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Fertilization of eggs from the African clawed frogXenopus laevisis characterized by an increase in cytosolic calcium, a phenomenon that is also observed in other vertebrates such as mammals and birds. During fertilization in mammals and birds, the transfer of the soluble PLCζ from sperm into the egg is thought to trigger the release of calcium from the endoplasmic reticulum (ER). Injecting sperm extracts into eggs reproduces this effect, reinforcing the hypothesis that a sperm factor is responsible for calcium release and egg activation. Remarkably, this occurs even when sperm extracts fromX. laevisare injected into mouse eggs, suggesting that mammals andX. laevisshare a sperm factor. However,X. laevislacks an annotatedPLCZ1gene, which encodes the PLCζ enzyme. In this study, we attempted to determine whether sperm fromX. laevisexpress an unannotatedPLCZ1ortholog. We identifiedPLCZ1orthologs in 11 amphibian species, including 5 that had not been previously characterized, but did not find any in eitherX. laevisor the closely relatedXenopus tropicalis. Additionally, we performed RNA sequencing on testes obtained from adultX. laevismales and did not identify potential PLCZ1 orthologs in our dataset or in previously collected ones. These findings suggest that PLCZ1 may have been lost in theXenopuslineage and raise the question of how fertilization triggers calcium release and egg activation in these species.

Metals are widespread environmental toxins that disrupt normal cellular processes through their interactions with proteins and other macromolecules. In this study, we developed the metalsensitive fluorescent protein mseGFP as a ratiometric reporter capable of binding heavy metals. We found that mseGFP bound mercury and lead tightly but had substantially lower sensitivity to other metals. By comparison, the redox sensor roGFP2 functioned as a ratiometric indicator for transition metals, with the highest sensitivity for copper, followed by nickel and cobalt. mseGFP and roGFP2 could also report metal binding through fluorescence quenching, and we used this effect to measure high affinity interactions for both proteins with copper and iron. Crystal structure analysis of mseGFP complexed with phenylarsine oxide revealed an unexpected mode of heavy metal interaction, with mseGFP binding PAO with 2:2 stoichiometry. Glutathione strongly inhibited most metal interactions with the fluorescent protein reporters, but increased the affinity of arsenic and cadmium for mseGFP. When expressed in HEK293T cells, mseGFP reported uptake of mercury and phenylarsine oxide from surrounding media. Glutathione depletion enhanced binding of phenylarsine oxide to mseGFP in cells, validating the importance of glutathione in modulating metal-protein interactions.

After fertilization, maternally contributed factors to the egg initiate the transition to pluripotency to give rise to embryonic stem cells, in large part by activating de novo transcription from the embryonic genome. Diverse mechanisms coordinate this transition across animals, suggesting that pervasive regulatory remodeling has shaped the earliest stages of development. Here, we show that maternal homologs of mammalian pluripotency reprogramming factors OCT4 and SOX2 divergently activate the two subgenomes of Xenopus laevis, an allotetraploid that arose from hybridization of two diploid species ~18 million years ago. Although most genes have been retained as two homeologous copies, we find that a majority of them undergo asymmetric activation in the early embryo. Chromatin accessibility profiling and CUT&RUN for modified histones and transcription factor binding reveal extensive differences in enhancer architecture between the subgenomes, which likely arose through genomic disruptions as a consequence of allotetraploidy. However, comparison with diploid X. tropicalis and zebrafish shows broad conservation of embryonic gene expression levels when divergent homeolog contributions are combined, implying strong selection to maintain dosage in the core vertebrate pluripotency transcriptional program, amid genomic instability following hybridization.

The Ca2+ activated Cl− channel formed by transmembrane member 16A (TMEM16A) is broadly expressed and regulates diverse processes. In addition to Ca2+, TMEM16A channels require the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to open. Like other channels regulated by PI(4,5)P2, TMEM16A-conducted currents recorded in excised patches slowly decay overtime. Here we assessed how intracellular Ca2+ alters the rate of this current rundown, using the channels endogenously expressed in oocytes from the African clawed frog, Xenopus laevis. We found that in excised, inside-out patches, the concentration of applied Ca2+ alters the rate of rundown, with high Ca2+ concentrations speeding rundown by activating membrane associated phospholipase C (PLC). Together, these results clarify our understanding of how Ca2+ regulates both TMEM16A directly, and targets PLC to regulate the membrane PI(4,5)P2 content.

