RESUMO
Previous cryo-electron micrographs suggested that the skeletal muscle Ca2+ release channel, ryanodine receptor (RyR)1, is regulated by intricate interactions between the EF hand Ca2+ binding domain and the cytosolic loop (S2-S3 loop). However, the precise molecular details of these interactions and functional consequences of the interactions remain elusive. Here, we used molecular dynamics simulations to explore the specific amino acid pairs involved in hydrogen bond interactions within the EF hand-S2-S3 loop interface. Our simulations unveiled two key interactions: (1) K4101 (EF hand) with D4730 (S2-S3 loop) and (2) E4075, Q4078, and D4079 (EF hand) with R4736 (S2-S3 loop). To probe the functional significance of these interactions, we constructed mutant RyR1 complementary DNAs and expressed them in HEK293 cells for [3H]ryanodine binding assays. Our results demonstrated that mutations in the EF hand, specifically K4101E and K4101M, resulted in reduced affinities for Ca2+/Mg2+-dependent inhibitions. Interestingly, the K4101E mutation increased the affinity for Ca2+-dependent activation. Conversely, mutations in the S2-S3 loop, D4730K and D4730N, did not significantly change the affinities for Ca2+/Mg2+-dependent inhibitions. Our previous finding that skeletal disease-associated RyR1 mutations, R4736Q and R4736W, impaired Ca2+-dependent inhibition, is consistent with the current results. In silico mutagenesis analysis aligned with our functional data, indicating altered hydrogen bonding patterns upon mutations. Taken together, our findings emphasize the critical role of the EF hand-S2-S3 loop interaction in Ca2+/Mg2+-dependent inhibition of RyR1 and provide insights into potential therapeutic strategies targeting this domain interaction for the treatment of skeletal myopathies.
Assuntos
Motivos EF Hand , Canal de Liberação de Cálcio do Receptor de Rianodina , Humanos , Cálcio/metabolismo , Células HEK293 , Músculo Esquelético/metabolismo , Mutação , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
AIMS: Action potential duration (APD) alternans is an established precursor or arrhythmia and sudden cardiac death. Important differences in fundamental electrophysiological properties relevant to arrhythmia exist between experimental models and the diseased in vivo human heart. To investigate mechanisms of APD alternans using a novel approach combining intact heart and cellular cardiac electrophysiology in human in vivo. METHODS AND RESULTS: We developed a novel approach combining intact heart electrophysiological mapping during cardiac surgery with rapid on-site data analysis to guide myocardial biopsies for laboratory analysis, thereby linking repolarization dynamics observed at the organ level with underlying ion channel expression. Alternans-susceptible and alternans-resistant regions were identified by an incremental pacing protocol. Biopsies from these sites (n = 13) demonstrated greater RNA expression in Calsequestrin (CSQN) and Ryanodine (RyR) and ion channels underlying IK1 and Ito at alternans-susceptible sites. Electrical restitution properties (n = 7) showed no difference between alternans-susceptible and resistant sites, whereas spatial gradients of repolarization were greater in alternans-susceptible than in alternans-resistant sites (P = 0.001). The degree of histological fibrosis between alternans-susceptible and resistant sites was equivalent. Mathematical modelling of these changes indicated that both CSQN and RyR up-regulation are key determinants of APD alternans. CONCLUSION: Combined intact heart and cellular electrophysiology show that regions of myocardium in the in vivo human heart exhibiting APD alternans are associated with greater expression of CSQN and RyR and show no difference in restitution properties compared to non-alternans regions. In silico modelling identifies up-regulation and interaction of CSQN with RyR as a major mechanism underlying APD alternans.
Assuntos
Arritmias Cardíacas/fisiopatologia , Técnicas Eletrofisiológicas Cardíacas , Sistema de Condução Cardíaco/fisiopatologia , Potenciais de Ação , Biópsia , Calsequestrina/metabolismo , Feminino , Humanos , Canais Iônicos/metabolismo , Masculino , Pessoa de Meia-Idade , Rianodina/metabolismoRESUMO
Neuronal Ca(2+) channels are rapidly inactivated by a mechanism that is termed Ca(2+)-dependent inactivation (CDI). In this study we investigated the influence of intracellular Ca(2+) release on CDI of high-voltage-activated Ca(2+) channels in rat thalamocortical relay neurons by combining voltage-clamp, Ca(2+) imaging and immunological techniques. Double-pulse protocols revealed CDI, which depended on the length of the conditioning pulses. Caffeine caused a concentration-dependent increase in CDI that was accompanied by an increase in the duration of Ca(2+) transients. Inhibition of ryanodine receptors and endoplasmic Ca(2+) pumps (by thapsigargin or cyclopiazonic acid) resulted in a reduction of CDI. In contrast, inhibition of inositol 1,4,5-tris-phosphate receptors by intracellular application of 2-aminoethoxy diphenyl borate or heparin did not influence CDI. The block of transient receptor potential channels by extracellular application of 2-aminoethoxy diphenyl borate, however, resulted in a significant reduction of CDI. The central role of L-type Ca(2+) channels was emphasized by the near-complete block of CDI by nifedipine, an effect only surpassed when Ca(2+) was replaced by Ba(2+) and chelated by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid (BAPTA). Trains of action potential-like stimuli induced a strong reduction in high-voltage-activated Ca(2+) current amplitude, which was significantly reduced when intracellular Ca(2+) stores were made inoperative by thapsigargin or Ba(2+)/BAPTA. Western blotting revealed expression of L-type Ca(2+) channels in thalamic and hippocampal tissue but not liver tissue. In summary, these results suggest a cross-signalling between L-type Ca(2+) channels and ryanodine receptors that controls the amount of Ca(2+) influx during neuronal activity.
