Identification of Novel and Natural High Affinity Peptide Inhibitors of KcsA by Phage-Display Reveals an Unexpected Mechanism of Pore Blockade

Abstract

Peptide neurotoxins are powerful tools for research, diagnosis and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high-affinity and specificity. Here we describe isolation of toxins for one such orphan target, KcsA, a K+ channel that has been fundamental to delineating the structural basis for ion channel function. A phage display strategy is employed whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We focus here on two of nine toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (Ki) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a Ki of 0.5 nM while Shaker, Kv1.2 and Kv1.3 channels are blocked over 200-fold less well. In contrast, HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2 and Kv1.3 channels with Ki of 1 to 4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys21 and Tyr22, are essential for high affinity binding. Unexpectedly, K+ ions traversing the channel from the inside confer voltage-sensitivity to the Hui1 off-rate via Arg23, indicating that Lys21 is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of high-affinity toxins for orphan receptors.