Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes Is a Prerequisite for Its Inhibition of Human Voltage-gated Sodium Channel NaV1.7

Abstract

ProTx-II is a disulfide-rich peptide toxin from tarantula venom able to inhibit the human voltage-gated sodium channel 1.7 (hNa V 1.7), a channel reported to be involved in nociception, and thus it might have potential as a pain therapeutic. ProTx-II acts by binding to the membrane-embedded voltage sensor domain of hNa V 1.7, but the precise peptide channel-binding site and the importance of membrane binding on the inhibitory activity of ProTx-II remain unknown. In this study, we examined the structure and membrane-binding properties of ProTx-II and several analogues using NMR spectroscopy, surface plasmon resonance, fluorescence spectroscopy, and molecular dynamics simulations. Our results show a direct correlation between ProTx-II membrane binding affinity and its potency as an hNa V 1.7 channel inhibitor. The data support a model whereby a hydrophobic patch on the ProTx-II surface anchors the molecule at the cell surface in a position that optimizes interaction of the peptide with the binding site on the voltage sensor domain. This is the first study to demonstrate that binding of ProTx-II to the lipid membrane is directly linked to its potency as an hNa V 1.7 channel inhibitor.Voltage-gated ion channels (VGICs) 4 are transmembrane proteins responsible for voltage-dependent movement of ions across cell membranes. They are involved in a wide range of physiological processes, including action potential generation in excitable cells, muscle and nerve relaxation, regulation of blood pressure, and sensory transduction. Many disorders are associated with VGIC abnormalities, and hence VGICs are actively pursued as drug targets for the treatment of a range of neuromuscular, neurological, or inflammatory disorders (1-3). For instance, the human voltage-gated sodium channel subtype 1.7 (hNa V 1.7) is involved in pain sensation, and molecules that selectively inhibit this channel might therefore be useful as leads for the development of novel analgesics (4). However, high sequence identity and structural homology exist between the nine subtypes of voltage-gated sodium channels, and given the critical role of Na V channels in the normal electrical activity of neurons, skeletal muscles, and cardiomyocytes, subtype selectivity is crucial but often difficult to achieve with small molecule inhibitors (2).Disulfide-rich peptide toxins isolated from animal venoms (e.g. spiders, snakes, and cone snails) have attracted much attention as potential analgesics because of their well defined three-dimensional structure and ability to inhibit voltage-gated sodium (Na V ), potassium (K V ), and calcium (Ca V ) ion channels with high potency and selectivity (5-9). Because of their stability, selectivity, and potency, these disulfide-rich peptides have been extensively characterized and are vigorously being pursued as drug leads as well as pharmacological tools to characterize VGICs (5).In general, peptide toxins that inhibit VGICs can be divided into two main groups based on their inhibitory strategy as fol-

Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has an increased inhibitory potency at human voltage-gated sodium channel hNa V 1.7

“…This structural feature has been proposed to promote not only GMT-voltage-gated ion channel interactions, but also GMT-lipid membrane interactions [12,13,17,18,48]. Several spider GMTs have been shown to interact with model membranes and the unique structures of these peptides have been proposed to facilitate a tri-molecular interaction involving the GMTs, the lipid membrane and target voltage-gated ion channels [14,47,[50][51][52].…”

“…Therefore, the neutrally charged, POPC/SM/CHOL (2.7:4:3.3) and anionic POPC/C1P/CHOL (2.7:4:3.3 molar ratio) model membranes were also studied [48,49].…”

“…Although we cannot exclude the possibility that an increased inhibitory potency on hNa V 1.7 and increased membrane binding are independent, we propose a model where gHwTx-IV acts by binding both to the membrane and to the target channel ( Figure 6B). A similar mechanism has been suggested for ProTx-I and ProTx-II, two GMTs that also seem to possess distinct surface regions responsible for membrane interaction and for channel interaction [18,48]. Furthermore, the fact the amino acid residues that seem to be involved in membrane binding are distinct from those involved in channel binding lead us to postulate that gHwTx-IV will show a similar selectivity profile to HwTx-IV, particularly against Na V 1.5 which is involved in cardiac muscle conduction [16].…”

Insensitivity to pain induced by a potent selective closed-state Nav1.7 inhibitor

“…The potency difference between ProTX-II-NH-Me and JNJ63955918 suggests that the binding contacts may be similar but not identical. W7 has recently been reported to be an important component of a lipid interaction surface that includes residues K4, W5, M6 and W7 along with supplementary contributions from Y1, W24 and K2729. Based on association/dissociation rates, parts of this face of ProTX-II were deemed likely to be important for membrane partitioning but not for direct interaction with the channel voltage sensor30.…”

Molecular simulations of venom peptide‐membrane interactions: Progress and challenges

“…Most GMs are 20–60 amino acids long and fold into a well‐defined secondary structure that is stabilized by multiple, highly conserved disulfide bonds . Some of the most studied examples include the spider venom peptides VsTx1, SGTx1, Hanatoxin, ProTx‐I, and ProTx‐II . The second group are disulfide‐rich peptides that act on mechanosensitive channels (MSCs).…”