C2CD6 regulates targeting and organization of the CatSper calcium channel complex in sperm flagella.

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here, we report identification of an uncharacterized protein, C2CD6, as a subunit of the mouse CatSper complex. C2CD6 contains a calcium-dependent, membrane-targeting C2 domain. C2CD6 associates with the CatSper calcium-selective, core-forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. CatSper currents are present but at a significantly lower level in C2CD6-deficient sperm. Transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 facilitates incorporation of the CatSper complex into the flagellar plasma membrane and may function as a calcium sensor. The identification of C2CD6 may enable the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

CatSper channels in sea urchin sperm.

Sea urchin sperm swimming is regulated by speract, a decapeptide released from egg jelly that induces chemotaxis and triggers membrane potential (Em) changes, intracellular increases in cyclic nucleotides (cGMP, cAMP), pH (pHi) and calcium concentration ([Ca2+]i). The identity of the ionic transporters associated with the [Ca2+]i changes required for chemotaxis is not fully known. CatSper, a sperm exclusive Ca2+ channel has been detected by proteomic analysis and immunofluorescence in sea urchin sperm and there is evidence for its involvement in chemotaxis. This work presents an electrophysiological characterization of a CatSper channel in sea urchin sperm. By swelling sperm suspending them in 10-fold diluted artificial sea water (ASW) we achieve on-cell patch-clamp recordings that document a mildly voltage and pHi dependent Na+ permeable channel (in absence of divalent ions in the pipette), sensitive to speract, and blocked by Mibefradil (Mibe), NNC55-0396 (NNC) and RU1968 (RU) resembling CatSper. We also recorded a voltage dependent Cl- channel inhibited by Niflumic Acid and the TMEM16A blocker.

Cdc42 localized in the CatSper signaling complex regulates cAMP-dependent pathways in mouse sperm.

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+ -dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.

Direct activation of the proton channel by albumin leads to human sperm capacitation and sustained release of inflammatory mediators by neutrophils.

Human voltage-gated proton channels (hHv1) extrude protons from cells to compensate for charge and osmotic imbalances due metabolism, normalizing intracellular pH and regulating protein function. Human albumin (Alb), present at various levels throughout the body, regulates oncotic pressure and transports ligands. Here, we report Alb is required to activate hHv1 in sperm and neutrophils. Dose-response studies reveal the concentration of Alb in semen is too low to activate hHv1 in sperm whereas the higher level in uterine fluid yields proton efflux, allowing capacitation, the acrosomal reaction, and oocyte fertilization. Likewise, Alb activation of hHv1 in neutrophils is required to sustain production and release of reactive oxygen species during the immune respiratory burst. One Alb binds to both voltage sensor domains (VSDs) in hHv1, enhancing open probability and increasing proton current. A computational model of the Alb-hHv1 complex, validated by experiments, identifies two sites in Alb domain II that interact with the VSDs, suggesting an electrostatic gating modification mechanism favoring the active "up" sensor conformation. This report shows how sperm are triggered to fertilize, resolving how hHv1 opens at negative membrane potentials in sperm, and describes a role for Alb in physiology that will operate in the many tissues expressing hHv1.

Regulatory mechanisms of mitochondrial BKCa channels.

The mitochondrial BKCa channel (mitoBKCa) is a splice variant of plasma membrane BKCa (Maxi-K, BKCa, Slo1, KCa1.1). While a high-resolution structure of mitoBKCa is not available yet, functional and structural studies of the plasma membrane BKCa have provided important clues on the gating of the channel by voltage and Ca2+, as well as the interaction with auxiliary subunits. To date, we know that the control of expression of mitoBKCa, targeting and voltage-sensitivity strongly depends on its association with its regulatory ß1-subunit, which overall participate in the control of mitochondrial Ca2+-overload in cardiac myocytes. Moreover, novel regulatory mechanisms of mitoBKCa such as ß-subunits and amyloid-ß have recently been proposed. However, major basic questions including how the regulatory BKCa-ß1-subunit reaches mitochondria and the mechanism through which amyloid-ß impairs mitoBKCa channel function remain to be addressed.

