The central hypothesis of this proposal is that the transverse tubular system (TTS) plays such a preponderant role in the overall properties of mammalian skeletal muscle that, in order to understand the pathophysiology of muscle channelopathies it will be necessary to carefully characterize the electrical properties of this membrane compartment. Changes in membrane potential of the TTS, which are mediated by the activation of ion channels, not only affect the electrical properties of the muscle fiber, but also are responsible for triggering the mechanisms of excitation-contraction coupling (ECC). We will use electrophysiological methods, state-of-the-art optical techniques (which permit to measure TTS voltage changes), and mathematical modeling of the radial spread of the depolarization in this compartment, in order to probe the detailed role that ionic conductances play in these processes. First, we will characterize the passive electrical properties and each of the major conductive pathways in normal mouse muscle fibers under voltage clamp conditions (Aim 1). We will then study the properties and limitations of the TTS propagation in fibers stimulated to elicit repetitive firing and test the effects that alterations in individual conductances (sodium and chloride in particular) have on these properties. The goal is to elucidate the potential role that K accumulation in the lumen of the TTS lumen may play in the phenomenology associated with channelopathies such as periodic paralysis and myotonia (Aim 2). Since a conundrum in the functional investigation of channelopathies is the tenuous demarcation between myotonia and paralysis, in Aim 3 we will investigate whether intricacies of the voltage regulation of the ECC can result in abolition or preservation of the Ca2+ release process depending on the pattern of electrical activity in the TTS. Finally, with the knowledge acquired in previous aims, we will investigate whether the pathogenesis observed in animal models of myotonia and hyperkaelemic periodic paralysis can be understood from alterations in the electrical propagation at the TTS (Aim 4). The knowledge gained with these investigations will not only be relevant towards the understanding of the pathophysiology of channelopathies, but since they will provide basic information about the physiological mechanisms of TTS electrical propagation, they will be of significance for understanding a number of muscle diseases.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MOSS-L (07))
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Boyce, Amanda T
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University of California Los Angeles
Schools of Medicine
Los Angeles
United States
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DiFranco, Marino; Yu, Carl; Quiñonez, Marbella et al. (2015) Inward rectifier potassium currents in mammalian skeletal muscle fibres. J Physiol 593:1213-38
DiFranco, Marino; Quiñonez, Marbella; Shieh, Perry et al. (2014) Action potential-evoked calcium release is impaired in single skeletal muscle fibers from heart failure patients. PLoS One 9:e109309
DiFranco, Marino; Yu, Carl; Quiñonez, Marbella et al. (2013) Age-dependent chloride channel expression in skeletal muscle fibres of normal and HSA(LR) myotonic mice. J Physiol 591:1347-71
DiFranco, Marino; Quinonez, Marbella; Vergara, Julio L (2012) The delayed rectifier potassium conductance in the sarcolemma and the transverse tubular system membranes of mammalian skeletal muscle fibers. J Gen Physiol 140:109-37
DiFranco, Marino; Herrera, Alvaro; Vergara, Julio L (2011) Chloride currents from the transverse tubular system in adult mammalian skeletal muscle fibers. J Gen Physiol 137:21-41
DiFranco, Marino; Tran, Philip; Quinonez, Marbella et al. (2011) Functional expression of transgenic 1sDHPR channels in adult mammalian skeletal muscle fibres. J Physiol 589:1421-42
Ermolova, Natalia; Kudryashova, Elena; DiFranco, Marino et al. (2011) Pathogenity of some limb girdle muscular dystrophy mutations can result from reduced anchorage to myofibrils and altered stability of calpain 3. Hum Mol Genet 20:3331-45
DiFranco, Marino; Vergara, Julio L (2011) The Na conductance in the sarcolemma and the transverse tubular system membranes of mammalian skeletal muscle fibers. J Gen Physiol 138:393-419
Capote, Joana; DiFranco, Marino; Vergara, Julio L (2010) Excitation-contraction coupling alterations in mdx and utrophin/dystrophin double knockout mice: a comparative study. Am J Physiol Cell Physiol 298:C1077-86
DiFranco, Marino; Quinonez, Marbella; Capote, Joana et al. (2009) DNA transfection of mammalian skeletal muscles using in vivo electroporation. J Vis Exp :

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