Heteropodatoxin 2: A specific gating modifier of Kv4 channels
Zarayskiy, Vladislav Vladimirovich
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Electrical activity in the heart follows a repeating cyclical pattern of depolarization followed by repolarization. During the cardiac cycle time dependent changes in ionic conductance occur that not only generate the different components of depolarization and repolarization but also lead to contraction. Membrane depolarization during phase 0 of the transmembrane action potential generates one of several K + currents, i.e. the transient outward K + current (I to ), which is altered in many heart diseases, such as arrhythmias. Lengthening of action potential duration and refractoriness can terminate reentrant arrhythmias. One unexplored approach of treating arrhythmias is that inhibition of I to , which would increase action potential duration and refractoriness, and possibly lead to an effective treatment of reentry. This concept is unexplored due to lack of specific I to blockers. Pharmacological susceptibility to an action of Heteropoda venatoria toxins (HpTx2 and HpTx3) distinguishes between subtypes of I to . Closely related in their amino acid sequence and electrophysiologically indistinguishable Kv4.2 and Kv4.3 voltage-gated potassium channels are strong candidates for being α subunits of rapidly recovering I to . HpTx2 is a 30 amino acid peptide reticulated by three disulfide bridges, which gives the peptide a compact structure known as "knottin." To obtain large quantities of the peptide we had produced a recombinant version in bacteria and purified it chromatographically. The purification resulted in a high yield of pure and physiologically active recombinant toxin. The recombinant toxin was applied to several potassium channels expressed in Xenopus oocytes. Using two-electrode voltage-clamp, we have found statistically significant difference in the amplitude of the current and gating characteristics such as time constants of activation and inactivation only for channels of Kv4 subfamily. The recombinant toxin shifts the threshold of activation to more depolarized voltages and its inhibition is voltage-dependent. K d for Kv4.1 is 1074 nM, K d for Kv4.3 is 760 nM which is lower than affinity of the natural HpTx2. The recombinant toxin accelerates deactivation and decelerates activation for both Kv4.1 and Kv4.3. A mathematical model of toxin-channel interaction was developed, which predicts voltage-dependent inhibition based on different fractional occupancy and can simulate activation and deactivation currents. According to the model, in Kv4.1 the toxin binds to closed-state and stabilizes predominantly closed-preopen state where all subunits are activated but the channel is still closed. In contrast, toxin bound to Kv4.3 the toxin stabilizes the closed-resting state.