as well as extraocular and facial muscles. In order to gain more insight into the pathophysiology of recessive RYR1-congential myopathies, we performed relative and absolute quantitative proteomic analysis of skeletal muscles from wild-type and transgenic mice carrying p.Q1970fsX16 and p.A4329D RyR1 mutations which were identified in a child with a severe congenital myopathy. Our in-depth proteomic analysis shows that recessive RYR1 mutations not only decrease the content of RyR1 protein in muscle, but change the expression of 1130, 753, and 967 proteins EDL, soleus and extraocular muscles, respectively. Specifically, recessive RYR1 mutations affect the expression level of proteins involved in calcium signaling, extracellular matrix, metabolism and ER protein quality control. This study also reveals the stoichiometry of major proteins involved in excitation contraction coupling and identifies novel potential pharmacological targets to treat RyR1-related congenital myopathies. Editor's evaluation This is a fundamental study reporting a comprehensive proteomic analysis in three skeletal muscle types from wild-type and RYR1-related myopathy mice. It adds quantitative stoichiometry of several excitation-contraction coupling-related proteins. This valuable work compares the disease-related proteomes of the different skeletal muscle groups. https://doi.org/10.7554/eLife.83618.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Skeletal muscles constitute the largest organ, accounting for approximately 60% of the total body mass; they are responsible for movement and posture and additionally, play a fundamental role in regulating metabolism. Furthermore, skeletal muscles are plastic and can respond to physiological stimuli such as increased workload and exercise by undergoing hypertrophy. Broadly speaking muscles can be subdivided into different types depending on their speed of contraction, namely slow twitch muscles are characterized by level of oxidative activity, while fast twitch muscles show high content of enzymes involved in glycolytic activity. Fast- and slow-twitch muscle can be also identified based on the expression of specific myosin heavy chain (MyHC) isoforms (Lieber, 2010; Schiaffino and Reggiani, 2011). Fast twitch muscles, also known as type II fibers, are specialized for rapid movements, are mainly glycolytic contain large glycogen stores and few mitochondria, fatigue rapidly and characteristically express the MyHC isoforms 2 X, 2B, and 2 A. They are also the first muscles to appear during development and are more severely impacted in patients with congenital myopathies; they also undergo more prominent age-related atrophy or sarcopenia (Lieber, 2010; Schiaffino and Reggiani, 2011; Buckingham et al., 2003; Jungbluth et al., 2005; Lawal et al., 2018; Nilwik et al., 2013). Slow twitch muscles (type 1 fibers) are mainly oxidative, contain many mitochondria and are fatigue resistant. Slow twitch muscle, such as soleus, contain muscle fibers expressing the MyHC 1 isoform in addition of muscle fibers expressing MyHC 2 A (Schiaffino and Reggiani, 2011). Type 1 fibers are generally less severely affected in patients with neuromuscular disorders such congenital myopathies. Although such a general classification based on MyHC isoform expression was used for many years by biochemists and physiologists, it has been recently improved thanks to the implementation of 'omic' approaches which have helped refine the phenotypic signature at the single fiber level. A great deal of data has shown that type 2 A fast fibers display a protein profile similar to type I fibers, namely a remarkable level of enzymes involved in oxidative metabolism. Interestingly, type 2 X fibers apparently encode proteins annotated to both oxidative and glycolytic pathways (Eggers et al., 2021; Murgia et al., 2021). There are also a number of functionally specialized muscles including extraocular muscles (EOM), jaw muscles and inner ear muscles that have a different embryonic origin and are made up of atypical fiber types (Schiaffino and Reggiani, 2011). For example, EOMs are the fastest contracting muscles yet they are fatigue resistant, contain many mitochondria and express most MyHC isoforms including type 1, embryonic and neonatal MyHC as well as EO-MyHC (Porter et al., 1995). EOMs are also specifically spared in patients with Duchenne Muscular Dystrophy yet they are affected in patients with some congenital myopathies, including patients with recessive RYR1 myopathies