Colocalization of dihydropyridine and ryanodine receptors in neonate rabbit heart using confocal microscopy

Because of undeveloped T tubules and sparse sarcoplasmic reticulum, Ca(2+)-induced Ca(2+) release (CICR) may not be the major mechanism providing contractile Ca(2+) in the neonatal heart. Spatial association of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), a key factor for CICR, was examined in isolated neonatal rabbit ventricular myocytes aged 3-20 days by double-labeling immunofluorescence and confocal microscopy. We found a significant increase (P < 0.0005) in the degree of colocalization of DHPR and RyR during development. The number of voxels containing DHPR that also contained RyR in the 3-day-old group (62 +/- 1.8%) was significantly lower than in the other age groups (76 +/- 1.3 in 6-day old, 75 +/- 1.2 in 10-day old, and 79 +/- 0.9% in 20-day old). The number of voxels containing RyR that also contained DHPR was significantly higher in the 20-day-old group (17 +/- 0.5%) compared with the other age groups (10 +/- 0.7 in 3-day old, 11 +/- 0.6 in 6-day old, and 11 +/- 0.5% in 10-day old). During this period, the pattern of colocalization changed from mostly peripheral to mostly internal couplings. Our results provide a structural basis for the diminished prominence of CICR in neonatal heart.     

Ryanodine and dihydropyridine receptor binding in ventricular cardiac muscle of fish with different temperature preferences

Ca(2+)-induced Ca2+ release (CICR) mechanism of cardiac excitation-contraction (e-c) coupling is dependent on the close apposition between the sarcolemmal dihydropyridine receptors (DHPR) and the sarcoplasmic reticulum (SR) ryanodine receptors (RyR). In particular, high RyR/DHPR ratio is considered to reflect strong dependence on SR Ca2+ stores for the intracellular Ca2+ transient. To indirectly evaluate the significance of CICR in fish hearts, densities of cardiac DHPRs and RyRs were compared in ventricular homogenates of three fish species (burbot, rainbow trout, and crucian carp) and adult rat by [3H] PN200-110 and [3H] ryanodine binding. The density of RyRs was significantly (P<0.05) higher in the adult rat (124+/-10 channels/microm3 myocyte volume) than in any of the fish species. Among the fish species, cold-acclimated (4 degrees C) trout had more RyRs than burbot, and crucian carp. The density of DHPRs was highest in the trout heart. RyR/DHPR ratio was significantly (P<0.05) higher in rat (4.1+/-0.5) than in the fish hearts (varying from 0.97+/-0.16 to 1.91+/-0.49) suggesting that "mammalian type" CICR is less important during e-c coupling in fish ventricular myocytes. In rainbow trout, acclimation to cold did not affect the RyR/DHPR ratio, while in crucian carp it was depressed in cold-acclimated animals (4 degrees C; 0.97+/-0.16) when compared to warm-acclimated fish (23 degrees C; 1.91+/-0.49). Although RyR/DHPR ratios were relatively low in fish hearts, there was a close correlation (r2=0.78) between the RyR/DHPR ratio and the magnitude of the Ry-sensitive component of contraction in ventricular muscle among the fish species examined in this study.     

Ca2+-dependent excitation-contraction coupling triggered by the heterologous cardiac/brain DHPR beta2a-subunit in skeletal myotubes

