Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

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. This paper describes isolation of toxins for one such orphan target, KcsA, a potassium channel that has been fundamental to delineating the structural basis for ion channel function. A phage-display strategy is presented 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 describe two 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 (K_i) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a K_i of 0.5 nM whereas Shaker, Kv1.2, and Kv1.3 channels are blocked over 200-fold less well. HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2, and Kv1.3 channels with K_i of 1–4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys₂₁ and Tyr₂₂, are essential for high-affinity binding. Unexpectedly, potassium 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.Venomous animals produce neurotoxic peptides for defense and to capture prey. With potencies in the nanomolar range, the peptides act by modulating the function of target receptors. Toxins isolated from venoms have been used to identify and purify ion channels, to clarify their roles in physiology, to elucidate the structural basis for their function, and, recently, to diagnose and treat disease. Given their utility, it is frustrating that natural toxins cross-react with related receptors (or have no known target) and that most receptors lack a specific, high-affinity toxin. This state of affairs is easily understood; the small amounts of toxins isolated from natural sources makes target identification a challenge and their purpose in the wild does not favor target specificity. Here, we advance our approach to overcoming these problems, that is, creation of expression libraries of toxins allowing cloning based on target binding (1), by seeking a specific, high-affinity ligand for an orphan channel receptor.Our strategy is to start with a known toxin and to design a phage-display library using the genetic database of its predicted homologs, in native and combinatorial fashion, so the encoded peptides share the same structural scaffold. As a proof of concept, we previously addressed a case of inadequate target discrimination by known natural toxins using a library of ∼11,200 peptides designed to share the fold in α-KTx scorpion toxins and a specific ligand for the human voltage-gated potassium channel Kv1.3 was isolated (1). Moka1, composed of domains from three scorpion species, blocks Kv1.3 with nanomolar affinity, allowing it to suppress T-cell-mediated immune responses, and is without unwanted side effects on gastrointestinal motility seen with natural toxins because it does not cross-inhibit Kv1.1 and Kv1.2. Supporting the design premise that encoded peptides are expressed, correctly folded, and accessible on the phage surface in a manner permissive of sorting based on target binding, the determined 3D structure of Moka1 revealed it to be constructed on an α-KTx scaffold. Here, we sought to extend our strategy by testing another scaffold and creation of a library sufficiently large to achieve isolation of peptides specific for a target with no known ligand.KcsA is a prokaryotic channel with high potassium conductance and selectivity (2). The first potassium channel visualized at high resolution (3), KcsA has a single ion conduction pathway on the central axis of symmetry formed by four identical subunits, each with two transmembrane segments and a reentrant pore-forming loop (TM1-P-TM2). The 3D structure of KcsA confirmed explanations for selective ion permeation and conduction pathway gating deduced in the period before crystallization and its continued interrogation has been key to delineating the mechanistic bases for channel function (4–6). Although described 20 y ago (7), KcsA remains an orphan target so that studies with peptide toxins have required production of mutant channels with multiple mutations in the pore domain (8) or chimeras such as Kv1.3-KcsA, where the entire KcsA pore domain is replaced by the one in Kv1.3 (9).To isolate toxins for KcsA, a peptide library was designed with ∼1,562,750 variants via combinatorial permutation of sequences related to the sea anemone type I (SAK1) toxin ShK. Phage sorting was performed on purified, wild-type KcsA channels. Peptides expressed on the enriched phage were synthesized and studied by surface plasmon resonance (SPR) to characterize their binding to purified KcsA and by voltage-clamp electrophysiology to assess channel blockade. Hui1, a novel and specific inhibitor of KcsA, HmK, a natural and promiscuous blocker, and Hui1 mutants were evaluated to identify toxin segments and residues responsible for specificity and affinity and to discern the mechanism of channel inhibition. The 3D structure of Hui1 determined by NMR, the 1:1 stoichiometry of KcsA inhibition via a pore-directed mechanism, and the role of two, canonical “dyad” residues (Lys₂₁ and Tyr₂₂) in high-affinity binding all met expectations for a SAK1-type toxin. In contrast, the influence of permeant trans ions (those traversing the channel after entering from the opposite side of the membrane) on dissociation of Hui1 from its external binding site indicated that Arg₂₃, a residue with a side chain too bulky to fit snugly into the potassium conduction pore (10), was responsible for the voltage dependence of block rather than Lys₂₁. This unexpected role for Hui1-Arg₂₃ could reflect a new SAK1 binding orientation for the novel toxin; however, some models have located the ShK dyad Lys in the outer pore vestibule of Kv1.3 (11) rather than in the narrow portion of the conduction pathway (12). We posit that Hui1 binds and blocks like some, and perhaps most, natural SAK1 toxins.