RESUMEN

Release of Ca(2+) from the sarcoplasmic reticulum (SR) through the cardiac ryanodine receptor (RyR2) is an essential step in cardiac excitation-contraction coupling. Excess Ca(2+) release due to overactive RyR2 can cause arrhythmia that can lead to cardiac arrest. Fragments derived from the carboxy-terminal domain of human glutathione transferase M2 (GSTM2C) specifically inhibit RyR2 activity. Our aim was to further improve this inhibition by mutagenesis and to assess the therapeutic potential of GSTM2C based peptides to treat Ca(2+) release-based arrhythmia. We generated several mutant variants of the C-terminal fragment GSTM2C H5-8 and from those mutant proteins we identified two (RM13 and SM2) that exhibited significantly greater inhibition of cardiac SR Ca(2+) release and single RyR2 channel activity. Flow cytometry analysis showed that these two mutant proteins as well as GSTM2C H5-8 are taken up by isolated adult mouse cardiomyocytes without the aid of any additional compounds, Ca(2+) imaging and isolated cell contraction measurements revealed that GSTM2C H5-8, SM2 and RM13 reduce the SR Ca(2+) release rate and the fractional shortening of adult mouse cardiomyocytes, while importantly increasing the rate of Ca(2+) removal from the sarcoplasm. These observations indicate that peptides derived from GSTM2C inhibit RyR2 at a cellular level and thus they may provide the basis for a novel therapeutic agent to treat arrhythmia and heart attack.

Asunto(s)

Bloqueadores de los Canales de Calcio/farmacología , Glutatión Transferasa/genética , Miocitos Cardíacos/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Cafeína/farmacología , Células Cultivadas , Dicroismo Circular , Escherichia coli/genética , Masculino , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Miocitos Cardíacos/metabolismo , Fragmentos de Péptidos/genética , Técnicas del Sistema de Dos Híbridos

RESUMEN

Junctin, a non-catalytic splice variant encoded by the aspartate-ß-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca(2+) store where it modifies Ca(2+) signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca(2+) release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca(2+) signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1-S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156.

Asunto(s)

Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Línea Celular , Perros , Unión Proteica , Estructura Terciaria de Proteína , Canal Liberador de Calcio Receptor de Rianodina/genética

RESUMEN

The Homer protein family allows clustering and/or functional modulation of many proteins from different calcium signalling complexes including those formed by the ryanodine receptor (RyR) Ca(2+) release channel in skeletal muscle and the heart. Homer1b/c and the cardiac RyR (RyR2) are strongly expressed in the heart and neurons where their interaction with each other may modulate Ca(2+) signalling. However, functional interactions between Homer1b and RyR2 have been poorly defined. Our preliminary data and similar consensus binding sites for Homer in RyR2 and skeletal RyR (RyR1) proteins, led to the hypothesis that Homer may similarly regulate both RyR isoforms. Single-channel and [(3)H]ryanodine binding data showed that RyR2 and RyR1 activity increased to a maximum with ~50-100 nM Homer1b and fell with Homer1b > 200 nM. Homer1b (50 nM) activated RyR2 and RyR1 at all cytosolic [Ca(2+)]; estimated EC(50) value of RyR2 diminished from ~2.8 microM Ca(2+) (control) to ~1.9 microM Ca(2+) in the presence of 50 nM Homer1b. Short Homer1 (lacking the coiled-coil multimerisation domain) and Homer1b similarly modulated RyR2, indicating an action through ligand binding, not mutimerisation. These actions of Homer were generally similar in RyR2 and RyR1. The strong functional interactions suggest that Homer1 is likely to be an endogenous modulator of RyR channels in the heart and neurons as well as in skeletal muscle.