carrying a hypomorphic or null allele (Porter et al., 1995; Fischer et al., 2002; Porter et al., 2003; Amburgey et al., 2013). Congenital Myopathies (CM) are a genetically heterogeneous group of early onset, non-dystrophic diseases preferentially affecting proximal and axial muscles. More than 20 genes have been implicated in CM, the most commonly affected being those encoding proteins involved in calcium homeostasis and excitation contraction coupling (ECC) and thin-thick filaments (Jungbluth et al., 2018). Mutations in RYR1, the gene encoding the ryanodine receptor 1 (RyR1) calcium channel of the sarcoplasmic reticulum, are found in approximately 30% of all CM patients, making it the most commonly mutated gene in human CM (Amburgey et al., 2013; Jungbluth et al., 2018). Within the group of patients carrying RYR1 mutations, those with the recessive form of the disease are more severely affected, present from birth, have axial and proximal muscle weakness as well as involvement of facial and EOM (Lawal et al., 2018; Amburgey et al., 2013; Jungbluth et al., 2018). A common finding is also the reduced content of RyR1 protein in muscle biopsies (Zhou et al., 2007; Monnier et al., 2008) which could be one of the causes leading to the weak muscle phenotype. To date, the pathomechanism of disease of recessive RYR1 mutations is not completely understood and for this reason we created a mouse model knocked in for compound heterozygous mutations identified in a severely affected child with RYR1-related congenital myopathy. The double knock in mouse, henceforth referred to as double heterozygous or dHT mouse, carries the RyR1 p.Q1970fsX16 mutation in one allele leading to the absence of a transcript due to nonsense-mediated decay of the allele carrying the frameshift mutation, and the mis-sense RyR1 p.A4329D mutation in the other allele (Elbaz et al., 2019). The muscle phenotype of the dHT mouse model closely resembles that of human patients carrying a hypomorphic allele plus a mis-sense RYR1 mutation, including reduced RyR1 protein content in skeletal muscles, the presence of cores and myofibrillar dis-array, mis-alignment of RyR1 and the dihydropyridine receptor and impaired EOM function (Elbaz et al., 2019; Eckhardt et al., 2020). Interestingly, beside a reduction in RyR1, the latter muscles also exhibited a significant decrease in mitochondrial number as well as changes in the expression and content of other proteins, including the almost complete absence of the EOM-specific MyHC isoform (Eckhardt et al., 2020). Such results imply that broad changes in protein expression caused by the mutation and/or reduced content of RyR1 channels, impact other signaling pathways, leading to altered muscle function. A corollary to this is that since not all muscles are equally affected (for example fast twitch muscles and EOMs are more affected than slow twitch muscles) there may be differences in how the RYR1 mutations affect the different muscle types. In order to establish how and if Ryr1 mutations differentially impinge on the expression and function of proteins specific for different muscle types, we performed qualitative and quantitative proteomic analysis of EDL, soleus and EOMs from wild-type and dHT mice. Results Figure 1 shows a diagram of our experimental workflow: three muscle types were isolated from 12 weeks old wild-type (WT)(n=5) and dHT (n=5) mice, samples were processed for Mass Spectrometry and the results obtained were analyzed against a protein database containing sequences of the predicted SwissProt entries of Mus musculus (https://www.ebi.ac.uk/, release date 2019/03/27), Myh2 and Myh13 from Trembl, the six calibration mix proteins (Ahrné et al., 2016) and commonly observed contaminants (in total 17,414 sequences) using the SpectroMine software. Results obtained from five muscles per group were averaged, filtered so that only changes in protein content ≥0.20 fold and showing a significance of q<0.05 or greater, were considered. In addition, proteins yielding only 1 peptide were not used for analysis and were filtered out. Figure 1 Download asset Open asset Schematic overview of the workflow. (A) Skeletal muscles from 12 weeks old WT (5 mice) and dHT littermates (5 mice) were isolated and flash frozen. Three different types of muscles were isolated per mouse, namely EDL, soleus and EOMs. On the day of the experiment, muscles were solubilized and processed for LC-MS. (B) For absolute protein quantification, synthetic peptides of