Affinity and Location of an Internal K⁺ Ion Binding Site in Shaker K Channels

We have examined the interaction between TEA and K⁺ ions in the pore of Shaker potassium channels. We found that the ability of external TEA to antagonize block of Shaker channels by internal TEA depended on internal K⁺ ions. In contrast, this antagonism was independent of external K⁺ concentrations between 0.2 and 40 mM. The external TEA antagonism of internal TEA block increased linearly with the concentration of internal K⁺ ions. In addition, block by external TEA was significantly enhanced by increases in the internal K⁺ concentration. These results suggested that external TEA ions do not directly antagonize internal TEA, but rather promote increased occupancy of an internal K⁺ site by inhibiting the emptying of that site to the external side of the pore. We found this mechanism to be quantitatively consistent with the results and revealed an intrinsic affinity of the site for K⁺ ions near 65 mM located ∼7% into the membrane electric field from the internal end of the pore. We also found that the voltage dependence of block by internal TEA was influenced by internal K⁺ ions. The TEA site (at 0 internal K⁺) appeared to sense ∼5% of the field from the internal end of the pore (essentially colocalized with the internal K⁺ site). These results lead to a refined picture of the number and location of ion binding sites at the inner end of the pore in Shaker K channels.