Pannexin-1 and CaV1.1 show reciprocal interaction during excitation–contraction and excitation–transcription coupling in skeletal muscle (original) (raw)
Related papers
Pannexin 1 channels in skeletal muscles
Frontiers in Physiology, 2014
Normal myotubes and adult innervated skeletal myofibers express the glycoprotein pannexin1 (Panx1). Six of them form a "gap junction hemichannel-like" structure that connects the cytoplasm with the extracellular space; here they will be called Panx1 channels. These are poorly selective channels permeable to ions, small metabolic substrate, and signaling molecules. So far little is known about the role of Panx1 channels in muscles but skeletal muscles of Panx1 −/− mice do not show an evident phenotype. Innervated adult fast and slow skeletal myofibers show Panx1 reactivity in close proximity to dihydropyridine receptors in the sarcolemma of T-tubules. These Panx1 channels are activated by electrical stimulation and extracellular ATP. Panx1 channels play a relevant role in potentiation of muscle contraction because they allow release of ATP and uptake of glucose, two molecules required for this response. In support of this notion, the absence of Panx1 abrogates the potentiation of muscle contraction elicited by repetitive electrical stimulation, which is reversed by exogenously applied ATP. Phosphorylation of Panx1 Thr and Ser residues might be involved in Panx1 channel activation since it is enhanced during potentiation of muscle contraction. Under denervation, Panx1 levels are upregulated and this partially explains the reduction in electrochemical gradient, however its absence does not prevent denervation-induced atrophy but prevents the higher oxidative state. Panx1 also forms functional channels at the cell surface of myotubes and their functional state has been associated with intracellular Ca 2+ signals and regulation of myotube plasticity evoked by electrical stimulation. We proposed that Panx1 channels participate as ATP channels and help to keep a normal oxidative state in skeletal muscles.
ATP Released by Electrical Stimuli Elicits Calcium Transients and Gene Expression in Skeletal Muscle
Journal of Biological Chemistry, 2009
ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca 2؉ concentration, with an EC 50 value of 7.8 ؎ 3.1 M. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 M suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y 2 receptor and pannexin-1. As reported previously for electrical stimulation, 500 M ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca 2؉ homeostasis and muscle physiology.
Neuropharmacology, 2013
During repetitive stimulation of skeletal muscle, extracellular ATP levels raise, activating purinergic receptors, increasing Ca 2þ influx, and enhancing contractile force, a response called potentiation. We found that ATP appears to be released through pannexin1 hemichannels (Panx1 HCs). Immunocytochemical analyses and function were consistent with pannexin1 localization to T-tubules intercalated with dihydropyridine and ryanodine receptors in slow (soleus) and fast (extensor digitorum longus, EDL) muscles. Isolated myofibers took up ethidium (Etd þ ) and released small molecules (as ATP) during electrical stimulation. Consistent with two glucose uptake pathways, induced uptake of 2-NBDG, a fluorescent glucose derivative, was decreased by inhibition of HCs or glucose transporter (GLUT4), and blocked by dual blockade. Adult skeletal muscles apparently do not express connexins, making it unlikely that connexin hemichannels contribute to the uptake and release of small molecules. ATP release, Etd þ uptake, and potentiation induced by repetitive electrical stimulation were blocked by HC blockers and did not occur in muscles of pannexin1 knockout mice. MRS2179, a P2Y 1 R blocker, prevented potentiation in EDL, but not soleus muscles, suggesting that in fast muscles ATP activates P2Y 1 but not P2X receptors. Phosphorylation on Ser and Thr residues of pannexin1 was increased during potentiation, possibly mediating HC opening. Opening of Panx1 HCs during repetitive activation allows efflux of ATP, influx of glucose and possibly Ca 2þ too, which are required for potentiation of contraction.
Skeletal Muscle, 2016
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, P2Y 2 receptor (P2Y 2 R), and dystrophin co-immunoprecipitated in the different preparations assessed. 2D blue native SDS/PAGE showed that DHPR, Panx1, P2Y 2 R and caveolin-3 (Cav3) belong to the same multiprotein complex. We observed co-localization and protein-protein proximity between DHPR, Panx1, P2Y 2 R, 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.
Cav1.1 controls frequency-dependent events regulating adult skeletal muscle plasticity
An important pending question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype as slow or fast twitch muscle fibers. We have previously shown that voltage-gated L-type calcium channel (Cav1.1) acts as a voltage sensor for activation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P 3 ]-dependent Ca 2+ signals that regulates gene expression. ATP released by muscle cells after electrical stimulation through pannexin-1 channels plays a key role in this process. We show now that stimulation frequency determines both ATP release and Ins(1,4,5)P 3 production in adult skeletal muscle and that Cav1.1 and pannexin-1 colocalize in the transverse tubules. Both ATP release and increased Ins(1,4,5)P 3 was seen in flexor digitorum brevis fibers stimulated with 270 pulses at 20 Hz, but not at 90 Hz. 20 Hz stimulation induced transcriptional changes related to fast-to-slow muscle fiber phenotype transition that required ATP release. Addition of 30 mM ATP to fibers induced the same transcriptional changes observed after 20 Hz stimulation. Myotubes lacking the Cav1.1-a1 subunit released almost no ATP after electrical stimulation, showing that Cav1.1 has a central role in this process. In adult muscle fibers, ATP release and the transcriptional changes produced by 20 Hz stimulation were blocked by both the Cav1.1 antagonist nifedipine (25 mM) and by the Cav1.1 agonist (-)S-BayK 8644 (10 mM). We propose a new role for Cav1.1, independent of its calcium channel activity, in the activation of signaling pathways allowing muscle fibers to decipher the frequency of electrical stimulation and to activate specific transcriptional programs that define their phenotype.