RyR1, Cav1.1, Stim1 and Orai1 were used. (C) Protein content in different muscle types and in the different mouse genotypes were analyzed and compared. Comparison of the proteome of EDL, soleus and EOM muscles from WT mice In order to perform their specific physiological functions, different muscle types express different protein isoforms or different amounts of specific proteins. For example, slow twitch muscles contain large amounts of the oxygen binding protein myoglobin and of carbonic anhydrase III the enzyme catalyzing the conversion of CO2 to H2CO3 and HCO3- (Garry et al., 1996; Dowling et al., 2021), while fast twitch muscles express large amounts of the calcium buffer protein parvalbumin (Celio and Heizmann, 1982) additionally, each muscle type contains specific isoforms of contractile and sarcomeric proteins (Schiaffino and Reggiani, 2011). Our first aim was to analyze the proteomes of WT mouse EDL, soleus and EOM muscles to establish their most important qualitative differences (Figure 2). Figure 2 Download asset Open asset Proteomic analysis of EDL, soleus and EOM muscles from WT mice confirms the significant difference in content if proteins involved in the TCA cycle and electron transport chain, fatty acid metabolism and muscle contraction. (A) Hierarchically clustered heatmaps of the relative abundance of proteins in EDL (columns 1–5) and soleus muscles (columns 6–10) from five mice. Blue blocks represent proteins which are increased in content, yellow blocks proteins which are decreased in content in EDL versus soleus muscles. Right pie chart shows overall number of increased (blue) and decreased (yellow) proteins. Areas are relative to their numbers. (B) Volcano plot of a total of 1866 quantified proteins which showed significant increased (blue) and decreased (yellow) content. The horizontal coordinate is the difference multiple (logarithmic transformation at the base of 2), and the vertical coordinate is the significant difference p value (logarithmic transformation at the base of 10). The proteins showing major change in content are abbreviated. Soleus: condition 2; EDL: condition 1(C) Reactome pathway analysis showing major pathways which differ between EDL and soleus muscles. (D) Hierarchically clustered heatmaps of the relative abundance of proteins in EDL (columns 1–5) and EOM muscles (columns 6–10) from five mice. Blue blocks represent proteins which are increased in content, yellow blocks proteins which are decreased in content in EDL versus EOM muscles. Right pie chart shows overall number of increased (blue) and decreased (yellow) proteins. Areas are relative to their numbers. (E) Volcano plot of a total of 1866 quantified proteins which showed significant increased (blue) and decreased (yellow) content. The horizontal coordinate is the difference multiple (logarithmic transformation at the base of 2), and the vertical coordinate is the significant difference p value (logarithmic transformation at the base of 10). The proteins showing major change in content are abbreviated. EOM: condition 2; EDL: condition 1 (F) Reactome pathway analysis showing major pathways which differ between EDL and EOM muscles. (G) Hierarchically clustered heatmaps of the relative abundance of proteins in soleus muscles (columns 1–5) and EOM (columns 6–10) from five mice. Blue blocks represent proteins which are increased in content, yellow blocks proteins which are decreased in content in soleus muscles versus EOM. Right pie chart shows overall number of increased (blue) and decreased (yellow) proteins. Areas are relative to their numbers. (H) Volcano plot of a total of 1866 quantified proteins which showed significant increased (blue) and decreased (yellow) content. The horizontal coordinate is the difference multiple (logarithmic transformation at the base of 2), and the vertical coordinate is the significant difference p value (logarithmic transformation at the base of 10). The proteins showing major change in content are abbreviated. EOM: condition 2; soleus: condition 1 (I) Reactome pathway analysis showing major pathways which differ between soleus and EOM muscles. A q-value of equal or less than 0.05 was used to filter significant changes prior to the pathway analyses. An additional filter was applied to the Heatmaps and Piecharts and only proteins showing a significant change ≥0.2 fold are included. Figure 2A shows that the content of more than 1800 proteins are differentially expressed (q<0.05) in soleus compared to EDL muscles from