Substitutions at F380 in S6 by Small Hydrophobic Amino Acids Makes the Opening Transition in BK Channels Rate Limiting

Abstract

Large conductance Ca2+ and voltage-activated K+ channels (BK) opening is mediated by a cross-talk of modular voltage and Ca2+ sensors. BK open probability is enhanced by increasing cytosolic Ca2+ concentration and/or depolarization. These stimuli activate sensors that are coupled by allosteric interactions to channel gating. The physical basis underlying the coupling between sensors activation and pore opening remains elusive. We found that replacement of the F380 residue in the transmembrane segment S6 by small hydrophobic amino acids, promotes a large and positive voltage shift of the open probability-voltage curve with minor changes in the gating charge-voltage curve. Using the Horrigan and Aldrich allosteric model, we show that these gating modifications are a consequence of large changes in the open-closed equilibrium and in the coupling between voltage and Ca2+ sensors with channel opening. In the presence of saturating internal Ca2+, the F380A mutant increases the energy barrier that separates closed from open states by about 16 kJ/mol compared to the wtBK. The allosteric factor describing interaction between channel opening and voltage sensor activation decreases from 19 in the wt BK channel to 6 in the F380A mutant, while the deactivation rate interpolated to 0 mV is 4.8 times faster in mutant. Molecular modeling suggest the existence of a hydrophobic ring formed by residues F380 and L377 of contiguous subunits that could function as a lever support site for coupling between both sensors and channel opening. Interestingly, the mutation (F380A) allows observing gating currents with minor contaminants of ionic currents, so it could be used for detailed voltage sensor studies in the presence of permeant cations.This work was supported by Fondecyt grant 1110430 to RL and Fondecyt 1131003 to FGN.