Skeletal muscle delimited myopathy and verapamil toxicity in SUR2 mutant mouse models of AIMS.

ABCC9-related intellectual disability and myopathy syndrome (AIMS) arises from loss-of-function (LoF) mutations in the ABCC9 gene, which encodes the SUR2 subunit of ATP-sensitive potassium (KATP ) channels. KATP channels are found throughout the cardiovascular system and skeletal muscle and couple cellular metabolism to excitability. AIMS individuals show fatigability, muscle spasms, and cardiac dysfunction. We found reduced exercise performance in mouse models of AIMS harboring premature stop codons in ABCC9. Given the roles of KATP channels in all muscles, we sought to determine how myopathy arises using tissue-selective suppression of KATP and found that LoF in skeletal muscle, specifically, underlies myopathy. In isolated muscle, SUR2 LoF results in abnormal generation of unstimulated forces, potentially explaining painful spasms in AIMS. We sought to determine whether excessive Ca2+ influx through CaV 1.1 channels was responsible for myopathology but found that the Ca2+ channel blocker verapamil unexpectedly resulted in premature death of AIMS mice and that rendering CaV 1.1 channels nonpermeable by mutation failed to reverse pathology; results which caution against the use of calcium channel blockers in AIMS.

The distal C terminus of the dihydropyridine receptor ß1a subunit is essential for tetrad formation in skeletal muscle.

The skeletal muscle dihydropyridine receptor (DHPR) ß1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitation­contraction (EC) coupling. Previously, we showed that all four vertebrate ß isoforms (ß1­ß4) facilitate α1S triad targeting and, except for ß3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488­7493 (2013)]. Consequently, ß3 failed to restore EC coupling despite the fact that both ß3 and ß1a restore tetrads. Thus, all ß-subunits are able to restore triad targeting, but only ß1a restores both tetrads and proper DHPR­RyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488­7493 (2013)]. To investigate the molecular region(s) of ß1a responsible for the tetradic arrangement of DHPRs and thus DHPR­RyR1 coupling, we expressed loss- and gain-of-function chimeras between ß1a and ß4, with systematically swapped domains in zebrafish strain relaxed (ß1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. ß1a/ß4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from ß4 showed complete restoration of SR Ca2+ release. However, chimera ß1a/ß4(C) with ß4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera ß4/ß1a(C) with ß1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of ß1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPR­RyR1 interaction, essential for skeletal muscle EC coupling.

Pore mutation N617D in the skeletal muscle DHPR blocks Ca2+ influx due to atypical high-affinity Ca2+ binding.

Skeletal muscle excitation-contraction (EC) coupling roots in Ca2+-influx-independent inter-channel signaling between the sarcolemmal dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) in the sarcoplasmic reticulum. Although DHPR Ca2+ influx is irrelevant for EC coupling, its putative role in other muscle-physiological and developmental pathways was recently examined using two distinct genetically engineered mouse models carrying Ca2+ non-conducting DHPRs: DHPR(N617D) (Dayal et al., 2017) and DHPR(E1014K) (Lee et al., 2015). Surprisingly, despite complete block of DHPR Ca2+-conductance, histological, biochemical, and physiological results obtained from these two models were contradictory. Here, we characterize the permeability and selectivity properties and henceforth the mechanism of Ca2+ non-conductance of DHPR(N617). Our results reveal that only mutant DHPR(N617D) with atypical high-affinity Ca2+ pore-binding is tight for physiologically relevant monovalent cations like Na+ and K+. Consequently, we propose a molecular model of cooperativity between two ion selectivity rings formed by negatively charged residues in the DHPR pore region.

The mechanism underlying transient weakness in myotonia congenita.

In addition to the hallmark muscle stiffness, patients with recessive myotonia congenita (Becker disease) experience debilitating bouts of transient weakness that remain poorly understood despite years of study. We performed intracellular recordings from muscle of both genetic and pharmacologic mouse models of Becker disease to identify the mechanism underlying transient weakness. Our recordings reveal transient depolarizations (plateau potentials) of the membrane potential to -25 to -35 mV in the genetic and pharmacologic models of Becker disease. Both Na+ and Ca2+ currents contribute to plateau potentials. Na+ persistent inward current (NaPIC) through NaV1.4 channels is the key trigger of plateau potentials and current through CaV1.1 Ca2+ channels contributes to the duration of the plateau. Inhibiting NaPIC with ranolazine prevents the development of plateau potentials and eliminates transient weakness in vivo. These data suggest that targeting NaPIC may be an effective treatment to prevent transient weakness in myotonia congenita.

