Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-ß1a subunit (CACNB1).

Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of ß1a subunit of the dihydropyridine receptor (DHPR), a channel essential for Ca2+ regulation in skeletal muscle. Here we evaluate the effect of the mutant variant V156A on the structure/function of DHPR ß1a subunit and assess its role on Ca2+ 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 ß1a subunit. Expression of the variant subunit in ß1-null mouse myotubes resulted in increased sensitivity to caffeine stimulation. Whole cell patch-clamp analysis of ß1a-V156A-expressing myotubes revealed a -2 mV shift in voltage dependence of channel activation, but no changes in Ca2+ conductance, current kinetics, or sarcoplasmic reticulum Ca2+ load were observed. Measurement of resting free Ca2+ and Na+ concentrations shows that both cations were significantly elevated in ß1a-V156A-expressing myotubes and that these changes were linked to increased rates of plasmalemmal Ca2+ entry through Na+/Ca2+ exchanger and/or transient receptor potential canonical channels. Overall, our data show that mutant variant V156A results in instability of protein subdomains of ß1a subunit leading to a phenotype of Ca2+ dysregulation that partly resembles that of other MH-linked mutations of DHPR α1S subunit. These data prove that homozygous expression of variant ß1a-V156A has the potential to be a pathological variant, although it may require other gene defects to cause a full MH phenotype.

A focus on extracellular Ca2+ entry into skeletal muscle.

The main task of skeletal muscle is contraction and relaxation for body movement and posture maintenance. During contraction and relaxation, Ca2+ in the cytosol has a critical role in activating and deactivating a series of contractile proteins. In skeletal muscle, the cytosolic Ca2+ level is mainly determined by Ca2+ movements between the cytosol and the sarcoplasmic reticulum. The importance of Ca2+ entry from extracellular spaces to the cytosol has gained significant attention over the past decade. Store-operated Ca2+ entry with a low amplitude and relatively slow kinetics is a main extracellular Ca2+ entryway into skeletal muscle. Herein, recent studies on extracellular Ca2+ entry into skeletal muscle are reviewed along with descriptions of the proteins that are related to extracellular Ca2+ entry and their influences on skeletal muscle function and disease.

Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor α1S Subunit Reveals Conformational Differences Induced by Binding of the ß1a 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. In dysgenic myotubes, energy transfer was observed from an N-terminal-fused YFP to a FRET acceptor, ReAsH (resorufin arsenical hairpin binder), targeted to each α1S intracellular loop, with the highest FRET efficiencies measured to the α1S II-III loop and C-terminal tail. However, in HEK-293T cells, FRET efficiencies from the α1S N terminus to the II-III and III-IV loops and the C-terminal tail were significantly lower, thus suggesting that these loop structures are influenced by the cellular microenvironment. The addition of the ß1a dihydropyridine receptor subunit enhanced FRET to the II-III loop, thus indicating that ß1a binding directly affects II-III loop conformation. This specific structural change required the C-terminal 36 amino acids of ß1a, which are essential to support EC coupling. Direct FRET measurements between α1S and ß1a confirmed that both wild type and truncated ß1a bind similarly to α1S These results provide new insights into the role of muscle-specific proteins on the structural arrangement of α1S intracellular loops and point to a new conformational effect of the ß1a subunit in supporting skeletal muscle excitation-contraction coupling.

Amino acid residues 489-503 of dihydropyridine receptor (DHPR) ß1a subunit are critical for structural communication between the skeletal muscle DHPR complex and type 1 ryanodine receptor.

The ß1a 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 ß1a 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. However, deletion of this domain did not affect expression/targeting or density (Qmax) of the DHPR-α1S subunit to the plasma membrane. Within this motif, triple alanine substitution of residues Leu(496), Leu(500), and Trp(503), which are thought to mediate direct ß1a-RyR1 interactions, weakened EC coupling but did not replicate the truncated phenotype. Therefore, these data demonstrate that an amino acid segment encompassing sequence (489)QVQVLTSLRRNLSFW(503) of ß1a contains critical determinant(s) for the physical link of DHPR and RyR1, further confirming a direct correspondence between DHPR positioning and DHPR/RyR functional interactions. In addition, our data strongly suggest that the motif Leu(496)-Leu(500)-Trp(503) within the ß1a C-terminal tail plays a nonessential role in the bidirectional DHPR/RyR1 signaling that supports skeletal-type EC coupling.

