A designer peptide toxin isolated by phage display that inhibits the human voltage-gated proton channel, Hv1

RUIMING ZHAO; GERARDO A. DE BLAS; LUIS S. MAYORGA; KELLEIGH KENNEDY; EDUARDO PEROZO; RODOLFO ARIAS; QUFEI LI; MARTÍN A. PAVAROTTI; STEVE A. N. GOLDSTEIN

New Orleans, Louisiana. USA

Congreso; Biophysical Society 61ST Annual Meeting; 2017

Biophysical Society

hHv1 is critical to immune defense, maintaining pH homeostasis in white blood cells by extruding protons during respiratory burst to compensate for reactive oxygen species (ROS) production, and during sperm capacitation causing cytosolic alkalization. Hindering basic and clinical research, hHv1 is an orphan receptor without a potent and specific blocker. hHv1 is homodimeric; each subunit has a conduction pathway and is homologous to the four membrane-spanning segments that form the voltage sensing domains (VSD) in KV and NaV channels.To produce a toxin ligand for hHv1, we employed a phage display strategy whereby ~1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) scaffold, a backbone stabilized by three disulfide bonds and found in nature to be rich in VSD-directed toxins. Designed by combinatorial permutation of 110 venom toxins, phage sorting was performed on purified recombinant hHv1 and specificity of binding validated by ELISA. Five novel peptides were identified (C2-C6), synthesized, and studied by external application to hHv1 channels expressed in HEK293T cells. Proton currents measured by whole cell patch clamp were inhibited by C6. Consistent with VSD trapping, C6 slows activation, accelerates deactivation, and shifts activation to more depolarized voltages. Inhibition by C6 is partial, showing at most 50% block at +40 mV (1 µM) with a Ki of 150 nM. Partial block was not due to partial occupancy of the dimer: two C6 were seen per dimer by single particle photobleaching using C6 labeled with 5,6-TAMRA and hHv1 tagged with TFP. Moreover, a monomeric hHv1 channel was also blocked by only 50% with a Ki of 110 nM and bound one fluorescent C6 peptide. Of note, a point mutation in the channel epitope where C6 binds increased toxin affinity and produced complete blockade (accompanying abstract by Zhao, Kennedy et al). C6 was isolated on purified hHv1 protein but shown to inhibit two cellular responses proposed to depend on native hHv1 function. Human whole blood cells stimulated by PMA release ROS and this was suppressed in a dose dependent manner by C6 (IC50 100 nM). C6 at 20 µM also specifically suppressed the cytosolic calcium increase and acrosome reaction triggered by progesterone in human sperm. Both ROS generation and capacitation were insensitive to 10 or 20 µM of scorpion toxin blockers of Kv1.3 K+ channels.