Assuntos
Vias Aferentes/metabolismo , Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Córtex Cerebral/citologia , Neurônios/fisiologia , Tálamo/citologia , Animais , Compostos de Boro/metabolismo , Bloqueadores dos Canais de Cálcio/metabolismo , Quelantes/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Inibidores Enzimáticos/metabolismo , Ativação do Canal Iônico/fisiologia , Neurônios/citologia , Nifedipino/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Long-Evans , Rianodina/metabolismo , Tapsigargina/metabolismoRESUMO
At the threshold concentration (1-10 pmol l(-1)), the neuropeptide hormones proctolin (PR) and the FLRFamide-like peptide (FLP) F(2) cause an increase in amplitude of electrically evoked contractions (each contraction is a brief tetanus) of lobster heart ostial muscle. At higher concentrations each peptide also induces an increase in tonus (contracture). The PR-induced contracture and augmentation of tetani are proportional to increases in [Ca2+]i. The rate of onset and recovery of peptide-induced effects on both tetani and contracture appeared to reduced by Ca2+ storage by the sarcoplasmic reticulum (SR). Enhanced tetani following a contracture may be due to enhanced voltage-gated Ca2+ current and sarcoplasmic reticular (SR) Ca2+ loading. The SR Ca2+ loading appears to be specific for PR and F2, since glutamic-acid-induced contractures are not followed by increased tetani. The prolonged elevation of [Ca2+]i during contracture causes a right-ward shift in the force-pCa curve indicating a decrease in myofibrillar sensitivity to Ca2+. Blocking voltage-gated Ca2+ channels with Cd2+, nifedipine or verapamil, while reducing tetani, does not prevent peptide-induced contracture and enhanced tetani. Opening SR Ca2+ channels and depleting SR Ca2+ with either caffeine or ryanodine blocked tetani but permitted accelerated peptide-induced contractures. We conclude that PR and F2 at low concentration enhance voltage-dependent Ca2+ induced Ca2+ release from the SR, while higher hormone levels directly gate Ca2+ entry across the sarcolemma.
Assuntos
Cálcio/metabolismo , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Nephropidae/metabolismo , Neuropeptídeos/metabolismo , Oligopeptídeos/metabolismo , Animais , Cádmio/metabolismo , Cafeína/metabolismo , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio/metabolismo , Fluorescência , Potenciais da Membrana , Microeletrodos , Neuropeptídeos/farmacologia , Nifedipino/metabolismo , Oligopeptídeos/farmacologia , Rianodina/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Verapamil/metabolismoRESUMO
The functional relevance of putative Ca(2+) binding motifs previously identified with Ca(2+) overlay binding analysis within the skeletal muscle ryanodine receptor isoform (RyR1) was examined using mutational analysis. EF hands between amino acid positions 4081 and 4092 (EF1) and 4116 and 4127 (EF2) were scrambled singly or in combination within the full-length rabbit RyR1 cDNA. These cDNAs were expressed in 1B5 RyR-deficient myotubes and channel function assessed using Ca(2+)-imaging techniques, [(3)H]ryanodine binding measurements, and single channel experiments. In intact myotubes, these mutations did not affect functional responses to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) compared with wtRyR1. However, in [(3)H]ryanodine binding measurements, both Ca(2+) activation and inhibition of the EF1 mutant was significantly altered compared with wtRyR1. No high affinity [(3)H]ryanodine binding was observed in membranes expressing the EF2 mutation, although in single channel measurements, the EF2-disrupted channel could be activated by micromolar Ca(2+) concentrations. In addition, micromolar levels of ryanodine placed these channels into the classical half-conductance state, thus indicating that occupancy of high affinity ryanodine binding sites is not required for ryanodine-induced subconductance states in RyR1. Disruption of three additional putative RyR1 calcium binding motifs located between amino acid positions 4254 and 4265 (EF3), 4407 and 4418 (EF4), or 4490 and 4502 (EF5) either singly or in combination (EF3-5) did not affect functional responses in 1B5 myotubes except that the EC(50) for caffeine activation for the EF3 construct was significantly increased compared with wtRyR1. However, in [(3)H]ryanodine binding experiments, the Ca(2+)-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected when compared with wtRyR1.
