Peptide-Membrane Interactions Affect the Inhibitory Potency and Selectivity of Spider Toxins ProTx-II and GpTx-1

Gating modifier toxins (GMTs) from spider venom can inhibit voltage gated sodium channels (Na_Vs) involved in pain signal transmission, including the Na_V1.7 subtype. GMTs have a conserved amphipathic structure that allow them to interact with membranes and also with charged residues in regions of Na_V that are exposed at the cell surface. ProTx-II and GpTx-1 are GMTs able to inhibit Na_V1.7 with high potency, but they differ in their ability to bind to membranes and in their selectivity over other Na_V subtypes. To explore these differences and gain detailed information on their membrane-binding ability and how this relates to potency and selectivity, we examined previously described Na_V1.7 potent/selective GpTx-1 analogues and new ProTx-II analogues designed to reduce membrane binding and improve selectivity for Na_V1.7. Our studies reveal that the number and type of hydrophobic residues as well as how they are presented at the surface determine the affinity of ProTx-II and GpTx-1 for membranes and that altering these residues can have dramatic effects on Na_V inhibitory activity. We demonstrate that strong peptide-membrane interactions are not essential for inhibiting Na_V1.7 and propose that hydrophobic interactions instead play an important role in positioning the GMT at the membrane surface proximal to exposed Na_V residues, thereby affecting peptide-channel interactions. Our detailed structure-activity relationship study highlights the challenges of designing GMT-based molecules that simultaneously achieve high potency and selectivity for Na_V1.7, as single mutations can induce local changes in GMT structure that can have a major impact on Na_V-inhibitory activity.