Proteomic profiling of impaired excitation-contraction coupling and abnormal calcium handling in muscular dystrophy.

The X-linked inherited neuromuscular disorder Duchenne muscular dystrophy is characterised by primary abnormalities in the membrane cytoskeletal component dystrophin. The almost complete absence of the Dp427-M isoform of dystrophin in skeletal muscles renders contractile fibres more susceptible to progressive degeneration and a leaky sarcolemma membrane. This in turn results in abnormal calcium homeostasis, enhanced proteolysis and impaired excitation-contraction coupling. Biochemical and mass spectrometry-based proteomic studies of both patient biopsy specimens and genetic animal models of dystrophinopathy have demonstrated significant changes in the concentration and/or physiological function of essential calcium-regulatory proteins in dystrophin-lacking voluntary muscles. Abnormalities include dystrophinopathy-associated changes in voltage sensing receptors, calcium release channels, calcium pumps and calcium binding proteins. This review article provides an overview of the importance of the sarcolemmal dystrophin-glycoprotein complex and the wider dystrophin complexome in skeletal muscle and its linkage to depolarisation-induced calcium-release mechanisms and the excitation-contraction-relaxation cycle. Besides chronic inflammation, fat substitution and reactive myofibrosis, a major pathobiochemical hallmark of X-linked muscular dystrophy is represented by the chronic influx of calcium ions through the damaged plasmalemma in conjunction with abnormal intracellular calcium fluxes and buffering. Impaired calcium handling proteins should therefore be included in an improved biomarker signature of Duchenne muscular dystrophy.

Physical interaction of junctophilin and the CaV1.1 C terminus is crucial for skeletal muscle contraction.

Close physical association of CaV1.1 L-type calcium channels (LTCCs) at the sarcolemmal junctional membrane (JM) with ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) is crucial for excitation-contraction coupling (ECC) in skeletal muscle. However, the molecular mechanism underlying the JM targeting of LTCCs is unexplored. Junctophilin 1 (JP1) and JP2 stabilize the JM by bridging the sarcolemmal and SR membranes. Here, we examined the roles of JPs in localization and function of LTCCs. Knockdown of JP1 or JP2 in cultured myotubes inhibited LTCC clustering at the JM and suppressed evoked Ca2+ transients without disrupting JM structure. Coimmunoprecipitation and GST pull-down assays demonstrated that JPs physically interacted with 12-aa residues in the proximal C terminus of the CaV1.1. A JP1 mutant lacking the C terminus including the transmembrane domain (JP1ΔCT) interacted with the sarcolemmal/T-tubule membrane but not the SR membrane. Expression of this mutant in adult mouse muscles in vivo exerted a dominant-negative effect on endogenous JPs, impairing LTCC-RyR coupling at triads without disrupting JM morphology, and substantially reducing Ca2+ transients without affecting SR Ca2+ content. Moreover, the contractile force of the JP1ΔCT-expressed muscle was dramatically reduced compared with the control. Taken together, JPs recruit LTCCs to the JM through physical interaction and ensure robust ECC at triads in skeletal muscle.

Congenital myopathy results from misregulation of a muscle Ca2+ channel by mutant Stac3.

Skeletal muscle contractions are initiated by an increase in Ca2+ released during excitation-contraction (EC) coupling, and defects in EC coupling are associated with human myopathies. EC coupling requires communication between voltage-sensing dihydropyridine receptors (DHPRs) in transverse tubule membrane and Ca2+ release channel ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3 and cysteine-rich domain 3) is an essential component of the EC coupling apparatus and a mutation in human STAC3 causes the debilitating Native American myopathy (NAM), but the nature of how Stac3 acts on the DHPR and/or RyR1 is unknown. Using electron microscopy, electrophysiology, and dynamic imaging of zebrafish muscle fibers, we find significantly reduced DHPR levels, functionality, and stability in stac3 mutants. Furthermore, stac3NAM myofibers exhibited increased caffeine-induced Ca2+ release across a wide range of concentrations in the absence of altered caffeine sensitivity as well as increased Ca2+ in internal stores, which is consistent with increased SR luminal Ca2+ These findings define critical roles for Stac3 in EC coupling and human disease.

