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Motility and fertilization potential of mammalian sperm are regulated by ion homeostasis which in turn is under tight control of ion channels and transporters. Sperm intracellular pH, membrane voltage and calcium concentration ([Ca2+]i) are all important for sperm activity within the female reproductive tract. While all mammalian sperm are united in their goal to find and fertilize an egg, the molecular mechanisms they utilize for this purpose are diverse and differ between species especially on the level of ion channels. Recent direct recording from sperm cells of different species indicate the differences between rodent, non-human primate, ruminant, and human sperm on the basic levels of their ion channel regulation. In this review we summarize the current knowledge about ion channel diversity of the animal kingdom and concentrate our attention on flagellar ion channels of mammalian sperm.

Mammalian spermatozoa gain competence to fertilize an oocyte as they travel through the female reproductive tract. This process is accompanied by an elevation of sperm intracellular calcium and a membrane hyperpolarization. The latter is evoked by K+ efflux; however, the molecular identity of the potassium channel of human spermatozoa (hKSper) is unknown. Here, we characterize hKSper, reporting that it is regulated by intracellular calcium but is insensitive to intracellular alkalinization. We also show that human KSper is inhibited by charybdotoxin, iberiotoxin, and paxilline, while mouse KSper is insensitive to these compounds. Such unique properties suggest that the Slo1 ion channel is the molecular determinant for hKSper. We show that Slo1 is localized to the sperm flagellum and is inhibited by progesterone. Inhibition of hKSper by progesterone may depolarize the spermatozoon to open the calcium channel CatSper, thus raising [Ca2+] to produce hyperactivation and allowing sperm to fertilize an oocyte. © Mannowetz et al.

Mammalian spermatozoa gain competence to fertilize an oocyte as they travel through the female reproductive tract. This process is accompanied by an elevation of sperm intracellular calcium and a membrane hyperpolarization. The latter is evoked by K(+) eff

To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5., Competing Interests: Declaration of interests We declare that one or more authors have a competing interest as defined by Nature Portfolio. The Krogan Laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche, and Rezo Therapeutics. N.J.K. has previously held financially compensated consulting agreements with the Icahn School of Medicine at Mount Sinai, New York, and Twist Bioscience Corp. He currently has financially compensated consulting agreements with Maze Therapeutics, Interline Therapeutics, Rezo Therapeutics, and GEn1E Lifesciences, Inc. He is on the Board of Directors of Rezo Therapeutics and is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics, and Interline Therapeutics., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In human spermatozoa, the electrochemical potentials across the mitochondrial and plasma membranes are related to sperm functionality and fertility, but the exact role of each potential has yet to be clarified. Impairing sperm mitochondrial function has been considered as an approach to creating male or unisex contraceptives, but it has yet to be shown whether this approach would ultimately block the ability of sperm to reach or fertilize an egg. To investigate whether the mitochondrial and plasma membrane potentials are necessary for sperm fertility, human sperm were treated with two small-molecule mitochondrial uncouplers (niclosamide ethanolamine and BAM15) that depolarize membranes by inducing passive proton flow, and evaluated the effects on a variety of sperm physiological processes. BAM15 specifically uncoupled human sperm mitochondria while niclosamide ethanolamine induced proton current in the plasma membrane in addition to depolarizing the mitochondria. In addition, both compounds significantly decreased sperm progressive motility with niclosamide ethanolamine having a more robust effect. However, these uncouplers did not reduce sperm adenosine triphosphate (ATP) content or impair other physiological processes, suggesting that human sperm can rely on glycolysis for ATP production if mitochondria are impaired. Thus, systemically delivered contraceptives that target sperm mitochondria to reduce their ATP production would likely need to be paired with sperm-specific glycolysis inhibitors. However, since niclosamide ethanolamine impairs sperm motility through an ATP-independent mechanism, and niclosamide is FDA approved and not absorbed through mucosal membranes, it could be a useful ingredient in on-demand, vaginally applied contraceptives., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm, providing the highest-resolution structural information to date. Our subtomogram averages reveal mammalian sperm-specific protein complexes within the microtubules, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm-specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm., (© 2023. The Author(s).)

Tmem95 encodes a sperm acrosomal membrane protein, whose knockout has a male-specific sterility phenotype in mice. Tmem95 knockout murine sperm can bind to, but do not fuse with, eggs. How TMEM95 plays a role in membrane fusion of sperm and eggs has remained elusive. Here, we utilize a sperm penetration assay as a model system to investigate the function of human TMEM95. We show that human TMEM95 binds to hamster egg membranes, providing evidence for a TMEM95 receptor on eggs. Using X-ray crystallography, we reveal an evolutionarily conserved, positively charged region of TMEM95 as a putative receptor-binding surface. Amino acid substitutions within this region of TMEM95 ablate egg-binding activity. We identify monoclonal antibodies against TMEM95 that reduce the number of human sperm fused with hamster eggs in sperm penetration assays. Strikingly, these antibodies do not block binding of sperm to eggs. Taken together, these results provide strong evidence for a specific, receptor-mediated interaction of sperm TMEM95 with eggs and suggest that this interaction may have a role in facilitating membrane fusion during fertilization.

