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Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the [Ca.sup.2+] release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice ([RYR1Y.sup.Y522S/WT]), the mutation causes SR [Ca.sup.2+] leak and MHS. Here, we identified mitochondrial-deficient core regions in skeletal muscle fibers from [RYR1.sup.Y522S/WT] knock-in mice and characterized the structural and temporal aspects involved in their formation. Mitochondrial swelling/disruption, the initial detectable structural change observed in young-adult [RYR1.sup.Y522S/WT] mice (2 months), does not occur randomly but rather is confined to discrete areas termed presumptive cores. This localized mitochondrial damage is followed by local disruption/loss of nearby SR and transverse tubules, resulting in early cores (2-4 months) and small contracture cores characterized by extreme sarcomere shortening and lack of mitochondria. At later stages (1 year), contracture cores are extended, frequent, and accompanied by areas in which contractile elements are also severely compromised (unstructured cores). Based on these observations, we propose a possible series of events leading to core formation in skeletal muscle fibers of [RYR1.sup.Y522S/WT] mice: Initial mitochondrial/SR disruption in confined areas causes significant loss of local [Ca.sup.2+] sequestration that eventually results in the formation of contractures and progressive degradation of the contractile elements. central core disease | excitation-contraction coupling | muscle disease | ryanodine receptor | mitochondria doi/10.1073/pnas.0911496106

Voltage-dependent calcium channels (Car) open in response to changes in membrane potential, but their activity is modulated by [Ca.sup.2+] binding to calmodulin (CAM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the [Ca.sub.v]1.2 [[alpha].sub.1] subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with [Ca.sup.2+].CaM in 2 distinct binding modes. The structure of the [Ca.sub.v]1.2 fragment is an unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 [Ca.sup.2+].CaMs interacting with the pre-IQ regions and a canonical [Ca.sub.v]1-IQ-[Ca.sup.2+]-CaM complex. Native [Ca.sub.v]1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces [Ca.sup.2+]-dependent inactivation of heterologously expressed [Ca.sub.v]1.2 channels. structure | function | voltage-gated calcium channel

Malignant hyperthermia (MH) is a life-threatening hypermetabolic condition caused by dysfunctional [Ca.sup.2+] homeostasis in skeletal muscle, which primarily originates from genetic alterations in the [Ca.sup.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.sup.Y522S]-knock-in mice. The voltage dependence of DHPR-triggered [Ca.sup.2+] release flux was left-shifted by [approximately equal to] 8 mV. As a consequence, the voltage window for steady-state [Ca.sup.2+] release extended to more negative holding potentials in muscle fibers of the [RyR1.sup.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 equal to]20 mV). The activation of the DHPR-mediated [Ca.sup.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 equal to]10 mV to more negative potentials, indicating a retrograde action of the RyR1 mutation on DHPR inactivation that limits window [Ca.sup.2+] release. This effect serves as a compensatory response to the lowered voltage threshold for [Ca.sup.2+] release caused by the Y522S mutation and represents a novel mechanism to counteract excessive [Ca.sup.2+] leak and store depletion in MH-susceptible muscle. dihydropyridine receptor | excitation-contraction coupling | malignant hyperthermia | mouse skeletal muscle | ryanodine receptor

The skeletal muscle [Ca.sup.2+] release channel (RyR1), a homotetramer, regulates the release of [Ca.sup.2+] from the sarcoplasmic reticulum to initiate muscle contraction. In this work, we have delineated the RyR1 monomer boundaries in a subnanometer-resolution electron cryomicroscopy (cryo-EM) density map. In the cytoplasmic region of each RyR1 monomer, 36 [alpha]-helices and 7 [beta]-sheets can be resolved. A [beta]-sheet was also identified close to the membrane-spanning region that resembles the cytoplasmic pore structures of inward rectifier [K.sup.+] channels. Three structural folds, generated for amino acids 12-565 using comparative modeling and cryo-EM density fitting, localize close to regions implicated in communication with the voltage sensor in the transverse tubules. Eleven of the 15 disease-related residues for these domains are mapped to the surface of these models. Four disease-related residues are found in a basin at the interfaces of these regions, creating a pocket in which the immunophilin FKBP12 can fit. Taken together, these results provide a structural context for both channel gating and the consequences of certain malignant hyperthermia and central core disease-associated mutations in RyR1. [Ca.sup.2+] release channels | cryo-EM | 3D Structure | modeling

