Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-β subunit (CACNB1).

Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of β subunit of the dihydropyridine receptor (DHPR), a channel essential for Ca regulation in skeletal muscle. Here we evaluate the effect of the mutant variant V156A on the structure/function of DHPR β subunit and assess its role on Ca metabolism of cultured myotubes. Using differential scanning fluorimetry, we show that mutation V156A causes a significant reduction in thermal stability of the Src homology 3/guanylate kinase core domain of β subunit.

A focus on extracellular Ca entry into skeletal muscle.

The main task of skeletal muscle is contraction and relaxation for body movement and posture maintenance. During contraction and relaxation, Ca in the cytosol has a critical role in activating and deactivating a series of contractile proteins. In skeletal muscle, the cytosolic Ca level is mainly determined by Ca movements between the cytosol and the sarcoplasmic reticulum.

Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor α1S Subunit Reveals Conformational Differences Induced by Binding of the β1a 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.

Amino acid residues 489-503 of dihydropyridine receptor (DHPR) β1a subunit are critical for structural communication between the skeletal muscle DHPR complex and type 1 ryanodine receptor.

The β1a subunit is a cytoplasmic component of the dihydropyridine receptor (DHPR) complex that plays an essential role in skeletal muscle excitation-contraction (EC) coupling. Here we investigate the role of the C-terminal end of this auxiliary subunit in the functional and structural communication between the DHPR and the Ca(2+) release channel (RyR1). Progressive truncation of the β1a C terminus showed that deletion of amino acid residues Gln(489) to Trp(503) resulted in a loss of depolarization-induced Ca(2+) release, a severe reduction of L-type Ca(2+) currents, and a lack of tetrad formation as evaluated by freeze-fracture analysis.

Whole body periodic acceleration is an effective therapy to ameliorate muscular dystrophy in mdx mice.

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by the absence of dystrophin in both skeletal and cardiac muscles. This leads to severe muscle degeneration, and dilated cardiomyopathy that produces patient death, which in most cases occurs before the end of the second decade. Several lines of evidence have shown that modulators of nitric oxide (NO) pathway can improve skeletal muscle and cardiac function in the mdx mouse, a mouse model for DMD.

Myoplasmic resting Ca2+ regulation by ryanodine receptors is under the control of a novel Ca2+-binding region of the receptor.

Passive SR (sarcoplasmic reticulum) Ca2+ leak through the RyR (ryanodine receptor) plays a critical role in the mechanisms that regulate [Ca2+]rest (intracellular resting myoplasmic free Ca2+ concentration) in muscle. This process appears to be isoform-specific as expression of either RyR1 or RyR3 confers on myotubes different [Ca2+]rest. Using chimaeric RyR3-RyR1 receptors expressed in dyspedic myotubes, we show that isoform-dependent regulation of [Ca2+]rest is primarily defined by a small region of the receptor encompassing amino acids 3770-4007 of RyR1 (amino acids 3620-3859 of RyR3) named as the CLR (Ca2+ leak regulatory) region.

Three-dimensional localization of the α and β subunits and of the II-III loop in the skeletal muscle L-type Ca2+ channel.

The L-type Ca(2+) channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α(1s) or Ca(V)1.1, α(2), β(1a), δ1, and γ), we created transgenic mice expressing a recombinant β(1a) subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively.

Triadin/Junctin double null mouse reveals a differential role for Triadin and Junctin in anchoring CASQ to the jSR and regulating Ca(2+) homeostasis.

Triadin (Tdn) and Junctin (Jct) are structurally related transmembrane proteins thought to be key mediators of structural and functional interactions between calsequestrin (CASQ) and ryanodine receptor (RyRs) at the junctional sarcoplasmic reticulum (jSR). However, the specific contribution of each protein to the jSR architecture and to excitation-contraction (e-c) coupling has not been fully established. Here, using mouse models lacking either Tdn (Tdn-null), Jct (Jct-null) or both (Tdn/Jct-null), we identify Tdn as the main component of periodically located anchors connecting CASQ to the RyR-bearing jSR membrane.

On the footsteps of Triadin and its role in skeletal muscle.

