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

• Published in: The Journal of physiology 2003-05, Vol.549 (1), p.107-120
• Main Authors: Thompson, Jill, Begenisich, Ted
• Format: Article
• Language: English
• Subjects: Animals; Antimony - pharmacology; Binding Sites - physiology; Cesium - pharmacology; Detergents - pharmacology; Membrane Potentials - drug effects; Membrane Potentials - physiology; Models, Chemical; Mutagenesis - physiology; Oocytes - physiology; Original; Potassium - metabolism; Potassium Channel Blockers - pharmacology; Potassium Channels - chemistry; Potassium Channels - genetics; Potassium Channels - physiology; Protein Conformation; Quaternary Ammonium Compounds - pharmacology; Rubidium - pharmacology; Shaker Superfamily of Potassium Channels; Tetraethylammonium - pharmacology; Xenopus laevis
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.2002.038646

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.