Probing the Allosteric Interactions that Mediate Voltage-Dependent BK (Slo1) Channel Activation with a Pore Blocker

Abstract

Like many transmembrane voltage-activated proteins, BK channels possess conserved voltage-sensing domains (VSD) which activate upon membrane depolarization. VSDs are structurally and functionally associated with the channel pore domain, such that VSD activation facilitates pore opening. BK channels are selectively blocked by Iberiotoxin (IbTX), a peptide component of scorpion venom which binds to the BK pore domain, preventing K+ flux. To elucidate the allosteric consequences of toxin binding to the BK channel pore, we probed the operation of the BK VSD using cut-open oocyte voltage clamp fluorometry, whereby a fluorophore labeling the BK VSD helix S0 reported the conformational rearrangements, and therefore the state of activation, of the VSD. This was performed in conducting channels, as well as channels blocked by external IbTX (10 μM) perfusion. The blockade regime caused a >95% decrease of macroscopic K+ conductance and an apparent functional impairment of the VSD, as suggested by the half-activation shift to depolarized potentials by ∼35 mV and effective charge reduction by ∼20%. Since IbTX is a BK channel pore blocker, we propose that the observed impairment of the VSD must be due to the allosteric coupling between the pore and the voltage sensing domains. To test and quantify this interpretation, we used a statistical-mechanical model of BK channel activation, which accounted for the blockade-induced impairment of the BK VSD by a large reduction (2.1x10−7 to 1.4x10−9) of the intrinsic equilibrium constant for channel opening at 0 mV (L0), suggesting that IbTX blockade caused the pore to “lock” into a shut state, which resulted in the observed perturbation of the voltage-sensing apparatus, mediated by the strong (−75 meV) allosteric linkage between the two domains.