Asunto(s)

Calcio/química , Proteínas Portadoras/química , Miocardio/química , Canal Liberador de Calcio Receptor de Rianodina/química , Retículo Sarcoplasmático/química , Animales , Proteínas de Andamiaje Homer , Unión Proteica , Conejos

RESUMEN

The muscle specific glutathione transferase GSTM2-2 inhibits the activity of cardiac ryanodine receptor (RyR2) calcium release channels with high affinity and activates skeletal RyR (RyR1) channels with lower affinity. To determine which overall region of the GSTM2-2 molecule supports binding to RyR2, we examined the effects of truncating GSTM2-2 on its ability to alter Ca(2+) release from sarcoplasmic reticulum (SR) vesicles and RyR channel activity. The C-terminal half of GSTM2-2 which lacks the critical GSH binding site supported the inhibition of RyR2, but did not support activation of RyR1. Smaller fragments of GSTM2-2 indicated that the C-terminal helix 6 was crucial for the action of GSTM2-2 on RyR2. Only fragments containing the helix 6 sequence inhibited Ca(2+) release from cardiac SR. Single RyR2 channels were strongly inhibited by constructs containing the helix 6 sequence in combination with adjacent helices (helices 5-8 or 4-6). Fragments containing helices 5-6 or helix 6 sequences alone had less well-defined effects. Chemical cross-linking indicated that C-terminal helices 5-8 bound to RyR2, but not RyR1. Structural analysis with circular dichroism showed that the helical content was greater in the longer helix 6 containing constructs, while the helix 6 sequence alone had minimal helical structure. Therefore the active centre of GSTM2-2 for inhibition of cardiac RyR2 involves the helix 6 sequence and the helical nature of this region is essential for its efficacy. GSTM2-2 helices 5-8 may provide the basis for RyR2-specific compounds for experimental and therapeutic use.

Asunto(s)

Glutatión Transferasa/metabolismo , Miocardio/enzimología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Calcio/metabolismo , Glutatión Transferasa/química , Glutatión Transferasa/genética , Humanos , Conejos , Retículo Sarcoplasmático/enzimología , Ovinos

RESUMEN

Alternative splicing of ASI residues (Ala(3481)-Gln(3485)) in the skeletal muscle ryanodine receptor (RyR1) is developmentally regulated: the residues are present in adult ASI(+)RyR1, but absent in the juvenile ASI(-)RyR1 which is over-expressed in adult myotonic dystrophy type 1 (DM1). Although this splicing switch may influence RyR1 function in developing muscle and DM1, little is known about the properties of the splice variants. We examined excitation-contraction (EC) coupling and the structure and interactions of the ASI domain (Thr(3471)-Gly(3500)) in the splice variants. Depolarisation-dependent Ca(2+) release was enhanced by >50% in myotubes expressing ASI(-)RyR1 compared with ASI(+)RyR1, although DHPR L-type currents and SR Ca(2+) content were unaltered, while ASI(-)RyR1 channel function was actually depressed. The effect on EC coupling did not depend on changes in ASI domain secondary structure. Probing RyR1 function with peptides possessing the ASI domain sequence indicated that the domain contributes to an inhibitory module in RyR1. The action of the peptide depended on a sequence of basic residues and their alignment in an alpha-helix adjacent to the ASI splice site. This is the first evidence that the ASI residues contribute to an inhibitory module in RyR1 that influences EC coupling. Implications for development and DM1 are discussed.

Asunto(s)

Empalme Alternativo/genética , Huesos/metabolismo , Contracción Muscular , Canal Liberador de Calcio Receptor de Rianodina/genética , Secuencia de Aminoácidos , Aminoácidos Básicos , Animales , Calcio/metabolismo , Activación del Canal Iónico , Cinética , Espectroscopía de Resonancia Magnética , Ratones , Datos de Secuencia Molecular , Fibras Musculares Esqueléticas/metabolismo , Péptidos/química , Retículo Sarcoplasmático/metabolismo , Análisis de Secuencia de Proteína

