RESUMO
We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the ryanodine receptor Ca2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly alpha-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1-10 microM) inhibited skeletal ryanodine receptor channels when the cytoplasmic Ca2+ concentration was 1 microM; but with 10 microM cytoplasmic Ca2+, skeletal ryanodine receptors were activated by < or = 1.0 microM peptide and inhibited by 10 microM peptide. Cardiac ryanodine receptors on the other hand were inhibited by all peptide concentrations, at both Ca2+ concentrations. When channels did open in the presence of the peptide, they were more likely to open to substate levels. The inhibition and increased fraction of openings to subconductance levels suggested that the domain peptide might destabilise inter-domain interactions that involve the C-terminal tail. We found that Homer 1b not only interacts with the channels, but reduces the inhibitory action of the C-terminal tail peptide, perhaps by stabilizing inter-domain interactions and preventing their disruption.
Assuntos
Proteínas de Transporte/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Proteínas de Arcabouço Homer , Músculo Esquelético/metabolismo , Peptídeos/antagonistas & inibidores , Peptídeos/química , Peptídeos/farmacologia , Estrutura Secundária de Proteína , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
A physical association between the II-III loop of the DHPR (dihydropryidine receptor) and the RyR (ryanodine receptor) is essential for excitation-contraction coupling in skeletal, but not cardiac, muscle. However, peptides corresponding to a part of the II-III loop interact with the cardiac RyR2 suggesting the possibility of a physical coupling between the proteins. Whether the full II-III loop and its functionally important 'C' region (cardiac DHPR residues 855-891 or skeletal 724-760) interact with cardiac RyR2 is not known and is examined in the present study. Both the cardiac DHPR II-III loop (CDCL) and cardiac peptide (C(c)) activated RyR2 channels at concentrations >10 nM. The skeletal DHPR II-III loop (SDCL) activated channels at < or =100 nM and weakly inhibited at > or =1 microM. In contrast, skeletal peptide (C(s)) inhibited channels at all concentrations when added alone, or was ineffective if added in the presence of C(c). Ca2+-induced Ca2+ release from cardiac sarcoplasmic reticulum was enhanced by CDCL, SDCL and the C peptides. The results indicate that the interaction between the II-III loop and RyR2 depends critically on the 'A' region (skeletal DHPR residues 671-690 or cardiac 793-812) and also involves the C region. Structure analysis indicated that (i) both C(s) and C(c) are random coil at room temperature, but, at 5 degrees C, have partial helical regions in their N-terminal and central parts, and (ii) secondary-structure profiles for CDCL and SDCL are similar. The data provide novel evidence that the DHPR II-III loop and its C region interact with cardiac RyR2, and that the ability to interact is not isoform-specific.
Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Miocárdio/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Cálcio/farmacologia , Canais de Cálcio Tipo L/genética , Dicroísmo Circular , Citoplasma/metabolismo , Espectroscopia de Ressonância Magnética , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Retículo Sarcoplasmático/metabolismo , Soluções , TemperaturaRESUMO
The actions of the recombinant skeletal dihydropyridine receptor II-III loop (SDCL), and the C region peptide (CS) on native skeletal muscle ryanodine receptor Ca2+ release channel (RyR1) have been examined. Three non conserved residues in the "C" region of the skeletal DHPR II-III loop were replaced by the equivalent cardiac residues in SDCLAFP-PTT (A739P, F741T and P742T) and single substitutions made in SDCLA-P, SDCLF-T and SDCLP-T. Wild type SDCL as well as SDCLF-T and SDCLP-T activated RyR1 in lipid bilayers with high affinity (10 nM to 1 microM). Wild type SDCL at higher concentrations inhibited RyR1. In contrast, SDCLAFP-PTT and SDCLA-P inhibited the channels at >or=10 nM. The inhibitory actions of these two skeletal loop mutants were distinctly different from the cardiac II-III loop (CDCL) which, like the wild-type SDCL, activated channels. In contrast to the full loop, the triple A739P, F741T and P742T mutation in peptide CS converted the peptides' function from skeletal-like to cardiac-like. The individual A739P mutation, but not F741T or P742T, reduced the functional efficacy of CS. None of the mutations significantly altered the NMR-based secondary structure of the C residues in SDCLAFP-PTT or CS. The CS peptide and its mutants, like the cardiac CC peptide, were all partially alpha helical at low temperatures. The results show that residue A739 is critical for the functional consequences of interactions between RyR1 and either the skeletal II-III loop or CS, but that none of A739, F741 or P742 are critical determinants of the structure of the C region.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/química , Células Cultivadas , Humanos , Músculo Esquelético/citologia , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/químicaRESUMO