Myotonia is a neuromuscular condition that causes problems with the relaxation of muscles following voluntary movements. One type of myotonia is Becker disease, also called recessive myotonia congenita. This is a genetic condition that causes muscle stiffness as a result of involuntary muscle activity. Patients may also suffer transient weakness for a few seconds or as long as several minutes after initiating a movement. The cause of these bouts of temporary weakness is still unclear, but there are hints that it could be linked to the muscle losing its excitability, the ability to respond to the stimuli that make it contract. However, this is at odds with findings that show that muscles in Becker disease are hyperexcitable. Muscle excitability depends on the presence of different concentrations of charged ions (positively charged sodium, calcium and potassium ions and negatively charged chloride ions) inside and outside of each muscle cells. These different concentrations of ions create an electric potential across the cell membrane, also called the 'membrane potential'. When a muscle cell gets stimulated, proteins on the cell membrane known as ion channels open. This allows the flow of ions between the inside and the outside of the cell, which causes an electrical current that triggers muscle contraction. To better understand the causes behind this muscle weakness, Myers et al. used mice that had either been genetically manipulated or given drugs to mimic Becker disease. By measuring both muscle force and the electrical currents that drive contraction, Myers et al. found that the mechanism underlying post-movement weakness involved a transient change in the concentrations of positively charged ions inside and outside the cells. Further experiments showed that proteins that regulate the passage of both sodium and calcium in and out of the cell ­ called sodium and calcium channels ­ contributed to this change in concentration. In addition, Myers et al. discovered that using a drug called ranolazine to stop sodium ions from entering the cell eliminated transient weakness in live mice. These findings suggest that in Becker disease, muscles cycle rapidly between being hyperexcited or not able to be excited, and that targeting the flow of sodium ions into the cell could be an effective treatment to prevent transient weakness in myotonia congenita. This study paves the way towards the development of new therapies to treat Becker disease as well as other muscle ion channel diseases with transient weakness such as periodic paralysis.

Divalent cations permeation in a Ca2+ non-conducting skeletal muscle dihydropyridine receptor mouse model.

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 (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα1S 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.

Ca2+-activated Cl- channel TMEM16A/ANO1 identified in zebrafish skeletal muscle is crucial for action potential acceleration.

The Ca2+-activated Cl- channel (CaCC) TMEM16A/Anoctamin 1 (ANO1) is expressed in gastrointestinal epithelia and smooth muscle cells where it mediates secretion and intestinal motility. However, ANO1 Cl- conductance has never been reported to play a role in skeletal muscle. Here we show that ANO1 is robustly expressed in the highly evolved skeletal musculature of the euteleost species zebrafish. We characterised ANO1 as bonafide CaCC which is activated close to maximum by Ca2+ ions released from the SR during excitation-contraction (EC) coupling. Consequently, our study addressed the question about the physiological advantage of implementation of ANO1 into the euteleost skeletal-muscle EC coupling machinery. Our results reveal that Cl- influx through ANO1 plays an essential role in restricting the width of skeletal-muscle action potentials (APs) by accelerating the repolarisation phase. Resulting slimmer APs enable higher AP-frequencies and apparently tighter controlled, faster and stronger muscle contractions, crucial for high speed movements.

Calcium Influx and Release Cooperatively Regulate AChR Patterning and Motor Axon Outgrowth during Neuromuscular Junction Formation.

Formation of synapses between motor neurons and muscles is initiated by clustering of acetylcholine receptors (AChRs) in the center of muscle fibers prior to nerve arrival. This AChR patterning is considered to be critically dependent on calcium influx through L-type channels (CaV1.1). Using a genetic approach in mice, we demonstrate here that either the L-type calcium currents (LTCCs) or sarcoplasmic reticulum (SR) calcium release is necessary and sufficient to regulate AChR clustering at the onset of neuromuscular junction (NMJ) development. The combined lack of both calcium signals results in loss of AChR patterning and excessive nerve branching. In the absence of SR calcium release, the severity of synapse formation defects inversely correlates with the magnitude of LTCCs. These findings highlight the importance of activity-dependent calcium signaling in early neuromuscular junction formation and indicate that both LTCC and SR calcium release individually support proper innervation of muscle by regulating AChR patterning and motor axon outgrowth.

