Abstract 948: An Allosteric Mechanism for Drug Block of a Cardiac Potassium Channel
Background: A common mechanism for block of ion channels is current inhibition by drug binding to residues - notably in S6 - within the permeating pore. Open state block is commonly observed.
Methods and Results: In this study, we used a combination of alanine/cysteine mutagenesis, patch clamp, and homology modeling to identify a novel allosteric mechanism for drug block of the human cardiac potassium channel KCNQ1 (the α-subunit encoding IKs). The initial mutagenesis experiments studied a total of 45 amino acid residues in the S6 segment (30 residues), and the proximal C-terminus (15 residues) of KCNQ1. Residues required for drug block were identified as alanine/cysteine mutants displaying <50% block of expressed current (n=4 – 6 each) upon exposure to drug concentrations inhibiting wild-type current by ≥90% (quinidine 100 μm, clofilium 30 μm and L-7 10 μm). Among the 30 residues in S6, there were five (F332, F335, S338, A341 and G348) displaying such reduced block and in the proximal C-terminus, 5/15 were less sensitive, including F351 which was near-totally resistant (current decrease = 4±3%). Homology modeling of KCNQ1 based on the Kv1.2 structure unexpectedly suggested that F351 faces away from the permeating pore, arguing against pore block as the mechanism for drug inhibition of KCNQ1 current. In the open channel model, F351 lines a pocket that also includes L251 and V254 in S4–S5. The model allows drug access to this pocket, and alanine substitutions at both sites yielded potassium currents that were resistant to drug block (↓34±4% for L251A and ↓40±5% for V254A).
Conclusion: These data strongly support a model in which open state block of this channel occurs not via binding to a site in the pore but rather by a novel allosteric mechanism, drug access to a side pocket generated in the open channel configuration and lined by S6 and S4/S5 residues.