Assuntos
Cálcio/química , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Cresóis/farmacologia , Análise Mutacional de DNA , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Fungicidas Industriais/farmacologia , Immunoblotting , Camundongos , Dados de Sequência Molecular , Músculos/patologia , Mutação , Inibidores de Fosfodiesterase/farmacologia , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Coelhos , Rianodina/química , Rianodina/metabolismo , Homologia de Sequência de AminoácidosRESUMO
A synthetic peptide (CaMBP) matching amino acids 3614-3643 of the skeletal ryanodine receptor (RyR1) binds to both Ca2+-free calmodulin (CaM) and Ca2+-bound CaM with nanomolar affinity [J. Biol. Chem. 276 (2001) 2069]. We report here that CaMBP increases [3H]ryanodine binding to RyR1 in a dose- and Ca2+-dependent manner; it also induces Ca2+ release from SR vesicles, and increases open probability (P(o)) of single RyR channels reconstituted in planar lipid bilayers. Further, CaMBP removes CaM associated with SR vesicles and increases [3H]ryanodine binding to purified RyR1, suggesting that its mechanism of action is two-fold: it removes endogenous inhibitors and also interacts directly with complementary regions in RyR1. Remarkably, the N-terminus of CaMBP activates RyRs while the C-terminus of CaMBP inhibits RyR activity, suggesting the presence of two discrete functional subdomains within this region. A ryr1 mutant lacking this region, RyR1-Delta3614-3643, was constructed and expressed in dyspedic myoblasts (RyR1-knockout). The depolarization-, caffeine- and 4-chloro-m-cresol (4-CmC)-induced Ca2+ transients in these cells were dramatically reduced compared with cells expressing wild type RyR1. Deletion of the 3614-3643 region also resulted in profound changes in unitary conductance and channel gating. We thus propose that the RyR1 3614-3643 region acts not only as the CaM binding site, but also as an important modulatory domain for RyR1 function.
Assuntos
Proteínas de Ligação a Calmodulina/fisiologia , Músculo Esquelético/metabolismo , Fragmentos de Peptídeos/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Ligação a Calmodulina/genética , Relação Dose-Resposta a Droga , Dados de Sequência Molecular , Músculo Esquelético/efeitos dos fármacos , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína , Rianodina/metabolismo , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo , SuínosRESUMO
Four ryanodine receptor type 1 and 2 chimeras (R4, R9, R10, and R16) and their respective wild-type ryanodine receptors (type 1 and 2; wtRyR1 and wtRyR2) were expressed in dyspedic 1B5 to identify possible negative regulatory modules of the Ca2+ release channel that are under the influence of the dihydropyridine receptor (DHPR). Responses of intact 1B5 myotubes expressing each construct to caffeine in the absence or presence of either La3+ and Cd2+ or the organic DHPR blocker nifedipine were determined by imaging single 1B5 myotubes loaded with fluo 4. The presence of La3+ and Cd2+ or nifedipine in the external medium at concentrations known to block Ca2+ entry through the DHPRs significantly decreased the caffeine EC50 of wtRyR1 (2.80 +/- 0.12 to 0.83 +/- 0.09 mM; P < 0.05). On the other hand, DHPR blockade did not significantly alter the caffeine EC50 values of wtRyR2, chimeras R10 and R16, whereas the caffeine EC50 values of chimeras R4 and R9 were significantly increased (1.27 +/- 0.05 to 2.60 +/- 0.16 mM, and 1.15 +/- 0.03 to 2.11 +/- 0.32 mM, respectively; P < 0.05). Despite the fact that all the chimeras form fully functional Ca2+ release channels in situ, sarcoplasmic reticulum (SR) containing R4, R10, and R16 did not possess high-affinity binding of [3H]ryanodine regardless of Ca2+ concentration. These results suggest the presence of an interaction between RyR1 and the DHPR, which is not present in RyR2, that contributes negative control of SR Ca2+ release induced by direct agonists such as caffeine. Although we were unable to define the negative module using RyR1-RyR2 chimeras, they further demonstrated that the RyR is very sensitive to long-range allosterism.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Regulação Alostérica , Animais , Cádmio/farmacologia , Cafeína/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/efeitos dos fármacos , Linhagem Celular , DNA Complementar/metabolismo , Lantânio/farmacologia , Potenciais da Membrana , Camundongos , Conformação Molecular , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Nifedipino/farmacologia , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/metabolismo , Fatores de TempoRESUMO
Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that segregates with >60 mutations within the MHS-1 locus on chromosome 19 coding for ryanodine receptor type 1 (RyR1). Although some MHRyR1s have been shown to enhance sensitivity to caffeine and halothane when expressed in non-muscle cells, their influence on EC coupling can only be studied in skeletal myotubes. We therefore expressed WTRyR1, six of the most common human MHRyR1s (R163C, G341R, R614C, R2163C, V2168M, and R2458H), and a newly identified C-terminal mutation (T4826I) in dyspedic myotubes to study their functional defects and how they influence EC coupling. Myotubes expressing any MHRyR1 were significantly more sensitive to stimulation by caffeine and 4-CmC than those expressing WTRyR1. The hypersensitivity of MH myotubes extended to K+ depolarization. MH myotubes responded to direct channel activators with maximum Ca2+ amplitudes consistently smaller than WT myotubes, whereas the amplitude of their responses to depolarization were consistently larger than WT myotubes. The magnitudes of responses attainable from myotubes expressing MHRyR1s are therefore related to the nature of the stimulus rather than size of the Ca2+ store. The functional changes of MHRyR1s were directly analyzed using [3H]ryanodine binding analysis of isolated myotube membranes. Although none of the MHRyR1s examined significantly altered EC50 for Ca2+ activation, many failed to be completely inhibited by a low Ca2+ (Assuntos
Hipertermia Maligna/genética
, Contração Muscular
, Músculo Esquelético/patologia
, Mutação
, Canal de Liberação de Cálcio do Receptor de Rianodina/genética
, Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia
, Animais
, Cafeína/metabolismo
, Cafeína/farmacologia
, Cálcio/metabolismo
, Membrana Celular/metabolismo
, Estimulantes do Sistema Nervoso Central/farmacologia
, Colágeno/farmacologia
, Cresóis/farmacologia
, DNA Complementar/metabolismo
, Relação Dose-Resposta a Droga
, Combinação de Medicamentos
, Herpesvirus Humano 1/metabolismo
, Immunoblotting
, Concentração Inibidora 50
, Laminina/farmacologia
, Magnésio/metabolismo
, Camundongos
, Músculos/citologia
, Cloreto de Potássio/metabolismo
, Cloreto de Potássio/farmacologia
, Estrutura Terciária de Proteína
, Proteoglicanas/farmacologia
, Coelhos
, Rianodina/metabolismo
RESUMO
4-Chloro-m-cresol (4-CmC) is a potent and specific activator of the intracellular Ca2+ release channel, the ryanodine receptor (RyR). We have previously shown that RyR1 expressed in dyspedic 1B5 myotubes is activated by 4-CmC, whereas RyR3 is not (Fessenden, J. D., Wang, Y., Moore, R. A., Chen, S. R. W., Allen, P. D., and Pessah, I. N. (2000) Biophys. J. 79, 2509-2525). To identify region(s) on RyR1 that are responsible for mediating activation by 4-CmC, we expressed RyR1-RyR3 chimeric proteins in dyspedic 1B5 myotubes and then measured 4-CmC-induced increases in intracellular Ca2+. Substitution of the C-terminal third of RyR1 into RyR3 imparted 4-CmC sensitivity to the resulting chimera, thus suggesting that determinants required for activation by 4-CmC are located in this region. We subdivided the C-terminal third of RyR1 into smaller segments and identified two overlapping regions of RyR1 (amino acids 3769-4180 and 4007-4382) that each imparted 4-CmC sensitivity to RyR3. Substitution of the 173 amino acids of RyR1 common to these two chimeras (amino acids 4007-4180) also weakly restored 4-CmC sensitivity in the resulting chimera. To confirm these findings, we created a complementary set of chimeras containing RyR3 substitutions in RyR1. Substitution of the RyR3 C terminus into RyR1 disrupted 4-CmC sensitivity in the resulting chimera. In addition, substitution of the corresponding RyR3 sequence into positions 4007-4180 of RyR1 disrupted 4-CmC sensitivity. Taken together, these results suggest that essential determinants required for activation of RyR1 by 4-CmC reside within a 173-amino acid region between residues 4007 and 4180.
