title: Protein kinase C-dependent regulation of ClC-1 channels in active human muscle and its effect on fast and slow gating.
abstract: Regulation of ion channel function during repeated firing of action potentials is commonly observed in excitable cells. Recently it was shown that muscle activity is associated with rapid, protein kinase C (PKC)-dependent ClC-1 Cl(-) channel inhibition in rodent muscle. While this PKC-dependent ClC-1 inhibition during muscle activity was shown to be important for the maintenance of contractile endurance in rat muscle it is unknown whether a similar regulation exists in human muscle. Also, the molecular mechanisms underlying the observed PKC-dependent ClC-1 inhibition are unclear. Here we present the first demonstration of ClC-1 inhibition in active human muscle fibres, and we determine the changes in ClC-1 gating that underlie the PKC-dependent ClC-1 inhibition in active muscle using human ClC-1 expressed in Xenopus oocytes. This activity-induced ClC-1 inhibition is suggested to represent a mechanism by which human muscle fibres maintain their excitability during sustained activity.
  
  
  
  Repeated firing of action potentials (APs) is known to trigger rapid, protein kinase C (PKC)-dependent inhibition of ClC-1 Cl(-) ion channels in rodent muscle and this inhibition is important for contractile endurance. It is currently unknown whether similar regulation exists in human muscle, and the molecular mechanisms underlying PKC-dependent ClC-1 inhibition are unclear. This study first determined whether PKC-dependent ClC-1 inhibition exists in active human muscle, and second, it clarified how PKC alters the gating of human ClC-1 expressed in Xenopus oocytes. In human abdominal and intercostal muscles, repeated AP firing was associated with 30-60% reduction of ClC-1 function, which could be completely prevented by PKC inhibition (1 μm GF109203X). The role of the PKC-dependent ClC-1 inhibition was evaluated from rheobase currents before and after firing 1000 APs: while rheobase current was well maintained after activity under control conditions it rose dramatically if PKC-dependent ClC-1 inhibition had been prevented with the inhibitor. This demonstrates that the ClC-1 inhibition is important for maintenance of excitability in active human muscle fibres. Oocyte experiments showed that PKC activation lowered the overall open probability of ClC-1 in the voltage range relevant for AP initiation in muscle fibres. More detailed analysis of this reduction showed that PKC mostly affected the slow gate of ClC-1. Indeed, there was no effect of PKC activation in C277S mutated ClC-1 in which the slow gate is effectively locked open. It is concluded that regulation of excitability of active human muscle fibres relies on PKC-dependent ClC-1 inhibition via a gating mechanism.
authors: Department of Biomedicine, Aarhus University, Ole Worms Allé 4, 8000, Aarhus C, Denmark.|Centre for Neuroscience and Department of Neurology, University of California, Davis, CA, 95618, USA.
authors: Anders
authors: 0000-0002-3725-5846
authors: A
authors: Riisager
authors: Department of Biomedicine, Aarhus University, Ole Worms Allé 4, 8000, Aarhus C, Denmark.|Department of Cardiothoracic and Vascular Surgery, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200, Aarhus N, Denmark.
authors: Frank Vincenzo
authors: FV
authors: de Paoli
authors: Centre for Neuroscience and Department of Neurology, University of California, Davis, CA, 95618, USA.
authors: Wei-Ping
authors: WP
authors: Yu
authors: Department of Biomedicine, Aarhus University, Ole Worms Allé 4, 8000, Aarhus C, Denmark.
authors: Thomas Holm
authors: TH
authors: Pedersen
authors: Centre for Neuroscience and Department of Neurology, University of California, Davis, CA, 95618, USA.
authors: Tsung-Yu
authors: TY
authors: Chen
authors: Department of Biomedicine, Aarhus University, Ole Worms Allé 4, 8000, Aarhus C, Denmark.
authors: Ole Baekgaard
authors: OB
authors: Nielsen
mesh_terms: D000009:Abdominal Muscles / Q000502:physiology*; D000200:Action Potentials; D000818:Animals; D018118:Chloride Channels / Q000235:genetics / Q000502:physiology*; D005260:Female; D006801:Humans; D007366:Intercostal Muscles / Q000502:physiology*; D015640:Ion Channel Gating / Q000502:physiology*; D009865:Oocytes; D011493:Protein Kinase C / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: C086321:CLC-1 channel; D018118:Chloride Channels; D011493:Protein Kinase C