Extracellular HEME Modulates Voltage-gating in CNBD Superfamily Cation Channels

Abstract

assess the significance of toxin residues. Here, we show that geneticallyencoded, 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-Lys 22 is shown to interact with K þ ions traversing the permeation pathway from the cytoplasm conferring voltage-dependence to the toxin offrate, a classic mechanism we observe as well for HmK peptide with both KcsA and K V 1.3 channels. In contrast, two related SAK1 toxins, Hui1 and ShK, block KcsA and K V 1.3, 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 þ channel pores.