Is lipid bilayer binding a common property of inhibitor cysteine knot ion-channel blockers?

Abstract

Recent studies of several ICK ion-channel blockers suggest that lipid bilayer interactions play a prominent role in their actions. Structural similarities led to the hypothesis that bilayer interactions are important for the entire ICK family. We have tested this hypothesis by performing direct measurements of the free energy of bilayer partitioning (DeltaG) of several peptide blockers using our novel quenching-enhanced fluorescence titration protocol. We show that various ICK peptides demonstrate markedly different modes of interaction with large unilamellar lipid vesicles. The mechanosensitive channel blocker, GsMTx4, and its active diastereomeric analog, D-GsMTx4, bind strongly to both anionic and zwitterionic membranes. One potassium channel gating modifier, rHpTx2gs, interacts negligibly with both types of vesicles at physiological pH, whereas another, SGTx1, interacts only with anionic lipids. The slope of DeltaG dependence on surface potential is very shallow for both GsMTx4 and D-GsMTx4, indicating complex interplay of their hydrophobic and electrostatic interactions with lipid. In contrast, a cell-volume regulator, GsMTx1, and SGTx1 exhibit a very steep DeltaG dependence on surface potential, resulting in a strong binding only for membranes rich in anionic lipids. The high variability of 5 kcal/mole in observed DeltaG shows that bilayer partitioning is not a universal property of the ICK peptides interacting with ion channels.

FIGURE 1

Quenching-enhanced fluorescence titration of two ICK blockers with LUV made of lipids specified on graphs (experimental details are described in Posokhov et al. (4)). Whereas GsMTx1 (A) requires high concentration of anionic POPG lipid for binding, rHpTx2gs (B) will only interact with vesicles at acidic pH (open symbols), but not at neutral pH (solid symbols).

FIGURE 2

Free energy of partitioning of four ICK blockers into membranes with various surface potential. GsMTx4 data were collected for (solid squares) POPC, 95POPC:5POPG, 75POPC:25POPG, and 25POPC:75POPG; (diamonds) PC and 25PC:75PS; and (open square) with 25POPC:75POPG in the presence of additional 450 mM KCl (all other samples contain 50 mM KI). D-GsMTx4 data (circles) were collected for POPC:POPG mixtures. SGTx1 (blue triangles) and GsMTx1 (olive triangles) data were collected for 25POPC:75POPG with (open symbols) or without (solid symbols) of additional 50 mM KCl. The effective charge for (D-)GsMTx4 peptides calculated from the slope of these dependences, Z_eff, is much smaller than the net charge of the peptide. A large Z_eff observed for SGTx1 and GsMTx1 is indicative of predominantly electrostatic interactions.

FIGURE 3

Primary amino acid sequence alignment of the four blockers used in this study, plus VsTx1 aligned along the positions of cysteines (blue). The rest of the residues are shaded in accordance with the Wimley-White hydrophobicity scale (10), with the most hydrophobic residues (W, Y, F, L, and I) in green, and the least hydrophobic (charged) in red.