Genetic and pharmacological studies of KV7 channels in excitability disorders

The KV7 (KCNQ) subfamily of voltage-gated potassium channels plays a key role in the regulation of neuronal excitability. By controlling the neuronal M-current, KV7 channels represent pharmacological targets for neurological disorders characterized by hyperexcitability, including epilepsy, developmental encephalopathies (DEEs), and pain. Accordingly, clinically approved KV7 channel openers such as retigabine and flupirtine have demonstrated anticonvulsant and analgesic efficacy, but their clinical use has been discontinued due to side effects unrelated to their primary mechanism of action. This thesis aims to investigate the role of KV7 channel modulation in neuronal excitability disorders through two approaches. First, it focuses on the functional consequences of a recurrent de novo KCNQ2 variant (A265V) identified in a proband with neonatal-onset DEE; second, it explores the analgesic potential of a novel retigabine analogue, compound 60 (C60), by means of functional, behavioral, and molecular assessments in naïve animals and in an oxaliplatin-induced mice model of neuropathic pain. Whole-cell patch-clamp recordings performed in a heterologous expression system revealed that KV7.2 A265V channels exhibited reduced current density and altered voltage-dependent activation, and caused a previously unreported use-dependent inactivation process during sustained depolarization, highlighting a novel pathogenetic mechanism underlying KCNQ2- related DEE. In the oxaliplatin-induced neuropathic pain model, acute administration of C60 dose- dependently reduced mechanical and cold allodynia similarly to retigabine. In chronic conditions, both drugs prevented the development of the model. Molecular analyses revealed that chronic KV7 activation counteracted oxaliplatin-induced alterations in KV7 subunit expression and in astrogliosis in pain-related regions. C60 and retigabine were tested in vitro in spinal cords from naïve mice, in which C60 displayed greater potency than retigabine in suppressing both non-nociceptive and nociceptive spinal reflexes. Overall, this work expands the understanding of KV7 channel dysfunction in KCNQ2-DEEs by identifying a novel potassium current inactivation pathomechanism, and demonstrates that pharmacological KV7 activation, particularly with C60, represents a promising non-opioid strategy for the treatment or prevention of acute and chronic pain.