共查询到20条相似文献,搜索用时 31 毫秒
1.
Du W McMahon TJ Zhang ZS Stiber JA Meissner G Eu JP 《The Journal of biological chemistry》2006,281(40):30143-30151
Excitation-contraction (EC) coupling in striated muscles is mediated by the cardiac or skeletal muscle isoform of voltage-dependent L-type Ca(2+) channel (Ca(v)1.2 and Ca(v)1.1, respectively) that senses a depolarization of the cell membrane, and in response, activates its corresponding isoform of intracellular Ca(2+) release channel/ryanodine receptor (RyR) to release stored Ca(2+), thereby initiating muscle contraction. Specifically, in cardiac muscle following cell membrane depolarization, Ca(v)1.2 activates cardiac RyR (RyR2) through an influx of extracellular Ca(2+). In contrast, in skeletal muscle, Ca(v)1.1 activates skeletal muscle RyR (RyR1) through a direct physical coupling that negates the need for extracellular Ca(2+). Since airway smooth muscle (ASM) expresses Ca(v)1.2 and all three RyR isoforms, we examined whether a cardiac muscle type of EC coupling also mediates contraction in this tissue. We found that the sustained contractions of rat ASM preparations induced by depolarization with KCl were indeed partially reversed ( approximately 40%) by 200 mum ryanodine, thus indicating a functional coupling of L-type channels and RyRs in ASM. However, KCl still caused transient ASM contractions and stored Ca(2+) release in cultured ASM cells without extracellular Ca(2+). Further analyses of rat ASM indicated that this tissue expresses as many as four L-type channel isoforms, including Ca(v)1.1. Moreover, Ca(v)1.1 and RyR1 in rat ASM cells have a similar distribution near the cell membrane in rat ASM cells and thus may be directly coupled as in skeletal muscle. Collectively, our data implicate that EC-coupling mechanisms in striated muscles may also broadly transduce diverse smooth muscle functions. 相似文献
2.
Ryanodine receptor expression is associated with intracellular Ca2+ release in rat parotid acinar cells 总被引:1,自引:0,他引:1
Zhang Xuejun; Wen Jiayu; Bidasee Keshore R.; Besch Henry R. Jr.; Rubin Ronald P. 《American journal of physiology. Cell physiology》1997,273(4):C1306
The ryanodinereceptor mediates intracellularCa2+ mobilization in muscle andnerve, but its physiological role in nonexcitable cells is less welldefined. Like adenosine 3',5'-cyclic monophosphate andinositol 1,4,5-trisphosphate, cyclic ADP-ribose (0.3-5 µM) andADP (1-25 µM) produced a concentration-dependent rise incytosolic Ca2+ in permeabilizedrat parotid acinar cells. Adenosine and AMP were less effective.Ryanodine markedly depressed theCa2+-mobilizing action of theadenine nucleotides and forskolin in permeabilized cells and waslikewise effective in depressing the action of forskolin in intactcells. Cyclic ADP-ribose-evoked Ca2+ release was enhanced bycalmodulin and depressed by W-7, a calmodulin inhibitor. Afluorescently labeled ligand,4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indacene-3-propionic acid-glycyl ryanodine, was synthesized to detect the expression anddistribution of ryanodine receptors. In addition, ryanodine receptorexpression was detected in rat parotid cells with a sequence highlyhomologous to a rat skeletal muscle type 1 and a novel brain type 1 ryanodine receptor. These findings demonstrate the presence of aryanodine-sensitive intracellularCa2+ store in rat parotid cellsthat shares many of the characteristics of stores in muscle and nerveand may mediate Ca2+-inducedCa2+ release or a modified form ofthis process. 相似文献
3.
Subtype specificity of the ryanodine receptor for Ca2+ signal amplification in excitation-contraction coupling. 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
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. 相似文献
4.