The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca 2+ gateway. Here, we applied cryo-electron tomography to visualize the higher-order organization of the native CatSper complex in intact mammalian sperm. The repeating CatSper units form long zigzag-rows along mouse and human sperm flagella. Above each tetrameric channel pore, most of the extracellular domains form a canopy that interconnects to a zigzag-shaped roof. Murine CatSper contains an additional wing-structure connected to the tetrameric channel. The intracellular domains link two neighboring channels to a diagonal array, suggesting a dimer formation. Fitting of an atomic model of isolated monomeric CatSper to the in situ map reveals supramolecular interactions and assembly of the CatSper complex. Loss of EFCAB9-CATSPERζ alters the architecture and interactions of the channels, resulting in fragmentation and misalignment of the zigzag-rows and disruption of flagellar movement in Efcab9 -/- sperm. This work offers unique insights into the structural basis for understanding CatSper regulation of sperm motility., (© 2022. The Author(s).)

The choroid plexus (CP) epithelium secretes cerebrospinal fluid and plays an important role in healthy homeostasis of the brain. CP function can be influenced by sex steroid hormones; however, the precise molecular mechanism of such regulation is not well understood. Here, using whole-cell patch-clamp recordings from male and female murine CP cells, we show that application of progesterone resulted in specific and strong potentiation of the inwardly rectifying potassium channel Kir7.1, an essential protein that is expressed in CP and is required for survival. The potentiation was progesterone specific and independent of other known progesterone receptors expressed in CP. This effect was recapitulated with recombinant Kir7.1, as well as with endogenous Kir7.1 expressed in the retinal pigment epithelium. Current-clamp studies further showed a progesterone-induced hyperpolarization of CP cells. Our results provide evidence of a progesterone-driven control of tissues in which Kir7.1 is present., (© 2021 Björkgren et al.)

Mammalian female fertility is defined by a successful and strictly periodic ovarian cycle, which is under the control of gonadotropins and steroid hormones, particularly progesterone and estrogen. The latter two are produced by the ovaries that are engaged in controlled follicular growth, maturation, and release of the eggs, i.e., ovulation. The steroid hormones regulate ovarian cycles via genomic signaling, by altering gene transcription and protein synthesis. However, despite this well-studied mechanism, steroid hormones can also signal via direct, non-genomic action, by binding to their membrane receptors. Here we show, that the recently discovered membrane progesterone receptor α/β hydrolase domain-containing protein 2 (ABHD2) is highly expressed in mammalian ovaries where the protein plays a novel regulatory role in follicle maturation and the sexual cycle of females. Ablation of Abhd2 caused a dysregulation of the estrous cycle rhythm with females showing shortened luteal stages while remaining in the estrus stage for a longer time. Interestingly, the ovaries of Abhd2 knockout (KO) females resemble polycystic ovary morphology (PCOM) with a high number of atretic antral follicles that could be rescued with injection of gonadotropins. Such a procedure also allowed Abhd2 KO females to ovulate a significantly increased number of mature and fertile eggs in comparison with their wild-type littermates. These results suggest a novel regulatory role of ABHD2 as an important factor in non-genomic steroid regulation of the female reproductive cycle., Competing Interests: LH is an inventor on patents of an electroporation-based CRISPR technology for mouse genome engineering and is a co-founder of a company to further develop this technology for mammalian genome editing. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Björkgren, Chung, Mendoza, Gabelev-Khasin, Petersen, Modzelewski, He and Lishko.)

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.

The choroid plexus (CP) is an epithelial tissue primarily responsible for the secretion of the cerebrospinal fluid (CSF). Choroid plexuses are found in each of the four brain ventricles: two laterals, third and fourth. They ensure continuous production of CSF to provide nutrients, remove waste products and provide a mechanical buffer to protect the brain. Tight junctions in the CP epithelium form a barrier between the blood plasma and the CSF, which allow channels and transporters in the CP to establish a highly regulated concentration gradient of ions between the two fluids, thereby controlling the composition of CSF. CP plays an important part in healthy brain homeostasis, as its failure to maintain adequate CSF perfusion is implicated in Alzheimer's disease and traumatic brain injury. And yet, the physiology of CP and the mechanism of its age-related functional decline is one of the most understudied areas of neurobiology. Here, we describe a protocol to isolate and identify individual choroid plexus epithelial cells (CPEC) from murine brain for whole-cell patch-clamp recordings and ion channel identification. Using the recording from the inwardly rectifying potassium channel K ir 7.1 and TRPM3 that are abundant in CP, we demonstrate a technique to study the regulators of ion channels in the choroid plexus., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A growing number of studies point to reduced fertility upon chronic exposure to endocrine-disrupting chemicals (EDCs) such as phthalates and plasticizers. These toxins are ubiquitous and are often found in food and beverage containers, medical devices, as well as in common household and personal care items. Animal studies with EDCs, such as phthalates and bisphenol A have shown a dose-dependent decrease in fertility and embryo toxicity upon chronic exposure. However, limited research has been conducted on the acute effects of these EDCs on male fertility. Here we used a murine model to test the acute effects of four ubiquitous environmental toxins: bisphenol A (BPA), di-2-ethylhexyl phthalate (DEHP), diethyl phthalate (DEP), and dimethyl phthalate (DMP) on sperm fertilizing ability and pre-implantation embryo development. The most potent of these toxins, di-2-ethylhexyl phthalate (DEHP), was further evaluated for its effect on sperm ion channel activity, capacitation status, acrosome reaction and generation of reactive oxygen species (ROS). DEHP demonstrated a profound hazardous effect on sperm fertility by producing an altered capacitation profile, impairing the acrosome reaction, and, interestingly, also increasing ROS production. These results indicate that in addition to its known chronic impact on reproductive potential, DEHP also imposes acute and profound damage to spermatozoa, and thus, represents a significant risk to male fertility., (Copyright © 2020 Khasin, Della Rosa, Petersen, Moeller, Kriegsfeld and Lishko.)

