Gene Cloning, Recombinant Expression And Functional Analysis Of BmK αIV, A Novel Modulator Of Sodium Channels

In this study, gene cloning, protein expression and functional analysis of BmKαIV, a novel sodium channel modulator, were investigated using molecular,cellular, biochemical, electrophysiological, toxicological and behavioraltechniques. The research includes two parts:1. Gene cloning, recombinant expression and functional analysisBmK αIV gene was cloned from venomous glands of the Chinese scorpionButhus martensii Karsch (BmK) and expressed successfully in Escherichiacoli. BmK αIV gene is composed of two exons separated by a 503 bp intron.The mature polypeptide contains 66 amino acid residues. BmK αIV haspotent toxicity in mice and cockroach. Surface plasmon resonance analysisfound that BmK αIV could bind to both rat cerebrocortical synaptosomes andcockroach neuronal membranes, and shared receptor sites on sodiumchannels with classical α-mammal neurotoxin (AaH II), β-like neurotoxin(BmK AS), the depressant insect-selective neurotoxin (BmK IT2) andtransitional neurotoxin (BmK abT), but not with α-like neurotoxin (BmK I).Two-electrode voltage clamp recordings on rNav1.2 channels expressed inXenopus oocytes revealed that BmK αIV increased the peak amplitude andprolonged the inactivation phase of Na+ currents. The structural andpharmacological properties compared to those of other scorpion α-toxinssuggest that BmK αIV represents a novel subgroup or functional hybrid ofα-toxins and might be an evolutionary intermediate neurotoxin for α-toxins.2. The epileptic seizures induced by BmK αIV and its possiblemechanisms The epileptic behavioral and electrical seizures could beinduced by intracerebroventrical(i.c.v)injection of BmK αIV (0.5-3 μg).Voltage clamp recordings revealed that BmK αIV could enhance thespontaneous firing and the amplitude of persistent currents and resurgentcurrents of rNav1.2 expressed in Xenopus Oocyte. The further experimentsshowed that BmK αIV could evoke glutamate release from rat cerebrocorticalnerve terminals (synaptosomes). The results of micro-fluorescence imagingshowed that BmK αIV could elevate the intracellular [Ca2+]i and [Na+]i inconcentration-dependent manners. The increase of intracellular Ca2+ and Na+,and glutamate release could be completely blocked by TTX. The molecularmechanisms for seizure induction may be ascribed firstly to the modulation ofBmK αIV on sodium channels (especially for rNav1.2) located on the neuronalterminals, thereby increase the concentration of intracellular Ca2+ and Na+ toevoke glutamate release from rat cerebrocortical synaptosomes. Thesechanges increased the excitability of brain cortical neurons and resulted inseizures. BmK αIV may thus be an available molecular to further ourunderstanding on epileptogenesis involved in sodium channels.