Scorpion toxin inhibits the voltage-gated proton channel using a Zn²⁺ -like long-range conformational coupling...

Blocking the voltage-gated proton channel H_V 1 is a promising strategy for the treatment of diseases like ischaemia stroke and cancer. However, few H_V 1 channel antagonists have been reported. Here, we have identified a novel H_V 1 channel antagonist from scorpion venom and have elucidated its action mechanism.

H_V 1 and NaV channels were heterologously expressed in mammalian cell lines and their currents recorded using whole-cell patch clamp. Site-directed mutagenesis was used to generate mutants. Toxins were recombinantly produced in Escherichia coli. AGAP/W38F-H_V 1 interaction was modelled by molecular dynamics simulations.

The scorpion toxin AGAP (anti-tumour analgesic peptide) potently inhibited H_V 1 currents. One AGAP mutant has reduced Na_V channel activity but intact H_V 1 activity (AGAP/W38F). AGAP/W38F inhibited H_V 1 channel activation by trapping its S4 voltage sensor in a deactivated state and inhibited H_V 1 currents with less pH dependence than Zn²⁺ . Mutation analysis showed that the binding pockets of AGAP/W38F and Zn²⁺ in H_V 1 channel partly overlapped (common sites are His140 and His193). The E153A mutation at the intracellular Coulombic network (ICN) in H_V 1 channel markedly reduced AGAP/W38F inhibition, as observed for Zn²⁺ . Experimental data and MD simulations suggested that AGAP/W38F inhibited H_V 1 channel using a Zn²⁺ -like long-range conformational coupling mechanism.

Our results suggest that the Zn²⁺ binding pocket in H_V 1 channel might be a hotspot for modulators and valuable for designing H_V 1 channel ligands. Moreover, AGAP/W38F is a useful molecular probe to study H_V 1 channel and a lead compound for drug development.