A Mutation in S6 of Shaker Potassium Channels Decreases the K+ Affinity of an Ion Binding Site Revealing Ion–Ion Interactions in the Pore

Under physiological conditions, potassium channels are extraordinarily selective for potassium over other ions. However, in the absence of potassium, certain potassium channels can conduct sodium. Sodium flux is blocked by the addition of low concentrations of potassium. Potassium affinity, and therefore the ability to block sodium current, varies among potassium channel subtypes (Korn, S.J., and S.R. Ikeda. 1995. Science. 269:410–412; Starkus, J.G., L. Kuschel, M.D. Rayner, and S.H. Heinemann. 1997. J. Gen. Physiol. 110:539–550). 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. Kuschel, M.D. Rayner, and S.H. Heinemann. 1997. J. Gen. Physiol. 110:539–550; Ogielska, E.M., and R.W. Aldrich. 1997. Biophys. J. 72:A233 [Abstr.]). 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. 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. 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. The decrease in the apparent potassium affinity in A463C channels allows further study of possible ion interactions in the pore. 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.

External barium block of Shaker potassium channels: evidence for two binding sites.

External barium ions inhibit K+ currents of Xenopus oocytes expressing ShH4 delta 6-46, the non-inactivating deletion of the Shaker K+ channel. At the macroscopic level, Ba2+ block comprises both a fast and a slow component. The fast component is less sensitive to Ba2+ (apparent dissociation constant at 0 mV, K(0), approximately 19.1 mM) than the slow component and is also less voltage dependent (apparent electrical distance, delta, approximately 0.14). The slow component (K(0), approximately 9.4 mM, delta approximately 0.25) is relieved by outward K+ current, which suggests that the corresponding binding site resides within the channel conduction pathway. At the single channel level, the fast component of block is evidenced as an apparent reduction in amplitude, suggesting an extremely rapid blocking and unblocking reaction. In contrast, the slow component appears to be associated with long blocked times that are present from the beginning of a depolarizing command. Installation of the slow component is much slower than a diffusion limited process; for example, the blocking time constant (tau) produced by 2 mM Ba2+ is approximately 159 s (holding potential, HP = -90 mV). However, the blocking rate of this slow component is not a linear function of external Ba2+ and tends to saturate at higher concentrations. This is inconsistent with a simple bi-molecular blocking reaction. These features of external Ba2+ block can be accounted for by a simple model of two sequential Ba2+ binding sites, where the deeper of the two sites produces the slow component of block.