Abstract
Dysfunction of the human voltage-gated K(+) channel Kv1.1 has been associated with epilepsy, multiple sclerosis, episodic ataxia, myokymia, and cardiorespiratory dysregulation. We report here that AETX-K, a sea anemone type I (SAK1) peptide toxin we isolated from a phage display library, blocks Kv1.1 with high affinity (K(i) ~ 1.6 pM) and notable specificity, inhibiting other Kv channels we tested a million-fold less well. Nuclear magnetic resonance (NMR) was employed both to determine the three-dimensional structure of AETX-K, showing it to employ a classic SAK1 scaffold while exhibiting a unique electrostatic potential surface, and to visualize AETX-K bound to the Kv1.1 pore domain embedded in lipoprotein nanodiscs. Study of Kv1.1 in Xenopus oocytes with AETX-K and point variants using electrophysiology demonstrated the blocking mechanism to employ a toxin-channel configuration we have described before whereby AETX-K Lys(23) , two positions away on the toxin interaction surface from the classical blocking residue, enters the pore deeply enough to interact with K(+) ions traversing the pathway from the opposite side of the membrane. The mutant channel Kv1.1-L(296) F is associated with pharmaco-resistant multifocal epilepsy in infants because it significantly increases K(+) currents by facilitating opening and slowing closure of the channels. Consistent with the therapeutic potential of AETX-K for Kv1.1 gain-of-function-associated diseases, AETX-K at 4 pM decreased Kv1.1-L(296) F currents to wild-type levels; further, populations of heteromeric channels formed by co-expression Kv1.1 and Kv1.2, as found in many neurons, showed a K(i) of ~10 nM even though homomeric Kv1.2 channels were insensitive to the toxin (K(i) > 2000 nM).