Title : Molecular mechanism underlying a traditional anticonvulsant: Synergistic KCNQ2/3 potassium channel activation by dual components of Mallotus oppositifolius extract
Abstract:
Voltage-gated potassium channels formed by KCNQ2 and KCNQ3 generate the phosphatidylinositol 4,5-bisphosphate (PIP2)-augmented M-current, which regulates neuronal excitability. Hyperexcitability of neuronal cells is associated with numerous neurological diseases, such as benign familial neonatal seizures (BFNS) and epileptic encephalopathy, diseases linked to mutations in the genes encoding KCNQ2 and KCNQ5. In the developing world, an estimated 80% of epilepsy patients use herbal remedies for primary healthcare, such as the Ghanaian shrub Mallotus oppositifolius. M. oppositifolius extract has been previously shown to delay the onset of seizures as well as reduce their frequency and duration in mouse models of epilepsy. However, the active components and the molecular basis for these anticonvulsant properties were unclear. Using two electrode voltage-clamp electrophysiology, we screened 10 compounds previously identified from M. oppositifolius extract against oocytes expressing KCNQ2/3 channels. Here, we report that mallotoxin (MTX) and isovaleric acid (IVA) activate neuronal KCNQ2/3 channels, with homomeric KCNQ2 exhibiting the greatest sensitivity to both, suggesting KCNQ2 as the primary molecular target. Strikingly, dual application of MTX and IVA to KCNQ2/3 channels produced a highly effective, synergistic KCNQ2/3 activation. In mouse pentylene tetrazole (PTZ) chemoconvulsant assays, MTX halved the clonic seizure incidence whereas IVA had no effect. MTX and IVA halved tonic seizure incidence when applied in combination. Most strikingly, MTX and IVA only increased survival in the seizure assay when administered in combination, tripling survival, compared to vehicle. Thus, MTX and IVA acted synergistically to reduce seizures and seizure-related mortality in mice, mirroring the observed effects on KCNQ2/3 activation in oocytes. Finally, co-administration of MTX and IVA with the modern, synthetic anticonvulsant retigabine (a KCNQ activator that favors KCNQ3) created a further synergy that locked KCNQ2/3 open, such that its activation was voltage-independent. Thus, leveraging the heteromeric composition of KCNQ2/3 channels in order to exert optimal synergistic effects on channel opening presents a novel approach to developing safe yet effective anticonvulsants by harnessing the synergy of ancient and modern medicines.