WT mice, of these 547 are present in lower amounts and 1319 are present in higher amounts in soleus compared to EDL muscles; Figure 2B shows a volcano plot of the log2 fold change of proteins in slow (condition 2) versus fast (condition 1) muscles. Reactome pathway analysis (Figure 2C) revealed that the pathways showing the greatest number of changes in annotated genes are those encoding proteins associated with mitochondrial function (fatty acid metabolism, TCA cycle, electron transport chain, complex 1 biogenesis, and ß-oxidation) which are significantly reduced in EDL muscles compared to soleus muscles. This is not unexpected considering that slow twitch muscles are made up type I and type IIa/IIx fibers which contain more mitochondria and oxidative enzymes than fast twitch type IIb fibers of fast twitch muscles. On the other hand, EDL muscles are significantly enriched in proteins annotated to muscle contraction, carbohydrate metabolism and glycolysis as well as collagen, integrins and extracellular matrix proteins compared to soleus muscles. Figure 2D shows that the content of more than 2500 proteins are differentially expressed (q<0.05) in EOM compared to EDL from WT mice, of these 508 are present in lower amounts and 2074 are present in higher amounts in EOM compared to EDL muscles. The volcano plot in Figure 2E shows the log2 fold change of proteins in EOM (condition 2) versus fast EDL (condition 1) muscles. Interestingly, Reactome pathway analysis (Figure 2F) revealed that EDL muscles contain a larger number of proteins annotated to adaptive immunity and MHC class I antigen presentation compared to EOMs, while the classes of proteins annotated to the citric acid cycle, electron transport chain and fatty acid ß-oxidation are significantly lower in EDL compared to EOMs. This result is in line with the fact that like soleus muscles, or cardiac muscles, EOMs are enriched in mitochondria (Porter et al., 1995; Fischer et al., 2002) to support continuous movements of the eyes. Figure 2H shows that the content of more than 2000 proteins are differentially expressed (q<0.05) in EOM compared to soleus from WT mice, of these 521 are present in lower amounts and 1663 are present in higher amounts in EOM compared to soleus muscles. The volcano plot in Figure 2H shows the log2 fold change of proteins in EOM (condition 2) versus slow soleus (condition 1) muscles. Reactome pathway analysis (Figure 2I) revealed that the most affected category is that containing genes annotated to muscle contraction (that were both up- and downregulated) followed by genes involved in MHC class I antigen presentation, translation and ubiquitin/proteasome degradation that are upregulated in soleus muscles compared to EOM muscles. Reactome pathway analysis as well as Genome Ontology pathway analysis are not sufficiently informative and probably miss important groups of proteins specific to skeletal muscle function; this observation prompted us to select specific proteins whose expression level is known to be different between fast, slow and EOM muscles. Focusing on the relative change in protein content between EDL and soleus muscles of contractile and sarcomeric proteins, our results confirm that the slow muscle Troponin I and C1 isoforms as well as the slow-MyHC 1 (encoded by Myh7) are enriched between 32 and 197-fold in soleus muscles, whereas α-actinin 3 and 4 and myomesin 1 are more abundant in EDL muscles and desmin is enriched in soleus muscles (Supplementary file 1a). Analysis of sarcoplasmic reticulum proteins involved in ECC show that the content of calsequestrin 2 and SERCA2 is 11- and 22-fold higher in soleus muscles, whereas the relative content of proteins of the junctional face membrane of the sarcoplasmic reticulum involved in ECC (Treves et al., 2009) including RyR1, the dihydropyridine (DHPR) complex (including the α1, β1, and α2δ subunits), Stac3, junctophilin-1 and triadin is more than 50% higher in EDL muscles compared to soleus, as is FKBP12 which binds to and stabilizes the RyR1 complex (Brillantes et al., 1994). Fast twitch muscles are also enriched in SERCA1, calsequestrin 1 and junctophilin 2. The abundance of protein annotated to calcium signaling and sarcoplasmic reticulum in EDL is consistent with the larger membrane volume of sarcotubular membrane in fast-twitch muscles compared to slow twitch muscles (Luff and Atwood, 1971). A similar approach was used to compare the relative content of specific proteins changing between EDL and EOMs