Starvation induces an increase in intracellular calcium and potentiates the progesterone-induced mouse sperm acrosome reaction.

We have recently reported two different methodologies that improve sperm functionality. The first method involved transient exposure to the Ca2+ ionophore A23187 , and the second required sperm incubation in the absence of energy nutrients (starvation). Both methods were associated with an initial loss of motility followed by a rescue step involving ionophore removal or addition of energy metabolites, respectively. In this work, we show that starvation is accompanied by an increase in intracellular Ca2+ ([Ca2+ ]i ). Additionally, the starved cells acquire a significantly enhanced capacity to undergo a progesterone-induced acrosome reaction. Electrophysiological measurements show that CatSper channel remains active in starvation conditions. However, the increase in [Ca2+ ]i was also observed in sperm from CatSper null mice. Upon starvation, addition of energy nutrients reversed the effects on [Ca2+ ]i and decreased the effect of progesterone on the acrosome reaction to control levels. These data indicate that both methods have common molecular features.

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

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 (Kd) 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.

CatSper channels are regulated by protein kinase A.

Mammalian sperm must undergo capacitation as a preparation for entering into hyperactivated motility, undergoing the acrosome reaction, and acquiring fertilizing ability. One of the initial capacitation events occurs when sperm encounter an elevated HCO3- concentration. This anion activates the atypical adenylyl cyclase Adcy10, increases intracellular cAMP, and stimulates protein kinase A (PKA). Moreover, an increase in intracellular Ca2+ concentration ([Ca2+] i ) is essential for sperm capacitation. Although a cross-talk between cAMP-dependent pathways and Ca2+ clearly plays an essential role in sperm capacitation, the connection between these signaling events is incompletely understood. Here, using three different approaches, we found that CatSper, the main sperm Ca2+ channel characterized to date, is up-regulated by a cAMP-dependent activation of PKA in mouse sperm. First, HCO3- and the PKA-activating permeable compound 8-Br-cAMP induced an increase in [Ca2+] i , which was blocked by the PKA peptide inhibitor PKI, and H89, another PKA inhibitor, also abrogated the 8-Br-cAMP response. Second, HCO3- increased the membrane depolarization induced upon divalent cation removal by promoting influx of monovalent cations through CatSper channels, which was inhibited by PKI, H89, and the CatSper blocker HC-056456. Third, electrophysiological patch clamp, whole-cell recordings revealed that CatSper activity is up-regulated by HCO3- and by direct cAMP injection through the patch-clamp pipette. The activation by HCO3- and cAMP was also blocked by PKI, H89, Rp-cAMPS, and HC-056456, and electrophysiological recordings in sperm from CatSper-KO mice confirmed CatSper's role in these activation modes. Our results strongly suggest that PKA-dependent phosphorylation regulates [Ca2+] i homeostasis by activating CatSper channel complexes.

CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization.

Ca(2+)-dependent mechanisms are critical for successful completion of fertilization. Here, we demonstrate that CRISP1, a sperm protein involved in mammalian fertilization, is also present in the female gamete and capable of modulating key sperm Ca(2+) channels. Specifically, we show that CRISP1 is expressed by the cumulus cells that surround the egg and that fertilization of cumulus-oocyte complexes from CRISP1 knockout females is impaired because of a failure of sperm to penetrate the cumulus. We provide evidence that CRISP1 stimulates sperm orientation by modulating sperm hyperactivation, a vigorous motility required for penetration of the egg vestments. Moreover, patch clamping of sperm revealed that CRISP1 has the ability to regulate CatSper, the principal sperm Ca(2+) channel involved in hyperactivation and essential for fertility. Given the critical role of Ca(2+) for sperm motility, we propose a novel CRISP1-mediated fine-tuning mechanism to regulate sperm hyperactivation and orientation for successful penetration of the cumulus during fertilization.

Electrophysiological evidence for the presence of cystic fibrosis transmembrane conductance regulator (CFTR) in mouse sperm.

