A retrograde signal from RyR1 alters DHP receptor inactivation and limits window Ca2+ release in muscle fibers of Y522S RyR1 knock-in mice.

Malignant hyperthermia (MH) is a life-threatening hypermetabolic condition caused by dysfunctional Ca(2+) homeostasis in skeletal muscle, which primarily originates from genetic alterations in the Ca(2+) release channel (ryanodine receptor, RyR1) of the sarcoplasmic reticulum (SR). Owing to its physical interaction with the dihydropyridine receptor (DHPR), RyR1 is controlled by the electrical potential across the transverse tubular (TT) membrane. The DHPR exhibits both voltage-dependent activation and inactivation. Here we determined the impact of an MH mutation in RyR1 (Y522S) on these processes in adult muscle fibers isolated from heterozygous RyR1(Y522S)-knock-in mice. The voltage dependence of DHPR-triggered Ca(2+) release flux was left-shifted by approximately 8 mV. As a consequence, the voltage window for steady-state Ca(2+) release extended to more negative holding potentials in muscle fibers of the RyR1(Y522S)-mice. A rise in temperature from 20 degrees to 30 degrees C caused a further shift to more negative potentials of this window (by approximately 20 mV). The activation of the DHPR-mediated Ca(2+) current was minimally changed by the mutation. However, surprisingly, the voltage dependence of steady-state inactivation of DHPR-mediated calcium conductance and release were also shifted by approximately 10 mV to more negative potentials, indicating a retrograde action of the RyR1 mutation on DHPR inactivation that limits window Ca(2+) release. This effect serves as a compensatory response to the lowered voltage threshold for Ca(2+) release caused by the Y522S mutation and represents a novel mechanism to counteract excessive Ca(2+) leak and store depletion in MH-susceptible muscle.

Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.

When sparks get old.

Sparks are transient local elevations of Ca ion concentration observed in different types of muscle cells. Such local Ca2+ signals can be provoked in skeletal muscle cells by altering the osmotic pressure of the extracellular solution. In this issue, Weisleder et al. (see p. 639) demonstrate that the Ca2+ response to osmotic stress is substantially altered in aged muscle. The study presents evidence for a link between this finding and a reduced expression of mitsugumin 29 (MG29), a small membrane protein of the sarcoplasmic reticulum (SR).

S100A1 promotes action potential-initiated calcium release flux and force production in skeletal muscle.

The role of S100A1 in skeletal muscle is just beginning to be elucidated. We have previously shown that skeletal muscle fibers from S100A1 knockout (KO) mice exhibit decreased action potential (AP)-evoked Ca(2+) transients, and that S100A1 binds competitively with calmodulin to a canonical S100 binding sequence within the calmodulin-binding domain of the skeletal muscle ryanodine receptor. Using voltage clamped fibers, we found that Ca(2+) release was suppressed at all test membrane potentials in S100A1(-/-) fibers. Here we examine the role of S100A1 during physiological AP-induced muscle activity, using an integrative approach spanning AP propagation to muscle force production. With the voltage-sensitive indicator di-8-aminonaphthylethenylpyridinium, we first demonstrate that the AP waveform is not altered in flexor digitorum brevis muscle fibers isolated from S100A1 KO mice. We then use a model for myoplasmic Ca(2+) binding and transport processes to calculate sarcoplasmic reticulum Ca(2+) release flux initiated by APs and demonstrate decreased release flux and greater inactivation of flux in KO fibers. Using in vivo stimulation of tibialis anterior muscles in anesthetized mice, we show that the maximal isometric force response to twitch and tetanic stimulation is decreased in S100A1(-/-) muscles. KO muscles also fatigue more rapidly upon repetitive stimulation than those of wild-type counterparts. We additionally show that fiber diameter, type, and expression of key excitation-contraction coupling proteins are unchanged in S100A1 KO muscle. We conclude that the absence of S100A1 suppresses physiological AP-induced Ca(2+) release flux, resulting in impaired contractile activation and force production in skeletal muscle.

Knockdown of TRPC3 with siRNA coupled to carbon nanotubes results in decreased insulin-mediated glucose uptake in adult skeletal muscle cells.

