RESUMO
Junctophilins (JPs) anchor the endo/sarcoplasmic reticulum to the plasma membrane, thus contributing to the assembly of junctional membrane complexes in striated muscles and neurons. Recent studies have shown that JPs may be also involved in regulating Ca2+ homeostasis. Here, we report that in skeletal muscle, JP1 and JP2 are part of a complex that, in addition to ryanodine receptor 1 (RyR1), includes caveolin 3 and the dihydropyridine receptor (DHPR). The interaction between JPs and DHPR was mediated by a region encompassing amino acids 230-369 and amino acids 216-399 in JP1 and JP2, respectively. Immunofluorescence studies revealed that the pattern of DHPR and RyR signals in C2C12 cells knocked down for JP1 and JP2 was rather diffused and characterized by smaller puncta in contrast to that observed in control cells. Functional experiments revealed that down-regulation of JPs in differentiated C2C12 cells resulted in a reduction of intramembrane charge movement and the L-type Ca2+ current accompanied by a reduced number of DHPRs at the plasma membrane, whereas there was no substantial alteration in Ca2+ release from the sterol regulatory element-binding protein. Altogether, these results suggest that JP1 and JP2 can facilitate the assembly of DHPR with other proteins of the excitation-contraction coupling machinery.
Assuntos
Canais de Cálcio Tipo L/química , Proteínas de Membrana/química , Músculo Esquelético/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Diferenciação Celular , Glutationa Transferase/metabolismo , Humanos , Masculino , Camundongos , Modelos Biológicos , Músculos/metabolismo , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
The sarcoplasmic reticulum (SR) of skeletal muscle cells is a complex network of tubules and cisternae that share a common lumen delimited by a single continuous membrane. The SR contains longitudinal and junctional domains characterized by distinctive patterns of protein localization, but how SR proteins reach and/or are retained at these sites is not known. Here, we report that the organization of longitudinal SR proteins is a slow process characterized by temporally distinct patterns of protein localization. In contrast, junctional SR proteins rapidly and synchronously assembled into clusters which, however, merged into mature triadic junctions only after completion of longitudinal SR protein organization. Fluorescence recovery after photobleaching experiments indicated that SR organization was accompanied by significant changes in the dynamic properties of longitudinal and junctional proteins. The decrease in mobility that accompanied organization of the longitudinal SR proteins ank1.5-GFP and GFP-InsP3R1 was abrogated by deletion of specific binding sites for myofibrillar or cytoskeletal proteins, respectively. Assembly of junctional SR domains was accompanied by a strong decrease in mobility of junctional proteins that in triadin appeared to be mediated by its intraluminal region. Together, the data suggest that the organization of specific SR domains results from a process of membrane reorganization accompanied by the establishment of multiple protein-protein interactions with intrinsic and extrinsic cues.
Assuntos
Junções Intercelulares/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/citologia , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/metabolismo , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Diferenciação Celular , Proteínas de Fluorescência Verde/metabolismo , Cinética , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/química , Células NIH 3T3 , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The sarcoplasmic reticulum (SR) of skeletal muscle cells is a convoluted structure composed of a variety of tubules and cisternae, which share a continuous lumen delimited by a single continuous membrane, branching to form a network that surrounds each myofibril. In this network, some specific domains basically represented by the longitudinal SR and the junctional SR can be distinguished. These domains are mainly dedicated to Ca(2+) homeostasis in relation to regulation of muscle contraction, with the longitudinal SR representing the sites of Ca(2+) uptake and storage and the junctional SR representing the sites of Ca(2+) release. To perform its functions, the SR takes contact with other cellular elements, the sarcolemma, the contractile apparatus and the mitochondria, giving rise to a number of interactions, most of which are still to be defined at the molecular level. This review will describe some of the most recent advancements in understanding the organization of this complex network and its specific domains. Furthermore, we shall address initial evidence on how SR proteins are retained at distinct SR domains.