Abstracts

Rationale: KCNQ2 and KCNQ3 encode voltage-dependent potassium channel subunits which co-assemble to form M-channels. Mutations in these genes cause an inherited epilepsy, benign familial neonatal seizures. We and others showed that KCNQ2 and KCNQ3 subunits are concentrated at the axon initial segments (AISs) of many types of CNS neurons, including dentate granule cells, hippocampal pyramidal cells, and cerebral cortical pyramidal cells. These findings imply that AIS KCNQ channels may contribute to controlling neuronal firing and that enhancing the opening of AIS KCNQ channels may represent a novel approach for correcting neuronal hyperexcitability. Methods: We performed whole cell voltage-clamp experiments on transfected HEK cells to characterize the pharmacological effects of two selective neuronal KCNQ openers, retigabine and CFPA. We used acutely dissociated cerebellar Purkinje neurons for parallel studies of native KCNQ channels. The AIS of these cells can be preserved during cell dissociation. This allows the relative contribution of channels restricted to the AIS to be discerned. We developed protocols allowing isolation of two classes of Purkinje cells: one possessing only the neuronal soma and very proximal dendrite (ie., lacking the AIS), the other possessing the soma, proximal dendrite, and AIS. We tested the effects of retigabine and CFPA on neuronal firing of both groups of cells. Results: When applied to HEK cells, retigabine and CFPA increased channel activation at resting and weakly depolarized membrane potentials by shifting the voltage-dependence of KCNQ2/3 channel activation to hyperpolarized potentials and slowing deactivation kinetics. Current clamp recording of Purkinje neurons possessing and lacking AIS exhibited spontaneous action potential firing (20 – 160Hz). Treatment of neurons possessing AISs with 5 µM retigabine or 5 µM CFPA reduced spontaneous action potential frequency by 20% and 50% respectively. By contrast, treatment of neurons lacking AISs with these drugs resulted in much smaller diminishment in neuronal firing rates. Conclusions: AIS-localized KCNQ channels appear to be critical for the ability of KCNQ channel openers to control the intrinsic excitability of Purkinje cells. KCNQ selective openers can enhance KCNQ current at AIS and consequently inhibit intrinsic neuronal excitability and responsiveness. It is expected that this is the case in neurons possessing KCNQ2/3 concentration at AIS from regions implicated in seizures and epileptogenesis. Regulation of intrinsic neuronal excitability via axonal KCNQ channel modulation may be a novel and general strategy for preventing and treating seizures and epilepsy.