Ion channels are highly diverse in the cnidarian model organism Nematostella vectensis (Anthozoa), but little is known about the evolutionary origins of this channel diversity and its conservation across Cnidaria. Here we examined the evolution of voltage-gated K+ channels in Cnidaria by comparing genomes and transcriptomes of diverse cnidarian species from Anthozoa and Medusozoa. We found an average of over 40 voltage-gated K+ channel genes per species, and phylogenetic reconstruction of the Kv, KCNQ and EAG gene families identified 28 voltage-gated K+ channels present in the last common ancestor of Anthozoa and Medusozoa (23 Kv, 1 KCNQ and 4 EAG). Thus, much of the diversification of these channels took place in the stem cnidarian lineage prior to the emergence of modern cnidarian classes. In contrast, the stem bilaterian lineage, from which humans evolved, contained no more than 9 voltage-gated K+ channels. These results hint at a complexity to electrical signaling in all cnidarians that contrasts with the perceived anatomical simplicity of their neuromuscular systems. These data provide a foundation from which the function of these cnidarian channels can be investigated, which will undoubtedly provide important insights into cnidarian physiology.

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.

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