Mapping the Binding Site of the Alkoxypsoralen PAP-1 in the Voltage-Gated K+ Channel Kv1.3

Abstract

Kv1.3 is widely regarded as an attractive drug target for the treatment of effector memory T cell-mediated autoimmune diseases such as multiple sclerosis, type-1 diabetes and psoriasis. Schmitz et al. (2005) identified 5-(4-phenoxybutoxy)psoralen (PAP-1) as a potent and selective small molecule Kv1.3 blocker. Unlike the classical Kv1 blocker tetraethylammonium, the nucleophilic PAP-1 blocks Kv1.3 with a 2:1 stoichiometry. Following a hypothesis that nucleophilic ligands can coordinate a metal ion in the channel pore, we used Monte Carlo-energy minimizations to search for possible complexes of two PAP-1 ligands with a K+ ion in the Kv1.2-based model of Kv1.3. In a predicted complex, the furocoumarin moieties of two ligands chelate a K+ ion at the focus of the P-helices in the central cavity, while the 4-phenoxybutoxy arms extend into the intrasubunit S5/S6 interfaces and reach the S4-S5 linkers. The model predicted ligand-sensing residues in the S4-S5 linker, S5, P-loop, and S6. We next tested the model by introducing single amino acid substitutions into Kv1.3 and exploring the biophysical properties of the mutants and their sensitivity to PAP-1 in whole-cell patch-clamp experiments. So far we have confirmed L335 in the S4-S5 linker, L353 in S5, and V417 and T419 in S6 as PAP-1 sensing residues. Among the mutants, V417L exhibited the largest change in IC50, 400 nM versus 2 nM for the wild-type channel. Interestingly, V417L and T419A also exhibit more of an open-channel type block rather than a C-type inactivated state block. The proposed model explains the actions of various nucleophilic ligands that block cationic channels with a Hill coefficient greater than 1, opening a new direction for structure-based design of ion channel drugs.Supported by CIHR, NIH, and HHMI.