Assuntos
Cresóis/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Expressão Gênica , Peso Molecular , Fibras Musculares Esqueléticas/metabolismo , Mutagênese Sítio-Dirigida , Cloreto de Potássio/farmacologia , Isoformas de Proteínas/genética , Coelhos , Proteínas Recombinantes de Fusão/química , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Relação Estrutura-Atividade , Transfecção , TrítioRESUMO
Radioligand binding assays evaluating directly the ability of a drug to interact with a defined molecular target is part of the drug discovery process. The need for a high throughput rate in screening drugs is actually leading to simplified experimental schemes that increase the probability of false negative results. Special concern involves voltage-gated ion channel drug discovery where a great care is required in designing assays because of frequent multiplicity of (interacting) binding sites. To clearly illustrate this situation, three different assays used in the academic drug discovery program of the authors were selected because they are rich of intrinsic artifacts: (I) (20 mmol/l caffeine almost duplicated [3H]ryanodine binding (89% higher than control) to rat heart microsomes at 0.3 mumol/l free calcium but did not exert any effect when using a high (107 mumol/l) free calcium, as mostly used in ryanodine binding assays; (II) An agonist for the ionotropic glutamate receptor of the kainate type can distinctly affect [3H]kainate binding to chicken cerebellum membranes depending on its concentration: unlabelled kainic acid per se either stimulated about 30% (at 50-100 nmol/l), had no effect (at 200 nmol/l) or even progressively decreased (at 0.3-2 mumol/l) the binding of 5 nmol/l [3H]kainate, emphasizing the risk of using a single concentration for screening a drug; (III) in a classical [3H]flunitrazepam binding assay, the stimulatory effect of a GABA (gamma-aminobutyric acid) agonist was only observed when using extensively washed rat brain synaptosomes (10 mumol/l GABA increased flunitrazepam binding by 90%). On the other hand, the inhibitory effect of a GABA antagonist was only observed when using crude synaptosomes (10 mumol/l bicuculine reduced [3H]flunitrazepam binding by 40%). It can be concluded that carefully designed radioligand assays which can be performed in an academic laboratory are appropriate for screening a small number of drugs, especially if these are potential hits because of their rational design. Therefore, the low throughput rate could be partially balanced by a higher performance when compared to what is done in a robotic high throughput screening where simplification of assay conditions can lead to false negative results.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Farmacologia/métodos , Ensaio Radioligante , Animais , Ligação Competitiva/efeitos dos fármacos , Cafeína/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Agonistas de Aminoácidos Excitatórios/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Flunitrazepam/metabolismo , Técnicas In Vitro , Ácido Caínico/metabolismo , Ácido Caínico/farmacologia , Membranas/metabolismo , Microssomos/metabolismo , Miocárdio/metabolismo , Ratos , Ratos Wistar , Rianodina/metabolismo , Espermina/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Cartap, a nereistoxin analogue pesticide, is reported to have no irritation to eyes in rabbits. However, we have demonstrated recently that cartap could actually cause acute death in rabbits via ocular exposure. Our preliminary study with isolated mouse phrenic nerve diaphragms has shown that instead of neuromuscular blockade, cartap caused muscular contracture. The objective of the study was to examine the effect of cartap on the neuromuscular junction in more detail and to investigate its possible underlying mechanism with isolated mouse phrenic nerve diaphragms and sarcoplasmic reticulum (SR) vesicles. Cartap or nereistoxin at various concentrations was added in the organ bath with isolated mouse phrenic nerve diaphragm and both nerve- and muscle-evoked twitches were recorded. Instead of blocking the neuromuscular transmission as nereistoxin did, cartap caused contracture in stimulated or quiescent isolated mouse phrenic nerve diaphragm. Both the cartap-induced muscular contracture force and the time interval to initiate the contracture were dose-dependent. The contracture induced by cartap was not affected by the pretreatment of the diaphragm with the acetylcholine receptor blocker alpha-bungarotoxin; the Na(+) channel blocker tetrodotoxin; or various Ca(2+) channel blockers, NiCl(2), verapamil, and nifedipine. On the contrary, the contracture was significantly inhibited when the diaphragm was pretreated with ryanodine or EGTA containing Ca(2+)-free Krebs solution or in combination. This suggested that both internal and extracellular Ca(2+) might participate in cartap-induced skeletal muscle contracture. Moreover, cartap inhibited the [(3)H]-ryanodine binding to the Ca(2+) release channel of SR in a dose-dependent manner. Additionally, cartap could induce a significant reduction in Ca(2+)-ATPase activity of SR vesicles at a relatively high dose. The results suggested that cartap might cause the influx of extracellular Ca(2+) and the release of internal Ca(2+), with subsequent induction of muscular contracture in the isolated mouse phrenic nerve diaphragm. Based on these findings, we propose that the acute death of rabbits following ocular exposure to cartap might have resulted from respiratory failure secondary to diaphragm contracture.
Assuntos
Inseticidas/farmacologia , Nervo Frênico/efeitos dos fármacos , Tiocarbamatos/farmacologia , Animais , Bungarotoxinas/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , ATPases Transportadoras de Cálcio/metabolismo , Diafragma/inervação , Estimulação Elétrica , Masculino , Toxinas Marinhas/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Bloqueio Neuromuscular , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/fisiologia , Nifedipino/farmacologia , Nervo Frênico/fisiologia , Rianodina/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologia , Tetrodotoxina/farmacologia , Verapamil/farmacologiaRESUMO
The purpose of the present investigation was to investigate the subcellular basis responsible for the loss of vasoreactivity in atherosclerotic vessels. We have chosen to focus on the potential of oxidized low density lipoprotein (oxLDL), an important atherogenic agent, to alter sarcoplasmic reticulum (SR) structure and function. Vascular smooth muscle cells (VSMCs) were exposed for 1 to 6 days to low concentrations of minimally oxidized LDL. ATP was used to probe SR function in VSMCs. ATP can increase [Ca(2+)](i) in control VSMCs because of a release of Ca(2+) from the SR. However, after chronic exposure to oxLDL, cells lose their ability to increase [Ca(2+)](i) in response to ATP. These cells also exhibit a depressed rise in [Ca(2+)](i) after exposure to ryanodine. These effects were associated with a decreased immunoreactivity for the ryanodine-sensitive Ca(2+)-release channels in the SR of oxLDL-treated cells. Immunohistochemical analysis of aortic sections obtained from rabbits fed a cholesterol-supplemented diet revealed a significant decrease in the immunoreactivity for ryanodine channels in the plaque and in the medial layer underlying the plaque. In summary, our data identify oxLDL as a component within the atherosclerotic milieu capable of inducing a decrease in smooth muscle ryanodine channel density. This alteration is associated with a significant defect in the ability of the SR within the smooth muscle cell to regulate Ca(2+). These lesions may contribute to the altered vasoreactivity exhibited by atherosclerotic vessels.