The Journal of …, 2006
In skeletal muscle, Ca 2+ is implicated in contraction, and in regulation of gene expression. An alteration of [Ca 2+ ] i homeostasis is responsible, at least partially, for the muscle degeneration that occurs after eccentric contractions in Duchenne muscular dystrophy, a disease characterized by the loss of the cytoskeletal protein dystrophin. Using patch clamp in the cell-attached configuration, we characterized the store-operated channels (SOCs) and the stretch-activated channels (SACs) present in isolated mouse skeletal muscle. SOCs were voltage independent, had a unitary conductance between 7 and 8 pS (110 mM Ca 2+ in the pipette), and their open probability increased when the sarcoplasmic reticulum was depleted by thapsigargin. These SOCs were identical to those previously described in the pathophysiology of Duchenne muscular dystrophy. Under the same experimental conditions, we detected a channel activity that was increased by applying a negative pressure to the patch electrode. The SACs responsible for this current had the same unitary conductance and current-voltage relationship as those observed for SOCs. SOCs and SACs had a similar sensitivity to pharmacological agents such as Gd 3+ , SKF-96365, 2-aminoethoxydiphenyl borate and GsMTx4 toxin. Moreover, stimulation with IGF-1 increased the occurrence of the activity of both channel types. Together, these observations suggest that SOCs and SACs might belong to the same population or share common constituents. From a functional point of view, treatment of soleus muscle with SKF-96365 or GsMTx4 toxin increased its sensitivity to a fatigue protocol, suggesting that the influx of Ca 2+ that occurs through these channels during contraction is also involved in force maintaining during repeated stimulations.
Excitation-transcription coupling in skeletal muscle: the molecular pathways of exercise
Biological Reviews, 2010
Muscle fibres have different properties with respect to force, contraction speed, endurance, oxidative/glycolytic capacity etc. Although adult muscle fibres are normally post-mitotic with little turnover of cells, the physiological properties of the pre-existing fibres can be changed in the adult animal upon changes in usage such as after exercise. The signal to change is mainly conveyed by alterations in the patterns of nerve-evoked electrical activity, and is to a large extent due to switches in the expression of genes. Thus, an excitation-transcription coupling must exist. It is suggested that changes in nerve-evoked muscle activity lead to a variety of activity correlates such as increases in free intracellular Ca 2+ levels caused by influx across the cell membrane and/or release from the sarcoplasmatic reticulum, concentrations of metabolites such as lipids and ADP, hypoxia and mechanical stress. Such correlates are detected by sensors such as protein kinase C (PKC), calmodulin, AMP-activated kinase (AMPK), peroxisome proliferator-activated receptor δ (PPARδ), and oxygen dependent prolyl hydroxylases that trigger intracellular signaling cascades. These complex cascades involve several transcription factors such as nuclear factor of activated T-cells (NFAT), myocyte enhancer factor 2 (MEF2), myogenic differentiation factor (myoD), myogenin, PPARδ, and sine oculis homeobox 1/eyes absent 1 (Six1/Eya1). These factors might act indirectly by inducing gene products that act back on the cascade, or as ultimate transcription factors binding to and transactivating/repressing genes for the fast and slow isoforms of various contractile proteins and of metabolic enzymes. The determination of size and force is even more complex as this involves not only intracellular signaling within the muscle fibres, but also muscle stem cells called satellite cells. Intercellular signaling substances such as myostatin and insulin-like growth factor 1 (IGF-1) seem to act in a paracrine fashion. Induction of hypertrophy is accompanied by the satellite cells fusing to myofibres and thereby increasing the capacity for protein synthesis. These extra nuclei seem to remain part of the fibre even during subsequent atrophy as a form of muscle memory facilitating retraining. In addition to changes in myonuclear number during hypertrophy, changes in muscle fibre size seem to be caused by alterations in transcription, translation (per nucleus) and protein degradation.
Biophysical Journal, 2005
The b 1a subunit of the skeletal muscle voltage-gated Ca 21 channel plays a fundamental role in the targeting of the channel to the tubular system as well as in channel function. To determine whether this cytosolic auxiliary subunit is also a regulatory protein of Ca 21 release from the sarcoplasmic reticulum in vivo, we pressure-injected the b 1a subunit into intact adult mouse muscle fibers and recorded, with Fluo-3 AM, the intracellular Ca 21 signal induced by the action potential. We found that the b 1a subunit significantly increased, within minutes, the amplitude of Ca 21 release without major changes in its time course. b 1a subunits with the carboxy-terminus region deleted did not show an effect on Ca 21 release. The possibility that potentiation of Ca 21 release is due to a direct interaction between the b 1a subunit and the ryanodine receptor was ruled out by bilayer experiments of RyR1 single-channel currents and also by Ca 21 flux experiments. Our data suggest that the b 1a subunit is capable of regulating E-C coupling in the short term and that the integrity of the carboxy-terminus region is essential for its modulatory effect.