Mutations in the cardiac ryanodine receptor 2 (RyR2) have been associated with catecholaminergic polymorphic ventricular tachycardia and a form of arrhythmogenic right ventricular dysplasia. To study the relationship between RyR2 function and these phenotypes, we developed knockin mice with the human disease-associated RyR2 mutation R176Q. Histologic analysis of hearts from RyR[2.sup.R176Q/+] mice revealed no evidence of fibrofatty infiltration or structural abnormalities characteristic of arrhythmogenic right ventricular dysplasia, but right ventricular end-diastolic volume was decreased in RyR[2.sup.R176Q/+] mice compared with controls, indicating subtle functional impairment due to the presence of a single mutant allele. Ventricular tachycardia (VT) was observed after caffeine and epinephrine injection in RyR[2.sup.R176Q/+], but not in WT, mice. Intracardiac electrophysiology studies with programmed stimulation also elicited VT in RyR[2.sup.R176Q/+] mice. Isoproterenol administration during programmed stimulation increased both the number and duration of VT episodes in RyR[2.sup.R176Q/+] mice, but not in controls. Isolated cardiomyocytes from RyR[2.sup.R176Q/+] mice exhibited a higher incidence of spontaneous [Ca.sup.2+] oscillations in the absence and presence of isoproterenol compared with controls. Our results suggest that the R176Q mutation in RyR2 predisposes the heart to catecholamine-induced oscillatory calcium-release events that trigger a calcium-dependent ventricular arrhythmia. arrhythmogenic right ventricular dysplasia | catecholaminergic polymorphic ventricular tachycardia | calcium-release channel

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a [micro]-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency ([k.sub.cat]/[K.sub.m]) at pH 4.5, whereas its catalytic rate constant ([k.sub.cat]) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the [pK.sub.a] of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-[Angstrom] resolution (final [R.sub.free] value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to [lambda] protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis. binuclear metal center | phosphate coordination

We used a combination of bioinformatics, electron cryomicroscopy, and biochemical techniques to identify an oxidoreductase-like domain in the skeletal muscle [Ca.sup.2+] release channel protein (RyR1). The initial prediction was derived from sequence-based fold recognition for the N-terminal region (41-420) of RyR1. The putative domain was computationally localized to the clamp domain in the cytoplasmic region of a 22[Angstrom] structure of RyR1. This localization was subsequently confirmed by difference imaging with a sequence specific antibody. Consistent with the prediction of an oxidoreductase domain, RyR1 binds [[sup.3]H]NA[D.sup.+], supporting a model in which RyR1 has a oxidoreductase-like domain that could function as a type of redox sensor.

There are five known subtypes of muscarinic receptors ([M.sub.1]-[M.sub.5]). We have used knockout mice lacking the [M.sub.1], [M.sub.2], or [M.sub.4] receptors to determine which subtypes mediate modulation of voltage-gated [Ca.sup.2+] channels in mouse sympathetic neurons. Muscarinic agonists modulate N- and L-type [Ca.sup.2+] channels in these neurons through two distinct G-protein-mediated mechanisms. One pathway is fast and membrane-delimited and inhibits N- and P/Q-type channels by shifting their activation to more depolarized potentials. The other is slow and voltage-independent and uses a diffusible cytoplasmic messenger to inhibit both [Ca.sup.2+] channel types. Using patch-clamp methods on acutely dissociated sympathetic neurons, we isolated each pathway by pharmacological and kinetic means and found that each one is nearly absent in a particular knockout mouse. The fast and voltage-dependent pathway is lacking in the [M.sub.2] receptor knockout mice; the slow and voltage-independent pathway is absent from the [M.sub.1] receptor knockout mice; and neither pathway is affected in the [M.sub.4] receptor knockout mice. The knockout effects are clean and are apparently not accompanied by compensatory changes in other muscarinic receptors.

Muscarinic acetylcholine receptors are members of the G protein-coupled receptor superfamily expressed in neurons, cardiomyocytes, smooth muscle, and a variety of epithelia. Five subtypes of muscarinic acetylcholine receptors have been discovered by molecular cloning, but their pharmacological similarities and frequent colocalization make it difficult to assign functional roles for individual subtypes in specific neuronal responses. We have used gene targeting by homologous recombination in embryonic stem cells to produce mice lacking the m1 receptor. These mice show no obvious behavioral or histological defects, and the m2, m3, and m4 receptors continue to be expressed in brain with no evidence of compensatory induction. However, the robust suppression of the M-current potassium channel activity evoked by muscarinic agonists in sympathetic ganglion neurons is completely lost in m1 mutant mice. In addition, both homozygous and heterozygous mutant mice are highly resistant to the seizures produced by systemic administration of the muscarinic agonist pilocarpine. Thus, the m1 receptor subtype mediates M current modulation in sympathetic neurons and induction of seizure activity in the pilocarpine model of epilepsy.

Identifies an oxidoreductase-like domain in the skeletal muscle Ca[sub 2+] release channel protein (RyR1). Description on the approaches for the identification; Function of the oxidoreductase domain; Possibility of the enzymatic domain of RyR1.

Voltage-dependent calcium channels (Ca(V)) open in response to changes in membrane potential, but their activity is modulated by Ca(2+) binding to calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the Ca(V)1.2 alpha(1) subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with Ca(2+).CaM in 2 distinct binding modes. The structure of the Ca(V)1.2 fragment is an unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 Ca(2+).CaMs interacting with the pre-IQ regions and a canonical Ca(V)1-IQ-Ca(2+).CaM complex. Native Ca(V)1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces Ca(2+)-dependent inactivation of heterologously expressed Ca(V)1.2 channels.