Influence of Hydrophobic Residues on BK Channel Gating

Abstract

Large-conductance calcium- and voltage-activated potassium (Slo1 BK) channels participate in the control of vascular tone and neurotransmitter release. A neuronal splice variant of BK channels with altered sequence in the S6/RCK1 linker exhibits increased open probability in 100 µM calcium at −150 mV (PoC) compared to “wild-type” BK channels (Soom et al. 2008, Channels 2:278-282). To identify amino-acid residues underlying this notable change in gating behavior, we expressed wild-type and mutant human Slo1 BK channels in HEK 293 cells and analyzed their calcium- and voltage-dependent gating in the inside-out configuration of the patch-clamp technique. Mutation G327L, located in the linker connecting the S6 helix to RCK1, most strongly increased PoC from 0.02 to 0.24. Systematic substitution at that site revealed that hydrophobicity is most important for channel opening at low voltages. PoC for G327F and G327Y were 0.19 and 0.01, respectively; addition of a single hydroxyl group (“F327Y”) decreased PoC by 0.18. The same change in hydrophobicity by mutation F315Y within S6 has qualitatively opposite effects (Lippiat et al. 2000, J Physiol 529:131-138). Phenylalanine scanning mutagenesis of the S6/RCK1-linker region revealed that the mutation K330F most strongly increased PoC to around 0.5. Introduction of an additional hydroxyl group at this site (“F330Y”) decreased PoC by 0.37 down to 0.13. We conclude that hydrophobicity within the S6/RCK1-linker region is a critical determinant of the calcium-dependent gating.