Fertilization of an egg by more than one sperm, a condition known as polyspermy, leads to gross chromosomal abnormalities and is embryonic lethal for most animals. Consequently, eggs have evolved multiple processes to stop supernumerary sperm from entering the nascent zygote. For external fertilizers, such as frogs and sea urchins, fertilization signals a depolarization of the egg membrane, which serves as the fast block to polyspermy. Sperm can bind to, but will not enter, depolarized eggs. In eggs from the African clawed frog, Xenopus laevis, the fast block depolarization is mediated by the Ca2+ activated Cl− channel TMEM16A. To do so, fertilization activates a phospholipase C, which generates IP3 to signal a Ca2+ release from the ER. Currently, the signaling pathway by which fertilization activates PLC remains unknown. Here, we sought to uncover this pathway by targeting the canonical activation of the PLC isoforms present in the X. laevis egg: PLCγ and PLCβ. We observed no changes to the fast block in X. laevis eggs inseminated in inhibitors of tyrosine phosphorylation, used to stop activation of PLCγ, or inhibitors of Gαq/11 pathways, used to stop activation of PLCβ. These data suggest that the PLC that signals the fast block depolarization in X. laevis is activated by a novel mechanism.

TransMEMbrane 16A (TMEM16A) is a Ca

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.

One of the earliest and most prevalent barriers to successful reproduction is polyspermy, or fertilization of an egg by multiple sperm. To prevent these supernumerary fertilizations, eggs have evolved multiple mechanisms. It has recently been proposed that zinc released by mammalian eggs at fertilization may block additional sperm from entering. Here, we demonstrate that eggs from amphibia and teleost fish also release zinc. Using Xenopus laevis as a model, we document that zinc reversibly blocks fertilization. Finally, we demonstrate that extracellular zinc similarly disrupts early embryonic development in eggs from diverse phyla, including Cnidaria, Echinodermata, and Chordata. Our study reveals that a fundamental strategy protecting human eggs from fertilization by multiple sperm may have evolved more than 650 million years ago., Fertilization of an egg by more than one sperm presents one of the earliest and most prevalent barriers to successful reproduction. This study shows that, as in mammals, eggs from diverse species release zinc upon fertilization; this extracellular zinc protects eggs from multiple fertilizations, revealing that this fundamental strategy may have evolved more than 650 million years ago.

Fertilization of an egg by more than one sperm presents one of the earliest and most prevalent obstacles to successful reproduction. As such, eggs employ multiple mechanisms to prevent sperm entry into the nascent zygote. The fast block to polyspermy is a depolarization of the egg membrane initiated by sperm entry and is employed by diverse external fertilizers including frogs and sea urchins. For some external fertilizers, sperm entry is associated with actin polymerization during the initiation of the fast block. We therefore sought to determine whether the fast block to polyspermy in the African clawed frog,Xenopus laevis, requires actin polymerization. Although actin polymerization is required for sperm entry into eggs from diverse external fertilizers, including sea urchins and zebrafish, here we demonstrate that actin polymerization is not required for the fast block to polyspermy inX. laevis.

The fast block to polyspermy is achieved in Xenopus laevis eggs by fertilization-induced depolarization. Wozniak et al. show that fertilization activates a signaling cascade involving phospholipase C, IP3, and intracellular Ca2+ release, which induces depolarization via Ca2+-activated Cl− efflux., The prevention of polyspermy is essential for the successful progression of normal embryonic development in most sexually reproducing species. In external fertilizers, the process of fertilization induces a depolarization of the egg’s membrane within seconds, which inhibits supernumerary sperm from entering an already-fertilized egg. This fast block requires an increase of intracellular Ca2+ in the African clawed frog, Xenopus laevis, which in turn activates an efflux of Cl− that depolarizes the cell. Here we seek to identify the source of this intracellular Ca2+. Using electrophysiology, pharmacology, bioinformatics, and developmental biology, we explore the requirement for both Ca2+ entry into the egg from the extracellular milieu and Ca2+ release from an internal store, to mediate fertilization-induced depolarization. We report that although eggs express Ca2+-permeant ion channels, blockade of these channels does not alter the fast block. In contrast, insemination of eggs in the presence of Xestospongin C—a potent inhibitor of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release from the endoplasmic reticulum (ER)—completely inhibits fertilization-evoked depolarization and increases the incidence of polyspermy. Inhibition of the IP3-generating enzyme phospholipase C (PLC) with U73122 similarly prevents fertilization-induced depolarization and increases polyspermy. Together, these results demonstrate that fast polyspermy block after fertilization in X. laevis eggs is mediated by activation of PLC, which increases IP3 and evokes Ca2+ release from the ER. This ER-derived Ca2+ then activates a Cl− channel to induce the fast polyspermy block. The PLC-induced cascade of events represents one of the earliest known signaling pathways initiated by fertilization.

