INVESTIGADORES
MAYORGA Luis Segundo
congresos y reuniones científicas
Título:
A designer peptide toxin isolated by phage display that inhibits the human voltage-gated proton channel, hHv1
Autor/es:
236. KENNEDY, K, ZHAO, R, , LI, Q, PEROZO, E, DE BLAS, GA, PAVAROTTI, MA, ARIAS, R, MAYORGA, LS, GOLDSTEIN, SAN
Lugar:
New Orleans
Reunión:
Congreso; 61st Annual Meeting of the Biophysical Society,; 2017
Institución organizadora:
Biophysical Soc
Resumen:
hHv1 is critical to immune defense, maintaining pH homeostasis in white blood cells by extruding protons duringrespiratory burst to compensate for reactive oxygen species (ROS) production, and during sperm capacitation causingcytosolic alkalization. Hindering basic and clinical research, hHv1 is an orphan receptor without a potent and specificblocker. hHv1 is homodimeric; each subunit has a conduction pathway and is homologous to the four membranespanningsegments 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 werefabricated on an inhibitor cysteine knot (ICK) scaffold, a backbone stabilized by three disulfide bonds and found innature to be rich in VSD-directed toxins. Designed by combinatorial permutation of 110 venom toxins, phage sortingwas performed on purified recombinant hHv1 and specificity of binding validated by ELISA. Five novel peptides wereidentified (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 slowsactivation, 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 ofthe dimer: two C6 were seen per dimer by single particle photobleaching using C6 labeled with 5,6-TAMRA and hHv1tagged with TFP. Moreover, a monomeric hHv1 channel was also blocked by only 50% with a Ki of 110 nM and boundone fluorescent C6 peptide. Of note, a point mutation in the channel epitope where C6 binds increased toxin affinity andproduced 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 nativehHv1 function. Human whole blood cells stimulated by PMA release ROS and this was suppressed in a dose dependentmanner by C6 (IC50 100 nM). C6 at 20 μM also specifically suppressed the cytosolic calcium increase and acrosomereaction triggered by progesterone in human sperm. Both ROS generation and capacitation were insensitive to 10 or 20μM of scorpion toxin blockers of Kv1.3 K+ channels.
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