The ATP required for potentiation of skeletal muscle contraction is released via pannexin hemichannels q Manuel A. Riquelme a , Luis A. Cea a, f , José L. Vega a, b, c , Mauricio P. Boric a , Hannah Monyer b, c , Michael V.L. Bennett d , Marina Frank e , Klaus Willecke e , Juan C. Sáez a, f, * a Departamento de Fisiología, Pontificia Universidad Católica de Chile, Santiago 8, Chile b Laboratorio de Fisiología Experimental (EPhyL), Instituto Antofagasta, Universidad de Antofagasta, Antofagasta, Chile c Department of Clinical Neurobioloy, University of Heidelberg, 6012 Heidelberg, Germany d Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA e Life and Medical Sciences Institute, Molecular Genetics, University of Bonn, 53115 Bonn, Germany f Instituto Milenio, Centro Interdisciplinario de Neurociencias de Valparaíso, Valparaíso, Chile article info Article history: Received 8 January 2013 Received in revised form 7 March 2013 Accepted 11 March 2013 Keywords: Pannexin Purinergic receptors Contractil force abstract During repetitive stimulation of skeletal muscle, extracellular ATP levels raise, activating purinergic re- ceptors, 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 dihy- dropyridine 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 up- take, 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. This article is part of the Special Issue Section entitled ‘Current Pharmacology of Gap Junction Channels and Hemichannels’. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction A single twitch of a vertebrate skeletal muscle can occur in the absence of extracellular Ca 2þ , and the required rise in intracellular free Ca 2þ , [Ca 2þ ] i , is released from intracellular stores (Araya et al., 2003). Repetitive twitches lead to potentiation of the contraction through accumulation of free Ca 2þ in the cytoplasm (Sandona et al., 2005; Zhi et al., 2005). ATP signaling is involved in both, fast and slow muscle potentiation. In slow skeletal muscles, potentiation depends on activation of purinergic P2X 4 receptors present in the T-tubule membrane and entry of extracellular Ca 2þ (Sandona et al., 2005). However, P2X 4 receptors are not found in fast skeletal muscle (Sandona et al., 2005), and potentiation in these muscles occurs without of extracellular Ca 2þ (Louboutin Abbreviations: Panx1 HCs, pannexin1 hemichannels; Etd þ , ethidium. q The data of this work was presented by Dr. Manuel A. Riquelme as partial fulfillment of the requirements to obtain the degree of Ph.D. in Physiological Sci- ences at the Pontificia Universidad Católica de Chile. This work was partially sup- ported by FONDECYT grants (1111033 to J.C.S. and 3120006 to J.L.V.) Anillo ACT71 (to J.C.S.) and a CONICYT Doctoral thesis grant (AT-23070155 to M.A.R.), an NIH grant (NS045287 to M.V.L.B) and DAAD-CONICYT grant (to J.C.S. and K.W.). * Corresponding author. Departamento de Fisiología, Pontificia Universidad Católica de Chile, casilla 114-D, Santiago 8, Chile. Tel.: þ56 2 6862862; fax: þ56 2 2225515. E-mail addresses: jsaez@bio.puc.cl, jcsaezc@gmail.com (J.C. Sáez). Contents lists available at SciVerse ScienceDirect Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.03.022 Neuropharmacology 75 (2013) 594e603