The involvement of Ca(2+) in the insulin-mediated signaling cascade, resulting in glucose uptake in skeletal muscle, is uncertain. Here, we test the hypothesis that Ca(2+) influx through canonical transient receptor potential 3 (TRPC3) channels modulates insulin-mediated glucose uptake in adult skeletal muscle. Experiments were performed on adult skeletal muscle cells of wild-type (WT) and obese, insulin-resistant ob/ob mice. Application of the diacylglycerol analog 1-oleyl-2-acetyl-sn-glycerol (OAG) induced a nonselective cation current, which was inhibited by the addition of anti-TRPC3 antibody in the patch pipette and smaller in ob/ob than in WT cells. Knockdown of TRPC3, using a novel technique based on small interfering RNA (siRNA) coupled to functionalized carbon nanotubes, resulted in pronounced (approximately 70%) decreases in OAG-induced Ca(2+) influx and insulin-mediated glucose uptake. TRPC3 and the insulin-sensitive glucose transporter 4 (GLUT4) coimmunoprecipitated, and immunofluorescence staining showed that they were colocalized in the proximity of the transverse tubular system, which is the predominant site of insulin-mediated glucose transport in skeletal muscle. In conclusion, our results indicate that TRPC3 interacts functionally and physically with GLUT4, and Ca(2+) influx through TRPC3 modulates insulin-mediated glucose uptake. Thus, TRPC3 is a potential target for treatment of insulin-resistant conditions.

Functional characterization of ryanodine receptor (RYR1) sequence variants using a metabolic assay in immortalized B-lymphocytes.

Mutations in the RYR1 gene are linked to malignant hyperthermia (MH), central core disease and multi-minicore disease. We screened by DHPLC the RYR1 gene in 24 subjects for mutations, and characterized functional alterations caused by some RYR1 variants. Three novel sequence variants and twenty novel polymorphisms were identified. Immortalized lymphoblastoid cell lines from patients with RYR1 variants and from controls were stimulated with 4-chloro-m-cresol (4-CmC) and the rate of extracellular acidification was recorded. We demonstrate that the increased acidification rate of lymphoblastoid cells in response to 4-CmC is mainly due to RYR1 activation. Cells expressing RYR1 variants in the N-terminal and in the central region of the protein (p.Arg530His, p.Arg2163Pro, p.Asn2342Ser, p.Glu2371Gly and p.Arg2454His) displayed higher activity compared with controls; this could account for the MH-susceptible phenotype. Cell lines harboring RYR1(Cys4664Arg) were significantly less activated by 4-CmC. This result indicates that the p.Cys4664Arg variant causes a leaky channel and depletion of intracellular stores. The functional changes detected corroborate the variants analyzed as disease-causing alterations and the acidification rate measurements as a means to monitor Ca(2+)-induced metabolic changes in cells harboring mutant RYR1 channels.

Local calcium signals induced by hyper-osmotic stress in mammalian skeletal muscle cells.

Strenuous activitiy of skeletal muscle leads to temporary osmotic dysbalance and isolated skeletal muscle fibers exposed to osmotic stress respond with characteristic micro-domain calcium signals. It has been suggested that osmotic stress targets transverse tubular (TT) dihydropyridine receptors (DHPRs) which normally serve as voltage-dependent activators of Ca release via ryanodine receptor (RyR1s) of the sarcoplasmic reticulum (SR). Here, we pursued this hypothesis by imaging the response to hyperosmotic solutions in both mouse skeletal muscle fibers and myotubes. Ca fluctuations in the cell periphery of fibers exposed to osmotic stress were accompanied by a substantial dilation of the peripheral TT. The Ca signals were completely inhibited by a conditioning depolarization that inactivates the DHPR. Dysgenic myotubes, lacking the DHP-receptor-alpha1-subunit, showed strongly reduced, yet not completely inhibited activity when stimulated with solutions of elevated tonicity. The results point to a modulatory, even though not essential, role of the DHP receptor for osmotic stress-induced Ca signals in skeletal muscle.

No voltage change at skeletal muscle SR membrane during Ca2+ release-just Mermaids on acid.

Calcium ions control multiple physiological functions by binding to extracellular and intracellular targets. One of the best-studied Ca2+-dependent functions is contraction of smooth and striated muscle tissue, which results from Ca2+ ligation to calmodulin and troponin C, respectively. Ca2+ signaling typically involves flux of the ion across membranes via specifically gated channel proteins. Because calcium ions are charged, they possess the ability to generate changes in the respective transmembrane voltage. Ca2+-dependent voltage alterations of the surface membrane are easily measured using microelectrodes. A well-known example is the characteristic plateau phase of the action potential in cardiac ventricular cells that results from the opening of voltage-gated L-type Ca2+ channels. Ca2+ ions are also released from intracellular storage compartments in many cells, but these membranes are not accessible to direct voltage recording with microelectrodes. In muscle, for example, release of Ca2+ from the sarcoplasmic reticulum (SR) to the myoplasm constitutes a flux that is considerably larger than the entry flux from the extracellular space. Whether this flux is accompanied by a voltage change across the SR membrane is an obvious question of mechanistic importance and has been the subject of many investigations. Because the tiny spaces enclosed by the SR membrane are inaccessible to microelectrodes, alternative methods have to be applied. In a study by Sanchez et al. (2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201812035) in this issue, modern confocal light microscopy and genetically encoded voltage probes targeted to the SR were applied in a new approach to search for changes in the membrane potential of the SR during Ca2+ release.

