Jerome Parness

Identification of a Dantrolene-binding Sequence on the Skeletal Muscle Ryanodine Receptor

Dantrolene is a drug that suppresses intracellular Ca2+ release from sarcoplasmic reticulum (SR) in skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Although its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1–1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca2+ release channel in SR, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene. Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]azidodantrolene, indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1 previously shown to affect RyR1 function in vitro andin vivo were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1–2s, peptides containing the amino acid sequence corresponding to RyR1 residues 590–609, were specifically labeled by [3H]azidodantrolene. A monoclonal anti-RyR1 antibody that recognizes RyR1 and its 1400-amino acid N-terminal fragment recognizes DP1 and DP1–2s in both Western blots and immunoprecipitation assays and specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in SR. Our results indicate that synthetic domain peptides can mimic a native, ligand-binding conformation in vitro and that the dantrolene-binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino acids 590–609.

Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

Interdomain interactions between N-terminal and central domains serving as a “domain switch” are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca2+ channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (KQ) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 °C and was unaffected by calmodulin. [3H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca2+ release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 °C, yet required the presence of calmodulin at 22 °C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca2+ channel opening rather than the domain switch itself.

Muscle aging is associated with compromised Ca2+ spark signaling and segregated intracellular Ca2+ release

Reduced homeostatic capacity for intracellular Ca2+ ([Ca2+]i) movement may underlie the progression of sarcopenia and contractile dysfunction during muscle aging. We report two alterations to Ca2+ homeostasis in skeletal muscle that are associated with aging. Ca2+ sparks, which are the elemental units of Ca2+ release from sarcoplasmic reticulum, are silent under resting conditions in young muscle, yet activate in a dynamic manner upon deformation of membrane structures. The dynamic nature of Ca2+ sparks appears to be lost in aged skeletal muscle. Using repetitive voltage stimulation on isolated muscle preparations, we identify a segregated [Ca2+]i reserve that uncouples from the normal excitation–contraction process in aged skeletal muscle. Similar phenotypes are observed in adolescent muscle null for a synaptophysin-family protein named mitsugumin-29 (MG29) that is involved in maintenance of muscle membrane ultrastructure and Ca2+ signaling. This finding, coupled with decreased expression of MG29 in aged skeletal muscle, suggests that MG29 expression is important in maintaining skeletal muscle Ca2+ homeostasis during aging.

Azumolene Inhibits a Component of Store-operated Calcium Entry Coupled to the Skeletal Muscle Ryanodine Receptor

Dantrolene reduces the elevated myoplasmic Ca2+ generated during malignant hyperthermia, a pharmacogenetic crisis triggered by volatile anesthetics. Although specific binding of dantrolene to the type 1 ryanodine receptor (RyR1), the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum, has been demonstrated, there is little evidence for direct dantrolene inhibition of RyR1 channel function. Recent studies suggest store-operated Ca2+ entry (SOCE) contributes to skeletal muscle function, but the effect of dantrolene on this pathway has not been examined. Here we show that azumolene, an equipotent dantrolene analog, inhibits a component of SOCE coupled to activation of RyR1 by caffeine and ryanodine, whereas the SOCE component induced by thapsigargin is not affected. Our data suggest that azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1.

The myotonias and susceptibility to malignant hyperthermia.

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle in which volatile anesthetics trigger a sustained increase in intramyoplasmic Ca2+ via release from sarcoplasmic reticulum and, possibly, entry from the extracellular milieu that leads to hypermetabolism, muscle rigidity, rhabdomyolysis, and death. Myotonias are a class of myopathies that result from gene mutations in various channels involved in skeletal muscle excitation-contraction coupling and sarcolemmal excitability, and unusual DNA sequence repeats that result in the inability of many proteins, including skeletal muscle channels that affect excitability, to undergo proper splicing. The suggestion has often been made that myotonic patients have an increased risk of developing MH. In this article, we review the physiology of muscle excitability and excitation-contraction coupling, the pathophysiology of MH and the myotonias, and review the clinical literature upon which the claims of MH susceptibility are based. We conclude that patients with these myopathies have a risk of developing MH that is equivalent to that of the general population with one potential exception, hypokalemic periodic paralysis. Despite the fact that there are no clinical reports of MH developing in patients with hypokalemic periodic paralysis, for theoretical reasons we cannot be as certain in estimating their risk of developing MH, even though we believe it is low.