Molecular determinants essential for skeletal-type excitation-contraction (EC) coupling have been described in the cytosolic loops of the dihydropyridine receptor (DHPR) alpha1S pore subunit and in the carboxyl terminus of the skeletal-specific DHPR beta1a-subunit. It is unknown whether EC coupling domains present in the beta-subunit influence those present in the pore subunit or if they act independent of each other. To address this question, we investigated the EC coupling signal that is generated when the endogenous DHPR pore subunit alpha1S is paired with the heterologous heart/brain DHPR beta2a-subunit. Studies were conducted in primary cultured myotubes from beta1 knockout (KO), ryanodine receptor type 1 (RyR1) KO, ryanodine receptor type 3 (RyR3) KO, and double RyR1/RyR3 KO mice under voltage clamp with simultaneous monitoring of confocal fluo-4 fluorescence. The beta2a-mediated Ca2+ current recovered in beta1 KO myotubes lacking the endogenous DHPR beta1a-subunit verified formation of the alpha1S/beta1a pair. In myotube genotypes which express no or low-density L-type Ca2+ currents, namely beta1 KO and RyR1 KO, beta2a overexpression recovered a wild-type density of nifedipine-sensitive Ca2+ currents with a slow activation kinetics typical of skeletal myotubes. Concurrent with Ca2+ current recovery, there was a drastic reduction of voltage-dependent, skeletal-type EC coupling and emergence of Ca2+ transients triggered by the Ca2+ current. A comparison of beta2a overexpression in RyR3 KO, RyR1 KO, and double RyR1/RyR3 KO myotubes concluded that both RyR1 and RyR3 isoforms participated in Ca2+-dependent Ca2+ release triggered by the beta2a-subunit. In beta1 KO and RyR1 KO myotubes, the Ca2+-dependent EC coupling promoted by beta2a overexpression had the following characteristics: 1), L-type Ca2+ currents had a wild-type density; 2), Ca2+ transients activated much slower than controls overexpressing beta1a, and the rate of fluorescence increase was consistent with the activation kinetics of the Ca2+ current; 3), the voltage dependence of the Ca2+ transient was bell-shaped and the maximum was centered at approximately +30 mV, consistent with the voltage dependence of the Ca2+ current; and 4), Ca2+ currents and Ca2+ transients were fully blocked by nifedipine. The loss in voltage-dependent EC coupling promoted by beta2a was inferred by the drastic reduction in maximal Ca2+ fluorescence at large positive potentials (DeltaF/Fmax) in double dysgenic/beta1 KO myotubes overexpressing the pore mutant alpha1S (E1014K) and beta2a. The data indicate that beta2a, upon interaction with the skeletal pore subunit alpha1S, overrides critical EC coupling determinants present in alpha1S. We propose that the alpha1S/beta pair, and not the alpha1S-subunit alone, controls the EC coupling signal in skeletal muscle.     

Role of the sequence surrounding predicted transmembrane helix M4 in membrane association and function of the Ca(2+) release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor isoform 1)

The role of the sequence surrounding M4 in ryanodine receptors (RyR) in membrane association and function was investigated. This sequence contains a basic, 19-amino acid M3/M4 loop, a hydrophobic 44-49 amino acid sequence designated M4 (or M4a/M4b), and a hydrophilic M4/M5 loop. Enhanced green fluorescent protein (EGFP) was inserted into RyR1 and truncated just after the basic sequence, just after M4, within the M4/M5 loop, just before M5 and just after M5. The A52 epitope was inserted into RyR2 and truncated just after M4a. Analysis of these constructs ruled out a M3/M4 transmembrane hairpin and narrowed the region of membrane association to M4a/M4b. EGFP inserted between M4a and M4b in full-length RyR2 was altered conformationally, losing fluorescence and gaining trypsin sensitivity. Although it was accessible to an antibody from the cytosolic side, tryptic fragments were membrane-bound. The expressed protein containing EGFP retained caffeine-induced Ca(2+) release channel function. These results suggest that M4a/M4b either forms a transmembrane hairpin or associates in an unorthodox fashion with the cytosolic leaflet of the membrane, possibly involving the basic M3/M4 loop. The expression of a mutant RyR1, Delta4274-4535, deleted in the sequence surrounding both M3 and M4, restored robust, voltage-gated L-type Ca(2+) currents and Ca(2+) transients in dyspedic myotubes, demonstrating that this sequence is not required for either orthograde (DHPR activation of sarcoplasmic reticulum Ca(2+) release) or retrograde (RyR1 increase in DHPR Ca(2+) channel activity) signals of excitation-contraction coupling. Maximal amplitudes of L-currents and Ca(2+) transients with Delta4274-4535 were larger than with wild-type RyR1, and voltage-gated sarcoplasmic reticulum Ca(2+) release was more sensitive to activation by sarcolemmal voltage sensors. Thus, this region may act as a negative regulatory module that increases the energy barrier for Ca(2+) release channel opening.     