The mammalian flagellum is a specific type of motile cilium required for sperm motility and male fertility. Effective flagellar movement is dependent on axonemal function, which in turn relies on proper ion homeostasis within the flagellar compartment. This ion homeostasis is maintained by the concerted function of ion channels and transporters that initiate signal transduction pathways resulting in motility changes. Advances in electrophysiology and super-resolution microscopy have helped to identify and characterize new regulatory modalities of the mammalian flagellum. Here, we discuss what is currently known about the regulation of flagellar ion channels and transporters that maintain sodium, potassium, calcium, and proton homeostasis. Identification of new regulatory elements and their specific roles in sperm motility is imperative for improving diagnostics of male infertility., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: PVL has a financial interest in YourChoice Therapeutics, Inc., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Varying pH of luminal fluid along the female reproductive tract is a physiological cue that modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize linear Ca 2+ signaling nanodomains along the sperm tail. Here, we identify EF-hand calcium-binding domain-containing protein 9 (EFCAB9) as a bifunctional, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CATSPERζ, is essential for pH-dependent and Ca 2+ -sensitive activation of the CatSper channel. In the absence of EFCAB9, sperm motility and fertility is compromised, and the linear arrangement of the Ca 2+ signaling domains is disrupted. EFCAB9 interacts directly with CATSPERζ in a Ca 2+ -dependent manner and dissociates at elevated pH. These observations suggest that EFCAB9 is a long-sought, intracellular, pH-dependent Ca 2+ sensor that triggers changes in sperm motility., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Vigorous sperm flagellar motility is essential for fertilization, and so the quantitative measurement of motility is a useful tool to assess the intrinsic fertility potential of sperm cells and explore how various factors can alter sperm's ability to reach the egg and penetrate its protective layers. Human sperm beat their flagella many times each second, and so recording and accurately quantifying this movement requires a high-speed camera. The aim of this protocol is to provide a detailed description of the tools required for quantitative beat frequency measurement of tethered human sperm at the single-cell level and to describe methods for investigating the effects of intracellular or extracellular factors on flagellar motion. This assay complements bulk measurements of sperm parameters using commercially-available systems for computer-assisted sperm analysis (CASA)., Competing Interests: Competing interests We declare there are no competing interests.

Study Question: Does a man (patient 1) with a previously described deficiency in principle cation channel of sperm (CatSper) function have a mutation in the CatSper-epsilon (CATSPERE) and/or CatSper-zeta (CATSPERZ) gene?, Summary Answer: Patient 1 has a homozygous in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE., What Is Known Already: CatSper is the principal calcium channel of mammalian spermatozoa. Spermatozoa from patient 1 had a specific loss of CatSper function and were unable to fertilize at IVF. Loss of CatSper function could not be attributed to genetic abnormalities in coding regions of seven CatSper subunits. Two additional subunits (CatSper-epsilon (CATPSERE) and CatSper-zeta (CATSPERZ)) were recently identified, and are now proposed to contribute to the formation of the mature channel complex., Study Design, Size, Duration: This was a basic medical research study analysing genomic data from a single patient (patient 1) for defects in CATSPERE and CATSPERZ., Participants/materials, Setting, Methods: The original exome sequencing data for patient 1 were analysed for mutations in CATSPERE and CATSPERZ. Sanger sequencing was conducted to confirm the presence of a rare variant., Main Results and the Role of Chance: Patient 1 is homozygous for an in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE that is predicted to be highly deleterious., Limitations, Reasons for Caution: The nature of the molecular deficit caused by the rs761237686 variant and whether it is exclusively responsible for the loss of CatSper function remain to be elucidated., Wider Implications of the Findings: Population genetics are available for a significant number of predicted deleterious variants of CatSper subunits. The consequence of homozygous and compound heterozygous forms on sperm fertilization potential could be significant. Selective targeting of CatSper subunit expression maybe a feasible strategy for the development of novel contraceptives., Study Funding/competing Interest(s): This study was funded by project grants from the MRC (MR/K013343/1 and MR/012492/1), Chief Scientist Office/NHS research Scotland. This work was also supported by NIH R01GM111802, Pew Biomedical Scholars Award 00028642 and Packer Wentz Endowment Will to P.V.L. C.L.R.B is the editor-in-chief of Molecular Human Reproduction, has received lecturing fees from Merck and Ferring, and is on the Scientific Advisory Panel for Ohana BioSciences. C.L.R.B was chair of the World Health Organization Expert Synthesis Group on Diagnosis of Male infertility (2012-2016).

Ion channels control sperm navigation within the female reproductive tract and, thus, are critical for their ability to find and fertilize an egg. The flagellar calcium channel CatSper controls sperm hyperactivated motility and is dependent on an alkaline cytoplasmic pH. The latter is accomplished by either proton transporters or, in human sperm, via the voltage-gated proton channel Hv1. To provide concerted regulation, ion channels and their regulatory proteins must be compartmentalized. Here, we describe flagellar regulatory nanodomains comprised of Hv1, CatSper, and its regulatory protein ABHD2. Super-resolution microscopy revealed that Hv1 is distributed asymmetrically within bilateral longitudinal lines and that inhibition of this channel leads to a decrease in sperm rotation along the long axis. We suggest that specific distribution of flagellar nanodomains provides a structural basis for the selective activation of CatSper and subsequent flagellar rotation. The latter, together with hyperactivated motility, enhances the fertility of sperm., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Ion channels control the ability of human sperm to fertilize the egg by triggering hyperactivated motility, which is regulated by membrane potential, intracellular pH, and cytosolic calcium. Previous studies unraveled three essential ion channels that regulate these parameters: (1) the Ca 2+ channel CatSper, (2) the K + channel KSper, and (3) the H + channel Hv1. However, the molecular identity of the sperm Na + conductance that mediates initial membrane depolarization and, thus, triggers downstream signaling events is yet to be defined. Here, we functionally characterize DSper, the Depolarizing Channel of Sperm, as the temperature-activated channel TRPV4. It is functionally expressed at both mRNA and protein levels, while other temperature-sensitive TRPV channels are not functional in human sperm. DSper currents are activated by warm temperatures and mediate cation conductance, that shares a pharmacological profile reminiscent of TRPV4. Together, these results suggest that TRPV4 activation triggers initial membrane depolarization, facilitating both CatSper and Hv1 gating and, consequently, sperm hyperactivation., Competing Interests: NM, MS, PL No competing interests declared, (© 2018, Mundt et al.)

