Anticonvulsant Effect Of Martentoxin And Molecular Mechanism Of Its Recognition On BK Channels

BK channels (large-conductance, calcium-activated potassium channels) widelydistributed in most cells and tissues are involved in regulating many physiologicalfunctions. The expression and activity of these channels are closely associated with avariety of human diseases. This study focuses on the anticonvulsant effect ofmartentoxin, an indigenous novel blocker of BK channels, in vivo and the molecularmechanisms of its recognition to BK channels.1. Anticonvulsant effects of martentoxin on PTZ-induced models: This studyfound that martentoxin could inhibit seizures and recurrences of PTZ-induced model.Intrahippocampal application of martentoxin could alleviate seizures in PTZtreatment rats. Low-dose application of martentoxin (0.05μg) prolonged the latencyto onset of the first seizure.0.05μg and0.5μg martentoxin could dose-dependentlyreduce the seizure duration and number, but had no significant effect on the seizurestage.0.05μg martentoxin could not only prolong the latency but also significantlyinhibit seizure-like behavior and reduced the seizure stage in PTZ-repeated injectionmodel.2. Molecular basis underlying recoginition of martentoxin on neuronal BKchannel subtype (α+β4): The neuronal BK channels (α+β4) were insensitive toclassical BK channel blockers, charybdotoxin and iberiotoxin, but could be potentlyblocked by martentoxin (MarTX， IC50=72nM). Here the alanine-scanningmutagenesis indicates that Y294located in the pore region of neuronal BK channelplays a crucial role in the inhibition of martentoxin while D261and E276are not asimportant as Y294. In addition, BK channels composed of α subunits alone could notaffected by martentoxin (even at1μM), strongly suggesting that β4subunit may contribute to the toxin recognition of the neuronal BK channel. Therefore,extracellular loops of β subunits between neuronal BK channels and BK channels(α+β1) were exchanged. Channels co-expressed with the β1subunit carrying theextracellular loop of the β4subunit (β1Lβ4) show toxin blocking characteristicsindistinguishable with the wild-type β4subunit, while β4Lβ1was enhanced bymartentoxin similar as the wild-type β1subunit. These results suggest that poreregion and β4subunit extracellular loop of BK (α+β4) channels might contribute tothe inhibition of martentoxin,3. Enhancement effects of martentoxin on glioma BK channel and BK channel(α+β1) subtypes: gBK channel (glioma BK channel) and BK channel (α+β1)possess higher Ca2+sensitivity than other known BK channel subtypes. The presentstudy investigated the modulatory characteristics of martentoxin on these two BKchannel subtypes by electrophysiological recordings, cell proliferation and Ca2+imaging. In the presence of cytoplasmic Ca2+, martentoxin could enhance theactivities of both gBK and BK channel (α+β1) subtypes in dose-dependent mannerwith EC50of46.7nM and495nM respectively, while not shifting the steady-stateactivation of these channels. The enhancement ratio of martentoxin on gBK and BKchannel (α+β1) was unrelated to the quantitive change of cytoplasmic Ca2+concentrations. Iberiotoxin, a selective BK pore blocker, could fully abolish theenhancement of these two BK subtypes induced by martentoxin, suggesting that theauxiliary β subunit might contribute to the docking for martentoxin. However, in theabsence of cytoplasmic Ca2+, the activity of gBK channel was surprisingly inhibitedby martentoxin while BK channel (α+β1) couldn’t be affected by the toxin. Thus, theresults shown here provide the novel evidence that martentoxin could increase thetwo Ca2+-hypersensitive BK channel subtypes activities in a new manner andindicate that β subunit of these BK channels plays a vital role in this enhancement bymartentoxin.4. Modulatory effects of martentoxin on β1-subunit N-deglycosylated BKchannels (α+β1): It remains elusive whether the glycosylation contributes to the regulation of BK channels by β1subunits. Here we combined theelectrophysiological approach with molecular mutations and biochemicalmanipulation to investigate the function roles of glycosylation in β1subunits. Theresults show that deglycosylation of β1subunits through double-site mutations (β1N80A/N142A or β1N80Q/N142Q) could significantly increase the inhibitorypotency of iberiotoxin, a specific BK channel blocker. The deglycosylated channelsalso have a different sensitivity to martentoxion. Contrary to the enhancing effects ofmartentoxin on glycosylated BK channels in the presence of cytoplasmic Ca2+, thedeglycosylated channels were not affected by the toxin. Interestingly, thedeglycosylated channels were inhibited by martentoxin in the absence ofcytoplasmic Ca2+, while the glycosylated channels were not inhibited under the samecondition. Meanwhile, the deglycosylated forms of the channels were activated at ahigher speed than the glycosylated ones though the V1/2and slope were not changedby the glycosylation. Therefore, the glycosylation of β1-subunits is an indispensabledeterminant of BK channel functional characteristic.The present work reveals that martentoxin is a novel anticonvulsant moleculetargeting on the neuronal BK channels (α+β4) and a unique probe to identify thesubunits of BK channels. The mechanism of its recognition to channels enriches theinteraction model of potassium channels and the ligands, which is crucial not only indeep-researching for physiological function of BK channel, but also in developingdrugs for BK channel-related diseases.