Lymphatic vessels display pressure-dependent contractions whose frequency is modulated by ion channel activity in lymphatic muscle. Zawieja et al. show that the Ca2+-activated Cl− channel Anoctamin 1 sets the lymphatic contraction rate by providing depolarizing current between action potentials., Lymphatic collecting vessels exhibit spontaneous contractions with a pressure-dependent contraction frequency. The initiation of contraction has been proposed to be mediated by the activity of a Ca2+-activated Cl− channel (CaCC). Here, we show that the canonical CaCC Anoctamin 1 (Ano1, TMEM16a) plays an important role in lymphatic smooth muscle pacemaking. We find that isolated murine lymphatic muscle cells express Ano1, and demonstrate functional CaCC currents that can be inhibited by the Ano1 inhibitor benzbromarone. These currents are absent in lymphatic muscle cells from Cre transgenic mouse lines targeted for Ano1 genetic deletion in smooth muscle. We additionally show that loss of functional Ano1 in murine inguinal-axillary lymphatic vessels, whether through genetic manipulation or pharmacological inhibition, results in an impairment of the pressure–frequency relationship that is attributable to a hyperpolarized resting membrane potential and a significantly depressed diastolic depolarization rate preceding each action potential. These changes are accompanied by alterations in action potential shape and duration, and a reduced duration but increased amplitude of the action potential–induced global “Ca2+ flashes” that precede lymphatic contractions. These findings suggest that an excitatory Cl− current provided by Ano1 is critical for mediating the pressure-sensitive contractile response and is a major component of the murine lymphatic action potential.

RNA sequencing (RNA-seq) is extensively used to quantify gene expression transcriptome-wide. Although often paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA), many applications require alternate approaches to counteract the high proportion of ribosomal RNA (rRNA) in total RNA. Recently, digestion using RNaseH and antisense DNA oligomers tiling target rRNAs has emerged as an alternative to commercial rRNA depletion kits. Here, we present a streamlined, more economical RNaseH-mediated rRNA depletion with substantially lower up-front costs, using shorter antisense oligos only sparsely tiled along the target RNA in a 5-min digestion reaction. We introduce a novel Web tool, Oligo-ASST, that simplifies oligo design to target regions with optimal thermodynamic properties, and additionally can generate compact, common oligo pools that simultaneously target divergent RNAs, e.g. across different species. We demonstrate the efficacy of these strategies by generating rRNA-depletion oligos for Xenopus laevis and for zebrafish, which expresses two distinct versions of rRNAs during embryogenesis. The resulting RNA-seq libraries reduce rRNA to5% of aligned reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion.

Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. Here, we combined inhibitor tests, germ-line–specific RNAi knockdown, and germ-line–specific CRISPR/Cas9 knockout to identify the Transient Receptor Potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. We observed a reduction in the proportion of eggs that hatched from trpm germ-line knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since a mouse ortholog of Trpm was recently reported also to be involved in calcium influx during egg activation and in further embryonic development, our results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation.

