Cholesterol inhibits human voltage-gated proton channel hHv1

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Significance

Our work identified membrane cholesterol as a new inhibitor of human voltage-gated proton channel hHv1. Using single-molecule fluorescence resonance energy transfer (smFRET), we further showed that cholesterol inhibits the hHv1 channel by stabilizing the voltage-sensing S4 segment at resting conformations, a similar mechanism also utilized by Zn ²⁺ inhibition. Our findings provided a new mechanism that the hHv1 channel regulates human fertilization by linking the cholesterol efflux with intracellular alkalization, thus sperm hyperactivation.

Abstract

Although human sperm is morphologically mature in the epididymis, it cannot fertilize eggs before capacitation. Cholesterol efflux from the sperm plasma membrane is a key molecular event essential for cytoplasmic alkalinization and hyperactivation, but the underlying mechanism remains unclear. The human voltage-gated proton (hHv1) channel functions as an acid extruder to regulate intracellular pHs of many cell types, including sperm. Aside from voltage and pH, Hv channels are also regulated by distinct ligands, such as Zn ²⁺ and albumin. In the present work, we identified cholesterol as an inhibitory ligand of the hHv1 channel and further investigated the underlying mechanism using the single-molecule fluorescence resonance energy transfer (smFRET) approach. Our results indicated that cholesterol inhibits the hHv1 channel by stabilizing the voltage-sensing S4 segment at resting conformations, a similar mechanism also utilized by Zn ²⁺. Our results suggested that the S4 segment is the central gating machinery in the hHv1 channel, on which voltage and distinct ligands are converged to regulate channel function. Identification of membrane cholesterol as an inhibitory ligand provides a mechanism by which the hHv1 channel regulates fertilization by linking the cholesterol efflux with cytoplasmic alkalinization, a change that triggers calcium influx through the CatSper channel. These events finally lead to hyperactivation, a remarkable change in the mobility pattern indicating fertilization competence of human sperm.

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Most cited references 47

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Progesterone activates the principal Ca2+ channel of human sperm.

Polina V. Lishko, Inna Botchkina, Yuriy Kirichok (2011)

Steroid hormone progesterone released by cumulus cells surrounding the egg is a potent stimulator of human spermatozoa. It attracts spermatozoa towards the egg and helps them penetrate the egg's protective vestments. Progesterone induces Ca(2+) influx into spermatozoa and triggers multiple Ca(2+)-dependent physiological responses essential for successful fertilization, such as sperm hyperactivation, acrosome reaction and chemotaxis towards the egg. As an ovarian hormone, progesterone acts by regulating gene expression through a well-characterized progesterone nuclear receptor. However, the effect of progesterone upon transcriptionally silent spermatozoa remains unexplained and is believed to be mediated by a specialized, non-genomic membrane progesterone receptor. The identity of this non-genomic progesterone receptor and the mechanism by which it causes Ca(2+) entry remain fundamental unresolved questions in human reproduction. Here we elucidate the mechanism of the non-genomic action of progesterone on human spermatozoa by identifying the Ca(2+) channel activated by progesterone. By applying the patch-clamp technique to mature human spermatozoa, we found that nanomolar concentrations of progesterone dramatically potentiate CatSper, a pH-dependent Ca(2+) channel of the sperm flagellum. We demonstrate that human CatSper is synergistically activated by elevation of intracellular pH and extracellular progesterone. Interestingly, human CatSper can be further potentiated by prostaglandins, but apparently through a binding site other than that of progesterone. Because our experimental conditions did not support second messenger signalling, CatSper or a directly associated protein serves as the elusive non-genomic progesterone receptor of sperm. Given that the CatSper-associated progesterone receptor is sperm specific and structurally different from the genomic progesterone receptor, it represents a promising target for the development of a new class of non-hormonal contraceptives.

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A voltage-gated proton-selective channel lacking the pore domain.