Assuntos
Canais Iônicos/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Rianodina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Cálcio/metabolismo , Células Cultivadas , Colesterol na Dieta/farmacologia , Endotelina-1/farmacologia , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Canais Iônicos/metabolismo , Microscopia Confocal , Músculo Liso Vascular/patologia , Concentração Osmolar , Coelhos , Rianodina/farmacologia , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismoRESUMO
The regulation of intracellular free Ca2+ concentration ([Ca2+]i) in B cells remains poorly understood and is presently explained almost solely by inositol 1,4,5-triphosphate (IP3)-mediated Ca2+ release, followed by activation of a store-operated channel mechanism. In fact, there are reports indicating that IP3 production does not always correlate with the magnitude of Ca2+ release. We demonstrate here that human B cells express a ryanodine receptor (RYR) that functions as a Ca2+ release channel during the B cell antigen receptor (BCR)-stimulated Ca2+ signaling process. Immunoblotting studies showed that both human primary CD19(+) B and DAKIKI cells express a 565-kDa immunoreactive protein that is indistinguishable in molecular size and immunoreactivity from the RYR. Selective reverse transcription-polymerase chain reaction, restriction fragment length polymorphism, and sequencing of cloned cDNA indicated that the major isoform of the RYR expressed in primary CD19(+) B and DAKIKI cells is identical to the skeletal muscle type (RYR1). Saturation analysis of [3H]ryanodine binding yielded Bmax = 150 fmol/mg of protein and Kd = 110 nM in DAKIKI cells. In fluo-3-loaded CD19(+) B and DAKIKI cells, 4-chloro-m-cresol, a potent activator of Ca2+ release mediated by the ryanodine-sensitive Ca2+ release channel, induced Ca2+ release in a dose-dependent and ryanodine-sensitive fashion. Furthermore, BCR-mediated Ca2+ release in CD19(+) B cells was significantly altered by 4-chloro-m-cresol and ryanodine. These results indicate that RYR1 functions as a Ca2+ release channel during BCR-stimulated Ca2+ signaling and suggest that complex Ca2+ signals that control the cellular activities of B cells may be generated by cooperation of the IP3 receptor and RYR1.
Assuntos
Linfócitos B/metabolismo , Sinalização do Cálcio , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sequência de Aminoácidos , Anticorpos/imunologia , Antígenos CD19/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Cresóis/farmacologia , DNA Complementar , Humanos , Imunoglobulina M/imunologia , Dados de Sequência Molecular , Ligação Proteica , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Homologia de Sequência de AminoácidosRESUMO
A rapid assay for high affinity [3H]ryanodine binding to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized recombinant or native Ca2+ release channel proteins (ryanodine receptor, RyR) was devised. The key to preservation of high affinity [3H]ryanodine binding sites in the presence of increasing concentrations of CHAPS was the addition of phosphatidylcholine. This assay was used to characterize the equilibrium and kinetic properties of [3H]ryanodine binding to recombinant skeletal (RyR1) and cardiac (RyR2) Ca2+ release channels and the effects on binding of physiological modulators including ATP, Ca2+, and Mg2+. Both RyR1 and RyR2 had a single high affinity ryanodine binding site and low affinity sites, but [3H]ryanodine binding to recombinant RyR2 was not sensitive to ATP activation or Ca2+ inactivation and was less sensitive to Mg2+ inhibition. The [3H]ryanodine binding assay was used to estimate the expression level of recombinant RyR2 and RyR1, and to show that RyR2 can be expressed at very high levels in HEK-293 cells. Analysis of the properties of recombinant RyR2 and RyR1 by measurement of intracellular Fura-2 fluorescence revealed that the different properties of RyR2 and RyR1 are retained in the recombinant expressed proteins.