Calcium is a crucial element for striated muscle function. As such, myoplasmic free Ca(2+) concentration is delicately regulated through the concerted action of multiple Ca(2+) pathways that relay excitation of the plasma membrane to the intracellular contractile machinery. In skeletal muscle, one of these major Ca(2+) pathways is Ca(2+) release from intracellular Ca(2+) stores through type-1 ryanodine receptor/Ca(2+) release channels (RyR1), which positions RyR1 in a strategic cross point to regulate Ca(2+) homeostasis.

Reduced gain of excitation-contraction coupling in triadin-null myotubes is mediated by the disruption of FKBP12/RyR1 interaction.

Several studies have suggested that triadin (Tdn) may be a critical component of skeletal EC-coupling. However, using Tdn-null mice we have shown that triadin ablation results in no significant disruption of skeletal EC-coupling. To analyze the role of triadin in EC-coupling signaling here we used whole-cell voltage clamp and simultaneous recording of intracellular Ca²+ release to characterize the retrograde and orthograde signaling between RyR1 and DHPR in cultured myotubes.

Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+.

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca(2+) ([Ca(2+)](rest)), suggesting a role for triadins in modulating global intracellular Ca(2+) homeostasis. To understand this mechanism, we study here how triadin alters [Ca(2+)](rest), Ca(2+) release, and Ca(2+) entry pathways using a combination of Ca(2+) microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca(2+), and Mn(2+) imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca(2+)](rest) that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity.

Ablation of triadin causes loss of cardiac Ca2+ release units, impaired excitation-contraction coupling, and cardiac arrhythmias.

Heart muscle excitation-contraction (E-C) coupling is governed by Ca(2+) release units (CRUs) whereby Ca(2+) influx via L-type Ca(2+) channels (Cav1.2) triggers Ca(2+) release from juxtaposed Ca(2+) release channels (RyR2) located in junctional sarcoplasmic reticulum (jSR). Although studies suggest that the jSR protein triadin anchors cardiac calsequestrin (Casq2) to RyR2, its contribution to E-C coupling remains unclear.

Enantiomeric specificity of (-)-2,2',3,3',6,6'-hexachlorobiphenyl toward ryanodine receptor types 1 and 2.

Polychlorinated biphenyls (PCBs) with unsymmetrical chlorine substitutions and multiple orthosubstitutions that restrict rotation around the biphenyl bond may exist in two stable enantiomeric forms.Stereospecific binding and functional modification of specific biological signaling targets have not been previously described for PCB atropisomers. We report that (-)-2,2',3,3',6,6'-hexachlorobiphenyl [(-)-PCB 136] enhances the binding of [3H]ryanodine to high-affinity sites on ryanodine receptors type 1(RyR1) and type 2 (RyR2) (EC50 values ~0.

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras.

We studied cation regulation of wild-type ryanodine receptor type 1 ((WT)RyR1), type 3 ((WT)RyR3), and RyR3/RyR1 chimeras (Ch) expressed in 1B5 dyspedic myotubes. Using [(3)H]ryanodine binding to sarcoplasmic reticulum (SR) membranes, Ca(2+) titrations with (WT)RyR3 and three chimeras show biphasic activation that is allosterically coupled to an attenuated inhibition relative to (WT)RyR1. Chimeras show biphasic Mg(2+) inhibition profiles at 3 and 10 microM Ca(2+), no observable inhibition at 20 microM Ca(2+) and monophasic inhibition at 100 microM Ca(2+).

Triadins modulate intracellular Ca(2+) homeostasis but are not essential for excitation-contraction coupling in skeletal muscle.

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype.

Bidirectional signaling between calcium channels of skeletal muscle requires multiple direct and indirect interactions.

We have defined regions of the skeletal muscle ryanodine receptor (RyR1) essential for bidirectional signaling with dihydropyridine receptors (DHPRs) and for the organization of DHPR into tetrad arrays by expressing RyR1-RyR3 chimerae in dyspedic myotubes. RyR1-RyR3 constructs bearing RyR1 residues 1-1681 restored wild-type DHPR tetrad arrays and, in part, skeletal-type excitation-contraction (EC) coupling (orthograde signaling) but failed to enhance DHPR Ca(2+) currents (retrograde signaling) to WT RyR1 levels. Within this region, the D2 domain (amino acids 1272-1455), although ineffective on its own, dramatically enhanced the formation of tetrads and EC coupling rescue by constructs that otherwise are only partially effective.

Trajectories associated to regional and extra-regional pollen transport in the southeast of Buenos Aires province, Mar del Plata (Argentina).