Differential regulation of skeletal muscle L-type Ca2+ current and excitation-contraction coupling by the dihydropyridine receptor beta subunit

The dihydropyridine receptor (DHPR) of skeletal muscle functions as a Ca2+ channel and is required for excitation-contraction (EC) coupling. Here we show that the DHPR beta subunit is involved in the regulation of these two functions. Experiments were performed in skeletal mouse myotubes selectively lacking a functional DHPR beta subunit. These beta-null cells have a low-density L-type current, a low density of charge movements, and lack EC coupling. Transfection of beta-null cells with cDNAs encoding for either the homologous beta1a subunit or the cardiac- and brain-specific beta2a subunit fully restored the L-type Ca2+ current (161 +/- 17 pS/pF and 139 +/- 9 pS/pF, respectively, in 10 mM Ca2+). We compared the Boltzmann parameters of the Ca2+ conductance restored by beta1a and beta2a, the kinetics of activation of the Ca2+ current, and the single channel parameters estimated by ensemble variance analysis and found them to be indistinguishable. In contrast, the maximum density of charge movements in cells expressing beta2a was significantly lower than in cells expressing beta1a (2.7 +/- 0.2 nC/microF and 6.7 +/- 0. 4 nC/microF, respectively). Furthermore, the amplitude of Ca2+ transient measured by confocal line-scans of fluo-3 fluorescence in voltage-clamped cells were 3- to 5-fold lower in myotubes expressing beta2a. In summary, DHPR complexes that included beta2a or beta1a restored L-type Ca2+ channels. However, a DHPR complex with beta1a was required for complete restoration of charge movements and skeletal-type EC coupling. These results suggest that the beta1a subunit participates in key regulatory events required for the EC coupling function of the DHPR.     

Multiple regions of RyR1 mediate functional and structural interactions with alpha(1S)-dihydropyridine receptors in skeletal muscle

Excitation-contraction (e-c) coupling in muscle relies on the interaction between dihydropyridine receptors (DHPRs) and RyRs within Ca(2+) release units (CRUs). In skeletal muscle this interaction is bidirectional: alpha(1S)DHPRs trigger RyR1 (the skeletal form of the ryanodine receptor) to release Ca(2+) in the absence of Ca(2+) permeation through the DHPR, and RyR1s, in turn, affect the open probability of alpha(1S)DHPRs. alpha(1S)DHPR and RyR1 are linked to each other, organizing alpha(1S)-DHPRs into groups of four, or tetrads. In cardiac muscle, however, alpha(1C)DHPR Ca(2+) current is important for activation of RyR2 (the cardiac isoform of the ryanodine receptor) and alpha(1C)-DHPRs are not organized into tetrads. We expressed RyR1, RyR2, and four different RyR1/RyR2 chimeras (R4: Sk1635-3720, R9: Sk2659-3720, R10: Sk1635-2559, R16: Sk1837-2154) in 1B5 dyspedic myotubes to test their ability to restore skeletal-type e-c coupling and DHPR tetrads. The rank-order for restoring skeletal e-c coupling, indicated by Ca(2+) transients in the absence of extracellular Ca(2+), is RyR1 > R4 > R10 > R16 > R9 > RyR2. The rank-order for restoration of DHPR tetrads is RyR1 > R4 = R9 > R10 = R16 > RyR2. Because the skeletal segment in R9 does not overlap with that in either R10 or R16, our results indicate that multiple regions of RyR1 may interact with alpha(1S)DHPRs and that the regions responsible for tetrad formation do not correspond exactly to the ones required for functional coupling.     

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.     

Subtype specificity of the ryanodine receptor for Ca2+ signal amplification in excitation-contraction coupling.

In excitable cells membrane depolarization is translated into intracellular Ca2+ signals. The ryanodine receptor (RyR) amplifies the Ca2+ signal by releasing Ca2+ from the intracellular Ca2+ store upon receipt of a message from the dihydropyridine receptor (DHPR) on the plasma membrane in striated muscle. There are two distinct mechanisms for the amplification of Ca2+ signalling. In cardiac cells depolarization-dependent Ca2+ influx through DHPR triggers Ca2+-induced Ca2+ release via RyR, while in skeletal muscle cells a voltage-induced change in DHPR is thought to be mechanically transmitted, without a requirement for Ca2+ influx, to RyR to cause it to open. In expression experiments using mutant skeletal myocytes lacking an intrinsic subtype of RyR (RyR-1), we demonstrate that RyR-1, but not the cardiac subtype (RyR-2), is capable of supporting skeletal muscle-type coupling. Furthermore, when RyR-2 was expressed in skeletal myocytes, we observed depolarization-independent spontaneous Ca2+ waves and oscillations, which suggests that RyR-2 is prone to regenerative Ca2+ release responses. These results demonstrate functional diversity among RyR subtypes and indicate that the subtype of RyR is the key to Ca2+ signal amplification.     

