Protein kinase C‐dependent regulation of ClC‐1 channels in active human muscle and its effect on fast and slow gating

Abstract

Key pointsr 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.r 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.r Also, the molecular mechanisms underlying the observed PKC-dependent ClC-1 inhibition are unclear.r 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.r This activity-induced ClC-1 inhibition is suggested to represent a mechanism by which human muscle fibres maintain their excitability during sustained activity.Abstract 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.

Role of physiological ClC-1 Cl− ion channel regulation for the excitability and function of working skeletal muscle

“…On this basis, it was suggested that there exists an optimum degree ClC-1 inhibition for maintenance of contractile endurance. Interestingly, this degree of ClC-1 inhibition is close to what has been observed to occur in both rodent and human muscles when muscles are activated repeatedly ( Pedersen et al, 2009a , b ; Riisager et al, 2014 , 2016 ).…”

“…This suggested that PKC activation affects the function of active channels by inhibiting ion permeation, effectively reducing single-channel conductance. However, more recent findings on ClC-1 channels expressed in Xenopus oocytes reported that the ClC-1 activation curve was shifted to more depolarized membrane potentials when exposed to phorbol esters ( Hsiao et al, 2010 ; Riisager et al, 2016 ). In the latter study, it was additionally shown that the PKC activation changed the voltage-dependent open probability through alterations of both the protopore and common gating mechanisms, whereas the maximum current was not affected.…”

“…Although PKC has been known for a long time to be a potent regulator of ClC-1 function and G Cl in native tissue, it is only recently that the physiological role of this regulation in active muscle has become clear. As described in detail below (Acute regulation of ClC-1 in the active muscle fiber), onset of repeated firing of action potentials triggers a PKC-dependent reduction in G Cl ( Pedersen et al, 2009a , b ; Riisager et al, 2016 ). This suggests that PKC-dependent ClC-1 inhibition has the physiological role of preserving muscle excitability during muscle activity.…”

“…Using inhibitors of myosin heavy chain II (BTS and blebbistatin) it has, however, become possible to almost abolish contractile activity while leaving other steps in excitation–contraction coupling of muscle fibers intact ( Macdonald et al, 2005 ; Pedersen et al, 2009b ). When exciting action potentials via one inserted microelectrode and recording the membrane voltage with one or two other electrodes, it has thus become possible to maintain electrodes inserted in muscle fibers while they fire thousands of action potentials ( Pedersen et al, 2009a ; Riisager et al, 2014 , 2016 ). Using this approach, experiments with intact rat, mouse, and human muscles have revealed that the ion channels that determine G M are highly regulated during muscle activity and that this regulation primarily occurs through modulation of ClC-1 channel function ( Pedersen et al, 2009a , b ; Riisager et al, 2016 ).…”

Defective Gating and Proteostasis of Human ClC-1 Chloride Channel: Molecular Pathophysiology of Myotonia Congenita

“…Furthermore, intensive exercise may cause muscle acidosis (74)(75)(76) as well as elevate intracellular calcium (Ca 2+ ) concentration that activates protein kinase C (PKC). Interestingly, both intracellular acidosis and PKC activation are known to inhibit ClC-1 channel activation (49,(77)(78)(79). This down-regulation of skeletal muscle membrane Cl − conductance, as well as the ensuing reduction in the membrane input conductance, effectively counteracts the effect of K + -induced slow inactivation of Na + channels, restoring muscle excitability and preventing muscle fatigue.…”