Long range transport of airborne pollen has been seldom studied in South America. Backward trajectories of Celtis and Nothofagus pollen grains trapped over a meteorological station outside Mar del Plata City were calculated in one-hour steps using the regional hybrid model developed by the NOAA (HYSPLIT 4.5) and the data of the NCEP filed in the NOAA server.

Efficient site-specific integration of large transgenes by an enhanced herpes simplex virus/adeno-associated virus hybrid amplicon vector.

We previously demonstrated that a herpes simplex virus type 1 (HSV-1)/adeno-associated virus (AAV) hybrid amplicon vector constructed by inserting the sequences of regulatory protein (rep) and inverted terminal repeats of AAV into an HSV amplicon vector resulted in the enhanced stability of transgene expression compared to the original HSV-1 amplicon vector. However, problems related to the expression of Rep compromised its therapeutic applications. We report here a new HSV/AAV hybrid amplicon vector system that not only solved problems associated with Rep expression but also markedly improved the stable transduction efficiency of this vector.

Expression levels of RyR1 and RyR3 control resting free Ca2+ in skeletal muscle.

To better understand the role of the transient expression of ryanodine receptor (RyR) type 3 (RyR3) on Ca(2+) homeostasis during the development of skeletal muscle, we have analyzed the effect of expression levels of RyR3 and RyR1 on the overall physiology of cultured myotubes and muscle fibers. Dyspedic myotubes were infected with RyR1 or RyR3 containing virions at 0.2, 0.

Amino acids 1-1,680 of ryanodine receptor type 1 hold critical determinants of skeletal type for excitation-contraction coupling. Role of divergence domain D2.

To identify domains of the ryanodine receptor (RyR1) that are functionally relevant for excitation-contraction (EC) coupling in vivo, we have studied the ability of RyR1/RyR3 chimera to rescue skeletal EC coupling in dyspedic myotubes. In this work we show that chimeric receptors containing amino acids 1-1,680 of RyR1 were able to render depolarization-induced Ca2+ release to RyR3. Within this region, residues 1,272-1,455, containing divergent domain D2 of RyR1, proved to be a critical element because the absence of this region selectively abolished depolarization-evoked Ca2+ transients without affecting chemically induced activation.

Identification of a key determinant of ryanodine receptor type 1 required for activation by 4-chloro-m-cresol.

4-Chloro-m-cresol (4-CmC) is a potent and specific activator of the intracellular Ca2+ release channel, the ryanodine receptor (RyR). We have previously shown that RyR1 expressed in dyspedic 1B5 myotubes is activated by 4-CmC, whereas RyR3 is not (Fessenden, J. D.

FKBP12 binding to RyR1 modulates excitation-contraction coupling in mouse skeletal myotubes.

The skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel or ryanodine receptor (RyR1) binds four molecules of FKBP12, and the interaction of FKBP12 with RyR1 regulates both unitary and coupled gating of the channel. We have characterized the physiologic effects of previously identified mutations in RyR1 that disrupt FKBP12 binding (V2461G and V2461I) on excitation-contraction (EC) coupling and intracellular Ca2+ homeostasis following their expression in skeletal myotubes derived from RyR1-knockout (dyspedic) mice. Wild-type RyR1-, V246I-, and V2461G-expressing myotubes exhibited similar resting Ca2+ levels and maximal responses to caffeine (10 mm) and cyclopiazonic acid (30 microm).

RyR1/RyR3 chimeras reveal that multiple domains of RyR1 are involved in skeletal-type E-C coupling.

Skeletal-type E-C coupling is thought to require a direct interaction between RyR1 and the alpha(1S)-DHPR. Most available evidence suggests that the cytoplasmic II-III loop of the dihydropyridine receptor (DHPR) is the primary source of the orthograde signal. However, identification of the region(s) of RyR1 involved in bidirectional signaling with the alpha(1S)-DHPR remains elusive.

Sea-land breeze in a coastal city and its effect on pollen transport.

This paper presents a statistical study of the sea-land breeze in Mar del Plata (Argentina) to characterize the periods of the year when the breeze affects pollen transport, particularly the dispersion of airborne Poaceae pollen between urban and rural areas. In order to analyse the sea breeze circulation, hourly data from coastal, urban and rural meteorological stations were used. The effect of the sea breeze on the particulate matter was analysed from syncronic hourly airborne pollen records from an urban and a rural area.