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 DHPRtriggered Ca 2؉ release flux was left-shifted by Ϸ8 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°to 30°C caused a further shift to more negative potentials of this window (by Ϸ20 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 Ϸ10 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.dihydropyridine receptor ͉ excitation-contraction coupling ͉ malignant hyperthermia ͉ mouse skeletal muscle ͉ ryanodine receptor C ontraction and relaxation of skeletal muscle fibers involve a carefully controlled release and reuptake of Ca 2ϩ ions stored in the sarcoplasmic reticulum (SR). Malignant hyperthermia (MH), a life-threatening hypermetabolic state accompanied by hyperthermia, hypoxia, hypercapnia, acidosis and muscle rigidity (1Ϫ3), results from uncontrolled release of Ca 2ϩ in skeletal muscle. Episodes of MH are triggered in genetically predisposed individuals by certain pharmaceutical agents, particularly volatile anesthetics and depolarizing muscle relaxants. A large number of different mutations leading to MH susceptibility has been identified (4). These mutations primarily reside in the gene coding for the skeletal muscle isoform of the ryanodine receptor (RyR1), the major Ca 2ϩ release channel of the SR. Activation of intracellular Ca 2ϩ release results from a conformational interaction between RyR1 channels in the SR and a specialized voltage-sensitive Ca 2ϩ channel (L-type Ca 2ϩ channel), the dihydropyridine receptor (DHPR), located in the adjacent membrane of the transverse tubular (TT) system. Upon membrane depolarization, the DHPR exhibits a fast reaction leading to Ca 2ϩ release (Ϸ10 milliseconds), a slower gating transition that activates the L-type Ca 2ϩ inward curre...

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca2+ 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 RYR1I4898T 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 Ca2+ content, and RYR1 Ca2+ release channel function using adult heterozygous Ryr1I4895T/+ 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 Ca2+ content, both electrically evoked and 4-chloro-m-cresol–induced Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca2+ ion permeation.

MF. S100A1 promotes action potential-initiated calcium release flux and force production in skeletal muscle. Am J Physiol Cell Physiol 299: C891-C902, 2010. First published August 4, 2010; doi:10.1152/ajpcell.00180.2010.-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 2ϩ transients, and that S100A1 binds competitively with calmodulin to a canonical S100 binding sequence within the calmodulinbinding domain of the skeletal muscle ryanodine receptor. Using voltage clamped fibers, we found that Ca 2ϩ release was suppressed at all test membrane potentials in S100A1 Ϫ/Ϫ 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 2ϩ binding and transport processes to calculate sarcoplasmic reticulum Ca 2ϩ 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 Ϫ/Ϫ 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 2ϩ release flux, resulting in impaired contractile activation and force production in skeletal muscle. S100; excitation-contraction coupling; calcium signaling; muscle IN SKELETAL AND CARDIAC MUSCLE, action potential (AP) depolarization triggers Ca 2ϩ release from the sarcoplasmic reticulum (SR), which in turn enables actomyosin interaction and contractile force generation in a process termed excitationcontraction (EC) coupling. The small Ca 2ϩ -binding protein S100A1 positively modulates EC coupling in both skeletal and cardiac muscle (34,39,40,48). In skeletal muscle, S100A1 localizes to sarcolemmal invaginations known as transverse tubules (t-tubules) and the adjacent junctional faces of the SR (jSR) (7,14,40). This region is termed the triad junction and houses the Ca 2ϩ release machinery of the muscle fiber (11, 16). S100A1 binds to the Ca 2ϩ release channel of the jSR ryanodine receptor type-1 (RyR1) and enhances RyR1-mediated Ca 2ϩ release (17,39,40,48). We have recently demonstrated that S100A1 competes with calmodulin (CaM) for the previously well-characterized CaM binding domain (CaMBD) on RyR1 (40, 55), a site documented to sensitize RyR1 to activation (43,49). Furthermore, s...

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.