We show that peptide fragments of the dihydropyridine receptor II-III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793-Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671-Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at > or = 10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 microM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations >1 mM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II-III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Cálcio/metabolismo , Fragmentos de Peptídeos/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Retículo Sarcoplasmático/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Canais de Cálcio Tipo L/química , Relação Dose-Resposta a Droga , Transporte de Íons , Bicamadas Lipídicas , Substâncias Macromoleculares , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Dados de Sequência Molecular , Contração Muscular , Proteínas Musculares/química , Proteínas Musculares/fisiologia , Músculo Esquelético/química , Miocárdio/química , Estrutura Secundária de Proteína , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Iron-deficiency remains a concern among low-income toddlers in the U.S. This formative study describes how primary care providers serving high-risk 1- to 3-year-old children in an urban ambulatory care setting approach anemia. Data collection included a retrospective review of randomly selected medical records (n=264) and semi-structured interviews with clinicians (n=41). Thirty-eight percent of the children presented with anemia (Hgb < 11.0 g/dl) at least once between 12 and 36 months of age. Just under half of these children were treated for anemia. Follow-up laboratories for iron-treated children were completed within 35 days in 16% of cases (median: 3 months). Interviews identified four key themes (iron-deficiency, communication, poverty, system) running through the two major categories of prevention and treatment. Treatment cut-points were variable. While providers felt clinically comfortable with anemia, they felt burdened and challenged by follow-up. Communication and system barriers weighed most heavily on perceived treatment outcomes.
Assuntos
Anemia Ferropriva/prevenção & controle , Acessibilidade aos Serviços de Saúde/economia , Pobreza/etnologia , Atenção Primária à Saúde/normas , Classe Social , Anemia Ferropriva/economia , Anemia Ferropriva/etnologia , Pré-Escolar , Comunicação , Connecticut , Feminino , Humanos , Lactente , Entrevistas como Assunto , Masculino , Auditoria Médica , Grupos Minoritários , Atenção Primária à Saúde/métodos , Pesquisa Qualitativa , Fatores SocioeconômicosRESUMO
The cysteine-rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract as well as in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionary diverse N-terminal domain and a characteristic cysteine-rich C-terminal domain, which we refer to as the Crisp domain. We presented the NMR solution structure of the Crisp domain of mouse Tpx-1, and we showed that it contains two subdomains, one of which has a similar fold to the ion channel regulators BgK and ShK. Furthermore, we have demonstrated for the first time that the ion channel regulatory activity of Crisp proteins is attributed to the Crisp domain. Specifically, the Tpx-1 Crisp domain inhibited cardiac ryanodine receptor (RyR) 2 with an IC(50) between 0.5 and 1.0 microM and activated the skeletal RyR1 with an AC(50) between 1 and 10 microM when added to the cytoplasmic domain of the receptor. This activity was nonvoltage-dependent and weakly voltage-dependent, respectively. Furthermore, the Tpx-1 Crisp domain activated both RyR forms at negative bilayer potentials and showed no effect at positive bilayer potentials when added to the luminal domain of the receptor. These data show that the Tpx-1 Crisp domain on its own can regulate ion channel activity and provide compelling evidence for a role for Tpx-1 in the regulation of Ca(2+) fluxes observed during sperm capacitation.
