Functional identification of ion binding sites at the internal end of the pore in Shaker K+ channels

The inner end of the pore in voltage-gated K+ channels is the site of conformational changes related to gating and contains binding sites for permeant ions and pore-blocking molecules including quaternary ammonium ions and drugs. In order to determine the location and affinity of ion binding sites we probed the Shaker K+ channel with the quaternary ammonium analogue, tetrabutyl antimony (TBSb), a compound that is sufficiently electron dense to have been observed to occupy the cavity site in the bacterial K+ channel, KcsA. TBSb has K+ channel blocking properties analogous to those of tetrabutyl ammonium (TBA), and kinetics slow enough to be reliably measured. In the presence of external TEA, the internal TBSb on-rate decreased with increased internal K+ concentration as if these permeant ions prevented TBSb access to its site in the pore. The TBSb off-rate in low K+ was increased with external TEA addition and then reduced with increased internal K+. We found several differences between the behaviour of internal TBSb and TEA suggesting these molecules bind to distinct but interacting sites in the pore. We also found several differences in how K+ and Rb+ ions occupy sites in the inner end of the pore. These data suggest the presence of three sites in the inner end of the pore: (1) a site near the cytoplasmic end that binds TEA and K+ (but not Rb+) ions; K+ ions binding to this site inhibit TBSb exit from the pore; (2) a TBSb site slightly more into the pore that is rarely occupied by K+ or Rb+ ions; (3) a site further into the pore that has a high affinity for K+ and Rb+ ions; occupancy of this site by these permeant ions increases the TBSb off-rate. These results provide information on the fine-structure of ion interactions with the inner end of the pore in K+ channels.