Mammalian sperm must undergo a maturational process, named capacitation, in the female reproductive tract to fertilize the egg. Sperm capacitation is regulated by a cAMP/protein kinase A (PKA) pathway and involves increases in intracellular Ca(2+), pH, Cl(-), protein tyrosine phosphorylation, and in mouse and some other mammals a membrane potential hyperpolarization. The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) channel modulated by cAMP/PKA and ATP, was detected in mammalian sperm and proposed to modulate capacitation. Our whole-cell patch-clamp recordings from testicular mouse sperm now reveal a Cl(-) selective component to membrane current that is ATP-dependent, stimulated by cAMP, cGMP, and genistein (a CFTR agonist, at low concentrations), and inhibited by DPC and CFTR(inh) -172, two well-known CFTR antagonists. Furthermore, the Cl(-) current component activated by cAMP and inhibited by CFTR(inh) -172 is absent in recordings on testicular sperm from mice possessing the CFTR ΔF508 loss-of-function mutation, indicating that CFTR is responsible for this component. A Cl(-) selective like current component displaying CFTR characteristics was also found in wild type epididymal sperm bearing the cytoplasmatic droplet. Capacitated sperm treated with CFTR(inh) -172 undergo a shape change, suggesting that CFTR is involved in cell volume regulation. These findings indicate that functional CFTR channels are present in mouse sperm and their biophysical properties are consistent with their proposed participation in capacitation.

Are TRP channels involved in sperm development and function?

Spermatozoa must translate information from their environment and the egg to achieve fertilization in sexually reproducing animals. These tasks require decoding a variety of signals in the form of intracellular Ca(2+) changes. As TRP channels constitute a large family of versatile multi-signal transducers, they are interesting subjects in which to explore their possible participation in sperm function. Here, we review the evidence for their presence and involvement in sperm motility, maturation, and the acrosome reaction, an exocytotic process required for sperm-egg fusion. Since store-operated Ca(2+) entry (SOCE) has been proposed to play an important role in these three functions, the main proteins responsible for this transport (STIM and ORAI) and their interaction with TRPs are also discussed. Improving our tools to solve infertility, improve animal breeding, and preserve biodiversity requires a better understanding of how Ca(2+) is regulated in spermatozoa.

Human spermatozoa possess a calcium-dependent chloride channel that may participate in the acrosomal reaction.

Motility, maturation and the acrosome reaction (AR) are fundamental functions of mammalian spermatozoa. While travelling through the female reproductive tract, spermatozoa must mature through a process named capacitation, so that they can reach the egg and undergo the AR, an exocytotic event necessary to fertilize the egg. Though Cl⁻ is important for sperm capacitation and for the AR, not much is known about the molecular identity of the Cl⁻ transporters involved in these processes.We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa.Our whole cell recordings revealed the presence of a Ca²âº-dependent Cl⁻ current. The biophysical characteristics of this current and its sensitivity to niflumic acid (NFA) and 4,4-diisothiocyano-2,2-stilbene disulphonic acid (DIDIS) are consistent with those displayed by the Ca²âº-dependent Cl⁻ channel from the anoctamin family (TMEM16). Whole cell patch clamp recordings in the cytoplasmic droplet of human spermatozoa corroborated the presence of these currents, which were sensitive to NFA and to a small molecule TMEM16A inhibitor (TMEM16Ainh, an aminophenylthiazole). Importantly, the human sperm AR induced by a recombinant human glycoprotein from the zona pellucida, rhZP3, displayed a similar sensitivity to NFA, DIDS and TMEM16Ainh as the sperm Ca²âº-dependent Cl⁻ currents. Our findings indicate the presence of Ca²âº-dependent Cl⁻ currents in human spermatozoa, that TMEM16A may contribute to these currents and also that sperm Ca²âº-dependent Cl⁻ currents may participate in the rhZP3-induced AR.

Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function.

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function.

TRPM8 in mouse sperm detects temperature changes and may influence the acrosome reaction.