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. Whole body periodic acceleration (pGz) is produced by applying sinusoidal motion to supine humans and in standing conscious rodents in a headward-footward direction using a motion platform. It adds small pulses as a function of movement frequency to the circulation thereby increasing pulsatile shear stress to the vascular endothelium, which in turn increases production of NO. In this study, we examined the potential therapeutic properties of pGz for the treatment of skeletal muscle pathology observed in the mdx mouse. We found that pGz (480 cpm, 8 days, 1 hr per day) decreased intracellular Ca(2+) and Na(+) overload, diminished serum levels of creatine kinase (CK) and reduced intracellular accumulation of Evans Blue. Furthermore, pGz increased muscle force generation and expression of both utrophin and the carboxy-terminal PDZ ligand of nNOS (CAPON). Likewise, pGz (120 cpm, 12 h) applied in vitro to skeletal muscle myotubes reduced Ca(2+) and Na(+) overload, diminished abnormal sarcolemmal Ca(2+) entry and increased phosphorylation of endothelial NOS. Overall, this study provides new insights into the potential therapeutic efficacy of pGz as a non-invasive and non-pharmacological approach for the treatment of DMD patients through activation of the NO pathway.

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. [Ca2+]rest regulation by the CLR region was associated with alteration of RyRs' Ca2+-activation profile and changes in SR Ca2+-leak rates. Biochemical analysis using Tb3+-binding assays and intrinsic tryptophan fluorescence spectroscopy of purified CLR domains revealed that this determinant of RyRs holds a novel Ca2+-binding domain with conformational properties that are distinctive to each isoform. Our data suggest that the CLR region provides channels with unique functional properties that modulate the rate of passive SR Ca2+ leak and confer on RyR1 and RyR3 distinctive [Ca2+]rest regulatory properties. The identification of a new Ca2+-binding domain of RyRs with a key modulatory role in [Ca2+]rest regulation provides new insights into Ca2+-mediated regulation of RyRs.

Three-dimensional localization of the α and ß 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), ß(1a), δ1, and γ), we created transgenic mice expressing a recombinant ß(1a) subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α(2)-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of Ca(V)1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the ß subunit in a single docking possibility that defines the α1-ß interaction. The ß subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α(1s) subunit to the membrane and its suggested role in excitation-contraction coupling.

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. Both proteins proved to be important for the structural organization of jSR cisternae and retention of CASQ within them, but with different degrees of impact. Our results also suggest that the presence of CASQ is responsible for the wide lumen of the jSR cisternae. Using Ca(2+) imaging and Ca(2+) selective microelectrodes we found that changes in e-c coupling, SR Ca(2+)content and resting [Ca(2+)] in Jct, Tdn and Tdn/Jct-null muscles are directly correlated to the effect of each deletion on CASQ content and its organization within the jSR. These data suggest that in skeletal muscle the disruption of Tdn/CASQ link has a more profound effect on jSR architecture and myoplasmic Ca(2+) regulation than Jct/CASQ association.

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. This major Ca(2+) traffic point appears to be highly sensitive to the intracellular environment, which senses through a plethora of chemical and protein-protein interactions. Among these modulators, perhaps one of the most elusive is Triadin, a muscle-specific protein that is involved in many crucial aspect of muscle function. This family of proteins mediates complex interactions with various Ca(2+) modulators and seems poised to be a relevant modulator of Ca(2+) signaling in cardiac and skeletal muscles. The purpose of this review is to examine the most recent evidence and current understanding of the role of Triadin in muscle function, in general, with particular emphasis on its contribution to 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. DHPR Ca²+ currents elicited by depolarization of Wt and Tdn-null myotubes displayed similar current densities and voltage dependence. However, kinetic analysis of the Ca²+ current shows that activation time constant of the slow component was slightly decreased in Tdn-null cells. Voltage-evoked Ca²+ transient of Tdn-null myotubes showed small but significant reduction in peak fluorescence amplitude but no differences in voltage dependence. This difference in Ca²+ amplitude was averted by over-expression of FKBP12.6. Our results show that bi-directional signaling between DHPR and RyR1 is preserved nearly intact in Tdn-null myotubes and that the effect of triadin ablation on Ca²+ transients appears to be secondary to the reduced FKBP12 binding capacity of RyR1 in Tdn-null myotubes. These data suggest that skeletal triadins do not play a direct role in skeletal EC-coupling.