To overcome egg protective vestments and ensure successful fertilization, mammalian spermatozoa switch symmetrical progressive motility to a powerful, whip-like flagellar motion, known as hyperactivation. The latter is triggered by a calcium influx through the sperm-specific, voltage-dependent, and alkalization-activated calcium channel of sperm - CatSper. The channel comprises nine subunits which together form a heteromeric complex. CatSper-deficient male mice and men with mutations in CatSper genes are infertile. This calcium channel is regulated by various endogenous compounds, such as steroids, prostaglandins, endocannabinoids, and intracellular pH. Being a sperm-specific ion channel that is not expressed anywhere else in the body, CatSper represents an ideal target for the development of female and even male contraceptives. In this review, we discuss the recent advances in studying CatSper functional properties and discuss future steps that are required to take in order to achieve a deep understanding of the molecular basis of CatSper function.

Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens.

The calcium channel of sperm (CatSper) is essential for sperm hyperactivated motility and fertility. The steroid hormone progesterone activates CatSper of human sperm via binding to the serine hydrolase ABHD2. However, steroid specificity of ABHD2 has not been evaluated. Here, we explored whether steroid hormones to which human spermatozoa are exposed in the male and female genital tract influence CatSper activation via modulation of ABHD2. The results show that testosterone, estrogen, and hydrocortisone did not alter basal CatSper currents, whereas the neurosteroid pregnenolone sulfate exerted similar effects as progesterone, likely binding to the same site. However, physiological concentrations of testosterone and hydrocortisone inhibited CatSper activation by progesterone. Additionally, testosterone antagonized the effect of pregnenolone sulfate. We have also explored whether steroid-like molecules, such as the plant triterpenoids pristimerin and lupeol, affect sperm fertility. Interestingly, both compounds competed with progesterone and pregnenolone sulfate and significantly reduced CatSper activation by either steroid. Furthermore, pristimerin and lupeol considerably diminished hyperactivation of capacitated spermatozoa. These results indicate that ( i ) pregnenolone sulfate together with progesterone are the main steroids that activate CatSper and ( ii ) pristimerin and lupeol can act as contraceptive compounds by averting sperm hyperactivation, thus preventing fertilization., Competing Interests: Conflict of interest statement: P.V.L. and N.M. are inventors on a patent application filed by University of California, Berkeley related to the work presented in this paper.

Targeting sperm ion channels and other sperm-specific proteins is an effective way to develop unisex contraceptives, as they should have decreased side effects. This Science & Society summarizes the current advances in human sperm physiology in attempts to evaluate what would be appropriate targets for unisex contraceptives., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Steroids regulate cell proliferation, tissue development, and cell signaling via two pathways: a nuclear receptor mechanism and genome-independent signaling. Sperm activation, egg maturation, and steroid-induced anesthesia are executed via the latter pathway, the key components of which remain unknown. Here, we present characterization of the human sperm progesterone receptor that is conveyed by the orphan enzyme α/β hydrolase domain-containing protein 2 (ABHD2). We show that ABHD2 is highly expressed in spermatozoa, binds progesterone, and acts as a progesterone-dependent lipid hydrolase by depleting the endocannabinoid 2-arachidonoylglycerol (2AG) from plasma membrane. The 2AG inhibits the sperm calcium channel (CatSper), and its removal leads to calcium influx via CatSper and ensures sperm activation. This study reveals that progesterone-activated endocannabinoid depletion by ABHD2 is a general mechanism by which progesterone exerts its genome-independent action and primes sperm for fertilization., (Copyright © 2016, American Association for the Advancement of Science.)

The basal fungus Allomyces macrogynus (A. macrogynus) produces motile male gametes displaying well-studied chemotaxis toward their female counterparts. This chemotaxis is driven by sirenin, a sexual pheromone released by the female gametes. The pheromone evokes a large calcium influx in the motile gametes, which could proceed through the cation channel of sperm (CatSper) complex. Herein, we report the total synthesis of sirenin in 10 steps and 8% overall yield and show that the synthetic pheromone activates the CatSper channel complex, indicated by a concentration-dependent increase in intracellular calcium in human sperm. Sirenin activation of the CatSper channel was confirmed using whole-cell patch clamp electrophysiology with human sperm. Based on this proficient synthetic route and confirmed activation of CatSper, analogues of sirenin can be designed as blockers of the CatSper channel that could provide male contraceptive agents.