RESUMEN

The aim of the present study was to examine residues that are variably spliced in the juvenile and adult isoforms of the skeletal-muscle RyR1 (type 1 ryanodine receptor). The juvenile ASI(-) splice variant is less active than the adult ASI(+) variant and is overexpressed in patients with DM (myotonic dystrophy) [Kimura, Nakamori, Lueck, Pouliquin, Aoike, Fujimura, Dirksen, Takahashi, Dulhunty and Sakoda (2005) Hum. Mol. Genet. 14, 2189-2200]. In the present study, we explore the ASI region using synthetic peptides corresponding to rabbit RyR1 residues Thr3471-Gly3500 either containing [PASI(+)] or lacking [PASI(-)] the ASI residues. Both peptides increased [3H]ryanodine binding to rabbit RyR1s, increased Ca2+ release from sarcoplasmic reti-culum vesicles and increased single RyR1 channel activity. The peptide PASI(-) was more active in each case than PASI(+). [3H]Ryanodine binding to recombinant ASI(+)RyR1 or ASI(-)-RyR1 was enhanced more by PASI(-) than PASI(+), with the greatest increase seen when PASI(-) was added to ASI(-)RyR1. The activation of the RyR channels is consistent with the hypo-thesis that the peptides interrupt an inhibitory inter-domain inter-action and that PASI(-) is more effective at interrupting this interaction than PASI(+). We therefore suggest that the ASI(-) sequence interacts more tightly than the ASI(+) sequence with its binding partner, so that the ASI(-)RyR1 is more strongly inhibited (less active) than the ASI(+)RyR1. Thus the affinity of the binding partners in this inter-domain interaction may deter-mine the activities of the mature and juvenile isoforms of RyR1 and the stronger inhibition in the juvenile isoform may contribute to the myopathy in DM.

Asunto(s)

Empalme Alternativo , Variación Genética , Músculo Esquelético/fisiología , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Secuencia de Aminoácidos , Animales , Exones , Cinética , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección

RESUMEN

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.

Asunto(s)

Proteínas Portadoras/farmacología , Canal Liberador de Calcio Receptor de Rianodina/química , Animales , Proteínas de Andamiaje Homer , Músculo Esquelético/metabolismo , Péptidos/antagonistas & inhibidores , Péptidos/química , Péptidos/farmacología , Estructura Secundaria de Proteína , Conejos , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/genética

RESUMEN

1. Our aim was to determine whether lipoamino acid conjugation of peptides that are high-affinity activators of ryanodine receptor (RyR) channels would (a) render the peptides membrane permeable, (b) alter their structure or (a) reduce their activity. The peptides correspond to the A region of the II-III loop of the skeletal dihydropyridine receptor. 2. The lipoamino acid conjugation increased the apparent permeability of the peptide across the Caco-2 cell monolayer by up to approximately 20-fold. 3. Nuclear magnetic resonance showed that the alpha-helical structure of critical basic residues, required for optimal activation of RyRs, was retained after conjugation. 4. The conjugated peptides were more effective in enhancing resting Ca2+ release, Ca2+-induced Ca2+ release and caffeine-induced Ca2+ release from isolated sarcoplasmic reticulum (SR) than their unconjugated counterparts, and significantly enhanced caffeine-induced Ca2+ release from mechanically skinned extensor digitorum longus (EDL) fibres. 5. The effect of both conjugated and unconjugated peptides on Ca2+ release from skeletal SR was 30-fold greater than their effect on either cardiac Ca2+ release or on the Ca2+ Mg2+ ATPase. 6. A small and very low affinity effect of the peptide in slowing Ca2+ uptake by the Ca2+, Mg2+ ATPase was exacerbated by lipoamino acid conjugation in both isolated SR and in skinned EDL fibres. 7. The results show that lipoamino acid conjugation of A region peptides increases their membrane permeability without impairing their structure or efficacy in activating skeletal and cardiac RyRs.

Asunto(s)

Permeabilidad de la Membrana Celular/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células CACO-2 , Cafeína/farmacología , Calcio/metabolismo , Técnicas de Cultivo de Célula , Permeabilidad de la Membrana Celular/fisiología , Relación Dosis-Respuesta a Droga , Electrofisiología , Humanos , Membrana Dobles de Lípidos , Datos de Secuencia Molecular , Estructura Molecular , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Estructura Secundaria de Proteína , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Estereoisomerismo , Relación Estructura-Actividad