Changes in the concentration of intracellular Ca(2+) ([Ca(2+) ]i) trigger and/or regulate principal sperm functions during fertilization, such as motility, capacitation, and the acrosome reaction (AR). Members of the large TRP channel family participate in a variety of Ca(2+) -dependent cell signaling processes. The eight TRPM channel members constitute one of the seven groups belonging to this family. Here we document using RT-PCR experiments the presence of Trpm2, 4, 7, and 8 in mouse spermatogenic cells. Trpm8 transcription is up-regulated after day 30. The localization of TRPM8 protein in mouse sperm was confirmed by immunocytochemistry and Western blots. Patch clamp recordings in testicular mouse sperm revealed TRPM8 agonist (menthol and icilin) activated currents sensitive to TRPM8 inhibitors N-(4-t-Butylphenyl)-4-(3-Chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC) and capsazepine. These findings are consistent with the presence of functional TRPM8 in mouse sperm. Furthermore, menthol induced a [Ca(2+) ]i increase and the AR in these cells, that were inhibited by capsazepine (20 µM) and BCTC (1.6 µM). Notably, the progesterone and zona pellucida-induced AR was significantly (>40%) inhibited by BCTC and capsazepine, suggesting the possible participation of TRPM8 channels in this reaction. TRPM family members present in sperm could be involved in other important signaling events, such as thermotaxis, chemotaxis, and mechanosensory transduction.

Dissection of the components for PIP2 activation and thermosensation in TRP channels.

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in the activation of several transient receptor potential (TRP) channels. The role of PIP2 on temperature gating of thermoTRP channels has not been explored in detail, and the process of temperature activation is largely unexplained. In this work, we have exchanged different segments of the C-terminal region between cold-sensitive (TRPM8) and heat-sensitive (TRPV1) channels, trying to understand the role of the segment in PIP2 and temperature activation. A chimera in which the proximal part of the C-terminal of TRPV1 replaces an equivalent section of TRPM8 C-terminal is activated by PIP2 and confers the phenotype of heat activation. PIP2, but not temperature sensitivity, disappears when positively charged residues contained in the exchanged region are neutralized. Shortening the exchanged segment to a length of 11 aa produces voltage-dependent and temperature-insensitive channels. Our findings suggest the existence of different activation domains for temperature, PIP2, and voltage. We provide an interpretation for channel-PIP2 interaction using a full-atom molecular model of TRPV1 and PIP2 docking analysis.

ThermoTRP channels as modular proteins with allosteric gating.

Ion channels activate by sensing stimuli such as membrane voltage, ligand binding or temperature and transduce this information into conformational changes that open the channel pore. Thus, a key question in understanding ion channel function is how do the protein domains involved in sensing stimuli (sensors) and opening the pore (gates) communicate. In this regard, transient receptor potential (TRP) channels that confer thermosensation [A. Dhaka, V. Viswanath, A. Patapoutian, TRP ion channels and temperature sensation, Annu. Rev. Neurosci. 29 (2006) 135-161; I.S. Ramsey, M. Delling, D.E. Clapham, An introduction to TRP channels, Annu. Rev. Physiol. 68 (2006) 619-647] (thermoTRP; Q(10)>10) are unique to the extent that they integrate a variety of physical and chemical stimuli. In some cases such as, for example, the vanilloid receptor TRPV1 [M.J. Caterina, M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, D. Julius, The capsaicin receptor: a heat-activated ion channel in the pain pathway, Nature 389 (1997) 816-824] and TRPA1 [G.M. Story, A.M. Peier, A.J. Reeve, S.R. Eid, J. Mosbacher, T.R. Hricik, T.J. Earley, A.C. Hergarden, D.A. Andersson, S.W. Hwang, P. McIntyre, T. Jegla, S. Bevan, A. Patapoutian, ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures, Cell 112 (2003) 819-829; S. Jordt, D. Julius, Molecular basis for species-specific sensitivity to "hot" chilli peppers, Cell 108 (2002) 421-430] the integration of these stimuli elicit pain [M. Tominaga, M.J. Caterina, A.B. Malmberg, T.A. Rosen, H. Gilbert, K. Skinner, B.E. Raumann, A.I. Basbaum, D. Julius, The cloned capsaicin receptor integrates multiple pain-producing stimuli, Neuron 21 (1998) 531-543; M. Bandell, A. Dubin, M. Petrus, A. Orth, J. Mathur, S. Hwang, A. Patapoutian, High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol, Nat. Neurosci. 9 (2006) 466-468; S. Zurborg, B. Yurgionas, JA. Jira, O. Caspani, P.A. Heppenstall, Direct activation of the ion channel TRPA1 by Ca(2+), Nat. Neurosci. 10 (2007) 277-279]. These stimuli include voltage, pH, agonist binding, and temperature. Understanding how each of these distinct physiological signals regulate channel opening will be informative about the mechanical linkages that can act either independently or in concert to influence channel activation. In this paper we show that thermoTRP channel-forming proteins are modular in the sense that certain structure or structures (modules) confer temperature-dependent regulation, whereas others confer voltage-dependent regulation. We also discuss the thermodynamic basis of heat and cold activation in an effort to elucidate what confer to these channels the capability to be gated by temperature directly.