Study Question: Are significant abnormalities of CatSper function present in IVF patients with normal sperm concentration and motility and if so what is their functional significance for fertilization success?, Summary Answer: Sperm with a near absence of CatSper current failed to respond to activation of CatSper by progesterone and there was fertilization failure at IVF., What Is Known Already: In human spermatozoa, Ca(2+) influx induced by progesterone is mediated by CatSper, a sperm-specific Ca(2+) channel. A suboptimal Ca(2+) influx is significantly associated with, and more prevalent in, men with abnormal semen parameters, and is associated with reduced fertilizing capacity. However, abnormalities in CatSper current can only be assessed directly using electrophysiology. There is only one report of a CatSper-deficient man who showed no progesterone potentiated CatSper current. A CatSper 2 genetic abnormality was present but there was no information on the [Ca(2+)]i response to CatSper activation by progesterone. Additionally, the semen samples had indicating significant abnormalities (oligoasthenoteratozoospermia) multiple suboptimal functional responses in the spermatozoon. As such it cannot be concluded that impaired CatSper function alone causes infertility or that CatSper blockade is a potential safe target for contraception., Study Design, Size, Duration: Spermatozoa were obtained from donors and subfertile IVF patients attending a hospital assisted reproductive techniques clinic between January 2013 and December 2014. In total 134 IVF patients, 28 normozoospermic donors and 10 patients recalled due to a history of failed/low fertilization at IVF took part in the study., Participants/materials, Setting, Methods: Samples were primarily screened using the Ca(2+) influx induced by progesterone and, if cell number was sufficient, samples were also assessed by hyperactivation and penetration into viscous media. A defective Ca(2+) response to progesterone was defined using the 99% confidence interval from the distribution of response amplitudes in normozoospermic donors. Samples showing a defective Ca(2+) response were further examined in order to characterize the potential CatSper abnormalities. In men where there was a consistent and robust failure of calcium signalling, a direct assessment of CatSper function was performed using electrophysiology (patch clamping), and a blood sample was obtained for genetic analysis., Main Results and the Role of Chance: A total of 101/102 (99%) IVF patients and 22/23 (96%) donors exhibited a normal Ca(2+) response. The mean (± SD) normalized peak response did not differ between donors and IVF patients (2.57 ± 0.68 [n = 34 ejaculates from 23 different donors] versus 2.66 ± 0.68 [n = 102 IVF patients], P = 0.63). In recall patients, 9/10 (90%) showed a normal Ca(2+) response. Three men were initially identified with a defective Ca(2+) influx. However, only one (Patient 1) had a defective response in repeat semen samples. Electrophysiology experiments on sperm from Patient 1 showed a near absence of CatSper current and exon screening demonstrated no mutations in the coding regions of the CatSper complex. There was no increase in penetration of viscous media when the spermatozoa were stimulated with progesterone and importantly there was failed fertilization at IVF., Limitations, Reasons for Caution: A key limitation relates to working with a specific functional parameter (Ca(2+) influx induced by progesterone) in fresh sperm samples from donors and patients that have limited viability. Therefore, for practical, technical and logistical reasons, some men (∼ 22% of IVF patients) could not be screened. As such the incidence of significant Ca(2+) abnormalities induced by progesterone may be higher than the ∼ 1% observed here. Additionally, we used a strict definition of a defective Ca(2+) influx such that only substantial abnormalities were selected for further study. Furthermore, electrophysiology was only performed on one patient with a robust and repeatable defective calcium response. This man had negligible CatSper current but more subtle abnormalities (e.g. currents present but significantly smaller) may have been present in men with either normal or below normal Ca(2+) influx., Wider Implications of the Findings: These data add significantly to the understanding of the role of CatSper in human sperm function and its impact on male fertility. Remarkably, these findings provide the first direct evidence that CatSper is a suitable and specific target for human male contraception., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

During transit through the female reproductive tract, mammalian spermatozoa are exposed to increasing concentrations of progesterone (P4) released by the cumulus oophorus. P4 triggers massive calcium influx into human sperm through activation of the sperm-specific calcium channel CatSper. These properties of human spermatozoa are thought to be unique since CatSper is not progesterone sensitive in rodent sperm. Here, by performing patch clamp recording from spermatozoa from rhesus macaque for the first time, we report that they express P4-sensitive CatSper channel identically to human sperm and react to P4 by inducing responsiveness to zona pellucida, unlike human sperm, which respond directly to P4. We have also determined the physiologic levels of P4 capable of inducing capacitation-associated changes in macaque sperm. Progesterone (1 μM) induced up to a 3-fold increase in the percentage of sperm undergoing the zona pellucida-induced acrosome reaction with the lowest threshold as low as 10 nM of P4. Submicromolar levels of P4 induced a dose-dependent increase in curvilinear velocity and lateral head displacement, while sperm protein tyrosine phosphorylation was not altered. Macaque spermatozoa exposed to 10 μM of P4 developed fully hyperactivated motility. Similar to human sperm, on approaching cumulus mass and binding to zona pellucida, macaque spermatozoa display hyperactivation and undergo an acrosome reaction that coincides with the rise in the sperm intracellular calcium. Taken together, these data indicate that P4 accelerates the completion of capacitation and provides evidence of spermatozoa "priming" as they move into a gradient of progesterone in search for the oocyte., (© 2015 by the Society for the Study of Reproduction, Inc.)

