The discovery of pathogenetic mechanisms is essential to identify new therapeutic approaches in Amyotrophic Lateral Sclerosis (ALS). Here we investigated the role of the most important ion channels in skeletal muscle of an ALS animal model (MLC/SOD1G93A) carrying a mutated SOD1 exclusively in this tissue, avoiding motor-neuron involvement. Ion channels are fundamental proteins for muscle function, and also to sustain neuromuscular junction and nerve integrity. By a multivariate statistical analysis, using machine learning algorithms, we identified the discriminant genes in MLC/SOD1G93A mice. Surprisingly, the expression of ClC-1 chloride channel, present only in skeletal muscle, was reduced. Also, the expression of Protein Kinase-C, known to control ClC-1 activity, was increased, causing its inhibition. The functional characterization confirmed the reduction of ClC-1 activity, leading to hyperexcitability and impaired relaxation. The increased expression of ion channel coupled AMPA-receptor may contribute to sustained depolarization and functional impairment. Also, the decreased expression of irisin, a muscle-secreted peptide protecting brain function, may disturb muscle-nerve connection. Interestingly, the in-vitro application of chelerythrine or acetazolamide, restored ClC-1 activity and sarcolemma hyperexcitability in these mice. These findings show that ion channel function impairment in skeletal muscle may lead to motor-neuron increased vulnerability, and opens the possibility to investigate on new compounds as promising therapy.