Abstract 929: The Molecular Basis of High-Affinity Binding of the Anti-Arrhythmic Compound, RSD1235, to the α-Subunit of Kv1.5 Channels
RSD1235 is a novel agent that converts atrial fibrillation rapidly and is well-tolerated in patients. Block of Kv1.5 channels that contribute to ultra-rapidly activating K+ currents is a feature of the activity of RSD1235 that prolongs refractoriness in the atria, and provides a significant component of its atria-selective antiarrhythmic action. RSD1235 blocks activated Kv1.5 channels from the intracellular side of the membrane with an IC50 of 13 μM.1 The purpose of this study was to define the location and detailed structure of the binding site(s) for RSD1235 on the Kv1.5 channel. Based on the conserved structure of the K+ channel pore region and the putative binding sites for other blockers, site-directed mutagenesis was used to target specific amino acid residues that have been previously shown to play a key role in antiarrhythmic drug binding to Kv channels. These residues, in the region T479 to V481 and A501 to P513 of Kv1.5, lie in the pore region of the channel and the S6 transmembrane domain near the activation gate. Wild-type residues were mutated to alanine or valine, and the IC50 for block by RSD1235 measured. Mutant channels were expressed in HEK293 cells and whole-cell patch clamped at 21–22°C. The I502A and V505A mutations significantly decreased RSD1235 binding to Kv1.5 channels, with IC50s of 280 ± 38 μM (n = 4 – 6) and 57 ± 4 μM (n = 3–5), respectively. In contrast to the diminished potency in these mutants, the mutation T479A increased the potency of RSD1235 binding to Kv1.5 (IC50 = 2 ± 0.2 μM, n = 4–7). Mutation V512L, a residue lying at the helix crossing, did not alter the IC50. These data identified T479, V502, and V505 as important residues for block of Kv1.5 by RSD1235. In addition, these results contrast with published data for flecainide and AVE0118 suggesting a unique binding site for RSD1235. Based on homology models, the key binding residues for RSD1235 are found at the deep inner pore and face towards the central cavity, overlapping only at the deep inner pore with binding sites for other blockers.