Protein Kinase C-Theta Controls the CLC-1 Chloride Channel Function and Skeletal Muscle Phenotype: A Biophysical and Gene Expression Study in Pkc-Theta Null Mice

Abstract

In skeletal muscle the resting chloride conductance (gCl), sustained by the ClC-1 chloride channel, controls the sarcolemma electrical stability. Resting gCl is negatively regulated by Protein Kinase C (PKC), since its pharmacological activation closes the ClC-1 channel. A reduced activity of PKC contributes to the low gCl typical of slow-twitch muscles compared to the fast ones. Different PKC isoforms are expressed in skeletal muscle, including the novel isoform PKC-theta, where it mediates various cellular responses. Here we investigated the role of PKC-theta in the regulation of ClC-1 channel activity and expression in extensor digitorum longus (EDL) and in soleus (Sol) muscles, using two models of PKC-theta null mice: a PKC-theta knockout model, in which the PKC-theta gene was inactivated and the mPKC-theta-K/R transgenic model, in which a dominant-negative mutant form of PKC-theta is expressed under a muscle-specific promoter control. Electrophysiological studies showed a gCl increase in Sol and EDL muscle of KO mice with respect to wild-type. Muscle excitability was reduced accordingly. Similar effects were observed in mPKC-theta-K/R mice. By using chelerythrine, a non-specific PKC inhibitor, we demonstrated that other PKC isoforms present in skeletal muscle of PKC-theta null mice are able to further modulate gCl. In parallel, we found that the expression of the ClC-1 channel, evaluated by RT-PCR, was not modified either in EDL or in Sol of PKC-theta-KO mice, demonstrating that PKC-theta does not control the ClC-1 expression but its activity. These results as well as the modification of calcineurin and myocyte-enhancer-factor-2 expression demonstrate the involvement of PKC-theta in the control of muscle phenotype. We conclude that PKC-theta plays a role in regulating ClC-1 chloride channel activity and skeletal muscle function. (ASI-OSMA).