Voltage modulates halothane-triggered Ca2+ release in malignant hyperthermia-susceptible muscle.

Malignant hyperthermia (MH) is a fatal hypermetabolic state that may occur during general anesthesia in susceptible individuals. It is often caused by mutations in the ryanodine receptor RyR1 that favor drug-induced release of Ca2+ from the sarcoplasmic reticulum. Here, knowing that membrane depolarization triggers Ca2+ release in normal muscle function, we study the cross-influence of membrane potential and anesthetic drugs on Ca2+ release. We used short single muscle fibers of knock-in mice heterozygous for the RyR1 mutation Y524S combined with microfluorimetry to measure intracellular Ca2+ signals. Halothane, a volatile anesthetic used in contracture testing for MH susceptibility, was equilibrated with the solution superfusing the cells by means of a vaporizer system. In the range 0.2 to 3%, the drug causes significantly larger elevations of free myoplasmic [Ca2+] in mutant (YS) compared with wild-type (WT) fibers. Action potential-induced Ca2+ signals exhibit a slowing of their time course of relaxation that can be attributed to a component of delayed Ca2+ release turnoff. In further experiments, we applied halothane to single fibers that were voltage-clamped using two intracellular microelectrodes and studied the effect of small (10-mV) deviations from the holding potential (-80 mV). Untreated WT fibers show essentially no changes in [Ca2+], whereas the Ca2+ level of YS fibers increases and decreases on depolarization and hyperpolarization, respectively. The drug causes a significant enhancement of this response. Depolarizing pulses reveal a substantial negative shift in the voltage dependence of activation of Ca2+ release. This behavior likely results from the allosteric coupling between RyR1 and its transverse tubular voltage sensor. We conclude that the binding of halothane to RyR1 alters the voltage dependence of Ca2+ release in MH-susceptible muscle fibers such that the resting membrane potential becomes a decisive factor for the efficiency of the drug to trigger Ca2+ release.

Loss of S100A1 expression leads to Ca2+ release potentiation in mutant mice with disrupted CaM and S100A1 binding to CaMBD2 of RyR1.

Calmodulin (CaM) and S100A1 fine-tune skeletal muscle Ca2+ release via opposite modulation of the ryanodine receptor type 1 (RyR1). Binding to and modulation of RyR1 by CaM and S100A1 occurs predominantly at the region ranging from amino acid residue 3614-3640 of RyR1 (here referred to as CaMBD2). Using synthetic peptides, it has been shown that CaM binds to two additional regions within the RyR1, specifically residues 1975-1999 and 4295-4325 (CaMBD1 and CaMBD3, respectively). Because S100A1 typically binds to similar motifs as CaM, we hypothesized that S100A1 could also bind to CaMBD1 and CaMBD3. Our goals were: (1) to establish whether S100A1 binds to synthetic peptides containing CaMBD1 and CaMBD3 using isothermal calorimetry (ITC), and (2) to identify whether S100A1 and CaM modulate RyR1 Ca2+ release activation via sites other than CaMBD2 in RyR1 in its native cellular context. We developed the mouse model (RyR1D-S100A1KO), which expresses point mutation RyR1-L3625D (RyR1D) that disrupts the modulation of RyR1 by CaM and S100A1 at CaMBD2 and also lacks S100A1 (S100A1KO). ITC assays revealed that S100A1 binds with different affinities to CaMBD1 and CaMBD3. Using high-speed Ca2+ imaging and a model for Ca2+ binding and transport, we show that the RyR1D-S100A1KO muscle fibers exhibit a modest but significant increase in myoplasmic Ca2+ transients and enhanced Ca2+ release flux following field stimulation when compared to fibers from RyR1D mice, which were used as controls to eliminate any effect of binding at CaMBD2, but with preserved S100A1 expression. Our results suggest that S100A1, similar to CaM, binds to CaMBD1 and CaMBD3 within the RyR1, but that CaMBD2 appears to be the primary site of RyR1 regulation by CaM and S100A1.

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.

Passive muscle stiffness may be influenced by active contractility of intramuscular connective tissue.