The Ca2+ influx through the mammalian skeletal muscle dihydropyridine receptor is irrelevant for muscle performance.

Skeletal muscle excitation-contraction (EC) coupling is initiated by sarcolemmal depolarization, which is translated into a conformational change of the dihydropyridine receptor (DHPR), which in turn activates sarcoplasmic reticulum (SR) Ca2+ release to trigger muscle contraction. During EC coupling, the mammalian DHPR embraces functional duality, as voltage sensor and L-type Ca2+ channel. Although its unique role as voltage sensor for conformational EC coupling is firmly established, the conventional function as Ca2+ channel is still enigmatic. Here we show that Ca2+ influx via DHPR is not necessary for muscle performance by generating a knock-in mouse where DHPR-mediated Ca2+ influx is eliminated. Homozygous knock-in mice display SR Ca2+ release, locomotor activity, motor coordination, muscle strength and susceptibility to fatigue comparable to wild-type controls, without any compensatory regulation of multiple key proteins of the EC coupling machinery and Ca2+ homeostasis. These findings support the hypothesis that the DHPR-mediated Ca2+ influx in mammalian skeletal muscle is an evolutionary remnant.In mammalian skeletal muscle, the DHPR functions as a voltage sensor to trigger muscle contraction and as a Ca2+ channel. Here the authors show that mice where Ca2+ influx through the DHPR is eliminated display no difference in skeletal muscle function, suggesting that the Ca2+ influx through this channel is vestigial.

The mammalian skeletal muscle DHPR has larger Ca2+ conductance and is phylogenetically ancient to the early ray-finned fish sterlet (Acipenser ruthenus).

The L-type Ca2+ channel or dihydropyridine receptor (DHPR) in vertebrate skeletal muscle is responsible for sensing sarcolemmal depolarizations and transducing this signal to the sarcoplasmic Ca2+ release channel RyR1 via conformational coupling to initiate muscle contraction. During this excitation-contraction (EC) coupling process there is a slow Ca2+ current through the mammalian DHPR which is fully missing in euteleost fishes. In contrast to ancestral evolutionary stages where skeletal muscle EC coupling is still depended on Ca2+-induced Ca2+-release (CICR), it is possible that the DHPR Ca2+ conductivity during mammalian (conformational) EC coupling was retained as an evolutionary remnant (vestigiality). Here, we wanted to test the hypothesis that due to the lack of evolutionary pressure in post-CICR species skeletal muscle DHPR Ca2+ conductivity gradually reduced as evolution progressed. Interestingly, we identified that the DHPR of the early ray-finned fish sterlet (Acipenser ruthenus) is phylogenetically positioned above the mammalian rabbit DHPR which retained robust Ca2+ conductivity, but below the euteleost zebrafish DHPR which completely lost Ca2+ conductivity. Remarkably, our results revealed that sterlet DHPR still retained the Ca2+ conductivity but currents are significantly reduced compared to rabbit. This decrease is due to lower DHPR membrane expression similar to zebrafish, as well as due to reduced channel open probability (Po). In both these fish species the lower DHPR expression density is partially compensated by higher efficacy of DHPR-RyR1 coupling. The complete loss of Po in zebrafish and other euteleost species was presumably based on the teleost specific 3rd round of genome duplication (Ts3R). Ts3R headed into the appearance of two skeletal muscle DHPR isoforms which finally, together with the radiation of the euteleost clade, fully lost the Po.

Domain cooperativity in the ß1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle.

The dihydropyridine receptor (DHPR) ß1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement--the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform ß3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in ß1-null zebrafish relaxed myotubes, unlike the other three vertebrate ß-isoforms (ß1a, ß2a, and ß4). Thus, we used ß3 for chimerization with ß1a to investigate whether any of the five distinct molecular regions of ß1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between ß1a and ß3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of ß1a in charge movement restoration. More interestingly, ß1a SH3 domain and C terminus, when simultaneously engineered into ß3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of ß1a (also of ß2a and ß4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which ß subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle.

Skeletal muscle excitation-contraction coupling is independent of a conserved heptad repeat motif in the C-terminus of the DHPRbeta(1a) subunit.