A hot-sensing cold receptor: C-terminal domain determines thermosensation in transient receptor potential channels.

Temperature transduction in mammals is possible because of the presence of a set of temperature-dependent transient receptor potential (TRP) channels in dorsal root ganglia neurons and skin cells. Six thermo-TRP channels, all characterized by their unusually high temperature sensitivity (Q10 > 10), have been cloned: TRPV1-4 are heat activated, whereas TRPM8 and TRPA1 are activated by cold. Because of the lack of structural information, the molecular basis for regulation by temperature remains unknown. In this study, we assessed the role of the C-terminal domain of thermo-TRPs and its involvement in thermal activation by using chimeras between the heat receptor TRPV1 and the cold receptor TRPM8, in which the entire C-terminal domain was switched. Here, we demonstrate that the C-terminal domain is modular and confers the channel phenotype regarding temperature sensitivity, channel gating kinetics, and PIP2 (phosphatidylinositol-4,5-bisphophate) modulation. Thus, thermo-TRP channels contain an interchangeable specific region, different from the voltage sensor, which allows them to sense temperature stimuli.

Occurrence of a tetrodotoxin-sensitive calcium current in rat ventricular myocytes after long-term myocardial infarction.

OBJECTIVE: To determine the characteristics of a TTX-sensitive Ca(2+) current that occurred only following remodelling after myocardial infarction in Wistar rat. METHODS: Using the whole-cell patch-clamp technique, we studied ionic inward current in myocytes isolated from four different ventricular regions of control Wistar rat hearts, or from hearts 4 to 6 months after ligation of the left coronary artery. Inward current characteristics were also analysed in Xenopus laevis oocytes that heterologously expressed the human sodium channel alpha-subunit Nav1.5. The effects of oxidative stress by hydrogen peroxide or tert-butyl-hydroxyperoxide as well as those of PKA-dependent phosphorylation, which partly mimic the pathological conditions, were investigated on control cardiomyocytes and Nav1.5-expressing oocytes. RESULTS: In Na-free solution, a low-threshold, tetrodotoxin-sensitive inward current was found in 20 out of 78 cells isolated from 16 post-myocardial infarcted (PMI) cardiomyocytes but not in cardiomyocytes from young and sham rat hearts. This current exhibited kinetics and pharmacological properties similar to the I(Ca(TTX)) current previously reported. I(Ca(TTX))-like current was critically dependent on extracellular Na(+) and was reduced by micromolar Na(+) concentrations. Neither in normal rat cardiomyocytes nor in Nav1.5-expressing oocytes could a I(Ca(TTX))-like current be elicited in Na(+)-free extracellular solution, even after oxidative stress or PKA-dependent phosphorylation. CONCLUSIONS: Our data suggest that I(Ca(TTX))-like current in PMI myocytes does not arise from classical Na(+) channels modified by oxidative stress or PKA phosphorylation and most probably represents a different Na(+) channel type re-expressed in some cells after remodelling.