The carbonyls form three ion binding sites within the narrow region, one external site and two internal sites that cannot be simultaneously occupied due to their close proximity. The narrow ion-selective region of KscA is lined by the backbone carbonyls from the signature sequence amino acids, while S6 helices form the wide internal vestibule of the channel ( Doyle et al., 1998).

The molecular fingerprint of a potassium channel is the highly conserved TXXTXGYG sequence found in the P region and it is revealed as the finely tuned selectivity filter. Although this is a two-transmembrane channel, it is highly homologous to voltage-gated potassium channels within the regions that form the ion conducting pore. The crystal structure of a Streptomyces lividans potassium channel (KscA), a proton-gated channel with two membrane spanning domains and a P region, has recently been determined ( Doyle et al., 1998). The loop between the fifth and sixth membrane spanning domains has been termed the P region because mutations in this loop affect several pore properties of the channel, including ion selectivity ( Armstrong and Hille, 1998). Voltage-gated potassium channels are tetramers ( MacKinnon, 1991 Liman et al., 1992 Kavanaugh et al., 1992 Doyle et al., 1998) with six membrane spanning regions (S1 through S6) in each of the four subunits. Our results indicate that sodium and potassium can occupy the pore simultaneously and that multiple occupancy results in interactions between ions in the channel pore. The decrease in the apparent potassium affinity in A463C channels allows further study of possible ion interactions in the pore. The A463C mutation decreases the internal barium affinity of the channel, as expected if barium blocks current by binding to a potassium site in the pore. Independent evidence that A463C decreases the apparent affinity of a binding site in the pore comes from a study of barium block of potassium currents. We show that changing a single residue in S6, A463C, decreases the apparent internal potassium affinity of the Shaker channel pore from the micromolar to the millimolar range, as determined from the ability of potassium to block the sodium currents. The Shaker potassium channel conducts sodium poorly in the presence of very low (micromolar) potassium due to its high potassium affinity (Starkus, J.G., L. Potassium affinity, and therefore the ability to block sodium current, varies among potassium channel subtypes (Korn, S.J., and S.R. Sodium flux is blocked by the addition of low concentrations of potassium. However, in the absence of potassium, certain potassium channels can conduct sodium.

Under physiological conditions, potassium channels are extraordinarily selective for potassium over other ions.

Quite revealing 1.8e serial