Assuntos
Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Rianodina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Linhagem Celular , DNA Complementar , Humanos , Cinética , Magnésio/metabolismo , Ligação Proteica , Coelhos , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Tapsigargina/farmacologia , Transfecção , TrítioRESUMO
The lobster skeletal muscle Ca2+ release channel, known also as the ryanodine receptor, is composed of four polypeptides of approximately 5000 amino acids each, like its mammalian counterparts. Clones encoding the carboxy-terminal region of the lobster ryanodine receptor were isolated from a lobster skeletal muscle cDNA library. Analysis of the deduced 1513 carboxy-terminal amino acid sequence suggests a cytoplasmic Ca2+ binding domain consisting of two EF-hand Ca2+ binding motifs (amino acid residues 594-656). The Ca2+ binding properties of this domain were assessed by preparing bacterial fusion proteins with sequences from the lobster Ca2+ binding domain and the corresponding sequences of the rabbit cardiac and skeletal muscle ryanodine receptors. The lobster skeletal muscle fusion protein bound 45Ca2+ in Ca2+ overlays, and bound two Ca2+ under equilibrium binding conditions with a Hill dissociation constant (KH) of 0.9 mM and coefficient (nH) of 1.4. Rabbit skeletal and cardiac fusion proteins bound two Ca2+ with KHs of 3.7 and 3.8 mM and nHs of 1.1 and 1.3, respectively. Similar to results previously reported for the mammalian RyRs, the lobster RyR was activated by micromolar Ca2+ and inhibited by millimolar Ca2+, as determined in single-channel and [3H]ryanodine binding measurements. These results suggest that the two EF-hand Ca2+ binding domain of the lobster Ca2+ release channel as well as the corresponding regions of the mammalian channels may play a role in Ca2+ inactivation of sarcoplasmic reticulum Ca2+ release.
Assuntos
Cálcio/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Glutationa Transferase/genética , Dados de Sequência Molecular , Nephropidae , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Homologia de Sequência de AminoácidosRESUMO
The effects of boldine [(S)-2,9-dihydroxy-1,10-dimethoxyaporphine], a major alkaloid in the leaves and bark of boldo (Peumus boldus Mol.), on skeletal muscle were studied using mouse diaphragm and isolated sarcoplasmic reticulum membrane vesicles. Boldine, at 10-200 microM, has little effect on the muscle-evoked twitches; however, the ryanodine-induced contracture was potentiated dose-dependently. At higher concentrations of 300 microM, boldine by itself induced muscle contracture of two phases, which were caused by the influx of extracellular Ca2+ and induction of Ca2+ release from the internal Ca2+ storage site, the sarcoplasmic reticulum, respectively. When tested with isolated sarcoplasmic reticulum membrane vesicles, boldine dose-dependently induced Ca2+ release from actively loaded sarcoplasmic reticulum vesicles isolated from skeletal muscle of rabbit or rat which was inhibited by ruthenium red, suggesting that the release was through the Ca2+ release channel, also known as the ryanodine receptor. Boldine also dose-dependently increased apparent [3H]-ryanodine binding with the EC50 value of 50 microM. In conclusion, we have shown that boldine could sensitize the ryanodine receptor and induce Ca2+ release from the internal Ca2+ storage site of skeletal muscle.
Assuntos
Aporfinas/farmacologia , Diafragma/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Fármacos Neuromusculares Despolarizantes/farmacologia , Retículo Sarcoplasmático/efeitos dos fármacos , Animais , Aporfinas/isolamento & purificação , Cálcio/metabolismo , Diafragma/metabolismo , Relação Dose-Resposta a Droga , Feminino , Transporte de Íons , Masculino , Camundongos , Camundongos Endogâmicos ICR , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Fármacos Neuromusculares Despolarizantes/isolamento & purificação , Plantas Medicinais/química , Coelhos , Ratos , Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Electron-microscopic analysis was used to show that invertebrate muscle has feetlike structures on the sarcoplasmic reticulum (SR) displaying the typical four-subunit appearance of the calcium (Ca2+) release channel/ryanodine receptor (RyR) observed in vertebrate skeletal muscle (K. E. Loesser, L. Castellani, and C. Franzini-Armstrong. J. Muscle Res. Cell Motil. 13: 161-173, 1992). SR vesicles from invertebrate muscle exhibited specific ryanodine binding and single channel currents that were activated by Ca2+, caffeine, and ATP and inhibited by ruthenium red. The single channel conductance of this invertebrate RyR was lower than that of the vertebrate RyR (49 and 102 pS, respectively). Activation of lobster and scallop SR Ca2+ release channel, in response to cytoplasmic Ca2+ (1 nM-10 mM), reflected a bell-shaped curve, as is found with the mammalian RyR. In contrast to a previous report (J.-H. Seok, L. Xu, N. R. Kramarcy, R. Sealock, and G. Meissner, J. Biol. Chem. 267: 15893-15901, 1992), our results show that regulation of the invertebrate and vertebrate RyRs is quite similar and suggest remarkably similar paths in these diverse organisms.
