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

Abstract

We show here that membrane-tethered toxins facilitate the biophysical study of the roles of toxin residues in K+ channel blockade to reveal two blocking mechanisms in the K+ channel pore. The structure of the sea anemone type I (SAK1) toxin HmK is determined by NMR. T-HmK residues are scanned by point mutation to map the toxin surface, and seven residues are identified to be critical to occlusion of the KcsA channel pore. T-HmK–Lys22 is shown to interact with K+ ions traversing the KcsA pore from the cytoplasm conferring voltage dependence on the toxin off rate, a classic mechanism that we observe as well with HmK in solution and for 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 and as free peptides.

Molecular determinants of inhibition of the human proton channel hHv1 by the designer peptide C6 and a bivalent derivative

“…The in vitro synthesis and folding of venom-toxin peptides can be technically challenging, costly, and time-consuming. To facilitate characterization of C6 by mutational screening, we employed a strategy whereby C6, or its variants, were expressed from a gene in tethered form on the extracellular surface of cells via a glycosyl phosphatidyl inositol (GPI)-anchored membrane-embedded leash, as we have before with other toxins ( 13 ). The construct included a signal peptide sequence, C6 or its variants, and a flexible linker, followed by a GPI targeting sequence ( Fig.…”

“…Using point mutations, we identified five residues in the hHv1 S3–S4 loop that alter the free energy of blockade (ΔΔG) by more than 2 kcal/mol and that we therefore characterize as important for C6 binding. Taking advantage of a membrane-tethered toxin method ( 13 ), we scanned 35 noncysteine residues in tethered C6 (T-C6) and identified 7 that also significantly decrease affinity. We also show that C6 partitions most readily into lipid membranes that contain negatively charged phospholipids.…”

A molecular lid mechanism of K+channel blocker action revealed by a cone peptide

“…An alternative binding mode that does not involve a pore-inserting lysine was deduced for Shk-Dap22, an analog with increased selectivity for Kv1.3, developed as immunosuppressant 39 . Recently, a similar mechanism was identified for the parental toxin, using a novel tethered-toxin methodology 40 . Further, the recent suggestion that the classical pore-blocker CTX wobbles between several bound conformations, further imply that the various K + blocking mechanisms portrayed above are not mutually exclusive, and may co-exist, albeit at different potencies, for a given peptide-channel interacting pair.…”

A Molecular Lid Mechanism of K+ Channel Blocker Action Revealed by a Cone Peptide

“…An alternative binding mode that does not involve a pore-inserting lysine was deduced for Shk-Dap22, an analog with increased selectivity for Kv1.3, developed as immunosuppressant 39 . Recently, a similar mechanism was identified for the parental toxin and for peptides designed on its scaffold, using phage-display and a novel tethered-toxin methodology 40,41 . Further, the recent suggestion that the classical pore-blocker CTX wobbles between several bound conformations, further imply that the various K + blocking mechanisms portrayed above are not mutually exclusive, and may co-exist, albeit at different potencies, for a given peptide-channel interacting pair.…”