Ca2+ handling abnormalities in early-onset muscle diseases: Novel concepts and perspectives.

The physiological process by which Ca2+ is released from the sarcoplasmic reticulum is called excitation-contraction coupling; it is initiated by an action potential which travels deep into the muscle fiber where it is sensed by the dihydropyridine receptor, a voltage sensing L-type Ca2+channel localized on the transverse tubules. Voltage-induced conformational changes in the dihydropyridine receptor activate the ryanodine receptor Ca2+ release channel of the sarcoplasmic reticulum. The released Ca2+ binds to troponin C, enabling contractile thick-thin filament interactions. The Ca2+ is subsequently transported back into the sarcoplasmic reticulum by specialized Ca2+ pumps (SERCA), preparing the muscle for a new cycle of contraction. Although other proteins are involved in excitation-contraction coupling, the mechanism described above emphasizes the unique role played by the two Ca2+ channels (the dihydropyridine receptor and the ryanodine receptor), the SERCA Ca2+ pumps and the exquisite spatial organization of the membrane compartments endowed with the proteins responsible for this mechanism to function rapidly and efficiently. Research over the past two decades has uncovered the fine details of excitation-contraction coupling under normal conditions while advances in genomics have helped to identify mutations in novel genes in patients with neuromuscular disorders. While it is now clear that many patients with congenital muscle diseases carry mutations in genes encoding proteins directly involved in Ca2+ homeostasis, it has become apparent that mutations are also present in genes encoding for proteins not thought to be directly involved in Ca2+ regulation. Ongoing research in the field now focuses on understanding the functional effect of individual mutations, as well as understanding the role of proteins not specifically located in the sarcoplasmic reticulum which nevertheless are involved in Ca2+ regulation or excitation-contraction coupling. The principal challenge for the future is the identification of drug targets that can be pharmacologically manipulated by small molecules, with the ultimate aim to improve muscle function and quality of life of patients with congenital muscle disorders. The aim of this review is to give an overview of the most recent findings concerning Ca2+ dysregulation and its impact on muscle function in patients with congenital muscle disorders due to mutations in proteins involved in excitation-contraction coupling and more broadly on Ca2+ homeostasis.

Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of CaV1.2 calcium channels in hippocampal neurons.

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes.

Effects of reduced muscle glycogen on excitation-contraction coupling in rat fast-twitch muscle: a glycogen removal study.

The aim of this study was to investigate the effects of an enzymatic removal of glycogen on excitation-contraction coupling in mechanically skinned fibres of rat fast-twitch muscles, with a focus on the changes in the function of Na+-K+-pump and ryanodine receptor (RyR). Glycogen present in the skinned fibres and binding to microsomes was removed using glucoamylase (GA). Exposure of whole muscle to 20 U mL-1 GA for 6 min resulted in a 72% decrease in the glycogen content. Six minutes of GA treatment led to an 18 and a 22% reduction in depolarization- and action potential-induced forces in the skinned fibres, respectively. There was a minor but statistically significant increase in the repriming period, most likely because of an impairment of the Na+-K+-pump function. GA treatment exerted no effect on the maximum Ca2+ release rate from the RyR in the microsomes and the myofibrillar Ca2+ sensitivity in the skinned fibres. These results indicate that reduced glycogen per se can decrease muscle performance due to the impairment of SR Ca2+ release and suggest that although Na+-K+-pump function is adversely affected by reduced glycogen, the extent of the impairment is not sufficient to reduce Ca2+ release from the sarcoplasmic reticulum. This study provides direct evidence that glycogen above a certain amount is required for the preservation of the functional events preceding Ca2+ release from the sarcoplasmic reticulum.

Structural and biophysical analyses of the skeletal dihydropyridine receptor ß subunit ß1a reveal critical roles of domain interactions for stability.

Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca2+ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α1 and ß subunits of DHPR are essential for this process. Here, we employed a series of techniques, including size-exclusion chromatography-multi-angle light scattering, differential scanning fluorimetry, and isothermal calorimetry, to characterize various biophysical properties of the skeletal DHPR ß subunit ß1a Removal of the intrinsically disordered N and C termini and the hook region of ß1a prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined ß isoforms, which consist of SH3 and guanylate kinase domains. However, transition melting temperatures derived from the differential scanning fluorimetry experiments indicated a significant difference in stability of ∼2-3 °C between the ß1a and ß2a constructs, and the addition of the DHPR α1s I-II loop (α-interaction domain) peptide stabilized both ß isoforms by ∼6-8 °C. Similar to other ß isoforms, ß1a bound with nanomolar affinity to the α-interaction domain, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and guanylate kinase domains play a role in the stability of ß1a while also providing a conduit for allosteric signaling events.

Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor α1S Subunit Reveals Conformational Differences Induced by Binding of the ß1a Subunit.

The skeletal muscle dihydropyridine receptor α1S subunit plays a key role in skeletal muscle excitation-contraction coupling by sensing membrane voltage changes and then triggering intracellular calcium release. The cytoplasmic loops connecting four homologous α1S structural domains have diverse functions, but their structural arrangement is poorly understood. Here, we used a novel FRET-based method to characterize the relative proximity of these intracellular loops in α1S subunits expressed in intact cells. In dysgenic myotubes, energy transfer was observed from an N-terminal-fused YFP to a FRET acceptor, ReAsH (resorufin arsenical hairpin binder), targeted to each α1S intracellular loop, with the highest FRET efficiencies measured to the α1S II-III loop and C-terminal tail. However, in HEK-293T cells, FRET efficiencies from the α1S N terminus to the II-III and III-IV loops and the C-terminal tail were significantly lower, thus suggesting that these loop structures are influenced by the cellular microenvironment. The addition of the ß1a dihydropyridine receptor subunit enhanced FRET to the II-III loop, thus indicating that ß1a binding directly affects II-III loop conformation. This specific structural change required the C-terminal 36 amino acids of ß1a, which are essential to support EC coupling. Direct FRET measurements between α1S and ß1a confirmed that both wild type and truncated ß1a bind similarly to α1S These results provide new insights into the role of muscle-specific proteins on the structural arrangement of α1S intracellular loops and point to a new conformational effect of the ß1a subunit in supporting skeletal muscle excitation-contraction coupling.

Excitation contraction uncoupling by high intracellular [Ca2+] in frog skeletal muscle: a voltage clamp study.

Raising the intracellular [Ca2+] ([Ca2+]i) was previously found to produce uncoupling between the electrical depolarization of the transverse tubules and contraction in skinned muscle fibers. Here we study the effect of elevated [Ca2+]i in voltage clamped cut fibers of frog skeletal muscle to establish how the charge movement, a measure of the activation of the dihydropyridine receptors (DHPR)-voltage sensors, and Ca2+ release, a consequence of the opening of the ryanodine receptor (RyR)-release channels, were affected. [Ca2+]i was raised by various procedures (pharmacological release from the sarcoplasmic reticulum, application of high [Ca2+]i intracellular solution, permeabilization of the plasma membrane by a Ca2+ ionophore) all of which produced impairment of excitation-contraction coupling. The charge movement was reduced from 20.2 ± 1.24 to 9.9 ± 0.94 nC/µF meanwhile the Ca2+ release flux was reduced from 13.5 + 0.7 to 2.2 ± 0.3 µM/ms (n = 33). This suggests that a significant fraction of the DHPRs that remained functional, could not activate RyRs, and were therefore presumably disconnected. These results are broadly consistent with the original reports in skinned fibers. Uncoupling was prevented by the addition to the intracellular solution of the protease inhibitor leupeptin. In approximately 40 % of the uncoupled cells we observed that the [Ca2+]i transient continued to rise after the voltage clamp pulse was turned off. This loss of control by membrane voltage suggests that the uncoupled release channels might have another mechanism of activation, likely by Ca2+.

Bridging the myoplasmic gap II: more recent advances in skeletal muscle excitation-contraction coupling.

In skeletal muscle, excitation-contraction (EC) coupling relies on the transmission of an intermolecular signal from the voltage-sensing regions of the L-type Ca(2+) channel (Ca(V)1.1) 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 Ca(V)1.1 and RyR1 as voltage sensor and SR Ca(2+) 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.

The L-type voltage-gated calcium channel modulates microglial pro-inflammatory activity.