I. Ramsey, Magdalene Moran, Jayhong Chong … (2006)

Voltage changes across the cell membrane control the gating of many cation-selective ion channels. Conserved from bacteria to humans, the voltage-gated-ligand superfamily of ion channels are encoded as polypeptide chains of six transmembrane-spanning segments (S1-S6). S1-S4 functions as a self-contained voltage-sensing domain (VSD), in essence a positively charged lever that moves in response to voltage changes. The VSD 'ligand' transmits force via a linker to the S5-S6 pore domain 'receptor', thereby opening or closing the channel. The ascidian VSD protein Ci-VSP gates a phosphatase activity rather than a channel pore, indicating that VSDs function independently of ion channels. Here we describe a mammalian VSD protein (H(V)1) that lacks a discernible pore domain but is sufficient for expression of a voltage-sensitive proton-selective ion channel activity. H(v)1 currents are activated at depolarizing voltages, sensitive to the transmembrane pH gradient, H+-selective, and Zn2+-sensitive. Mutagenesis of H(v)1 identified three arginine residues in S4 that regulate channel gating and two histidine residues that are required for extracellular inhibition of H(v)1 by Zn2+. H(v)1 is expressed in immune tissues and manifests the characteristic properties of native proton conductances (G(vH+)). In phagocytic leukocytes, G(vH+) are required to support the oxidative burst that underlies microbial killing by the innate immune system. The data presented here identify H(v)1 as a long-sought voltage-gated H+ channel and establish H(v)1 as the founding member of a family of mammalian VSD proteins.

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The CatSper channel mediates progesterone-induced Ca2+ influx in human sperm.

Timo Strünker, Normann Goodwin, Christoph Brenker … (2011)

In the oviduct, cumulus cells that surround the oocyte release progesterone. In human sperm, progesterone stimulates a Ca(2+) increase by a non-genomic mechanism. The Ca(2+) signal has been proposed to control chemotaxis, hyperactivation and acrosomal exocytosis of sperm. However, the underlying signalling mechanism has remained mysterious. Here we show that progesterone activates the sperm-specific, pH-sensitive CatSper Ca(2+) channel. We found that both progesterone and alkaline pH stimulate a rapid Ca(2+) influx with almost no latency, incompatible with a signalling pathway involving metabotropic receptors and second messengers. The Ca(2+) signals evoked by alkaline pH and progesterone are inhibited by the Ca(v) channel blockers NNC 55-0396 and mibefradil. Patch-clamp recordings from sperm reveal an alkaline-activated current carried by mono- and divalent ions that exhibits all the hallmarks of sperm-specific CatSper Ca(2+) channels. Progesterone substantially enhances the CatSper current. The alkaline- and progesterone-activated CatSper current is inhibited by both drugs. Our results resolve a long-standing controversy over the non-genomic progesterone signalling. In human sperm, either the CatSper channel itself or an associated protein serves as the non-genomic progesterone receptor. The identification of CatSper channel blockers will greatly facilitate the study of Ca(2+) signalling in sperm and help to define further the physiological role of progesterone and CatSper.

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Author and article information

Journal

Journal ID (nlm-ta): Proc Natl Acad Sci U S A

Journal ID (iso-abbrev): Proc Natl Acad Sci U S A

Journal ID (hwp): pnas

Journal ID (publisher-id): PNAS

Title: Proceedings of the National Academy of Sciences of the United States of America

Publisher: National Academy of Sciences

ISSN (Print): 0027-8424

ISSN (Electronic): 1091-6490

Publication date (Electronic): 29 August 2022

Publication date (Print): 6 September 2022

Publication date PMC-release: 1 March 2023

Volume: 119

Issue: 36

Electronic Location Identifier: e2205420119

Affiliations

[1] ^aDivision of Biological and Biomedical Systems, School of Science and Engineering, University of Missouri-Kansas City , Kansas City, MO 64110;

[2] ^bDepartment of Biomedical Sciences, School of Medicine, University of Missouri-Kansas City , Kansas City, MO 64108

Author notes

¹To whom correspondence may be addressed. Email: wangshizhen@ 123456umkc.edu .

Edited by Ehud Isacoff, University of California, Berkeley, CA; received March 28, 2022; accepted July 20, 2022

Author contributions: S.W. conceived the study; S.W., S.H., and X.-P.C. designed research; S.H., S.P., J.V., R.G., P.G., and X.-P.C. performed research; S.H., X.-P.C., and S.W. analyzed data; and S.W. wrote the paper.