Structural basis for the inhibition of voltage-dependent K+ channel by gating modifier toxin

Abstract

Voltage-dependent K+ (Kv) channels play crucial roles in nerve and muscle action potentials. Voltage-sensing domains (VSDs) of Kv channels sense changes in the transmembrane potential, regulating the K+-permeability across the membrane. Gating modifier toxins, which have been used for the functional analyses of Kv channels, inhibit Kv channels by binding to VSD. However, the structural basis for the inhibition remains elusive. Here, fluorescence and NMR analyses of the interaction between VSD derived from KvAP channel and its gating modifier toxin, VSTx1, indicate that VSTx1 recognizes VSD under depolarized condition. We identified the VSD-binding residues of VSTx1 and their proximal residues of VSD by the cross-saturation (CS) and amino acid selective CS experiments, which enabled to build a docking model of the complex. These results provide structural basis for the specific binding and inhibition of Kv channels by gating modifier toxins.

A complicated complex: Ion channels, voltage sensing, cell membranes and peptide inhibitors

“…This value would represent the equilibrium dissociation constant between the lipid partitioned ligand and the lipid embedded receptor. Remarkably, however, the value is in good agreement with equilibrium dissociation constants measured between micelle embedded receptors and free ligands [48,55,64].…”

“…Similarly, GMTs such as hanatoxin [59,60], ProTx-II [61], HHN2B-G7W [53] and SGTx [62] have been shown to bind to lipid bilayers. These and several other studies have also consistently shown that these cationic GMTs have an increased affinity for anionic lipids [48,55,63,64]. The distribution of the basic residues on the GMTs forms a basic patch surrounded by hydrophobic residues creating an amphipathic structure which has been suggested to mediates membrane association by several biophysical studies as well as molecular dynamics simulations [48,[64][65][66].…”

Disulfide mapping the voltage-sensing mechanism of a voltage-dependent potassium channel

“…In order to investigate the voltage-dependent conformational change of VSD, we used the V119C mutant, in which the Cys119 residue was chemically modified by a fluorescent dye, monobromobimane (mBBr), as previously reported20 (Fig. 2a).…”

“…The cells were grown at 37 °C to an A 600 of 0.4–0.6, and protein expression was then induced with 1.0 mM isopropyl-β-D-thiogalactopyranoside for 3–6 hours at 37 °C. Purification of the MBP-VSD was described elsewhere20. For the reconstitution of the purified proteins, the buffer was exchanged with “liposome buffer”, containing 20 mM Hepes-NaOH, pH 8.0, 0.10 mM KCl, 149.9 mM NaCl, and 10%(v/v) glycerol, in the presence of 2 mM DM.…”

“…Monobromobimane-modified VSD was prepared according to the published procedure20. The fluorescence analysis was conducted with membrane potential buffer (Table 1).…”