title: Characterization of a multiprotein complex involved in excitation-transcription coupling of skeletal muscle.
abstract: BACKGROUND
  Electrical activity regulates the expression of skeletal muscle genes by a process known as "excitation-transcription" (E-T) coupling. We have demonstrated that release of adenosine 5'-triphosphate (ATP) during depolarization activates membrane P2X/P2Y receptors, being the fundamental mediators between electrical stimulation, slow intracellular calcium transients, and gene expression. We propose that this signaling pathway would require the proper coordination between the voltage sensor (dihydropyridine receptor, DHPR), pannexin 1 channels (Panx1, ATP release conduit), nucleotide receptors, and other signaling molecules. The goal of this study was to assess protein-protein interactions within the E-T machinery and to look for novel constituents in order to characterize the signaling complex.
  
  
  METHODS
  Newborn derived myotubes, adult fibers, or triad fractions from rat or mouse skeletal muscles were used. Co-immunoprecipitation, 2D blue native SDS/PAGE, confocal microscopy z-axis reconstruction, and proximity ligation assays were combined to assess the physical proximity of the putative complex interactors. An L6 cell line overexpressing Panx1 (L6-Panx1) was developed to study the influence of some of the complex interactors in modulation of gene expression.
  
  
  RESULTS
  Panx1, DHPR, P2Y2 receptor (P2Y2R), and dystrophin co-immunoprecipitated in the different preparations assessed. 2D blue native SDS/PAGE showed that DHPR, Panx1, P2Y2R and caveolin-3 (Cav3) belong to the same multiprotein complex. We observed co-localization and protein-protein proximity between DHPR, Panx1, P2Y2R, and Cav3 in adult fibers and in the L6-Panx1 cell line. We found a very restricted location of Panx1 and Cav3 in a putative T-tubule zone near the sarcolemma, while DHPR was highly expressed all along the transverse (T)-tubule. By Panx1 overexpression, extracellular ATP levels were increased both at rest and after electrical stimulation. Basal mRNA levels of the early gene cfos and the oxidative metabolism markers citrate synthase and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were significantly increased by Panx1 overexpression. Interleukin 6 expression evoked by 20-Hz electrical stimulation (270 pulses, 0.3 ms each) was also significantly upregulated in L6-Panx1 cells.
  
  
  CONCLUSIONS
  We propose the existence of a relevant multiprotein complex that coordinates events involved in E-T coupling. Unveiling the molecular actors involved in the regulation of gene expression will contribute to the understanding and treatment of skeletal muscle disorders due to wrong-expressed proteins, as well as to improve skeletal muscle performance.
authors.affiliation: Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453 Chile ; Instituto de Investigación en Ciencias Odontológicas, Facultad de Odontología, Universidad de Chile, Sergio Livingstone Pohlhammer 943, 8380492 Santiago, Chile.
authors.forename: Manuel
authors.initials: M
authors.lastname: Arias-Calderón
authors.affiliation: Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453 Chile.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Almarza
authors.affiliation: Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453 Chile.
authors.forename: Alexis
authors.initials: A
authors.lastname: Díaz-Vegas
authors.affiliation: Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453 Chile.
authors.forename: Ariel
authors.initials: A
authors.lastname: Contreras-Ferrat
authors.affiliation: Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453 Chile.
authors.forename: Denisse
authors.initials: D
authors.lastname: Valladares
authors.affiliation: Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453 Chile.
authors.forename: Mariana
authors.initials: M
authors.lastname: Casas
authors.affiliation: Programa de Biología Molecular y Celular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453 Chile.
authors.forename: Héctor
authors.initials: H
authors.lastname: Toledo
authors.affiliation: Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453 Chile ; Programa de Biología Molecular y Celular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453 Chile.
authors.forename: Enrique
authors.initials: E
authors.lastname: Jaimovich
authors.affiliation: Instituto de Investigación en Ciencias Odontológicas, Facultad de Odontología, Universidad de Chile, Sergio Livingstone Pohlhammer 943, 8380492 Santiago, Chile.
authors.forename: Sonja
authors.identifier: 0000-0002-2286-2918
authors.initials: S
authors.lastname: Buvinic
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism; D051244:Caveolin 3 / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D017630:Connexins / Q000235:genetics / Q000378:metabolism; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism; D004558:Electric Stimulation; D005786:Gene Expression Regulation; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D046912:Multiprotein Complexes; D009119:Muscle Contraction; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017208:Rats, Wistar; D058473:Receptors, Purinergic P2Y2 / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D020746:Calcium Channels, L-Type; C495548:Cav3 protein, mouse; C495549:Cav3 protein, rat; D051244:Caveolin 3; D017630:Connexins; C090839:Dmd protein, rat; D016189:Dystrophin; D046912:Multiprotein Complexes; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; C503631:Panx1 protein, mouse; D058473:Receptors, Purinergic P2Y2; C085339:apo-dystrophin 1; C479936:pannexin 1, rat; D000255:Adenosine Triphosphate
keywords: Dihydropyridine receptor; Excitation-transcription coupling; Multiprotein complex; Nucleotide receptors; Pannexin 1; Skeletal muscle plasticity