Characterization of a multiprotein complex involved in excitation-transcription coupling of skeletal muscle

Abstract

BackgroundElectrical 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.MethodsNewborn 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.ResultsPanx1, 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.ConclusionsWe 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.

Expression of Pannexin 1 and Pannexin 3 during skeletal muscle development, regeneration, and Duchenne muscular dystrophy

“…The ATP release mediated by Panx1 channels after electrical stimulation was shown to play a key role in transcription of the slow‐type Troponin (TnI) gene (Jorquera et al, ), a signature of the fast‐to‐slow muscle fiber phenotype transition (Casas et al, ). Panx1 was also recently shown to be part of a multiprotein complex, involving DHPR (dihydropyridine receptor), P2Y 2 receptor, dystrophin, and caveolin‐3, relevant to this excitation‐transcription coupling process (Arias‐Calderon et al, ). In addition to their role in the potentiation of contraction and skeletal muscle plasticity, Cea et al () have proposed that Panx1 channels may also help maintain the normal oxidative state of skeletal muscle.…”

“…Panx1 was also recently shown to be part of a multiprotein complex, involving DHPR (dihydropyridine receptor), P2Y 2 receptor, dystrophin, and caveolin-3, relevant to this excitation-transcription coupling process (Arias-Calderon et al, 2016). In addition to their role in the potentiation of contraction and skeletal muscle plasticity, Cea et al (2013) have proposed that Panx1 channels may also help maintain the normal oxidative state of skeletal muscle.…”

Electrical pulse stimulation of cultured skeletal muscle cells as a model for in vitro exercise – possibilities and limitations

“…, Arias‐Calderon et al . ), which all applied only very short‐term EPS (80 ms–10 min). Interestingly, Thelen et al .…”

Endogenous purines modulate K+‐evoked ACh secretion at the mouse neuromuscular junction

“…On the other hand, it cannot be ruled out that K + depolarization also provokes secretion of purines from other cells (i.e., muscle cells, Schwann cells), contributing to the concentration of purines in the extracellular space. In this regard, pannexin 1 was recently proposed as a possible pathway for ATP release from skeletal muscle (Arias‐Calderón et al, ; Buvinic et al, ; Cea, Riquelme, Vargas, Urrutia, & Sáez, ; Riquelme et al, ), and among other stimuli, high extracellular K + increases the activity of pannexin 1 channels (D'Hondt et al, ). However, pannexin 1 is localized in the sarcolemma of the T‐tubules (Cea et al, ; Cea, Riquelme, Vargas, Urrutia, & Sáez, ; Jorquera et al, ; Riquelme et al, ) far from the synaptic area.…”