The female steroid hormone progesterone regulates ovulation and supports pregnancy, but also controls human sperm function within the female reproductive tract. Progesterone causes elevation of sperm intracellular Ca(2+) leading to sperm hyperactivation, acrosome reaction, and perhaps chemotaxis toward the egg. Although it has been suggested that progesterone-dependent Ca(2+) influx into human spermatozoa is primarily mediated by cationic channel of sperm (CatSper), the principal flagellar Ca(2+) channel of sperm, conclusive loss-of-function genetic evidence for activation of CatSper by progesterone has yet to be provided. Moreover, it is not clear whether the responsiveness of CatSper to progesterone is an innate property of human spermatozoa or is acquired as the result of exposure to the seminal plasma. Here, by recording ionic currents from spermatozoa of an infertile CatSper-deficient patient, we demonstrate that CatSper is indeed the principal Ca(2+) channel of human spermatozoa, and that it is strongly potentiated by progesterone. In addition, by recording CatSper currents from human epididymal and testicular spermatozoa, we show that CatSper sensitivity to progesterone arises early in sperm development and increases gradually to a peak when spermatozoa are ejaculated. These results unambiguously establish an important role of CatSper channel in human sperm nongenomic progesterone signaling and demonstrate that the molecular mechanism responsible for activation of CatSper by progesterone arises early in sperm development concurrently with the CatSper channel itself.

Mitochondrial uncoupling protein 1 (UCP1) is responsible for nonshivering thermogenesis in brown adipose tissue (BAT). Upon activation by long-chain fatty acids (LCFAs), UCP1 increases the conductance of the inner mitochondrial membrane (IMM) to make BAT mitochondria generate heat rather than ATP. Despite being a member of the family of mitochondrial anion carriers (SLC25), UCP1 is believed to transport H(+) by an unusual mechanism that has long remained unresolved. Here, we achieved direct patch-clamp measurements of UCP1 currents from the IMM of BAT mitochondria. We show that UCP1 is an LCFA anion/H(+) symporter. However, the LCFA anions cannot dissociate from UCP1 due to hydrophobic interactions established by their hydrophobic tails, and UCP1 effectively operates as an H(+) carrier activated by LCFA. A similar LCFA-dependent mechanism of transmembrane H(+) transport may be employed by other SLC25 members and be responsible for mitochondrial uncoupling and regulation of metabolic efficiency in various tissues., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The sperm connecting piece is a complex structure that, from a mechanical perspective, appears to play a role in stabilizing the proximal part of the sperm tail. We report the three-dimensional structure of the intact bovine sperm connecting piece, revealing an intricate, asymmetrical architecture with the segmented columns held together by filamentous linkages. The columns fuse, at the proximal end, with each other into structures that form the centriolar vault, and at the distal end, with the outer dense fibers (ODFs). The grouping of the fibers into these structures is consistent with bending only in the plane of the head. Structures reminiscent of the proximal centriole were observed in the vault, while the association of a novel bar structure with ODFs 3 and 8 organizes the distal centriolar vault. It has been proposed that the elastic compliance of the connecting piece provides the underlying mechanism behind initiation of the sperm beat cycle and bend propagation. According to the basal sliding theory of sperm movement, distortion of the connecting piece may store energy that initiates a new beat. The intersegment linkers could serve as mechanosensitive elements that regulate alternation of the sperm tail's bending direction in the beat cycle in addition to providing structural stabilization for the connecting piece segmented structures. On the other hand, our video recordings of the bull sperm movement show little bending of the head with respect to the tail, so it appears that there may be normally little strain within the connecting piece.

Ion channels control the sperm ability to fertilize the egg by regulating sperm maturation in the female reproductive tract and by triggering key sperm physiological responses required for successful fertilization such as hyperactivated motility, chemotaxis, and the acrosome reaction. CatSper, a pH-regulated, calcium-selective ion channel, and KSper (Slo3) are core regulators of sperm tail calcium entry and sperm hyperactivated motility. Many other channels had been proposed as regulating sperm activity without direct measurements. With the development of the sperm patch-clamp technique, CatSper and KSper have been confirmed as the primary spermatozoan ion channels. In addition, the voltage-gated proton channel Hv1 has been identified in human sperm tail, and the P2X2 ion channel has been identified in the midpiece of mouse sperm. Mutations and deletions in sperm-specific ion channels affect male fertility in both mice and humans without affecting other physiological functions. The uniqueness of sperm ion channels makes them ideal pharmaceutical targets for contraception. In this review we discuss how ion channels regulate sperm physiology.

Upon ejaculation, mammalian spermatozoa have to undergo a sequence of physiological transformations within the female reproductive tract that will allow them to reach and fertilize the egg. These include initiation of motility, hyperactivation of motility and perhaps chemotaxis toward the egg, and culminate in the acrosome reaction that permits sperm to penetrate the protective vestments of the egg. These physiological responses are triggered through the activation of sperm ion channels that cause elevations of sperm intracellular pH and Ca(2+) in response to certain cues within the female reproductive tract. Despite their key role in sperm physiology and their absolute requirement for the process of fertilization, sperm ion channels remain poorly understood due to the extreme difficulty in application of the patch-clamp technique to spermatozoa. This review covers the topic of sperm ion channels in the following order: first, we discuss how the intracellular Ca(2+) and pH signaling mediated by sperm ion channels controls sperm behavior during the process of fertilization. Then, we briefly cover the history of the methodology to study sperm ion channels, which culminated in the recent development of a reproducible whole-cell patch-clamp technique for mouse and human cells. We further discuss the main approaches used to patch-clamp mature mouse and human spermatozoa. Finally, we focus on the newly discovered sperm ion channels CatSper, KSper (Slo3) and HSper (H(v)1), identified by the sperm patch-clamp technique. We conclude that the patch-clamp technique has markedly improved and shifted our understanding of the sperm ion channels, in addition to revealing significant species-specific differences in these channels. This method is critical for identification of the molecular mechanisms that control sperm behavior within the female reproductive tract and make fertilization possible.

