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

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. 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, Lys 21 and Tyr 22 , are essential for highaffinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg 23 , indicating that Lys 21 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 highaffinity toxins for orphan receptors.Hui1 toxin | ShK toxin | HmK toxin | sea anemone | NMR

Tethered peptide neurotoxins display two blocking mechanisms in the K + channel pore as do their untethered analogs

“…Thus, injection of 0.25 ng of cRNA of T-Hui1 with KcsA-Shaker inhibited ~82% of the current, consistent with an apparent K i of 0.77 ± 0.09 nM. Whereas WT T-Hui1 block of KcsA-Shaker was sensitive to voltage with a z of ∼0.32, similar to the z of peptide Hui1 (z ~0.3) that we previously reported (11), and Lys neutralization in T-Hui1-Lys 21 Asn did not alter the z (~0.31), T-Hui1-Arg 23 Ala eliminated the voltage dependence of toxin blockade ( fig. S3B).…”

“…In contrast, we previously observed the chimeric SAK1 toxin peptide Hui1 to interact with trans-ions in the KcsA-Shaker via Arg 23 , reflecting a new blocking mechanism (11), a finding that we recapitulate here using T-Hui1 and variants (fig. S3); Hui1 does not inhibit hKv1.3 (11). Here, we find that ShK blocks hKv1.3 by a similar, Arg-dependent mechanism (Fig.…”

“…T-HmK and HmK peptide also block hKv1.3 with similar affinity and voltage dependence via Lys 22 . In contrast, ShK (stichodactyla toxin) peptide (and T-ShK) reveal the Arg-dependent mechanism, in disagreement with prior mechanistic assertions (15) and similar to the chimeric toxin Hui1 (11). The fact that the two natural SAK1 toxins, HmK and ShK, block hKv1.…”

“…It was one of the 150 SAK1 toxins that we used to design a phage display library comprising 1.5 million combinatorial SAK1 variants and was subsequently isolated by sorting the library on purified KcsA protein. HmK peptide inhibited KcsA-Shaker with a K i ~1 nM (11). T-HmK was constructed by cloning the sequence of HmK into a T-toxin plasmid between a signal peptide sequence and a flexible linker with an embedded c-Myc epitope that was followed by a GPI targeting sequence ( Fig.…”

“…Since SAK1 toxin blockade of the K + channel pore is a bimolecular interaction (11), the apparent K i of T-HmK block of KcsA-Shaker channels can be calculated after 0.5-ng cRNA injection to be 1.1 ± 0.1 nM using the unblocked fraction of the current at equilibrium (F un ) and the effective toxin concentration [Tx] ( Fig. 1E) according to Eq.…”