Vukcevic M Spagnoli GC Iezzi G Zorzato F Treves S 《The Journal of biological chemistry》2008,283(50):34913-34922
Dendritic cells express the skeletal muscle ryanodine receptor (RyR1), yet little is known concerning its physiological role and activation mechanism. Here we show that dendritic cells also express the Ca(v)1.2 subunit of the L-type Ca(2+) channel and that release of intracellular Ca(2+) via RyR1 depends on the presence of extracellular Ca(2+) and is sensitive to ryanodine and nifedipine. Interestingly, RyR1 activation causes a very rapid increase in expression of major histocompatibility complex II molecules on the surface of dendritic cells, an effect that is also observed upon incubation of mouse BM12 dendritic cells with transgenic T cells whose T cell receptor is specific for the I-A(bm12) protein. Based on the present results, we suggest that activation of the RyR1 signaling cascade may be important in the early stages of infection, providing the immune system with a rapid mechanism to initiate an early response, facilitating the presentation of antigens to T cells by dendritic cells before their full maturation. 相似文献
5.
Ca2+-dependent excitation-contraction coupling triggered by the heterologous cardiac/brain DHPR beta2a-subunit in skeletal myotubes
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
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. 相似文献
6.
Dynamic alterations in myoplasmic Ca2+ in malignant hyperthermia and central core disease 总被引:4,自引:0,他引:4
Lyfenko AD Goonasekera SA Dirksen RT 《Biochemical and biophysical research communications》2004,322(4):1256-1266
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. 相似文献
7.
FKBP12 binding to RyR1 modulates excitation-contraction coupling in mouse skeletal myotubes 总被引:2,自引:0,他引:2
Avila G Lee EH Perez CF Allen PD Dirksen RT 《The Journal of biological chemistry》2003,278(25):22600-22608
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). However, maximal voltage-gated Ca2+ release in V2461G-expressing myotubes was reduced by approximately 50% compared with that attributable to wild-type RyR1 (deltaF/Fmax = 1.6 +/- 0.2 and 3.1 +/- 0.4, respectively). Dyspedic myotubes expressing the V2461I mutant protein, that binds FKBP12.6 but not FKBP12, exhibited a comparable reduction in voltage-gated SR Ca2+ release (deltaF/Fmax = 1.0 +/- 0.1). However, voltage-gated Ca2+ release in V2461I-expressing myotubes was restored to a normal level (deltaF/Fmax = 2.9 +/- 0.6) following co-expression of FKBP12.6. None of the mutations that disrupted FKBP binding to RyR1 significantly affected RyR1-mediated enhancement of L-type Ca2+ channel activity (retrograde coupling). These data demonstrate that FKBP12 binding to RyR1 enhances the gain of skeletal muscle EC coupling. 相似文献
8.
Ruehr ML Russell MA Ferguson DG Bhat M Ma J Damron DS Scott JD Bond M 《The Journal of biological chemistry》2003,278(27):24831-24836
Protein kinase A anchoring proteins (AKAPs) tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The muscle AKAP, mAKAP, co-localizes with the sarcoplasmic reticulum Ca2+ release channel or ryanodine receptor (RyR). The purpose of this study was to determine whether anchoring of PKA by mAKAP regulates RyR function. Either mAKAP or mAKAP-P, which is unable to anchor PKA, was expressed in CHO cells stably expressing the skeletal muscle isoform of RyR (CHO-RyR1). Immunoelectron microscopy showed that mAKAP co-localized with RyR1 in disrupted skeletal muscle. Following the addition of 10 microm forskolin to activate adenylyl cyclase, RyR1 phosphorylation in CHO-RyR1 cells expressing mAKAP increased by 42.4 +/- 6.6% (n = 4) compared with cells expressing mAKAP-P. Forskolin treatment alone did not increase the amplitude of the cytosolic Ca2+ transient in CHO-RyR1 cells expressing mAKAP or mAKAP-P; however, forskolin plus 10 mm caffeine elicited a cytosolic Ca2+ transient, the amplitude of which increased by 22% (p < 0.05) in RyR1/mAKAP-expressing cells compared with RyR1/mAKAP-P-expressing cells. Therefore, localization of PKA by mAKAP at RyR1 increases both PKA-dependent RyR phosphorylation as well as efflux of Ca2+ through the RyR. Therefore, RyR1 function is regulated by mAKAP targeting of PKA, implying an important functional role for PKA phosphorylation of RyR in skeletal muscle. 相似文献
9.