Arg(615)Cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca(2+) release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr(671)-Leu(690); 36 microM) enhanced the rate of Ca(2+) release from normal SR (SR(N)) and from SR of MH-susceptible muscle (SR(MH)) by 10 +/- 3.2 nmole/mg/min and 76 +/- 9.7 nmole/mg/min, respectively. Ca (2+) release from SR(N) or SR(MH) was not increased by control peptide NB (Gly(689)-Lys(708)). AS (scrambled A sequence; 36 microM) did not alter Ca (2+) release from SR(N), but increased release from SR(MH) by 29 +/- 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR(MH)) were up to about fourfold more strongly activated by peptide A (> or =1 nM) than normal RyR channels (RyR(N)) at -40 mV. Neither NB or AS activated RyR(N). RyR(MH) showed an approximately 1.8-fold increase in mean current with 30 microM AS. Inhibition at +40 mV was stronger in RyR(MH) and seen with peptide A (> or = 0.6 microM) and AS (> or = 0.6 microM), but not NB. These results show that the Arg(615)Cys substitution in RyR(MH) has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca(2+) release from SR in MH-susceptible muscle.     

不同频率慢性电刺激膈神经对兔膈肌骨骼肌型二氢吡啶受体α1亚单位、ryanodine受体mRNA和蛋白表达的影响

测定不同频率慢性电刺激(chronic electrical stimulation,CES)膈神经5周后对兔膈肌钙释放单位中骨骼肌型二氢吡啶受体(DHPR)α1亚单位和ryanodine受体(RyRs)的mRNA和蛋白表达水平的影响,探讨CES后兔膈肌钙释放单位结构组成的变化和可能的临床应用价值.封闭群日本大耳白兔30只,随机分为正常对照组、10、20、50和100Hz,每组6只;以10和20 Hz为慢性低频电刺激组,50和100Hz为慢性高频电刺激组.CES参数为波宽0.2 ms 3～6个波/次,45次/min,电压10～20 V.刺激时间2×2 h/日,每周刺激6 d,连续刺激5周.分别采用RT-PCR和免疫组织化学法测定兔膈肌骨骼肌型DHPRα1-亚单位和RyR1、RyR2和RyR3的mRNA和蛋白表达.结果显示与对照组比较,慢性低频电刺激10和20 Hz组骨骼肌型DHPRα1、RyR的mRNA和蛋白表达明显降低(P＜0.01),有低度的RyR,mRNA的表达出现;慢性高频电刺激50和100Hz组骨骼肌型DHPRα1、RyR1的mRNA和蛋白表达明显升高(P＜0.01),未检测到RyR2mRNA的阳性表达.本实验提示慢性低频电刺激膈神经5周后,膈肌质膜上DHPR与RyRs之间的信号转导方式已从变构耦联为主转变为以Ca2+诱导Ca2+释放耦联为主.     

Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).     

Dynamics of tight and adherens junctions under EGTA treatment

The dynamics of tight junctions (TJs) and adherens junctions (AJs) under EGTA treatment were investigated in Madin Darby canine kidney (MDCK) cells. Detailed information about the behavior of TJ and AJ proteins during the opening and resealing of TJs and AJs is still scarce. By means of the "calcium chelation" method, the distribution and colocalization of junctional proteins were studied with confocal laser scanning microscopy using a deconvolution algorithm for high-resolution images. Colocalization was analyzed for pairs of the following proteins: ZO-1, occludin, claudin-1, E-cadherin and F-actin. Significant differences were found for the analyzed pairs in control cells compared to EGTA-treated cells with respect to the position of the colocalization maxima within the cell monolayers as well as with respect to the amount of colocalized voxels. Under EGTA treatment, colocalization for ZO-1/occludin, ZO-1/claudin-1, claudin-1/occludin, E-cadherin/occludin and E-cadherin/claudin-1 dropped below 35% of the control value. Only for the ZO-1/E-cadherin pair, the amount of colocalized voxels increased and a shift to a more basal position was observed. During the opening of TJs and AJs, ZO-1 colocalized with E-cadherin in the lateral membrane region, whereas in controls, ZO-1 colocalized with occludin and claudin-1 in the junctional complex. The combination of deconvolution with colocalization analysis of confocal data sets offers a powerful tool to investigate the spatial relationship of TJ and AJ proteins during assembly and disassembly of cell-cell contacts.     