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. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn(2+) quench rates under resting conditions indicate that triadin-null cells also have higher Ca(2+) entry rates and lower sarcoplasmic reticulum Ca(2+) load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca(2+) entry, recovering sarcoplasmic reticulum Ca(2+) content levels, and restoring near normal [Ca(2+)](rest). Exogenous FKBP12.6 also reduced the RyR1 channel P(o) but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P(o) nor recovered multiple subconductance gating. These data suggest that elevated [Ca(2+)](rest) in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca(2+) entry at rest.

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. Here, we identify the role of triadin using mice with ablation of the Trdn gene (Trdn(-/-)). The structure and protein composition of the cardiac CRU is significantly altered in Trdn(-/-) hearts. jSR proteins (RyR2, Casq2, junctin, and junctophilin 1 and 2) are significantly reduced in Trdn(-/-) hearts, whereas Cav1.2 and SERCA2a remain unchanged. Electron microscopy shows fragmentation and an overall 50% reduction in the contacts between jSR and T-tubules. Immunolabeling experiments show reduced colocalization of Cav1.2 with RyR2 and substantial Casq2 labeling outside of the jSR in Trdn(-/-) myocytes. CRU function is impaired in Trdn(-/-) myocytes, with reduced SR Ca(2+) release and impaired negative feedback of SR Ca(2+) release on Cav1.2 Ca(2+) currents (I(Ca)). Uninhibited Ca(2+) influx via I(Ca) likely contributes to Ca(2+) overload and results in spontaneous SR Ca(2+) releases upon beta-adrenergic receptor stimulation with isoproterenol in Trdn(-/-) myocytes, and ventricular arrhythmias in Trdn(-/-) mice. We conclude that triadin is critically important for maintaining the structural and functional integrity of the cardiac CRU; triadin loss and the resulting alterations in CRU structure and protein composition impairs E-C coupling and renders hearts susceptible to ventricular arrhythmias.

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 ~0.95 microM), whereas (+)-PCB 136 is inactive at < or =10 microM.(-)-PCB 136 induces a rapid release of Ca2+ from microsomal vesicles by selective sensitization of RyRs, an effect not antagonized by (+)-PCB 136. (-)-PCB 136 (500nM) enhances the activity of reconstituted RyR1 channels 3-fold by stabilizing the open and destabilizing the closed conformational states. The enantiomeric specificity is also demonstrated in intact HEK 293 cells expressing RyR1 where exposure to (-)-PCB 136 (100 nM; 12 h) sensitizes responses to caffeine, whereas (+)-PCB 136 does not. These data show enantiomeric specificity of (-)-PCB 136 toward a broadly expressed family of microsomal Ca2+ channels that may extend to other chiral noncoplanar PCBs and related structures.Evidence for enantioselective enrichment of PCBs in biological tissues that express RyR1 and RyR2channels may provide new mechanistic leads about their toxicological impacts on human health

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+). Ca(2+) imaging of intact myotubes expressing Ch-4 exhibit caffeine-induced Ca(2+) transients with inhibition kinetics that are significantly slower than those expressing (WT)RyR1 or (WT)RyR3. Four new aspects of RyR regulation are evident: (1) high affinity (H) activation and low affinity (L) inhibition sites are allosterically coupled, (2) Ca(2+) facilitates removal of the inherent Mg(2+) block, (3) (WT)RyR3 exhibits reduced cooperativity between H activation sites when compared to (WT)RyR1, and (4) uncoupling of these sites in Ch-4 results in decreased rates of inactivation of caffeine-induced Ca(2+) transients.