Assuntos
Moluscos , Músculos/química , Nephropidae , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Cafeína/farmacologia , Cálcio/farmacologia , Canais de Cálcio/metabolismo , Condutividade Elétrica , Bicamadas Lipídicas/metabolismo , Microscopia Eletrônica , Músculos/fisiologia , Músculos/ultraestrutura , Rutênio Vermelho/farmacologia , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/análise , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/fisiologia , Retículo Sarcoplasmático/ultraestruturaRESUMO
We present an in-depth analysis of the structural and functional properties of Imperatoxin I (IpTxi), an approximately 15-kDa protein from the venom of the scorpion Pandinus imperator that inhibits Ca2+ release channel/ryanodine receptor (RyR) activity (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and Coronado, R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12185-12189). A cDNA library was prepared from the venomous glands of this scorpion and used to clone the gene encoding IpTxi. From a single continuous messenger RNA, the information coding for the toxin is translated into two mature polypeptide subunits after elimination of a basic pentapeptide. The IpTxi dimer consists of a large subunit (104-amino acid residues) with phospholipase A2 (PLA2) activity covalently linked by a disulfide bond to a smaller (27 amino acid residues), structurally unrelated subunit. Thus, IpTxi is a heterodimeric protein with lipolytic action, a property that is only shared with beta-bungarotoxins, a group of neurotoxins from snake venoms. The enzymatic subunit of IpTxi is highly homologous to PLA2 from bee (Apis mellifera) and lizard (Heloderma horridum) venoms. The small subunit has no significant similarity to any other known peptide, including members of the Kunitz protease inhibitors superfamily that target the lipolytic effect of beta-bungarotoxins. A synthetic peptide with amino acid sequence identical to that of the small subunit failed to inhibit RyR. On the other hand, treatment of IpTxi with p-bromophenacylbromide, a specific inhibitor of PLA2 activity, greatly reduced the capacity of IpTxi to inhibit RyRs. These results suggested that a lipid product of PLA2 activity, more than a direct IpTxi-RyR interaction, was responsible for RyR inhibition.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Proteínas Musculares/fisiologia , Venenos de Escorpião/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Complementar , Biblioteca Gênica , Cinética , Bicamadas Lipídicas , Substâncias Macromoleculares , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas Musculares/efeitos dos fármacos , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Fosfolipases A/química , Fosfolipases A/isolamento & purificação , Fosfolipases A/metabolismo , Fosfolipases A2 , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina , Retículo Sarcoplasmático/metabolismo , Venenos de Escorpião/biossíntese , Venenos de Escorpião/isolamento & purificação , Escorpiões , Homologia de Sequência de Aminoácidos , SuínosRESUMO
An oligonucleotide probe to a conserved 3' region within the three identified ryanodine receptor-calcium release channel isoforms hybridized to a single clone from a rabbit kidney cDNA library. The kidney clone encoded the carboxyl-terminal 338 amino acids within the putative transmembrane domain of the type 2 ryanodine receptor sequence. Reverse transcriptase-polymerase chain reaction with isoform-specific oligonucleotide primers demonstrated the presence of the type 2 ryanodine receptor transcript in rabbit kidney, as well as in a non-excitable cell line, LLC-RK1, derived from rabbit kidney epithelial cells. Amplification by rapid amplification of 5' cDNA ends indicated the kidney type 2 ryanodine receptor transcript extended >7000 base pairs from the stop codon and is therefore not homologous to the short RyR-1 transcript of approximately 2500 base pairs previously observed in rabbit brain. [3H]Ryanodine binding and immunoblot analysis with a type 2 ryanodine receptor-specific antibody demonstrated that the native type 2 ryanodine receptor protein is expressed in the kidney. These observations suggest that the type 2 ryanodine receptor isoform may play a functional role in regulating intracellular calcium homeostasis in non-excitable cells.
Assuntos
Canais de Cálcio/genética , Córtex Renal/metabolismo , Proteínas Musculares/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Encéfalo/metabolismo , Clonagem Molecular , DNA Complementar , Células Epiteliais , Epitélio/metabolismo , Córtex Renal/citologia , Dados de Sequência Molecular , RNA Mensageiro/genética , Coelhos , Ensaio Radioligante , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina , Homologia de Sequência de Aminoácidos , TrítioRESUMO
Ryanodine receptors (RyRs) are a family of intracellular calcium release channels. Three cDNAs encoding different isoforms of RyR have been identified and cloned. We report the complete sequence of the mink RyR3 cDNA and the characterization of three alternative spliced regions. The first two splicing sites are represented by insertions of five and six amino acids, respectively. The third site is represented by a mutually exclusive splicing. The tissue distribution of the alternatively spliced transcripts revealed a ubiquitous expression of splicing site I and a differential distribution of sites II and III, indicating that a further level of complexity in RyR3 expression may result from alternative splicings in this gene.