Rapid activation of the cardiac ryanodine receptor by submillisecond calcium stimuli

The local control concept of excitation-contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca(2+) entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (<0.5 ms) Ca(2+) elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca(2+) signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca(2+) generated by flash photolysis of DM-nitrophen. Application of such Ca(2+) spikes (amplitude approximately 10-30 microM, duration approximately 0.1-0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope approximately 2.5) with spike amplitude. The time constants of RyR activation were 0.07-0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a power n approximately 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca(2+)-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca(2+) triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca(2+) ions in accordance with the tetrameric organization of the channel protein.     

A component of excitation-contraction coupling triggered in the absence of the T671-L690 and L720-Q765 regions of the II-III loop of the dihydropyridine receptor alpha(1s) pore subunit.

We conducted a deletion analysis of two regions identified in the II-III loop of alpha(1S), residues 671-690, which were shown to bind to ryanodine receptor type 1 (RyR1) and stimulate RyR1 channels in vitro, and residues 720-765 or the narrower 724-743 region, which confer excitation-contraction (EC) coupling function to chimeric dihydropyridine receptors (DHPRs). Deletion mutants were expressed in dysgenic alpha(1S)-null myotubes and analyzed by voltage-clamp and confocal fluo-4 fluorescence. Immunostaining of the mutant subunits using an N-terminus tag revealed abundant protein expression in all cases. Furthermore, the maximum recovered charge movement density was >80% of that recovered by full-length alpha(1S) in all cases. Delta671-690 had no effect on the magnitude of voltage-evoked Ca(2+) transients or the L-type Ca(2+) current density. In contrast, Delta720-765 or Delta724-743 abolished Ca(2+) transients entirely, and L-type Ca(2+) current was reduced or absent. Surprisingly, Ca(2+) transients and Ca(2+) currents of a moderate magnitude were recovered by the double deletion mutant Delta671-690/Delta720-765. A simple explanation for this result is that Delta720-765 induces a conformation change that disrupts EC coupling, and this conformational change is partially reverted by Delta671-690. To test for Ca(2+)-entry independent EC coupling, a pore mutation (E1014K) known to entirely abolish the inward Ca(2+) current was introduced. alpha(1S) Delta671-690/Delta720-765/E1014K expressed Ca(2+) transients with Boltzmann parameters identical to those of the Ca(2+)-conducting double deletion construct. The data strongly suggest that skeletal-type EC coupling is not uniquely controlled by alpha(1S) 720-765. Other regions of alpha(1S) or other DHPR subunits must therefore directly contribute to the activation of RyR1 during EC coupling.     

兔膈肌骨骼肌型DHPRα1亚单位和RyRs的mRNA与蛋白表达测定

本文测定了兔膈肌钙释放单位中骨骼肌型ＤＨＰＲα１－亚单位和ＲｙＲｓ的ｍＲＮＡ与蛋白表达水平,探讨了兔膈肌钙释放单位的结构组成特征。采用ＲＴ－ＰＣＲ、原位杂交和免疫组织化学技术,分别测定兔膈肌骨骼肌型ＤＨＰＲα１－亚单位和ＲｙＲ１、ＲｙＲ２及ＲｙＲ３的ｍＲＮＡ与蛋白表达。结果显示,兔膈肌可见较高水平的骨骼肌型α１－亚单位和ＲｙＲ１ｍＲＮＡ与蛋白表达;较低水平的ＲｙＲ３ ｍＲＮＡ与蛋白表达。表明兔膈肌     

Dynamic alterations in myoplasmic Ca2+ in malignant hyperthermia and central core disease

Ca2+ ions play a pivotal role in a wide array of cellular processes ranging from fertilization to cell death. In skeletal muscle, a mechanical interaction between plasma membrane dihydropyridine receptors (DHPRs, L-type Ca2+ channels) and Ca2+ release channels (ryanodine receptors, RyR1s) of the sarcoplasmic reticulum orchestrates a complex, bi-directional Ca2+ signaling process that converts electrical impulses in the sarcolemma into myoplasmic Ca2+ transients during excitation-contraction coupling. Mutations in the genes that encode the two proteins that coordinate this electrochemical conversion process (the DHPR and RyR1) result in a variety of skeletal muscle disorders including malignant hyperthermia (MH), central core disease (CCD), multiminicore disease, nemaline rod myopathy, and hypokalemic periodic paralysis. Although RyR1 and DHPR disease mutations are thought to alter excitability and Ca2+ homeostasis in skeletal muscle, only recently has research begun to probe the molecular mechanisms by which these genetic defects lead to distinct clinical and histopathological manifestations. This review focuses on recent advances in determining the impact of MH and CCD mutations in RyR1 on muscle Ca2+ signaling and how these effects contribute to disease-specific aspects of these disorders.     