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. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca(2+) imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca(2+) transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca(2+).[(3)H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca(2+) activation or Ca(2+) and Mg(2+) inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca(2+) homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.

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. These findings suggest that the orthograde signal and DHPR tetrad formation require the contributions of numerous RyR regions. Surprisingly, we found that RyR3, although incapable of supporting EC coupling or tetrad formation, restored a significant level of Ca(2+) current, revealing a functional interaction with the skeletal muscle DHPR. Thus, our data support the hypotheses that (i) the structural/functional link between RyR1 and the skeletal muscle DHPR requires multiple interacting regions, (ii) the D2 domain of RyR1 plays a key role in stabilizing this interaction, and (iii) a form of retrograde signaling from RyR3 to the DHPR occurs in the absence of direct protein-protein interactions.

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. Results showed that the observed trajectories agree with the location of vegetation sources of the collected tree species. In the case of Celtis, the transport was associated to anticyclones located east of the city, generating winds with a N-NE component, which produce pollen cloud advection from the Celtis forests located some tens of kilometers to the N and NE of the city. The sources of Nothofagus pollen correspond to a narrow strip on the Andes slopes between 39 degrees and 55 degrees S, at least 1100 km to the SW of Mar del Plata. The transport was associated to eastward displacement of the troughs corresponding to the Westerlies circulation and the presence of an anticyclone system that brings back Nothofagus pollen towards Mar del Plata area.

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. This new HSV/AAV vector is designed in a way that little or no Rep would be expressed in packaging cells, but it can be expressed in transduced cells if Cre recombinase is provided. Furthermore, Rep expression will be automatically suppressed as a consequence of Rep-mediated integration. Our results showed that the new hybrid amplicon vector yielded titers comparable to those of standard amplicon vectors. When Cre-expressing 293 cells were transduced, a low level of Rep expression was detected, and stable transduction was achieved in approximately 22% of transduced cells; of those cells, approximately 70% transduction was achieved by Rep-mediated site-specific integration. In the majority of the stably transduced cells, Rep expression was no longer observed. Our results also proved that this vector system is capable of efficiently accommodating and site-specifically integrating large transgenes, such as the full-length dystrophin expression cassette. Thus, the new HSV/AAV vector demonstrated unique advantages in safe and effective delivery of long-lasting transgene expression into human cells.

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, 0.4, 1.0, and 4.0 moieties of infection (MOI), and analysis of their pattern of expression, caffeine sensitivity, and resting free Ca(2+) concentration ([Ca(2+)](r)) was performed. Although increased MOI resulted in increased expression of each receptor isoform, it did not significantly affect the immunopattern of RyRs or the expression levels of calsequestrin, triadin, or FKBP-12. Interestingly, myotubes expressing RyR3 always had significantly higher [Ca(2+)](r) and lower caffeine EC(50) than did cells expressing RyR1. Although some of the increased sensitivity of RyR3 to caffeine could be attributed to the higher [Ca(2+)](r) in RyR3-expressing cells, studies of [(3)H]ryanodine binding demonstrated intrinsic differences in caffeine sensitivity between RyR1 and RyR3. Tibialis anterior (TA) muscle fibers at different stages of postnatal development exhibited a transient increase in [Ca(2+)](r) coordinately with their level of RyR3 expression. Similarly, adult soleus fibers, which also express RyR3, had higher [Ca(2+)](r) than did adult TA fibers, which exclusively express RyR1. These data show that in skeletal muscle, RyR3 increases [Ca(2+)](r) more than RyR1 does at any expression level. These data suggest that the coexpression of RyR1 and RyR3 at different levels may constitute a novel mechanism by which to regulate [Ca(2+)](r) in skeletal muscle.

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. Although the D2 domain by itself failed to restore skeletal EC coupling to RyR3, the addition of the D2 region resulted in a dramatic enhancement of EC coupling restored by an RyR3 chimera containing amino acids 1,681-3,770 of RyR1. These results suggest that although the D2 domain of RyR1 plays a key role during EC coupling, additional region(s) from the N-terminal end of RyR1 as well as previously identified regions of the central portion of the receptor are needed in order to allow normal EC coupling.