J H Seok L Xu N R Kramarcy R Sealock G Meissner 《The Journal of biological chemistry》1992,267(22):15893-15901
The 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized ryanodine receptor (RyR) of lobster skeletal muscle has been isolated by rate density centrifugation as a 30 S protein complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified 30 S receptor revealed a single high molecular weight protein band with a mobility intermediate between those of the mammalian skeletal and cardiac M(r) 565,000 RyR polypeptides. Immunoblot analysis showed no or only minimal cross-reactivity with the rabbit skeletal and canine cardiac RyR polypeptides. By immunofluorescence the lobster RyR was localized to the junctions of the A-I bands. Following planar lipid bilayer reconstitution of the purified 30 S lobster RyR, single channel K+ and Ca2+ currents were observed which were modified by ryanodine and optimally activated by millimolar concentrations of cis (cytoplasmic) Ca2+. Vesicle-45Ca2+ flux measurements also indicated an optimal activation of the lobster Ca2+ channel by millimolar Ca2+, whereas 45Ca2+ efflux from mammalian skeletal and cardiac muscle sarcoplasmic reticulum (SR) vesicles is optimally activated by micromolar Ca2+. Further, mammalian muscle SR Ca2+ release activity is modulated by Mg2+ and ATP, whereas neither ligand appreciably affected 45Ca2+ efflux from lobster SR vesicles. These results suggested that lobster and mammalian muscle express immunologically and functionally distinct SR Ca2+ release channel protein complexes. 相似文献
10.
Ca2+ activation of RyR1 is not necessary for the initiation of skeletal-type excitation-contraction coupling
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Although an elevation in myoplasmic Ca2+ can activate the skeletal muscle ryanodine receptor (RyR1), the function of this Ca2+ activation is unclear because extracellular Ca2+ influx is unnecessary for skeletal-type EC coupling. To determine whether Ca2+ activation of RyR1 is necessary for the initiation of skeletal-type EC coupling, we examined the behavior of RyR1 with glutamate 4032 mutated to alanine (E4032A-RyR1) because this mutation had been shown to dramatically reduce activation by Ca2+. Proc. Natl. Acad. Sci. USA. 98:2865-2870). Analysis after reconstitution into planar lipid bilayers revealed that E4032A-RyR1 was negligibly activated by 100 microM Ca2+ (P(o) too low to be measured). Even in the presence of both 2 mM caffeine and 2 mM ATP, P(o) remained low for E4032A-RyR1 (ranging from <0.0001 in 100 microM free Ca2+ to 0.005 in 2 mM free Ca2+). Thus, the E4032A mutation caused a nearly complete suppression of activation of RyR1 by Ca2+. Depolarization of E4032A-RyR1-expressing myotubes elicited L-type Ca2+ currents of approximately normal size and myoplasmic Ca2+ transients that were skeletal-type, but about fivefold smaller than those for wild-type RyR1. The reduced amplitude of the Ca2+ transient is consistent either with the possibility that Ca2+ activation amplifies Ca2+ release during EC coupling, or that the E4032A mutation generally inhibits activation of RyR1. In either case, Ca2+ activation of RyR1 does not appear to be necessary for the initiation of Ca2+ release during EC coupling in skeletal muscle. 相似文献
11.
Six chimeras of the skeletal muscle (RyR1) and cardiac muscle (RyR2) Ca(2+) release channels (ryanodine receptors) previously used to identify RyR1 dihydropyridine receptor interactions [Nakai et al. (1998) J. Biol. Chem. 273, 13403] were expressed in HEK293 cells to assess their Ca(2+) dependence in [(3)H]ryanodine binding and single channel measurements. The results indicate that the C-terminal one-fourth has a major role in Ca(2+) activation and inactivation of RyR1. Further, our results show that replacement of RyR1 regions with corresponding RyR2 regions can result in loss and/or reduction of [(3)H]ryanodine binding affinity while maintaining channel activity. 相似文献
12.
