Selective block of human Kv1.1 channels and an epilepsy‐associated gain‐of‐function mutation by AETX‐K peptide

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).