Using a de novo peptide inhibitor, Corza6 (C6), we demonstrate that the human voltage-gated proton channel (hHv1) is the main pathway for H+ efflux that allows capacitation in sperm and permits sustained reactive oxygen species (ROS) production in white blood cells (WBCs). C6 was identified by a phage-display strategy whereby ∼1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) scaffold and sorting on purified hHv1 protein. Two C6 peptides bind to each dimeric channel, one on the S3–S4 loop of each voltage sensor domain (VSD). Binding is cooperative with an equilibrium affinity (Kd) of ∼1 nM at −50 mV. As expected for a VSD-directed toxin, C6 inhibits by shifting hHv1 activation to more positive voltages, slowing opening and speeding closure, effects that diminish with membrane depolarization.

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compartment, so that the plug and pore ring could again seal the channel. Here we prevented the intermediate from either backsliding or exiting through the lateral gate by (i) stalling a highly charged helix, the voltage sensor domain S4 of the potassium channel KvAP in the SecYEG pore and (ii) locking it between the ribosome on one side of the membrane and calmodulin on the other side. Dc was unable to gate this complex. We observed several conductivity levels, suggesting that the S4 helix was free to sample between the aqueous environment of the pore and the lipid interior. This observation (i) confirms the general view of how the translocon distinguishes membrane proteins from secretory proteins and (ii) disproves the hypothesis that the pore ring acts to maintain the membrane barrier in an active translocon. The work was supported by a grant from the Austrian science fund to Peter Pohl (P28213). 1. Saparov SM, et al. (2007) Determining the conductance of the SecY protein translocation channel for small molecules. Mol Cell 26(4):501-509. 2. Park E & Rapoport TA (2011) Preserving the membrane barrier for small molecules during bacterial protein translocation.