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Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor.

ABSTRACT: 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 (K d) 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.

SUBMITTER: Zhao R

PROVIDER: S-EPMC6294887 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor.

Zhao Ruiming R Kennedy Kelleigh K De Blas Gerardo A GA Orta Gerardo G Pavarotti Martín A MA Arias Rodolfo J RJ de la Vega-Beltrán José Luis JL Li Qufei Q Dai Hui H Perozo Eduardo E Mayorga Luis S LS Darszon Alberto A Goldstein Steve A N SAN

Proceedings of the National Academy of Sciences of the United States of America 20181126 50

Using a de novo peptide inhibitor, Corza6 (C6), we demonstrate that the human voltage-gated proton channel (hHv1) is the main pathway for H<sup>+</sup> 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 ...[more]

PMID: 30478045

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Project description:Sperm capacitation is a complex and indispensable physiological process that spermatozoa must undergo in order to acquire fertilization capability. Spermatozoa from several mammalian species, including mice, exhibit a capacitation-associated plasma membrane hyperpolarization, which is necessary for the acrosome reaction to occur. Despite its importance, this hyperpolarization event has not been adequately examined in human sperm. In this report we used flow cytometry to show that a subpopulation of human sperm indeed undergo a plasma membrane hyperpolarization upon in vitro capacitation. This hyperpolarization correlated with two other well-characterized capacitation parameters, namely an increase in intracellular pH and Ca(2+) concentration, measured also by flow cytometry. We found that sperm membrane hyperpolarization was completely abolished in the presence of a high external K(+) concentration (60 mM), indicating the participation of K(+) channels. In order to identify, which of the potential K(+) channels were involved in this hyperpolarization, we used different K(+) channel inhibitors including charybdotoxin, slotoxin and iberiotoxin (which target Slo1) and clofilium (a more specific blocker for Slo3). All these K(+) channel antagonists inhibited membrane hyperpolarization to a similar extent, suggesting that both members of the Slo family may potentially participate. Two very recent papers recorded K(+) currents in human sperm electrophysiologically, with some contradictory results. In the present work, we show through immunoblotting that Slo3 channels are present in the human sperm membrane. In addition, we found that human Slo3 channels expressed in CHO cells were sensitive to clofilium (50 μM). Considered altogether, our data indicate that Slo1 and Slo3 could share the preponderant role in the capacitation-associated hyperpolarization of human sperm in contrast to what has been previously reported for mouse sperm, where Slo3 channels are the main contributors to the hyperpolarization event.

Dataset Information

Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor.

Publications

Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor.

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