Assuntos
Sinalização do Cálcio , Glicoproteínas/química , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Sequência de Aminoácidos , Animais , Moléculas de Adesão Celular , Dissulfetos/química , Glicoproteínas/fisiologia , Espectroscopia de Ressonância Magnética , Masculino , Proteínas de Membrana , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Espermatozoides/fisiologiaRESUMO
Imperatoxin A is a high affinity activator of ryanodine receptors. The toxin contains a positively charged surface structure similar to that of the A fragment of skeletal dihydropyridine receptors (peptide A), suggesting that the toxin and peptide could bind to a common site on the ryanodine receptor. However, the question of a common binding site has not been resolved, and the concentration dependence of the actions of the toxin has not been fully explored. We characterize two novel high affinity actions of the toxin on the transient gating of cardiac and skeletal channels, in addition to the well documented lower affinity induction of prolonged substates. Transient activity was (a) enhanced with 0.2-10 nm toxin and (b) depressed by >50 nm toxin. The toxin at >/=1 nm enhanced Ca(2+) release from SR in a manner consistent with two independent activation processes. The effects of the toxin on transient activity, as well as the toxin-induced substate, were independent of cytoplasmic Ca(2+) or Mg(2+) concentrations or the presence of adenine nucleotide and were seen in diisothiocyanostilbene-2',2'-disulfonic acid-modified channels. Peptide A activated skeletal and cardiac channels with 100 nm cytoplasmic Ca(2+) and competed with Imperatoxin A in the high affinity enhancement of transient channel activity and Ca(2+) release from SR. In contrast to transient activity, prolonged substate openings induced by the toxin were not altered in the presence of peptide A. The results suggest that Imperatoxin A has three independent actions on ryanodine receptor channels and competes with peptide A for at least one action.
Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Venenos de Escorpião/metabolismo , Animais , Sítios de Ligação , Bicamadas Lipídicas , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Retículo Sarcoplasmático/metabolismoRESUMO
Enhanced sensitivity to caffeine is part of the standard tests for susceptibility to malignant hyperthermia (MH) in humans and pigs. The caffeine sensitivity of skeletal muscle contraction and Ca(2+) release from the sarcoplasmic reticulum is enhanced, but surprisingly, the caffeine sensitivity of purified porcine ryanodine receptor Ca(2+)-release channels (RyRs) is not affected by the MH mutation (Arg(615)Cys). In contrast, we show here that native malignant hyperthermic pig RyRs (incorporated into lipid bilayers with RyR-associated lipids and proteins) were activated by caffeine at 100- to 1000-fold lower concentrations than native normal pig RyRs. In addition, the results show that the mutant ryanodine receptor channels were less sensitive to high-affinity activation by a peptide (C(S)) that corresponds to a part of the II-III loop of the skeletal dihydropyridine receptor (DHPR). Furthermore, subactivating concentrations of peptide C(S) enhanced the response of normal pig and rabbit RyRs to caffeine. In contrast, the caffeine sensitivity of MH RyRs was not enhanced by the peptide. These novel results showed that in MH-susceptible pig muscles 1). the caffeine sensitivity of native RyRs was enhanced, 2). the sensitivity of RyRs to a skeletal II-III loop peptide was depressed, and 3). an interaction between the caffeine and peptide C(S) activation mechanisms seen in normal RyRs was lost.
Assuntos
Cafeína/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Hipertermia Maligna/fisiopatologia , Inibidores de Fosfodiesterase/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Animais , Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Hipertermia Maligna/metabolismo , Contração Muscular/fisiologia , Fragmentos de Peptídeos/farmacologia , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/isolamento & purificação , SuínosRESUMO
An alpha-helical II-III loop segment of the dihydropyridine receptor activates the ryanodine receptor calcium-release channel. We describe a novel manipulation in which this agonist's activity is increased by modifying its surface structure to resemble that of a toxin molecule. In a unique system, native beta-sheet scorpion toxins have been reported to activate skeletal muscle ryanodine receptor calcium channels with high affinity by binding to the same site as the lower-affinity alpha-helical dihydropyridine receptor segment. We increased the alignment of basic residues in the alpha-helical peptide to mimic the spatial orientation of active residues in the scorpion toxin, with a consequent 2-20-fold increase in the activity of the alpha-helical peptide. We hypothesized that, like the native peptide, the modified peptide and the scorpion toxin may bind to a common site. This was supported by (i) similar changes in ryanodine receptor channel gating induced by the native or modified alpha-helical peptide and the beta-sheet toxin, a 10-100-fold reduction in channel closed time, with a < or = 2-fold increase in open dwell time and (ii) a failure of the toxin to further activate channels activated by the peptides. These results suggest that diverse structural scaffolds can present similar conformational surface properties to target common receptor sites.