and soleus and EOMs. Importantly, the results of the mass spectroscopy approach reported here validate a great deal of experimental observations including the fact that EOMs express high levels of Myhc13, a specific extra-ocular muscle MyHC isoform (MyHC-EO), as well as more cardiac muscle specific protein isoforms. For example, within the contractile and sarcomeric protein category, compared to EDL muscles, EOMs are particularly enriched in MyHC-slow (24-fold), MyHC-EO (29-fold) and Troponin C1 (slow and cardiac muscle isoform, 31-fold), whereas they contain very low amounts of α-actinin 3 (0.02-fold), MyHC 2b (0.07-fold) and MyHC 2 X (0.61-fold). Within the ECC coupling category, EOMs are enriched in calsequestrin 2 (21-fold), SERCA2 (3.6-fold) and junctin/junctate/aspartyl-ß-hydroxylase (3.5-fold) whereas their content of RyR1, the α-1 subunit of the dihydropyridine receptor (DHPRα1s), calsequestrin 1, Stac3, junctophilin-1 and triadin is significantly reduced by more than 50% compared to EDL muscles (Supplementary file 1b). Similarly, soleus muscles and EOMs vary in their content of a large number of proteins. Within the contractile and sarcomeric protein category, EOMs are enriched in embryonic MyHC (Myhc3, 49-fold), MyHC-EO (20-fold) and cardiac troponin T (3.10-fold), whereas compared to soleus muscles they contain very low amounts of slow- MyHC (0.0096-fold), myosin light chain 2 (0.01-fold), myozenin-2 (0.017-fold) and α-actinin 2 (0.017-fold). In the ECC category, EOMs are enriched in a number of proteins including SERCA1 (eightfold) and SERCA3 (sevenfold), Stim1 (fourfold), Junctin/junctate/Aspartyl-ß-hydroxylase (threefold), DHPRα1s (1.4-fold) and junctophilin-1 (1.4-fold), whereas they contain very low amounts of SERCA2 and >50% lower amounts of Mitsugumin 53 (Supplementary file 1c). Interestingly compared to EDL and soleus muscles, EOMs are enriched more than twofold in Stim1, junctin/junctate/aspartylß-hydroxlase. Furthermore, compared to soleus muscles and EOMs, EDLs are enriched in parvalbumin and in proteins annotated to calcium-dependent signaling' via the calcium /calmodulin dependent protein kinase IIα and IIγ, whereas soleus and EOM muscles are enriched in S100A1. Altogether, the results of the mass spectrometry analysis not only confirm known differences between muscle types (Schiaffino and Reggiani, 2011; Porter et al., 1995; Fischer et al., 2002; Celio and Heizmann, 1982; Luff and Atwood, 1971) but also reveal new molecular signatures of EDL, soleus and EOMs. In this context, it is worth mentioning that more than 10 heat shock proteins are more abundant in soleus muscles and EOMs compared to EDL muscles, including Hspb6 (16-fold higher in soleus compared to EDL) and Hspa12a (7-fold higher in EOM vs soleus). Hspb6 has been implicated in protection against atrophy, ischemia, hypertensive stress, and metabolic dysfunction (Dreiza et al., 2010). Importantly, a great deal of data has shown that muscles from patients with several neuromuscular disorders including those caused by RYR1 mutations show fiber type 1 predominance (Jungbluth et al., 2005; Lawal et al., 2018) and heat shock proteins have been suggested to have a against muscle caused by calcium and of mitochondrial chain et al., as well as against in et al., Interestingly, the content of Mitsugumin 53 (encoded by a protein involved in muscle membrane et al., 2009) is higher in slow twitch muscles compared to fast twitch muscles. on the these observations we the that increased expression of Mitsugumin with a of heat shock proteins (Dreiza et al., 2010; et al., 2003; et al., et al., be in muscle fiber type 1 associated with the presence of recessive RYR1 mutations or with other type of To this we the proteome of fast and slow twitch muscles in a mouse model of congenital muscle disorders carrying the p.Q1970fsX16 mutation in one allele and the mis-sense p.A4329D mutation in the other allele (Elbaz et al., 2019). Comparison of muscles isolated from WT and RyR1 dHT mice In the the proteome of three different muscles from dHT mice vs those of WT mice were compared. Figure and shows that in EDL muscles a total of proteins are significantly (q<0.05) in dHT in and proteins are up- or only in the EDLs of dHT mice compared to WT mice, respectively. Reactome pathway (Figure analysis revealed that proteins involved in homeostasis of the extracellular matrix, including and chain and are in EDLs from WT compared to dHT mice. also compared the proteome of soleus muscles from