Molecular Mechanism Underlying The Antiepileptic Effects Of Martentoxin Via Targeting Neuronal BK Channel

Large-conductance Ca2+-and voltage-activated potassium(BK)channels are widely distributed in both excitable and non-excitable cells.They play multiple roles in physiological processes including regulation of neuronal excitability and neurotransmitter release.Martentoxin(MarTX)is a 37-residue short-chain peptide from East-Asian scorpion(Buthus martensi Karsch,BmK)venom.The dynamic expression and distribution of BK channel and its auxiliary subunit in pentylenetetrazole(PTZ)-induced acute/chronic model and kainic acid(KA)-induced TLE model were investigated,and epileptogenesis mechanism involving BK channel were also explored in this paper.The following results were obtained:1.BK channel was exceptional expressed in the brain of acute seizure model(acute model)and chronic kindling model(chronic model)induced by PTZ,and the exceptional expression of BK channel could be lessened by VPA intervention and kindling failure.The exceptional BK channel was mainly located in VGIuT1-positive pyramidal neurons and distributed in the entire hippocampus and the cortex,which was not detected in dentate granule cells.The expression and distribution of auxiliary subunit β2 and β4 were also changed in acute model and chronic model.The expression of β2 subunit were both elevated in the hippocampus and cortex of acute model and chronic model,and β2 subunit was not co-localized to VGIuTl.The immunoreaction of p4 subunit was increased in the hippocampus and cortex of acute model,which had fallen back in chronic model,and β4 subunit was most localized in VGIuT1-positive neurons.2.MarTX could effectively relief the recurrent seizure of acute model and chronic model evoked by PTZ.Intrahippocampal applications of MarTX in low-dose(0.1μg)and high-dose(0.5μg)could prolong the latency to onset of seizure recurrence and dose-dependently reduce the seizure duration and number,and they eased the severity of seizure-like behavier in acute model and chronic model.The electroencephalogram(EEG)showed that 0.1 μg MarTX could depress the energy surge of local field potential(LFP)in epileptiform discharge induced by PTZ,and suppress the promotion of β rhythm percentage.Furthermore,by using wavelet packet decomposition and short-time Fourier analysis,MarTX could suppress the elevated power of the high-frequency rhythm,such as α rhythm,βrhythm,and γ rhythm,and weaken the entire power change of LFP.The electrophysiologaical recordings showed that MarTX could enhance the current threshold of action potential spike and reduce the resting potential to decrease excitability of hippocampal neurons。3.LRRC55,a newly discovered auxiliary subunit of the BK channel,is ubiquitously expressed in the hippocampus of normal rats,and its expression was reduced in the course of KA-TLE model。The co-immunoprecipitation showed that LRRC55 binding to BK channel a subunit on the membrane of hippocampal pyramidal neuron.Down-regulated expression of LRRC55 could significantly enhance the current density and alter the activation kinetic of BK channel,which increased the excitability of hippocampal neurons.The animals that was down-regulated LRRC55 showed hypersensitivity to subconvulsive dose of PTZ(35 mg/kg).4.Previous studies showed that BmK I,a site-3 toxin of voltage-gated sodium channels(VGSCs)and BmK IT2,a site-4 toxin of VGSCs,exhibited opposite pharmacological effects on the excitability of neurons.A stronger pain-related behavior has been previously recognized in BmK venom(containing BmK I and BmK IT2)and not in BmK I alone.In the present study,the amplitude of peak current of VGSC on ND7-23 cells was significantly increased and the activation of sodium channel was enhanced by joint application of BmK I and BmK IT2.Larger peak current and more negative half-activation voltage were elicited by joint application of BmK I and BmK IT2 than by application of BmK I or BmK IT2 alone.co-applied BmK I and BmK IT2 also significantly prolonged the time constant of inactivation of sodium channel.The present works revealed that:(1)The exceptional expression of BK channel is supposed to be the pathological basis of ictogenesis or epileptogenesis,and most of the exceptional BK channel should be type Ⅱ BK channel(α+β4),namely the neuronal BK channel。BKβ2 subunit might be related to change of synaptic plasticity in the chronic phase of epilepsy.(2)MarTX has been proved to have antiepileptic effects on acute model and chronic model induced by PTZ.(3)LRRC55 is the auxiliary subunit of BK channel on the hippocampal pyramidal neuron.LRRC55 regulated the excitability of pyramidal neurons by effect the endoplasmic trafficking on surface expression of BK channel.(4)BmK IT2 can enhance the pharmacological effect of BmK I on VGSCs synergistically,which may bring a novel perspective for exploring the underlying mechanisms of scorpion sting-induced pain.This study will help to understand the functional changes of BK channels in related diseases such as epilepsy,and provide important basis for the development of follow-up drugs such as antiepileptic drugs.