A designer peptide toxin isolated by phage display that inhibits the human voltage-gated proton channel, hHv1

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

New Orleands, Louisiana

Congreso; 61st Annual Meeting of the Biophysical Society,; 2017

Biophysical sociaety

hHv1 is critical to immune defense, maintaining pH homeostasis in whiteblood cells by extruding protons during respiratory burst to compensate forreactive oxygen species (ROS) production, and during sperm capacitationcausing cytosolic alkalization. Hindering basic and clinical research, hHv1 isan orphan receptor without a potent and specific blocker. hHv1 is homodimeric;each subunit has a conduction pathway and is homologous to the fourmembrane-spanning segments that form the voltage sensing domains (VSD)in KV and NaV channels. To produce a toxin ligand for hHv1, we employeda phage display strategy whereby ~1 million novel peptides were fabricatedonan inhibitor cysteine knot (ICK) scaffold, a backbone stabilized by three disulfidebonds and found in nature to be rich in VSD-directed toxins. Designed bycombinatorial permutation of 110 venom toxins, phage sorting was performedon purified recombinant hHv1 and specificity of binding validated by ELISA.Five novel peptides were identified (C2-C6), synthesized, and studied byexternal application to hHv1 channels expressed in HEK293T cells. Protoncurrents measured by whole cell patch clamp were inhibited by C6. Consistentwith VSD trapping, C6 slows activation, accelerates deactivation, and shiftsactivation to more depolarized voltages. Inhibition by C6 is partial, showingat most 50% block at þ40 mV (1 mM) with a Ki of 150 nM. Partial blockwas not due to partial occupancy of the dimer: two C6 were seen per dimerby single particle photobleaching using C6 labeled with 5,6-TAMRA andhHv1 tagged with TFP. Moreover, a monomeric hHv1 channel was alsoblocked 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 increasedtoxin affinity and produced complete blockade (accompanying abstract byZhao, Kennedy et al). C6 was isolated on purified hHv1 protein but shownto inhibit two cellular responses proposed to depend on native hHv1 function.Human whole blood cells stimulated by PMA release ROS and this was suppressedin a dose dependent manner by C6 (IC50 100 nM). C6 at 20 mM alsospecifically suppressed the cytosolic calcium increase and acrosome reactiontriggered by progesterone in human sperm. Both ROS generation and capacitationwere insensitive to 10 or 20 mM of scorpion toxin blockers of Kv1.3 Kþchannels.