WT and dHT mice. Figure and show that the overall number of proteins showing significant changes in their relative content between dHT and WT mice, is than that observed in EDL muscles. In we found that and proteins are up- or only in the soleus muscles of dHT mice compared to those from WT mice, respectively. to EDL muscles, Reactome pathway analysis to a preferentially affected Figure 3 with 1 all Download asset Open asset Proteomic analysis of muscles from dHT and WT mice. and Hierarchically clustered heatmaps of the relative abundance of proteins in EDL soleus muscles (C) and EOMs (E) from three to five mice. Blue blocks represent proteins which are increased in content, yellow blocks proteins which are decreased in content in WT (columns in A and in versus dHT in A and in Right pie chart shows overall number of increased and decreased (yellow) proteins. Areas are relative to their numbers. and Volcano of total quantified proteins showing significant increased (blue) and decreased (yellow) content in dHT (condition 2) versus WT (condition 1) EDL soleus (D) and EOMs The horizontal coordinate is the difference multiple (logarithmic transformation at the base of 2), and the vertical coordinate is the significant difference p value (logarithmic transformation at the base of 10). The proteins showing major change in content are abbreviated. A q-value of equal or less than 0.05 was used to filter significant changes prior to the pathway analyses. An additional filter was applied to the Heatmaps and Piecharts and only proteins showing a significant change are included. is a common observed in patients affect by congenital myopathies to recessive RYR1 mutations (Lawal et al., 2018; Amburgey et al., 2013; Jungbluth et al., we also the proteome of EOMs from dHT and WT mice. Figure and shows that and proteins are up- or only in the EOM of dHT mice compared to WT mice, respectively. Interestingly, Reactome pathway analysis that genes encoding proteins involved in the citric acid cycle and electron transport chain, and protein to heat are upregulated in dHT vs WT EOMs (Figure The diagram (Figure shows that the three muscle types from the dHT mice a number of proteins whose content or It also shows that there are a number of proteins whose content or in a specific muscle type namely proteins in EDL, proteins in soleus and proteins in EOMs. analyzed these proteins to they were annotated to specific pathways but the results were not sufficiently informative as as skeletal muscle ECC and calcium homeostasis are In analysis showed that the genes encoding the proteins that were or upregulated specifically in dHT EDL, soleus and EOM were annotated to the category, response to and process and metabolic response to and of process in EDL muscles from dHT mice (Figure and and metabolic process and metabolic process and metabolic process in soleus muscles from dHT mice (Figure and and metabolic process and process and metabolic process and of quality in EOM muscles from dHT mice (Figure and Figure 4 with 1 all Download asset Open asset in protein content in EDL, soleus and EOM between dHT vs WT mice. (A) diagram showing significantly decreased proteins and increased proteins in the three muscle types. (B) process analysis of common proteins that are and (C) upregulated in muscle from dHT mice. common proteins showing significant changes in content in both EDL and soleus muscles. common proteins showing significant changes in content in EDL and common proteins showing significant changes in content in EOM and soleus muscles. (D) of the proteins whose content is increased in EDL, soleus and EOMs in dHT mice. (E) analysis annotated to of the proteins that are increased in muscles from dHT mice. we and analyzed protein a role in skeletal muscle muscle contraction, and heat shock protein and calcium-dependent that a significantly different (q<0.05) content between the mouse 1 shows that several proteins involved in skeletal muscle ECC are in EDLs from dHT mice, including the RyR1 as well as binding protein and 1 whose relative content by more than and respectively. The content of which different proteins including and (Treves et al., almost whereas in EDLs from dHT mice. the expression of type 2 fibers is impacted since MyHC 2 X and 2B as well 3

Quantitative proteomic analysis shows that recessive Ryr1 mutations not only decrease the content of RyR1 protein in muscle, but also affect the content of many other proteins involved in a variety of biological processes.