Characterization and expression of the rat heart sarcoplasmic reticulum Ca2+-ATPase mRNA 总被引:3,自引:0,他引:3
Sarcoplasmic reticulum Ca2+-ATPase cDNA clones have been isolated from an adult rat heart cDNA library and the nucleotide sequence of the Ca2+-ATPase mRNA determined. The sequence has an open reading frame of 997 codons. It is identical to a cDNA isolated from a rat stomach cDNA library and 90% isologous to the rabbit and human slow/cardiac cDNAs. Nuclease S1 mapping analysis indicates that this sequence corresponds to the main Ca2+-ATPase mRNA present in heart and in slow skeletal muscle and that it is expressed in various proportions in smooth and non-muscle tissues, together with another isoform which differs from the cardiac form in the sequence of its 3'-end. 相似文献
13.
The ryanodine-sensitive calcium channels, also called ryanodine receptors, are intracellular Ca(2+)-release channels that have been shown to bind the neutral plant alkaloid ryanodine with nanomolar affinity. The activity of the skeletal muscle (RyR1), cardiac muscle (RyR2), and brain (RyR3) ryanodine receptor isoforms have been shown to be highly regulated by physiological factors including pH, temperature, and ionic strength; endogenous compounds including Ca(2+), Mg(2+), and adenosine triphosphate (ATP); and pharmacological agents including caffeine, ruthenium red, and neomycin. RyR3 is reportedly expressed in diverse tissues including lung; however, specific [(3)H]ryanodine binding sites in mammalian lung tissue have not been characterized. In this study, hamster lung ryanodine binding proteins were shown to specifically bind [(3)H]ryanodine with an affinity similar to that of RyR isoforms found in other tissues and this binding was shown to be sensitive to Ca(2+) concentration, stimulation by caffeine and spermine, and inhibition by Mg(2+), ruthenium red, and neomycin. The solubilized, intact ryanodine binding protein from hamster lung demonstrated approximately the same 30S sedimentation coefficient as RyR1 and RyR2, but a putative ryanodine receptor subunit from hamster lung was not found to cross-react with antibodies specific for the three known isoforms. We conclude that the hamster lung ryanodine binding protein demonstrates sedimentation and binding characteristics that are similar to those of the known RyR isoforms, but may exhibit antigenic dissimilarity from the typical RyR isoforms found in muscle and brain. 相似文献
14.
Weisleder N Ferrante C Hirata Y Collet C Chu Y Cheng H Takeshima H Ma J 《Cell calcium》2007,42(6):548-555
Ca2+ sparks are localized intracellular Ca2+ release events from the sarcoplasmic reticulum in muscle cells that result from synchronized opening of ryanodine receptors (RyR). In mammalian skeletal muscle, RyR1 is the predominant isoform present in adult skeletal fibers, while some RyR3 is expressed during development. Functional studies have revealed a differential role for RyR1 and RyR3 in the overall Ca2+ signaling in skeletal muscle, but the contribution of these two isoforms to Ca2+ sparks in adult mammalian skeletal muscle has not been fully examined. When enzyme-disassociated, individual adult skeletal muscle fibers are exposed to an osmotic shock, the resting fiber converts from a quiescent to a highly active Ca2+ release state where Ca2+ sparks appear proximal to the sarcolemmal membrane. These osmotic shock-induced Ca2+ sparks occur in ryr3(-/-) muscle with a spatial distribution similar to that seen in wild type muscle. Kinetic analysis reveals that systemic ablation of RyR3 results in significant changes to the initiation, duration and amplitude of individual Ca2+ sparks in muscle fibers. These changes may reflect the adaptation of the muscle Ca2+ signaling or contractile machinery due to the loss of RyR3 expression in distal tissues, as biochemical assays identify significant changes in expression of myosin heavy chain protein in ryr3(-/-) muscle. 相似文献
15.
