Development of charybdotoxin Q18F variant as a selective peptide blocker of neuronal BK(α + β4) channel for the treatment of epileptic seizures.

Epilepsy is the results from the imbalance between inhibition and excitation in neural circuits, which is mainly treated by some chemical drugs with side effects. Gain-of-function of BK channels or knockout of its β4 subunit associates with spontaneous epilepsy. Currently, few reports were published about the efficacy of BK(α + β4) channel modulators in epilepsy prevention. Charybdotoxin is a non-specific inhibitor of BK and other K channels. Here, by nuclear magnetic resonance (NMR) and other biochemical techniques, we found that charybdotoxin might interact with the extracellular loop of human β4 subunit (i.e., hβ4-loop) of BK(α + β4) channel at a molar ratio 4:1 (hβ4-loop vs. charybdotoxin). Charybdotoxin enhanced its ability to prevent K current of BK(α + β4 H101Y) channel. The charybdotoxin Q18F variant selectively reduced the neuronal spiking frequency and increased interspike intervals of BK(α + β4) channel by π-π stacking interactions between its residue Phe and residue His of hβ4-loop. Moreover, intrahippocampal infusion of charybdotoxin Q18F variant significantly increased latency time of seizure, reduced seizure duration and seizure numbers on pentylenetetrazole-induced pre-sensitized rats, inhibited hippocampal hyperexcitability and c-Fos expression, and displayed neuroprotective effects on hippocampal neurons. These results implied that charybdotoxin Q18F variant could be potentially used for intractable epilepsy treatment by therapeutically targeting BK(α + β4) channel.