Side pockets provide the basis for a new mechanism of Kv channel-specific inhibition.

Most known small-molecule inhibitors of voltage-gated ion channels have poor subtype specificity because they interact with a highly conserved binding site in the central cavity. Using alanine-scanning mutagenesis, electrophysiological recordings and molecular modeling, we have identified a new drug-binding site in Kv1.x channels. We report that Psora-4 can discriminate between related Kv channel subtypes because, in addition to binding the central pore cavity, it binds a second, less conserved site located in side pockets formed by the backsides of S5 and S6, the S4-S5 linker, part of the voltage sensor and the pore helix. Simultaneous drug occupation of both binding sites results in an extremely stable nonconducting state that confers high affinity, cooperativity, use-dependence and selectivity to Psora-4 inhibition of Kv1.x channels. This new mechanism of inhibition represents a molecular basis for the development of a new class of allosteric and selective voltage-gated channel inhibitors.

Temperature-sensitive defects in paramyotonia congenita mutants R1448C and T1313M.

The biophysical origins of paramyotonia congenita and its exacerbation in cold temperatures were examined. Human skeletal muscle voltage-gated sodium channels were expressed in Xenopus oocytes and macroscopic currents were recorded from cell-attached patches. Wild-type (hNaV1.4) channels were compared to two mutant channel isoforms, T1313M and R1448C. The voltage dependence and temperature sensitivity of activation, fast-inactivation onset and recovery, and deactivation were studied. Although activation and the onset of fast-inactivation were temperature sensitive in all three isoforms, and although these properties in mutant channels differed from those in wild-type channels, they did not account for cold-exacerbation. Deactivation, however, was disproportionately slower in R1448C, but not in T1313M, than in hNaV1.4. These defects may, at least in part, account for the clinical symptoms of paramyotonia congenita and its exacerbation by cold, and provide a basis for studies into the therapeutic alleviation of these symptoms.