Functional consequences of a decreased potassium affinity in a potassium channel pore. Ion interactions and C-type inactivation.

Ions bound near the external mouth of the potassium channel pore impede the C-type inactivation conformational change (Lopez-Barneo, J., T. Hoshi, S. Heinemann, and R. Aldrich. 1993. Receptors Channels. 1:61- 71; Baukrowitz, T., and G. Yellen. 1995. Neuron. 15:951-960). In this study, we present evidence that the occupancy of the C-type inactivation modulatory site by permeant ions is not solely dependent on its intrinsic affinity, but is also a function of the relative affinities of the neighboring sites in the potassium channel pore. The A463C mutation in the S6 region of Shaker decreases the affinity of an internal ion binding site in the pore (Ogielska, E.M., and R.W. Aldrich, 1998). However, we have found that this mutation also decreases the C-type inactivation rate of the channel. Our studies indicate that the C-type inactivation effects observed with substitutions at position A463 most likely result from changes in the pore occupancy of the channel, rather than a change in the C-type inactivation conformational change. We have found that a decrease in the potassium affinity of the internal ion binding site in the pore results in lowered (electrostatic) interactions among ions in the pore and as a result prolongs the time an ion remains bound at the external C-type inactivation site. We also present evidence that the C-type inactivation constriction is quite local and does not involve a general collapse of the selectivity filter. Our data indicate that in A463C potassium can bind within the selectivity filter without interfering with the process of C-type inactivation.

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.

Cooperative subunit interactions in C-type inactivation of K channels.

C-type inactivation of potassium channels is distinct from N-terminal mediated (N-type) inactivation and involves a closing of the outer mouth of the channel. We have investigated the role of the individual subunits of the tetrameric channel in the C-type inactivation conformational change by comparing the inactivation rates of channels constructed from different combinations of subunits. The relationship between the inactivation rate and the number of fast subunits is exponential, as would be predicted by a cooperative mechanism where the C-type conformational change involves all four subunits, and rules out a mechanism where a conformational change in any of the individual subunits is sufficient for inactivation. Subunit interactions in C-type inactivation are further supported by an interaction between separate mutations affecting C-type inactivation when in either the same or separate subunits.