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.

Elevations of sperm intracellular pH and Ca(2+) regulate sperm motility, chemotaxis, capacitation and the acrosome reaction, and play a vital role in the ability of the sperm cell to reach and fertilise the egg. In human spermatozoa, the flagellar voltage-gated proton channel Hv1 is the main H(+) extrusion pathway that controls sperm intracellular pH, and the pH-dependent flagellar Ca²(+) channel CatSper is the main pathway for Ca²(+) entry as measured by the whole-cell patch clamp technique. Hv1 and CatSper channels are co-localized within the principal piece of the sperm flagellum. Hv1 is dedicated to proton extrusion from flagellum and is activated by membrane depolarisation, an alkaline extracellular environment, the endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. The CatSper channel is strongly potentiated by intracellular alkalinisation. Since Hv1 and CatSper channels are located in the same subcellular domain, proton extrusion via Hv1 channels should induce intraflagellar alkalinisation and activate CatSper ion channels. Therefore the combined action of Hv1 and CatSper channels in human spermatozoa can induce elevation of both intracellular pH and Ca²(+) required for sperm activation in the female reproductive tract. Here, we discuss how Hv1 and CatSper channels regulate human sperm physiology and the differences in control of sperm intracellular pH and Ca²(+) between species.

Human spermatozoa are quiescent in the male reproductive system and must undergo activation once introduced into the female reproductive tract. This process is known to require alkalinization of sperm cytoplasm, but the mechanism responsible for transmembrane proton extrusion has remained unknown because of the inability to measure membrane conductance in human sperm. Here, by successfully patch clamping human spermatozoa, we show that proton channel Hv1 is their dominant proton conductance. Hv1 is confined to the principal piece of the sperm flagellum, where it is expressed at unusually high density. Robust flagellar Hv1-dependent proton conductance is activated by membrane depolarization, an alkaline extracellular environment, endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. Hv1 allows only outward transport of protons and is therefore dedicated to inducing intracellular alkalinization and activating spermatozoa. The importance of Hv1 for sperm activation makes it an attractive target for controlling male fertility., (2010 Elsevier Inc. All rights reserved.)

Two isoforms of the GTPase-activating protein, regulator of G protein signaling 9 (RGS9), control such fundamental functions as vision and behavior. RGS9-1 regulates phototransduction in rods and cones, and RGS9-2 regulates dopamine and opioid signaling in the basal ganglia. To determine their functional differences in the same intact cell, we replaced RGS9-1 with RGS9-2 in mouse rods. Surprisingly, RGS9-2 not only supported normal photoresponse recovery under moderate light conditions but also outperformed RGS9-1 in bright light. This versatility of RGS9-2 results from its ability to inactivate the G protein, transducin, regardless of its effector interactions, whereas RGS9-1 prefers the G protein-effector complex. Such versatility makes RGS9-2 an isoform advantageous for timely signal inactivation across a wide range of stimulus strengths and may explain its predominant representation throughout the nervous system.

Transient receptor potential (TRP) proteins are cation channels composed of a transmembrane domain flanked by large N- and C-terminal cytoplasmic domains. All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD). The ARD of mammalian TRPV6, an important regulator of calcium uptake and homeostasis, is essential for channel assembly and regulation. The 1.7 A crystal structure of the TRPV6-ARD reveals conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third finger loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. Size exclusion chromatography and crystal packing indicate that the TRPV6-ARD does not assemble as a tetramer and is monomeric in solution. Adenosine triphosphate-agarose and calmodulin-agarose pull-down assays show that the TRPV6-ARD does not interact with either ligand, indicating a different functional role for the TRPV6-ARD than in the paralogous thermosensitive TRPV1 channel. Similar biochemical findings are also presented for the highly homologous mammalian TRPV5-ARD. The implications of the structural and biochemical data on the role of the ankyrin repeats in different TRPV channels are discussed.

TRPV1 plays a key role in nociception, as it is activated by heat, low pH, and ligands such as capsaicin, leading to a burning pain sensation. We describe the structure of the cytosolic ankyrin repeat domain (ARD) of TRPV1 and identify a multiligand-binding site important in regulating channel sensitivity within the TRPV1-ARD. The structure reveals a binding site that accommodates triphosphate nucleotides such as ATP, and biochemical studies demonstrate that calmodulin binds the same site. Electrophysiology experiments show that either ATP or PIP2 prevent desensitization to repeated applications of capsaicin, i.e., tachyphylaxis, while calmodulin plays an opposing role and is necessary for tachyphylaxis. Mutations in the TRPV1-ARD binding site eliminate tachyphylaxis. We present a model for the calcium-dependent regulation of TRPV1 via competitive interactions of ATP and calmodulin at the TRPV1-ARD-binding site and discuss its relationship to the C-terminal region previously implicated in interactions with PIP2 and calmodulin.

Ion channels are often modulated by intracellular calcium levels. TRPV1, a channel responsible for the burning pain sensation in response to heat, acid or capsaicin, is desensitized at high intracellular calcium concentrations. We recently identified a multiligand-binding site in the N-terminal ankyrin repeat domain (ARD) of TRPV1 that binds ATP and sensitizes the channel. Calcium-calmodulin binds the same site and is necessary for calcium-mediated TRPV1 desensitization. Here, we examine in more detail the conservation of this TRPV1 multiligand-binding site in other species. Furthermore, using sequence analysis, we determine that the unusually twisted shape of the TRPV1-ARD is likely conserved in other TRPV channels, but not in the ARDs of other TRP subfamilies.