The article introduces the hypothesis that intramuscular connective tissue, in particular the fascial layer known as the perimysium, may be capable of active contraction and consequently influence passive muscle stiffness, especially in tonic muscles. Passive muscle stiffness is also referred to as passive elasticity, passive muscular compliance, passive extensibility, resting tension, or passive muscle tone. Evidence for the hypothesis is based on five indications: (1) tonic muscles contain more perimysium and are therefore stiffer than phasic muscles; (2) the specific collagen arrangement of the perimysium is designed to fit a load-bearing function; (3) morphological considerations as well as histological observations in our laboratory suggest that the perimysium is characterized by a high density of myofibroblasts, a class of fibroblasts with smooth muscle-like contractile kinetics; (4) in vitro contraction tests with fascia have demonstrated that fascia, due to the presence of myofibroblasts, is able to actively contract, and that the resulting contraction forces may be strong enough to influence musculoskeletal dynamics; (5) the pronounced increase of the perimysium in muscle immobilization and in the surgical treatment of distraction osteogenesis indicates that perimysial stiffness adapts to mechanical stimulation and hence influences passive muscle stiffness. In conclusion, the perimysium seems capable of response to mechanostimulation with a myofibroblast facilitated active tissue contraction, thereby adapting passive muscle stiffness to increased tensional demands, especially in tonic musculature. If verified, this new concept may lead to novel pharmaceutical or mechanical approaches to complement existing treatments of pathologies which are accompanied by an increase or decrease of passive muscle stiffness (e.g., muscle fibroses such as torticollis, peri-partum pelvic pain due to pelvic instability, and many others). Methods for testing this new concept are suggested, including histological examinations and specific in vitro contraction tests.

Fast-to-Slow Transition of Skeletal Muscle Contractile Function and Corresponding Changes in Myosin Heavy and Light Chain Formation in the R6/2 Mouse Model of Huntington's Disease.

Huntington´s disease (HD) is a hereditary neurodegenerative disease resulting from an expanded polyglutamine sequence (poly-Q) in the protein huntingtin (HTT). Various studies report atrophy and metabolic pathology of skeletal muscle in HD and suggest as part of the process a fast-to-slow fiber type transition that may be caused by the pathological changes in central motor control or/and by mutant HTT in the muscle tissue itself. To investigate muscle pathology in HD, we used R6/2 mice, a common animal model for a rapidly progressing variant of the disease expressing exon 1 of the mutant human gene. We investigated alterations in the extensor digitorum longus (EDL), a typical fast-twitch muscle, and the soleus (SOL), a slow-twitch muscle. We focussed on mechanographic measurements of excised muscles using single and repetitive electrical stimulation and on the expression of the various myosin isoforms (heavy and light chains) using dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole muscle and single fiber preparations. In EDL of R6/2, the functional tests showed a left shift of the force-frequency relation and decrease in specific force. Moreover, the estimated relative contribution of the fastest myosin isoform MyHC IIb decreased, whereas the contribution of the slower MyHC IIx isoform increased. An additional change occurred in the alkali MyLC forms showing a decrease in 3f and an increase in 1f level. In SOL, a shift from fast MyHC IIa to the slow isoform I was detectable in male R6/2 mice only, and there was no evidence of isoform interconversion in the MyLC pattern. These alterations point to a partial remodeling of the contractile apparatus of R6/2 mice towards a slower contractile phenotype, predominantly in fast glycolytic fibers.

Altered inactivation of Ca2+ current and Ca2+ release in mouse muscle fibers deficient in the DHP receptor gamma1 subunit.

Functional impacts of the skeletal muscle-specific Ca2+ channel subunit gamma1 have previously been studied using coexpression with the cardiac alpha1C polypeptide in nonmuscle cells and primary-cultured myotubes of gamma1-deficient mice. Data from single adult muscle fibers of gamma-/- mice are not yet available. In the present study, we performed voltage clamp experiments on enzymatically isolated mature muscle fibers of the m. interosseus obtained from gamma+/+ and gamma-/- mice. We measured L-type Ca2+ inward currents and intracellular Ca2+ transients during 100-ms step depolarizations from a holding potential of -80 mV. Ratiometric Ca2+ transients were analyzed with a removal model fit approach to calculate the flux of Ca2+ from the sarcoplasmic reticulum. Ca2+ current density, Ca2+ release flux, and the voltage dependence of activation of both Ca2+ current and Ca2+ release were not significantly different. By varying the holding potential and recording Ca2+ current and Ca2+ release flux induced by 100-ms test depolarizations to +20 mV, we studied quasi-steady-state properties of slow voltage-dependent inactivation. For the Ca2+ current, these experiments showed a right-shifted voltage dependence of inactivation. Importantly, we could demonstrate that a very similar shift occurred also in the inactivation curve of Ca2+ release. Voltages of half maximal inactivation were altered by 16 (current) and 14 mV (release), respectively. Muscle fiber bundles, activated by elevated potassium concentration (120 mM), developed about threefold larger contracture force in gamma-/- compared with gamma+/+. This difference was independent of the presence of extracellular Ca2+ and likely results from the lower sensitivity to voltage-dependent inactivation of Ca2+ release. These results demonstrate a specific alteration of voltage-dependent inactivation of both Ca2+ entry and Ca2+ release by the gamma1 subunit of the dihydropyridine receptor in mature muscle fibers of the mouse.