Under pathological conditions, microglia, the resident CNS immune cells, become reactive and release pro-inflammatory cytokines and neurotoxic factors. We investigated whether this phenotypic switch includes changes in the expression of the L-type voltage-gated calcium channel (VGCC) in a rat model of N-methyl-D-aspartate-induced hippocampal neurodegeneration. Double immunohistochemistry and confocal microscopy evidenced that activated microglia express the L-type VGCC. We then analyzed whether BV2 microglia express functional L-type VGCC, and investigated the latter's role in microglial cytokine release and phagocytic capacity. Activated BV2 microglia express the CaV1.2 and CaV1.3 subunits of the L-type VGCC determined by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry. Depolarization with KCl induced a Ca2+ entry facilitated by Bay k8644 and partially blocked with nifedipine, which also reduced TNF-α and NO release by 40%. However, no nifedipine effect on BV2 microglia viability or phagocytic capacity was observed. Our results suggest that in CNS inflammatory processes, the L-type VGCC plays a specific role in the control of microglial secretory activity.

Skeletal muscle-specific T-tubule protein STAC3 mediates voltage-induced Ca2+ release and contractility.

Excitation-contraction (EC) coupling comprises events in muscle that convert electrical signals to Ca(2+) transients, which then trigger contraction of the sarcomere. Defects in these processes cause a spectrum of muscle diseases. We report that STAC3, a skeletal muscle-specific protein that localizes to T tubules, is essential for coupling membrane depolarization to Ca(2+) release from the sarcoplasmic reticulum (SR). Consequently, homozygous deletion of src homology 3 and cysteine rich domain 3 (Stac3) in mice results in complete paralysis and perinatal lethality with a range of musculoskeletal defects that reflect a blockade of EC coupling. Muscle contractility and Ca(2+) release from the SR of cultured myotubes from Stac3 mutant mice could be restored by application of 4-chloro-m-cresol, a ryanodine receptor agonist, indicating that the sarcomeres, SR Ca(2+) store, and ryanodine receptors are functional in Stac3 mutant skeletal muscle. These findings reveal a previously uncharacterized, but required, component of the EC coupling machinery of skeletal muscle and introduce a candidate for consideration in myopathic disorders.

Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane.

Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein dysferlin. Studies of dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but dysferlin's association with calcium (Ca(2+)) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca(2+)] or blocking L-type Ca(2+) channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of dysferlin as a t-tubule protein that stabilizes stress-induced Ca(2+) signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.

Cardiac-specific disruption of Bin1 in mice enables a model of stress- and age-associated dilated cardiomyopathy.

Non-compensated dilated cardiomyopathy (DCM) leading to death from heart failure is rising rapidly in developed countries due to aging demographics, and there is a need for informative preclinical models to guide the development of effective therapeutic strategies to prevent or delay disease onset. In this study, we describe a novel model of heart failure based on cardiac-specific deletion of the prototypical mammalian BAR adapter-encoding gene Bin1, a modifier of age-associated disease. Bin1 deletion during embryonic development causes hypertrophic cardiomyopathy and neonatal lethality, but there is little information on how Bin1 affects cardiac function in adult animals. Here we report that cardiomyocyte-specific loss of Bin1 causes age-associated dilated cardiomyopathy (DCM) beginning by 8-10 months of age. Echocardiographic analysis showed that Bin1 loss caused a 45% reduction in ejection fraction during aging. Younger animals rapidly developed DCM if cardiac pressure overload was created by transverse aortic constriction. Heterozygotes exhibited an intermediate phenotype indicating Bin1 is haplo-insufficient to sustain normal heart function. Bin1 loss increased left ventricle (LV) volume and diameter during aging, but it did not alter LV volume or diameter in hearts from heterozygous mice nor did it affect LV mass. Bin1 loss increased interstitial fibrosis and mislocalization of the voltage-dependent calcium channel Cav 1.2, and the lipid raft scaffold protein caveolin-3, which normally complexes with Bin1 and Cav 1.2 in cardiomyocyte membranes. Our findings show how cardiac deficiency in Bin1 function causes age- and stress-associated heart failure, and they establish a new preclinical model of this terminal cardiac disease.

