Tethered Peptide Neurotoxins Facilitate Biophysical Study and Reveal Two Voltage-dependent Blocking Mechanisms for SAK1 Toxins in the K+ Channel Pore

Abstract

Peptide toxins are powerful tools for studying membrane receptors, and for the diagnosis, and treatment of disease. Determining the structural and mechanistic bases for toxin action has required the synthesis of many peptide variants to assess the significance of toxin residues. Here, we show that genetically-encoded, membrane-tethered toxins (T-toxins) allow rapid screening of the residues to determine the mechanistic basis for toxin-ion channel interaction, including kinetics parameters of interaction, through study of two channels, KcsA and Kv1.3. First, the structure of the sea anemone type I (SAK1) toxin HmK is determined by NMR. Then, T-HmK residues are scanned by point mutation to identify the seven residues in close contact with the KcsA pore. T-HmK-Lys22 is shown to interact with K+ ions traversing the permeation pathway from the cytoplasm conferring voltage-dependence to the toxin off-rate, a classic mechanism we observe as well for HmK peptide with both KcsA and KV1.3 channels. In contrast, two related SAK1 toxins, Hui1 and ShK, block KcsA and KV1.3, respectively, via an arginine rather than the canonical lysine, when tethered as well as free peptides. Our study changes a long-held conclusion about the voltage-dependent mechanism of ShK-Kv1.3 interaction, and demonstrates that there are two orientations for SAK1 toxins in K+ channel pores.