Altered Ca(2+) signaling in skeletal muscle fibers of the R6/2 mouse, a model of Huntington's disease.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat within the gene encoding the protein huntingtin. The resulting elongated glutamine (poly-Q) sequence of mutant huntingtin (mhtt) affects both central neurons and skeletal muscle. Recent reports suggest that ryanodine receptor-based Ca(2+) signaling, which is crucial for skeletal muscle excitation-contraction coupling (ECC), is changed by mhtt in HD neurons. Consequently, we searched for alterations of ECC in muscle fibers of the R6/2 mouse, a mouse model of HD. We performed fluorometric recordings of action potentials (APs) and cellular Ca(2+) transients on intact isolated toe muscle fibers (musculi interossei), and measured L-type Ca(2+) inward currents on internally dialyzed fibers under voltage-clamp conditions. Both APs and AP-triggered Ca(2+) transients showed slower kinetics in R6/2 fibers than in fibers from wild-type mice. Ca(2+) removal from the myoplasm and Ca(2+) release flux from the sarcoplasmic reticulum were characterized using a Ca(2+) binding and transport model, which indicated a significant reduction in slow Ca(2+) removal activity and Ca(2+) release flux both after APs and under voltage-clamp conditions. In addition, the voltage-clamp experiments showed a highly significant decrease in L-type Ca(2+) channel conductance. These results indicate profound changes of Ca(2+) turnover in skeletal muscle of R6/2 mice and suggest that these changes may be associated with muscle pathology in HD.

Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum.

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca(2+) release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation-contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1(I4898T) mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca(2+) content, and RYR1 Ca(2+) release channel function using adult heterozygous Ryr1(I4895T/+) knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca(2+) content, both electrically evoked and 4-chloro-m-cresol-induced Ca(2+) release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4-6-mo-old IT/+ mice. The sensitivity of the SR Ca(2+) release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca(2+) permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca(2+) release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca(2+) ion permeation.

The auxiliary subunit gamma 1 of the skeletal muscle L-type Ca2+ channel is an endogenous Ca2+ antagonist.

Ca2+ channels play crucial roles in cellular signal transduction and are important targets of pharmacological agents. They are also associated with auxiliary subunits exhibiting functions that are still incompletely resolved. Skeletal muscle L-type Ca2+ channels (dihydropyridine receptors, DHPRs) are specialized for the remote voltage control of type 1 ryanodine receptors (RyR1) to release stored Ca2+. The skeletal muscle-specific gamma subunit of the DHPR (gamma 1) down-modulates availability by altering its steady state voltage dependence. The effect resembles the action of certain Ca2+ antagonistic drugs that are thought to stabilize inactivated states of the DHPR. In the present study we investigated the cross influence of gamma 1 and Ca2+ antagonists by using wild-type (gamma+/+) and gamma 1 knockout (gamma-/-) mice. We studied voltage-dependent gating of both L-type Ca2+ current and Ca2+ release and the allosteric modulation of drug binding. We found that 10 microM diltiazem, a benzothiazepine drug, more than compensated for the reduction in high-affinity binding of the dihydropyridine agent isradipine caused by gamma 1 elimination; 5 muM devapamil [(-)D888], a phenylalkylamine Ca2+ antagonist, approximately reversed the right-shifted voltage dependence of availability and the accelerated recovery kinetics of Ca2+ current and Ca2+ release. Moreover, the presence of gamma 1 altered the effect of D888 on availability and strongly enhanced its impact on recovery kinetics demonstrating that gamma 1 and the drug do not act independently of each other. We propose that the gamma 1 subunit of the DHPR functions as an endogenous Ca2+ antagonist whose task may be to minimize Ca2+ entry and Ca2+ release under stress-induced conditions favoring plasmalemma depolarization.