The molecular interaction of heart LIM protein (HLP) with RyR2 and caveolin-3 is essential for Ca(2+)-induced Ca(2+) release in the heart.

The heart LIM protein (HLP) is a LIM-only protein family member that mediates protein-protein interactions. To date, no studies have yet been conducted regarding its function in the heart. In the present study, we have identified that HLP binds the cytosolic region of RyR2 in the heart using a bacterial two-hybrid system, LC-MS/MS, co-immunoprecipitation, and GST-pull down assays. Microscopy revealed that HLP forms a triple complex with RyR2 and caveolin-3. siRNA and adenovirus-mediated KD of HLP decreased the electrically evoked Ca(2+) release from the sarcoplasmic reticulum without directly affecting SERCA2 and RyR2 activities. Collectively, the HLP-RyR2 interaction in the cell surface caveolae region may be essential for efficient excitation-contraction coupling in the heart.

Congenital Myopathy as a Phenotypic Expression of CACNA1S Gene Mutation: Case Report and Systematic Review of the Literature.

BACKGROUND: Congenital myopathies are a group of clinically, genetically, and histologically heterogeneous diseases caused by mutations in a large group of genes. One of these is CACNA1S, which is recognized as the cause of Dihydropyridine Receptor Congenital Myopathy. METHODS: To better characterize the phenotypic spectrum of CACNA1S myopathy, we conducted a systematic review of cases in the literature through three electronic databases following the PRISMA guidelines. We selected nine articles describing 23 patients with heterozygous, homozygous, or compound heterozygous mutations in CACNA1S and we added one patient with a compound heterozygous mutation in CACNA1S (c.1394-2A>G; c.1724T>C, p.L575P) followed at our Institute. We collected clinical and genetic data, muscle biopsies, and muscle MRIs when available. RESULTS: The phenotype of this myopathy is heterogeneous, ranging from more severe forms with a lethal early onset and mild-moderate forms with a better clinical course. CONCLUSIONS: Our patient presented a phenotype compatible with the mild-moderate form, although she presented peculiar features such as a short stature, myopia, mild sensorineural hearing loss, psychiatric symptoms, and posterior-anterior impairment gradient on thigh muscle MRI.

Biophysical reviews top five: voltage-dependent charge movement in nerve and muscle.

The discovery of gating currents and asymmetric charge movement in the early 1970s represented a remarkable leap forward in our understanding of the biophysical basis of voltage-dependent events that underlie electrical signalling that is vital for nerve and muscle function. Gating currents and charge movement reflect a fundamental process in which charged amino acid residues in an ion channel protein move in response to a change in the membrane electrical field and therefore activate the specific voltage-dependent response of that protein. The detection of gating currents and asymmetric charge movement over the past 50 years has been pivotal in unraveling the multiple molecular and intra-molecular processes which lead to action potentials in excitable tissues and excitation-contraction (EC) coupling in skeletal muscle. The recording of gating currents and asymmetric charge movement remains an essential component of investigations into the basic molecular mechanisms of neuronal conduction and muscle contraction.

Advances in CaV1.1 gating: New insights into permeation and voltage-sensing mechanisms.

The 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.

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.

Influence of cannabinoids upon nerve-evoked skeletal muscle contraction.

Endocannabinoids play important roles in regulating CNS synaptic function and peripheral metabolism, but cannabinoids can also act acutely to modulate contraction strength in skeletal muscle. Nerve terminals and the skeletal muscle sarcolemma express components of the cannabinoid signaling system. Endocannabinoids, N-arachidonylethanolamine (anandamide, AEA) and 2-arachidonoyl-glycerol (2-AG), are produced by skeletal muscle. They may be involved in the acute regulation of neuromuscular transmission, by adjusting the parameters for quantal acetylcholine release from the motor nerve terminal. Downstream of neuromuscular transmission, cannabinoids may also act to limit the efficiency of excitation-contraction coupling. Improved understanding of the distinct signaling actions of particular cannabinoid compounds and their receptor/transduction systems will help advance our understanding of the role of endocannabinoids in skeletal muscle physiology. Cannabinoids might also offer the potential to develop new pharmacotherapeutics to treat neuromuscular disorders that affect muscle strength.