458 Effects on Ion Permeation with Hydrophobic Substitutions at a Residue in Shaker S6 That Interacts with a Signature Sequence Amino Acid PAUL C. ZEI, a EVA M. OGIELSKA, a,c TOSHINORI HOSHI, b AND RICHARD W. ALDRICH a a Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA b Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242, USA lthough potassium channels differ in their gating properties, they all select for K + with an extremely high fidelity and without compromising high flux rates. Based on reversal potential measurements, potassium channels display a relative selectivity sequence of K + > Rb + > NH4 + > Cs + >> Na + . 1–3 Potassium channels are tetramers, and each subunit contributes equally to the formation of the ion-conducting pore, which is formed along the central axis of the protein. 4–7 Voltage-gated K + channels have six transmembrane domains (S1–S6) in each subunit, as well as a membrane-spanning region (P-region) between S5 and S6. The P-region possesses a highly conserved sequence (TXTTXGYG) required for K + selectivity. 3–8 The prokaryotic K + channel, KscA, has only two membrane-spanning domains, but these are homologous to the S5 and S6 regions of the voltage-gated K + channels. The crystal structure of KscA has revealed that ion binding sites in the pore are formed by the backbone carbonyls of the signature sequence amino acids, while the S6-like helices cradle the selectivity filter and line the internal vestibule of the channel. 7 In accordance with the idea that S6 helices of voltage-gated K + channels also form part of the channel pore are the observations that mutations in this region can alter several pore properties. 9–14 Substitutions at position A463 in the S6 of Shaker are known to alter K + affinity, the rate of C-type inactivation, internal blocker efficacy, and the interaction of external permeant ions with channel closing. 15–18 In the KscA channel a methionine occupies position 463, and the crystal structure has revealed that this residue inhabits a region of tight protein packing. 7 In this study we examined the effect of hydrophobic substitutions of varying bulkiness at posi- tion 463 on the single-channel conductance and the relative K + selectivity of the Shaker channel. c Corresponding author. Phone: 650-723-7557; fax: 650-725-4463; e-mail: ogielski@cmgm.stanford. edu A