Morrissette J Xu L Nelson A Meissner G Block BA 《American journal of physiology. Regulatory, integrative and comparative physiology》2000,279(5):R1889-R1898
Two distinct skeletal muscle ryanodine receptors (RyR1s) are expressed in a fiber type-specific manner in fish skeletal muscle (11). In this study, we compare [(3)H]ryanodine binding and single channel activity of RyR1-slow from fish slow-twitch skeletal muscle with RyR1-fast and RyR3 isolated from fast-twitch skeletal muscle. Scatchard plots indicate that RyR1-slow has a lower affinity for [(3)H]ryanodine when compared with RyR1-fast. In single channel recordings, RyR1-slow and RyR1-fast had similar slope conductances. However, the maximum open probability (P(o)) of RyR1-slow was threefold less than the maximum P(o) of RyR1-fast. Single channel studies also revealed the presence of two populations of RyRs in tuna fast-twitch muscle (RyR1-fast and RyR3). RyR3 had the highest P(o) of all the RyR channels and displayed less inhibition at millimolar Ca(2+). The addition of 5 mM Mg-ATP or 2.5 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the channels increased the P(o) and [(3)H]ryanodine binding of both RyR1s but also caused a shift in the Ca(2+) dependency curve of RyR1-slow such that Ca(2+)-dependent inactivation was attenuated. [(3)H]ryanodine binding data also showed that Mg(2+)-dependent inhibition of RyR1-slow was reduced in the presence of AMP-PCP. These results indicate differences in the physiological properties of RyRs in fish slow- and fast-twitch skeletal muscle, which may contribute to differences in the way intracellular Ca(2+) is regulated in these muscle types. 相似文献
16.
Stange M Xu L Balshaw D Yamaguchi N Meissner G 《The Journal of biological chemistry》2003,278(51):51693-51702
Phosphorylation of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptors has been reported to modulate channel activity. Abnormally high phosphorylation levels (hyperphosphorylation) at Ser-2843 in RyR1 and Ser-2809 in RyR2 and dissociation of FK506-binding proteins from the receptors have been implicated as one of the causes of altered calcium homeostasis observed during human heart failure. Using site-directed mutagenesis, we prepared recombinant RyR1 and RyR2 mutant receptors mimicking constitutively phosphorylated and dephosphorylated channels carrying a Ser/Asp (RyR1-S2843D and RyR2-S2809D) and Ser/Ala (RyR1-S2843A and RyR2-S2809A) substitution, respectively. Following transient expression in human embryonic kidney 293 cells, the effects of Ca2+, Mg2+, and ATP on channel function were determined using single channel and [3H]ryanodine binding measurements. In both assays, neither the skeletal nor cardiac mutants showed significant differences compared with wild type. Similarly essentially identical caffeine responses were observed in Ca2+ imaging measurements. Co-immunoprecipitation and Western blot analysis showed comparable binding of FK506-binding proteins to wild type and mutant receptors. Finally metabolic labeling experiments showed that the cardiac ryanodine receptor was phosphorylated at additional sites. Taken together, the results did not support the view that phosphorylation of a single site (RyR1-Ser-2843 and RyR2-Ser-2809) substantially changes RyR1 and RyR2 channel function. 相似文献
17.
