GsMTx4: Mechanism of Inhibiting Mechanosensitive Ion Channels

Abstract

GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We synthesized six analogs with single lysine-to-glutamate substitutions and tested them against Piezo1 channels in outside-out patches and independently measured lipid binding. Four analogs had ∼20% lower efficacy than the wild-type (WT) peptide. The equilibrium constants calculated from the rates of inhibition and washout did not correlate with the changes in inhibition. The lipid association strength of the WT GsMTx4 and the analogs was determined by tryptophan autofluorescence quenching and isothermal calorimetry with membrane vesicles and showed no significant differences in binding energy. Tryptophan fluorescence-quenching assays showed that both WT and analog peptides bound superficially near the lipid-water interface, although analogs penetrated deeper. Peptide-lipid association, as a function of lipid surface pressure, was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure. Analogs with compromised efficacy had pressure-area isotherms with steeper slopes in this region, suggesting tighter peptide association. The pressure-dependent redistribution of peptide between "deep" and "shallow" binding modes was supported by molecular dynamics (MD) simulations of the peptide-monolayer system under different area constraints. These data suggest a model placing GsMTx4 at the membrane surface, where it is stabilized by the lysines, and occupying a small fraction of the surface area in unstressed membranes. When applied tension reduces lateral pressure in the lipids, the peptides penetrate deeper acting as "area reservoirs" leading to partial relaxation of the outer monolayer, thereby reducing the effective magnitude of stimulus acting on the MSC gate.

Piezo channels and GsMTx4: Two milestones in our understanding of excitatory mechanosensitive channels and their role in pathology

“…And the recent crystal structure of TRAAK [46] supports a model where inner monolayer tension gates K2P channels. Our tests with GsMTx4 also support this model, as GsMTx4 potentiates TREK1 MSC currents when applied to outside-out patches suggesting peptide relief of outer monolayer tension increases tension on the inner monolayer [74]. This may be due to a combination of GsMTx4 inducing negative curvature in the resting patches and/or acting as an outer monolayer tension clamp when pressure is applied.…”

“…To better understand GsMTx4’s effects on membranes we synthesized six lysine-glutamate peptide analogs and tested them against Piezo1 channels in patches [74]. We found several analogs with compromised inhibitory activity.…”

Spatial Relationship and Functional Relevance of Three Lipid Domain Populations at the Erythrocyte Surface

“…To further test the potential link between lipid domains and calcium exchange kinetics upon RBC deformation, we used the peptide GsMTx4, a validated inhibitor of the mechanoactivated ion channels [37] which are mostly responsible for the calcium entry upon RBC deformation [1]. We showed that this treatment abolished calcium entry as well as GM1-and SM-enriched domain increase without inducing detectable toxicity (Suppl.…”

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

“…The free energy profiles from the all‐atom and CG simulations for the wild‐type peptide were within 6 kJ mol −1 . The Δ G b predicted from the CG simulations ranged from −18 kJ mol −1 to −27 kJ mol −1 while the corresponding values reported from Trp quenching experiments were −25 kJ mol −1 to −26 kJ mol −1 . The ΔΔ G b for the K‐E mutants predicted from simulations ranged from −2 kJ mol −1 to 4 kJ mol −1 while the ΔΔ G b from experiments was <1 kJ mol −1 for all mutants.…”

“…Nishizawa et al used both all‐atom and CG simulations to calculate the free energy for the binding of GsMTx4 and analogues thereof to a POPC membrane. The aim was to investigate the relationship between membrane binding properties of the peptide and activity on the MSC Piezo1 . For this, the simulations studied the effect of K to E mutations on the insertion depths and orientation of the peptide in the membrane and their binding affinities.…”