RESUMEN
Striated muscle represents one of the best models for studies on Ca(2+) signalling. However, although much is known on the localisation and molecular interactions of the ryanodine receptors (RyRs), far less is known on the localisation and on the molecular interactions of the inositol trisphosphate receptors (InsP(3)Rs) in striated muscle cells. Recently, members of the Homer protein family have been shown to cluster type 1 metabotropic glutamate receptors (mGluR1) in the plasma membrane and to interact with InsP(3)R in the endoplasmic reticulum of neurons. Thus, these scaffolding proteins are good candidates for organising plasma membrane receptors and intracellular effector proteins in signalosomes involved in intracellular Ca(2+) signalling. Homer proteins are also expressed in skeletal muscle, and the type 1 ryanodine receptor (RyR1) contains a specific Homer-binding motif. We report here on the relative sub-cellular localisation of InsP(3)Rs and Homer proteins in skeletal muscle cells with respect to the localisation of RyRs. Immunofluorescence analysis showed that both Homer and InsP(3)R proteins present a staining pattern indicative of a localisation at the Z-line, clearly distinct from that of RyR1. Consistent herewith, in sub-cellular fractionation experiments, Homer proteins and InsP(3)R were both found in the fractions enriched in longitudinal sarcoplasmic reticulum (LSR) but not in fractions of terminal cisternae that are enriched in RyRs. Thus, in skeletal muscle, Homer proteins may play a role in the organisation of a second Ca(2+) signalling compartment containing the InsP(3)R, but are apparently not involved in the organisation of RyRs at triads.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas Portadoras/metabolismo , Músculo Esquelético/metabolismo , Neuropéptidos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Retículo Sarcoplasmático/metabolismo , Anticuerpos/inmunología , Calcio/metabolismo , Canales de Calcio/inmunología , Proteínas Portadoras/inmunología , Técnica del Anticuerpo Fluorescente , Proteínas de Andamiaje Homer , Receptores de Inositol 1,4,5-Trifosfato , Fibras Musculares Esqueléticas/metabolismo , Neuropéptidos/inmunología , Receptores Citoplasmáticos y Nucleares/inmunología , Canal Liberador de Calcio Receptor de Rianodina/inmunología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The action of cyclic-ADP-ribose was studied on calcium release from sarcoplasmic reticulum of skeletal muscles of neonatal and adult wild-type and RyR3-deficient mice. cADPR increased calcium efflux from microsomes, enhanced caffeine-induced calcium release, and, in 20% of the tests, triggered calcium release in single muscle fibers. These responses occurred only in the diaphragm of adult RyR3-deficient mice. cADPR action was abolished by ryanodine, ruthenium red, and 8-brome-cADPR. These results strongly favor a specific action of cADPR on RyR1. The responsiveness of RyR1 appears in adult muscles when RyR3 is lacking.
Asunto(s)
Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/farmacología , Calcio/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , ADP-Ribosa Cíclica , Ratones , Ratones Noqueados , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Permeabilidad , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
Point mutations in the ryanodine receptor (RYR1) gene are associated with malignant hyperthermia, an autosomal dominant disorder triggered in susceptible people (MHS) by volatile anaesthetics and depolarising skeletal muscle relaxants. To date, 17 missense point mutations have been identified in the human RYR1 gene by screening of the cDNA obtained from muscle biopsies. Here we report single strand conformation polymorphism (SSCP) screening for nine of the most frequent RYR1 mutations using genomic DNA isolated from MHS patients. In addition, the Argl63Cys mutation was analysed by restriction enzyme digestion. We analysed 57 unrelated patients and detected seven of the known RYR1 point mutations. Furthermore, we found a new mutation, Arg2454His, segregating with the MHS phenotype in a large pedigree and a novel amino acid substitution at position 2436 in another patient, indicating a 15.8% frequency of these mutations in Italian patients. A new polymorphic site in intron 16 that causes the substitution of a G at position -7 with a C residue was identified.
Asunto(s)
Hipertermia Maligna/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Cafeína/metabolismo , Cartilla de ADN , Femenino , Halotano/metabolismo , Humanos , Italia , Masculino , Músculos/metabolismo , Miopatías Nemalínicas/genética , Linaje , Mutación Puntual , Polimorfismo Genético , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
Incubation of normal mitochondria at 45 degrees C results in increases of respiration and of total apparent proton conductance (TAPC, respiration/proton motive force) and in an upward shift of the flow-force relationships. Similar effects are observed during operation of the redox proton pumps at different sites of the respiratory chain. These effects are accompanied by an almost equivalent increase of the passive proton conductance (PPC, proton leakage/proton motive force). In mitochondria from 3,3,5-triiodo-L-thyronine (T3)-treated rats there are also increases of respiration and of TAPC and an upward shift of flow-force relationships, more pronounced at the level of the cytochrome oxidase proton pump. However, at variance from the incubation at 45 degrees C, in mitochondria from T3-treated rats there is only a slight increase of PPC. Addition of bovine serum albumin to normal mitochondria incubated at 45 degrees C results in a marked depression of TAPC in the nonlinear range of the flow-force relationships. An equivalent effect is not observed in mitochondria from T3-treated rats. The experimental results have been compared with computer simulations obtained on the basis of a chemiosmotic model of energy transduction. The increase of TAPC following incubation at high temperature is apparently due to changes of the proton conductance mainly at the level of PPC, while the increase of TAPC following T3 administration is rather due to changes presumably at the level of the redox or ATPase proton pumps.
Asunto(s)
Hipertiroidismo/metabolismo , Mitocondrias Hepáticas/metabolismo , Fosforilación Oxidativa , Consumo de Oxígeno , Adenosina Trifosfato/metabolismo , Animales , Membranas Intracelulares/metabolismo , Membranas Intracelulares/fisiología , Cinética , Masculino , Potenciales de la Membrana , Proteínas de la Membrana/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Potasio/metabolismo , Ratas , Ratas Endogámicas , Temperatura , Termodinámica , Triyodotironina/farmacologíaRESUMEN
T3 administration increases the extent of non-linearity in the flow-force relationship between pump proton conductance and protonmotive force. The effect is present also at the ATPase proton pump. These effects are not accompanied by changes in passive proton conductance. Incubation of mitochondria at 45 degrees C also causes an increased non-linearity, accompanied by a partial increase of proton conductance. It appears that the increase of respiratory activity following T3 administration is due to loss of thermodynamic control within or at the proton pumps, an effect which might be attributed to increased slipping.