Formation of triads without the dihydropyridine receptor alpha subunits in cell lines from dysgenic skeletal muscle

Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR.     

Immunolocalization of sarcolemmal dihydropyridine receptor and sarcoplasmic reticular triadin and ryanodine receptor in rabbit ventricle and atrium

The subcellular distribution of sarcolemmal dihydropyridine receptor (DHPR) and sarcoplasmic reticular triadin and Ca2+ release channel/ryanodine receptor (RyR) was determined in adult rabbit ventricle and atrium by double labeling immunofluorescence and laser scanning confocal microscopy. In ventricular muscle cells the immunostaining was observed primarily as transversely oriented punctate bands spaced at approximately 2-micron intervals along the whole length of the muscle fibers. Image analysis demonstrated a virtually complete overlap of the staining patterns of the three proteins, suggesting their close association at or near dyadic couplings that are formed where the sarcoplasmic reticulum (SR) is apposed to the surface membrane or its infoldings, the transverse (T-) tubules. In rabbit atrial cells, which lack an extensive T-tubular system, DHPR-specific staining was observed to form discrete spots along the sarcolemma but was absent from the interior of the fibers. In atrium, punctate triadin- and RyR-specific staining was also observed as spots at the cell periphery and image analysis indicated that the three proteins were co- localized at, or just below, the sarcolemma. In addition, in the atrial cells triadin- and RyR-specific staining was observed to form transverse bands in the interior cytoplasm at regularly spaced intervals of approximately 2 micron. Electron microscopy suggested that this cytoplasmic staining was occurring in regions where substantial amounts of extended junctional SR were present. These data indicate that the DHPR codistributes with triadin and the RyR in rabbit ventricle and atrium, and furthermore suggest that some of the SR Ca2+ release channels in atrium may be activated in the absence of a close association with the DHPR.     

Redox modulation of diaphragm contractility: Interaction between DHPR and RyR channels

Previous reports indicate that reactive oxygen species (ROS) may modulate contractility in skeletal muscle. Although Ca(2+)-sensitivity of the contractile apparatus appears to be a primary site of regulation, dihydropyridine receptor (DHPR or L-type Ca(2+) channels) and calcium efflux in isolated sarcoplasmic reticulum (SR) vesicles appear to be redox sensitive as well. However, DHPR as a target is poorly understood in intact muscles at body temperature, particularly in the diaphragm, a muscle more dependent on external Ca(2+) than locomotor muscles. Previously, we reported that oxidant challenge via xanthine oxidase (XO) alters the K(+) contractures in diaphragm fiber bundles, suggestive of a role of L-type Ca(2+) channels. Contractility of isolated rat diaphragm fiber bundles revealed a biphasic response to ROS challenge that was dose and time dependent. Potentiation of twitch and low-frequency diaphragm fiber bundle contractility with 0.02 U?ml(-1) XO was reversible or partially preventable with washout, dithiothreitol, and the SOD/catalase mimetic EUK-134. The RyR antagonist ruthenium red inhibited xanthine oxidase-induced potentiation, while the RyR agonist caffeine elevated diaphragm twitch and low-frequency tension in a non-additive manner by 55% when introduced simultaneously with ROS challenge. The DHPR antagonist nitrendipine (15 μM) inhibited elevation in low-frequency diaphragm tension produced by ROS challenge. Caffeine threshold tension curves were shifted to the left with 0.02 U?ml(-1) XO, but this effect was partially reversed with 15 μM nitrendipine. These results are consistent with the hypothesis that DHPR redox state and RyR function are modulated in an interactive manner, affecting contractility in intact diaphragm fiber bundles.     

The β(1a) subunit of the skeletal DHPR binds to skeletal RyR1 and activates the channel via its 35-residue C-terminal tail

Although it has been suggested that the C-terminal tail of the β(1a) subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca(2+) release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β(1a) bound to RyR1 in affinity chromatography. The full-length β(1a) subunit and the C-terminal peptide increased [(3)H]ryanodine binding and RyR1 channel activity with an AC(50) of 450-600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca(2+), ATP, and Mg(2+) concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg(2+) inhibition or addition of 100 nM Ca(2+) (without ATP). Maximum increases were seen with 1-10 μM Ca(2+), in the absence of Mg(2+) inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [(3)H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β(1a) subunit and RyR1 may support an in vivo function of β(1a) during voltage-activated Ca(2+) release.