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. key words: C-type inactivation • ion interactions • potassium affinity • Shaker i n t r o d u c t i o n In response to prolonged membrane depolarization, voltage-gated potassium channels restrict the movement of ions through the ion-selective pore by the process of inactivation. The Shaker potassium channel displays two types of inactivation, Nand C-type, that are mediated by distinct regions of the channel protein. N-type inactivation occurs within a few milliseconds after channel opening in the Shaker B channel variant and results from the physical occlusion of the cytoplasmic entrance to the pore by the amino terminus of the protein (Hoshi et al., 1990; Zagotta et al., 1990; Demo and Yellen, 1991). C-type inactivation occurs over a period of seconds in the Shaker B channel and involves a structural rearrangement of the outer mouth of the pore (Yellen et al., 1994; Liu et al., 1996). The C-type inactivation rate is also dependent on the occupancy of an external ion binding site, which must be empty before the C-type conformational change can proceed (LopezBarneo et al., 1993; Baukrowitz and Yellen, 1995). The external site is nearly saturated in physiological solutions, due to outward potassium flux through the channel that creates a local accumulation of ions (Baukrowitz and Yellen, 1995). Prevention of potassium efflux by internal blockers increases the C-type inactivation rate by preventing the external site from being refilled after the last ion has exited from the pore to the external solution (Baukrowitz and Yellen, 1995). In the absence of added potassium, the Shaker channel conducts sodium (Starkus et al., 1997; Ogielska and Aldrich, 1998); however, the rate of C-type inactivation is extremely rapid in symmetrical sodium solutions (Starkus et al., 1997), indicating that either sodium does not interfere with the C-type inactivation process or that its dwell time at the C-type inactivation controlling site is short compared with potassium. The C-type inactivation rate is also sensitive to amino acid substitutions in the pore-forming regions of the channel (Lopez-Barneo et al., 1993; Heginbotham et al., 1994; Yang et al., 1997), but how these mutations affect the inactivation rate of the channel is not well understood. Amino acid substitutions could either directly affect the energetics of the C-type inactivation conformational change, or alternatively they could affect the ion occupancy of the C-type modulatory site in the pore. Portions of this work were previously published in abstract form (Ogielska, E.M., and R.W. Aldrich. 1998. Biophys. J. 74:A19). Address correspondence to Dr. Richard W. Aldrich, Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305. Fax: 650-725-4463; E-mail: [email protected] on Sptem er 0, 2007 ww.jgp.org Doloaded rom