Antiparallel Segregation of Notch Components in the Immunological Synapse Directs Reciprocal Signaling in Allogeneic Th:DC Conjugates

Direct T cell allorecognition underlies the development of a vigorous immune response in the clinical setting of acute graft rejection. The Notch pathway is an important regulator of Th immune responses, yet the molecular underpinnings of directional Notch signaling, otherwise critical for binary cell fate decisions, are unknown during autologous or allogeneic Th:DC interactions. Using the development of immune synapses (IS) in the allogeneic, human physiological Th:DC interaction, we demonstrate that Th-Notch1 receptor and DC-Notch ligands (Delta-like1, Jagged1) cluster in their apposed central-supramolecular-activation-clusters (cSMAC), whereas DC-Notch1 receptor and Th-Notch ligands cluster in their apposed peripheral-SMAC (pSMAC). Numb, a negative regulator of Notch, is excluded from the IS-microdomains where Notch1 receptor accumulates. This antiparallel arrangement across the partnering halves of the IS supports reciprocal Notch signal propagation in the DC-to-Th direction via the cSMAC and Th-to-DC direction via the pSMAC. As a result, processed Notch1 receptor (Notch-intracellular-domain, NICD1) and its ligands, as well as their downstream targets, HES-1 and phosphorylated-STAT3, accumulate in the nuclei of both cell-types. There is also enhancement of GLUT1 expression in both cell-types, as well as increased production of Th-IFN-γ. Significantly, neutralizing Notch1R Ab inhibits NICD1 and HES-1 nuclear translocation, and production of IFN-γ. In contrast, the IS formed during Ag-nonspecific, autologous Th:DC interaction is immature, resulting in failure of Notch1 receptor segregation and subsequent nuclear translocation of NICD1. Our results provide the first evidence for the asymmetric recruitment of Notch components in the Th:DC immunological synapse, which regulates the bidirectional Notch signal propagation.

Increased Store-Operated Ca2+ Entry in Skeletal Muscle with Reduced Calsequestrin-1 Expression

Store-operated Ca(2+) entry (SOCE) contributes to Ca(2+) handling in normal skeletal muscle function, as well as the progression of muscular dystrophy and sarcopenia, yet the mechanisms underlying the change in SOCE in these states remain unclear. Previously we showed that calsequestrin-1 (CSQ1) participated in retrograde regulation of SOCE in cultured skeletal myotubes. In this study, we used small-hairpin RNA to determine whether knockdown of CSQ1 in adult mouse skeletal muscle can influence SOCE activity and muscle function. Small-hairpin RNA against CSQ1 was introduced into flexor digitorum brevis muscles using electroporation. Transfected fibers were isolated for SOCE measurements using the Mn(2+) fluorescence-quenching method. At room temperature, the SOCE induced by submaximal depletion of the SR Ca(2+) store was significantly enhanced in CSQ1-knockdown muscle fibers. When temperature of the bathing solution was increased to 39 degrees C, CSQ1-knockdown muscle fibers displayed a significant increase in Ca(2+) permeability across the surface membrane likely via the SOCE pathway, and a corresponding elevation in cytosolic Ca(2+) as compared to control fibers. Preincubation with azumolene, an analog of dantrolene used for the treatment of malignant hyperthermia (MH), suppressed the elevated SOCE in CSQ1-knockdown fibers. Because the CSQ1-knockout mice develop similar MH phenotypes, this inhibitory effect of azumolene on SOCE suggests that elevated extracellular Ca(2+) entry in skeletal muscle may be a key factor for the pathophysiological changes in intracellular Ca(2+) signaling in MH.

Antibody probe study of Ca2+ channel regulation by interdomain interaction within the ryanodine receptor.

N-terminal and central domains of ryanodine receptor 1 (RyR1), where many reported malignant hyperthermia (MH) mutations are localized, represent putative channel regulatory domains. Recent domain peptide (DP) probe studies led us to the hypothesis that these domains interact to stabilize the closed state of channel (zipping), while weakening of domain-domain interactions (unzipping) by mutation de-stabilizes the channel, making it leaky to Ca2+ or sensitive to the agonists of RyR1. As shown previously, DP1 (N-terminal domain peptide) and DP4 (central domain peptide) produced MH-like channel activation/sensitization effects, presumably by peptide binding to sites critical to stabilizing domain-domain interactions and resultant loss of conformational constraints. Here we report that polyclonal anti-DP1 and anti-DP4 antibodies also produce MH-like channel activation and sensitization effects as evidenced by about 4-fold enhancement of high affinity [3H]ryanodine binding to RyR1 and by a significant left-shift of the concentration-dependence of activation of sarcoplasmic reticulum Ca2+ release by polylysine. Fluorescence quenching experiments demonstrate that the accessibility of a DP4-directed, conformationally sensitive fluorescence probe linked to the RyR1 N-terminal domain is increased in the presence of domain-specific antibodies, consistent with the view that these antibodies produce unzipping of interacting domains that are of hindered accessibility to the surrounding aqueous environment. Our results suggest that domain-specific antibody binding induces a conformational change resulting in channel activation, and are consistent with the hypothesis that interacting N-terminal and central domains are intimately involved in the regulation of RyR1 channel function.

Future Directions in Malignant Hyperthermia Research and Patient Care

Malignant hyperthermia (MH) is a complex pharmacogenetic disorder of muscle metabolism. To more closely examine the complexities of MH and other related muscle disorders, the Malignant Hyperthermia Association of the United States (MHAUS) recently sponsored a scientific conference at which an interdisciplinary group of experts gathered to share new information and ideas. In this Special Article, we highlight key concepts and theories presented at the conference along with exciting new trends and challenges in MH research and patient care.

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 μM and a peak-plateau signal at 500 μM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.