The expression excitation-contraction (EC) coupling in skeletal muscle was coined in 1952 (Sandow A. Yale J Biol Med 25: 176-201, 1952). The term evolved narrowly to include only the processes at the triad that intervene between depolarization of the transverse tubular (T-tubular) membrane and Ca2+ release from the sarcoplasmic reticulum (SR). From 1970 to 1988, the foundation of EC coupling was elucidated. The channel through which Ca2+ was released during activation was located in the SR by its specific binding to the plant insecticide ryanodine. This channel was called the ryanodine receptor (RyR). The RyR contained four subunits that together constituted the "SR foot" structure that traversed the gap between the SR and the T-tubular membrane. Ca2+ channels, also called dihydropyridine receptors (DHPRs), were located in the T-tubular membrane at the triadic junction and shown to be essential for EC coupling. There was a precise relationship between the two channels. Four DHPRs, organized as tetrads, were superimposed on alternate RyRs. This structure was consistent with the proposal that EC coupling was mediated via a movement of intramembrane charge in the T-tubular system. The speculation was that the DHPR acted as a voltage sensor transferring information to the RyRs of the SR by protein-protein interaction causing the release of Ca2+ from the SR. A great deal of progress was made by 1988 toward understanding EC coupling. However, the ultimate question of how voltage sensing is coupled to the opening of the SR Ca2+ release channel remains unresolved.NEW & NOTEWORTHY The least understood part of the series of events in excitation-contraction coupling in skeletal muscle was how information was transmitted from the transverse tubules to the sarcoplasmic (SR) and how Ca2+ was released from the SR. Through an explosion of technical approaches including physiological, biochemical, structural, pharmacological, and molecular genetics, much was discovered between 1970 and 1988. By the end of 1988, the foundation of EC coupling in skeletal muscle was established.

The Ca(V)1.4 voltage-gated calcium channel is predominantly expressed in the retina, and mutations to this channel have been associated with human congenital stationary night blindness type-2. The L-type Ca(V)1.4 channel displays distinct properties such as absence of calcium-dependent inactivation (CDI) and slow voltage-dependent inactivation (VDI) due to the presence of an autoinhibitory domain (inhibitor of CDI) in the distal C terminus. We hypothesized that native Ca(V)1.4 is subjected to extensive alternative splicing, much like the other voltage-gated calcium channels, and employed the transcript scanning method to identify alternatively spliced exons within the Ca(V)1.4 transcripts isolated from the human retina. In total, we identified 19 alternative splice variations, of which 16 variations have not been previously reported. Characterization of the C terminus alternatively spliced exons using whole-cell patch clamp electrophysiology revealed a splice variant that exhibits robust CDI. This splice variant arose from the splicing of a novel alternate exon (43*) that can be found in 13.6% of the full-length transcripts screened. Inclusion of exon 43* inserts a stop codon that truncates half the C terminus. The Ca(V)1.4 43* channel exhibited robust CDI, a larger current density, a hyperpolarized shift in activation potential by ∼10 mV, and a slower VDI. Through deletional experiments, we showed that the inhibitor of CDI was responsible for modulating channel activation and VDI, in addition to CDI. Calcium currents in the photoreceptors were observed to exhibit CDI and are more negatively activated as compared with currents elicited from heterologously expressed full-length Ca(V)1.4. Naturally occurring alternative splice variants may in part contribute to the properties of the native Ca(V)1.4 channels.

ABSTRACTSkeletal muscle excitation-contraction (EC) coupling is independent of calcium influx. In fact, alternative splicing of the voltage-gated calcium channel CaV1.1 actively suppresses calcium currents in mature muscle. Whether this is necessary for normal development and function of muscle is not known. However, splicing defects that cause aberrant expression of the calcium-conducting developmental CaV1.1e splice variant correlate with muscle weakness in myotonic dystrophy. Here, we deleted CaV1.1 (Cacna1s) exon 29 in mice. These mice displayed normal overall motor performance, although grip force and voluntary running were reduced. Continued expression of the developmental CaV1.1e splice variant in adult mice caused increased calcium influx during EC coupling, altered calcium homeostasis, and spontaneous calcium sparklets in isolated muscle fibers. Contractile force was reduced and endurance enhanced. Key regulators of fiber type specification were dysregulated and the fiber type composition was shifted toward slower fibers. However, oxidative enzyme activity and mitochondrial content declined. These findings indicate that limiting calcium influx during skeletal muscle EC coupling is important for the secondary function of the calcium signal in the activity-dependent regulation of fiber type composition and to prevent muscle disease.

Voltage-dependent transitions between resting and activated states of the four Cav1.1 voltage sensors observed in MD simulation.