In skeletal muscle excitation-contraction (EC) coupling the sarcolemmal L-type Ca(2+) channel or 1,4-dihydropyridine receptor (DHPR) transduces the membrane depolarization signal to the sarcoplasmic Ca(2+) release channel RyR1 via protein-protein interaction. While it is evident that the pore-forming and voltage-sensing DHPRalpha(1S) subunit is essential for this process, the intracellular DHPRbeta(1a) subunit was also shown to be indispensable. We previously found that the beta(1a) subunit is essential to target the DHPR into groups of four (tetrads) opposite the RyR1 homotetramers, a prerequisite for skeletal muscle EC coupling. Earlier, a unique hydrophobic heptad repeat motif (Lcdots, three dots, centeredVcdots, three dots, centeredV) in the C-terminus of beta(1a) was postulated by others to be essential for skeletal muscle EC coupling, as substitution of these residues with alanines resulted in 80% reduction of RyR1 Ca(2+) release. Therefore, we wanted to address the question if the proposed beta(1a) heptad repeat motif could be an active element of the DHPR-RyR1 signal transduction mechanism or already contributes at the ultrastructural level i.e. DHPR tetrad arrangement. Surprisingly, our experiments revealed full tetrad formation and an almost complete restoration of EC coupling in beta(1)-null zebrafish relaxed larvae and isolated myotubes upon expression of a beta(1a)-specific heptad repeat mutant (LVV to AAA) and thus contradict the earlier results.

Non-Ca2+-conducting Ca2+ channels in fish skeletal muscle excitation-contraction coupling.

During skeletal muscle excitation-contraction (EC) coupling, membrane depolarizations activate the sarcolemmal voltage-gated L-type Ca(2+) channel (Ca(V)1.1). Ca(V)1.1 in turn triggers opening of the sarcoplasmic Ca(2+) release channel (RyR1) via interchannel protein-protein interaction to release Ca(2+) for myofibril contraction. Simultaneously to this EC coupling process, a small and slowly activating Ca(2+) inward current through Ca(V)1.1 is found in mammalian skeletal myotubes. The role of this Ca(2+) influx, which is not immediately required for EC coupling, is still enigmatic. Interestingly, whole-cell patch clamp experiments on freshly dissociated skeletal muscle myotubes from zebrafish larvae revealed the lack of such Ca(2+) currents. We identified two distinct isoforms of the pore-forming Ca(V)1.1alpha(1S) subunit in zebrafish that are differentially expressed in superficial slow and deep fast musculature. Both do not conduct Ca(2+) but merely act as voltage sensors to trigger opening of two likewise tissue-specific isoforms of RyR1. We further show that non-Ca(2+) conductivity of both Ca(V)1.1alpha(1S) isoforms is a common trait of all higher teleosts. This non-Ca(2+) conductivity of Ca(V)1.1 positions teleosts at the most-derived position of an evolutionary trajectory. Though EC coupling in early chordate muscles is activated by the influx of extracellular Ca(2+), it evolved toward Ca(V)1.1-RyR1 protein-protein interaction with a relatively small and slow influx of external Ca(2+) in tetrapods. Finally, the Ca(V)1.1 Ca(2+) influx was completely eliminated in higher teleost fishes.

Proper restoration of excitation-contraction coupling in the dihydropyridine receptor beta1-null zebrafish relaxed is an exclusive function of the beta1a subunit.

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) beta(1a) subunit. Lack of beta(1a) results in (i) reduced membrane expression of the pore forming DHPR alpha(1S) subunit, (ii) elimination of alpha(1S) charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of beta(1a) from rather general functions of beta isoforms. Zebrafish and mammalian beta(1a) subunits quantitatively restored alpha(1S) triad targeting and charge movement as well as intracellular Ca(2+) release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal beta(2a) as the phylogenetically closest, and the ancestral housefly beta(M) as the most distant isoform to beta(1a) also completely recovered alpha(1S) triad expression and charge movement. However, both revealed drastically impaired intracellular Ca(2+) transients and very limited tetrad formation compared with beta(1a). Consequently, larval motility was either only partially restored (beta(2a)-injected larvae) or not restored at all (beta(M)). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested beta subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the beta(1a) isoform. Consequently, we postulate a model that presents beta(1a) as an allosteric modifier of alpha(1S) conformation enabling skeletal muscle-type EC coupling.