Photoreceptor cells have a remarkable capacity to adapt the sensitivity and speed of their responses to ever changing conditions of ambient illumination. Recent studies have revealed that a major contributor to this adaptation is the phenomenon of light-driven translocation of key signaling proteins into and out of the photoreceptor outer segment, the cellular compartment where phototransduction takes place. So far, only two such proteins, transducin and arrestin, have been established to be involved in this mechanism. To investigate the extent of this phenomenon we examined additional photoreceptor proteins that might undergo light-driven translocation, focusing on three Ca(2+)-binding proteins, recoverin and guanylate cyclase activating proteins 1 (GCAP1) and GCAP2. The changes in the subcellular distribution of each protein were assessed quantitatively using a recently developed technique combining serial tangential sectioning of mouse retinas with Western blot analysis of the proteins in the individual sections. Our major finding is that light causes a significant reduction of recoverin in rod outer segments, accompanied by its redistribution toward rod synaptic terminals. In both cases the majority of recoverin was found in rod inner segments, with approximately 12% present in the outer segments in the dark and less than 2% remaining in that compartment in the light. We suggest that recoverin translocation is adaptive because it may reduce the inhibitory constraint that recoverin imposes on rhodopsin kinase, an enzyme responsible for quenching the photo-excited rhodopsin during the photoresponse. To the contrary, no translocation of rhodopsin kinase itself or either GCAP was identified.

DEP (for Disheveled, EGL-10, Pleckstrin) homology domains are present in numerous signaling proteins, including many in the nervous system, but their function remains mostly elusive. We report that the DEP domain of a photoreceptor-specific signaling protein, RGS9 (for regulator of G-protein signaling 9), plays an essential role in RGS9 delivery to the intracellular compartment of its functioning, the rod outer segment. We generated a transgenic mouse in which RGS9 was replaced by its mutant lacking the DEP domain. We then used a combination of the quantitative technique of serial tangential sectioning-Western blotting with electrophysiological recordings to demonstrate that mutant RGS9 is expressed in rods in the normal amount but is completely excluded from the outer segments. The delivery of RGS9 to rod outer segments is likely to be mediated by the DEP domain interaction with a transmembrane protein, R9AP (for RGS9 anchoring protein), known to anchor RGS9 on the surface of photoreceptor membranes and to potentiate RGS9 catalytic activity. We show that both of these functions are also abolished as the result of the DEP domain deletion. These findings indicate that a novel function of the DEP domain is to target a signaling protein to a specific compartment of a highly polarized neuron. Interestingly, sequence analysis of R9AP reveals the presence of a conserved R-SNARE (for soluble N-ethylmaleimide-sensitive factor attachment protein receptor) motif and a predicted overall structural homology with SNARE proteins involved in vesicular trafficking and fusion. This presents the possibility that DEP domains might serve to target various DEP-containing proteins to the sites of their intracellular action via interactions with the members of extended SNARE protein family.

The complex between the short splice variant of the ninth member of the RGS protein family and the long splice variant of type 5 G protein beta subunit (RGS9-Gbeta5L) plays a critical role in regulating the duration of the light response in vertebrate photoreceptors by activating the GTPase activity of the photoreceptor-specific G protein, transducin. RGS9-Gbeta5L is tightly associated with the membranes of photoreceptor outer segments; however, the nature of this association remains unknown. Here we demonstrate that rod outer segment membranes contain a limited number of sites for high affinity RGS9-Gbeta5L binding, which are highly sensitive to proteolysis. In membranes isolated from bovine rod outer segments, all of these sites are occupied by the endogenous RGS9-Gbeta5L, which prevents the binding of exogenous recombinant RGS9-Gbeta5L to these sites. However, treating membranes with urea or high pH buffers causes either removal or denaturation of the endogenous RGS9-Gbeta5L, allowing for high affinity binding of recombinant RGS9-Gbeta5L to these sites. This binding results in a striking approximately 70-fold increase in the RGS9-Gbeta5L ability to activate transducin GTPase. The DEP (disheveled/EGL-10/pleckstrin) domain of RGS9 plays a crucial role in the RGS9-Gbeta5L membrane attachment, as evident from the analysis of membrane-binding properties of deletion mutants lacking either N- or C-terminal parts of the RGS9 molecule. Our data indicate that specific association of RGS9-Gbeta5L with photoreceptor disc membranes serves not only as a means of targeting it to an appropriate subcellular compartment but also serves as an important determinant of its catalytic activity.

We have found that the platelet activating factor antagonist (BN52021) is an effective blocker of the glycine (Gly) receptor-mediated responses in the hippocampal pyramidal neurons of rat. Using the whole-cell voltage clamp and concentration clamp recording techniques, we investigated the mechanism underlying the inhibitory action of this terpenoid on the glycine-induced chloride current. BN52021 selectively and reversibly inhibits glycine current in a non-competitive and voltage-dependent fashion. The antagonistic effect of this substance is more pronounced at positive membrane potentials. At holding potential -70mV and in the presence of 200 microM glycine IC50 value for the blocking action of BN52021 was 270+/-10nM. Repetitive applications of BN52021 reveal the use-dependence of its blocking action. When co-applied with strychnine (STR), a competitive glycine receptor antagonist, BN52021 does not alter the IC50 value for strychnine. The inhibitory effect of BN52021 on gamma-aminobutyric acid (GABA) current is at least 25 times less potent than the effect on glycine current. This substance fails to affect AMPA and NMDA responses. It may be concluded that BN52021 inhibits glycine-gated Cl- channels by interacting with the pore region and does not compete for the strychnine-binding centre.