Effects of perchlorate on the molecules of excitation-contraction coupling of skeletal and cardiac muscle
下载免费PDF全文
![点击此处可从《The Journal of general physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
《The Journal of general physiology》1993,102(3):423-448
To understand the nature of the transmission process of excitation- contraction (EC) coupling, the effects of the anion perchlorate were investigated on the voltage sensor (dihydropyridine receptor, DHPR) and the Ca release channel (ryanodine receptor, RyR) of the sarcoplasmic reticulum (SR). The molecules, from rabbit skeletal muscle, were either separated in membrane vesicular fractions or biochemically purified so that the normal EC coupling interaction was prevented. Additionally, the effect of ClO4- was investigated on L-type Ca2+ channel gating currents of guinea pig ventricular myocytes, as a native DHPR not in the physiological interaction of skeletal muscle. At 20 mM, ClO4- had minor effects on the activation of ionic currents through Ca channels from skeletal muscle transverse tubular (T) membranes fused with planar bilayers: a +7-mV shift in the midpoint voltage, V, with no change in kinetics of activation or deactivation. This is in contrast with the larger, negative shift that ClO4- causes on the distribution of intramembrane charge movement of skeletal muscle. At up to 100 mM it did not affect the binding of the DHP [3H]PN200-110 to triad-enriched membrane fractions (TR). At 8 mM it did not affect the kinetics or the voltage distribution of gating currents of Ca channels in heart myocytes. These negative results were in contrast to the effects of ClO4- on the release channel. At 20 mM it increased several-fold the open probability of channels from purified RyR incorporated in planar bilayers and conducting Ba2+, an effect seen on channels first closed by chelation of Ca2+ or by the presence of Mg2+. It significantly increased the initial rate of efflux of 45Ca2+ from TR vesicles (by a factor of 1.75 at 20 mM and 4.5 at 100 mM). ClO4- also increased the binding of [3H]ryanodine to TR fractions. The relative increase in binding was 50-fold at the lowest [Ca2+] used (1 microM) and then decayed to much lower values as [Ca2+] was increased. The increase was due entirely to an increase in the association rate constant of ryanodine binding. The chaotropic ions SCN- and I- increased the association rate constant to a similar extent. The binding of ryanodine to purified RyR protein reconstituted into liposomes had a greater affinity than to TR fractions but was similarly enhanced by ClO4-. The reducing agent dithiothreitol (5 mM) did not reduce the effect of ClO4- , and 5% polyethylene glycol, with an osmolarity equivalent to 20 mM ClO4-, did not change ryanodine binding.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
18.
Bastide B Snoeckx K Mounier Y 《Biochemical and biophysical research communications》2002,292(5):1267-1271
The Ca(2+) mobilizing metabolite cyclic ADP-ribose has been shown to release Ca(2+) from intracellular ryanodine sensitive stores in many cells. However, the activation of the ryanodine receptor of skeletal muscle by cADP-ribose (cADPr) and its precursor and metabolite (beta-NAD(+) and ADPr) remains to be discussed. We studied the effect of ADPr on the Ca(2+) release channel of skeletal muscle RyR1 after incorporation of microsomes isolated from fast muscles of rat in planar lipid bilayers. We observed an increase in the electrophysiological activity of the channel after addition of ADPr (10 microM) at micromolar Ca(2+) concentrations, characterized by a time-lag. The increase in P(o) is mainly due to an increase in the open frequency. The long time course observed for the development of the ADPr effect may indicate that this activation induces a change in the conformation of the RyR1 channel, which increases its sensitivity to calcium. 相似文献
19.
Functional properties of the ryanodine receptor type 3 (RyR3) Ca2+ release channel. 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A Sonnleitner A Conti F Bertocchini H Schindler V Sorrentino 《The EMBO journal》1998,17(10):2790-2798
Single-channel analysis of sarcoplasmic reticulum vesicles prepared from diaphragm muscle, which contains both RyR1 and RyR3 isoforms, revealed the presence of two functionally distinct ryanodine receptor calcium release channels. In addition to channels with properties typical of RyR1 channels, a second population of ryanodine-sensitive channels with properties distinct from those of RyR1 channels was observed. The novel channels displayed close-to-zero open-probability at nanomolar Ca2+ concentrations in the presence of 1 mM ATP, but were shifted to the open conformation by increasing Ca2+ to micromolar levels and were not inhibited at higher Ca2+ concentrations. These novel channels were sensitive to the stimulatory effects of cyclic adenosine 5'-diphosphoribose (cADPR). Detection of this second population of RyR channels in lipid bilayers was always associated with the presence of the RyR3 isoform in muscle preparations used for single-channel measurements and was abrogated by the knockout of the RyR3 gene in mice. Based on the above, we associated the novel population of channels with the RyR3 isoform of Ca2+ release channels. The functional properties of the RyR3 channels are in agreement with a potential qualitative contribution of this channel to Ca2+ release in skeletal muscle and in other tissues. 相似文献