Transmembrane member 16A (TMEM16A) is a widely expressed Ca(2+)-activated Cl(−) channel with various physiological functions ranging from mucosal secretion to regulating smooth muscle contraction. Understanding how TMEM16A controls these physiological processes and how its dysregulation may cause disease requires a detailed understanding of how cellular processes and second messengers alter TMEM16A channel gating. Here we assessed the regulation of TMEM16A gating by recording Ca(2+)-evoked Cl(−) currents conducted by endogenous TMEM16A channels expressed in Xenopus laevis oocytes, using the inside-out configuration of the patch clamp technique. During continuous application of Ca(2+), we found that TMEM16A-conducted currents decay shortly after patch excision. Such current rundown is common among channels regulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Thus, we sought to investigate a possible role of PIP(2) in TMEM16A gating. Consistently, synthetic PIP(2) rescued the current after rundown, and the application of PIP(2) modulating agents altered the speed kinetics of TMEM16A current rundown. First, two PIP(2) sequestering agents, neomycin and anti-PIP(2), applied to the intracellular surface of excised patches sped up TMEM16A current rundown to nearly twice as fast. Conversely, rephosphorylation of phosphatidylinositol (PI) derivatives into PIP(2) using Mg-ATP or inhibiting dephosphorylation of PIP(2) using β-glycerophosphate slowed rundown by nearly 3-fold. Our results reveal that TMEM16A regulation is more complicated than it initially appeared; not only is Ca(2+) necessary to signal TMEM16a opening, but PIP(2) is also required. These findings improve our understanding of how the dysregulation of these pathways may lead to disease and suggest that targeting these pathways could have utility for potential therapies.

Fertilization of an egg by multiple sperms, polyspermy, is lethal to most sexually reproducing species. To combat the entry of additional sperm into already fertilized eggs, organisms have developed various polyspermy blocks. One such barrier, the fast polyspermy block, uses a fertilization-activated depolarization of the egg membrane to electrically inhibit supernumerary sperm from entering the egg. The fast block is commonly used by eggs of oviparous animals with external fertilization. In this review, we discuss the history of the fast block discovery, as well as general features shared by all organisms that use this polyspermy block. Given the diversity of habitats of external fertilizers, the fine details of the fast block-signaling pathways differ drastically between species, including the identity of the depolarizing ions. We highlight the known molecular mediators of these signaling pathways in amphibians and echinoderms, with a fine focus on ion channels that signal these fertilization-evoked depolarizations. We also discuss the investigation for a fast polyspermy block in mammals and teleost fish, and we outline potential fast block triggers. Since the first electrical recordings made on eggs in the 1950s, the fields of developmental biology and electrophysiology have substantially matured, and yet we are only now beginning to discern the intricate molecular mechanisms regulating the fast block to polyspermy.

In their preceding paper, Wozniak et al. show that fertilization increases intracellular Ca2+ in Xenopus laevis eggs by activating an IP3 signaling cascade. Here, they reveal that Ca2+ subsequently opens the Cl− channel TMEM16A to allow Cl− efflux, cell depolarization, and fast block to polyspermy., In externally fertilizing animals, such as sea urchins and frogs, prolonged depolarization of the egg immediately after fertilization inhibits the entry of additional sperm—a phenomenon known as the fast block to polyspermy. In the African clawed frog Xenopus laevis, this depolarization is driven by Ca2+-activated Cl− efflux. Although the prominent Ca2+-activated Cl− currents generated in immature X. laevis oocytes are mediated by X. laevis transmembrane protein 16a (xTMEM16A) channels, little is known about the channels that contribute to the fast block in mature eggs. Moreover, the gamete undergoes a gross transformation as it develops from an immature oocyte into a fertilization-competent egg. Here, we report the results of our approach to identify the Ca2+-activated Cl− channel that triggers the fast block. By querying published proteomic and RNA-sequencing data, we identify two Ca2+-activated Cl− channels expressed in fertilization-competent X. laevis eggs: xTMEM16A and X. laevis bestrophin 2A (xBEST2A). By exogenously expressing xTMEM16A and xBEST2A in axolotl cells lacking endogenous Ca2+-activated currents, we characterize the effect of inhibitors on currents mediated by these channels. None of the inhibitors tested block xBEST2A currents specifically. However, 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9) and N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA) each reduce xTMEM16A currents by more than 70% while only nominally inhibiting those generated by xBEST2A. Using whole-cell recordings during fertilization, we find that Ani9 and MONNA effectively diminish fertilization-evoked depolarizations. Additionally, these inhibitors lead to increased polyspermy in X. laevis embryos. These results indicate that fertilization activates TMEM16A channels in X. laevis eggs and induces the earliest known event triggered by fertilization: the fast block to polyspermy.

At fertilization, the terminally differentiated egg begins a new life as a totipotent zygote. Egg activation is an important process in this transformation to totipotency, as activation represents the time when immature oocytes develop from a state of cellular quiescence into one ready for embryogenesis. In PNAS, Hu and Wolfner demonstrate that the Trpm ion channel conducts a Ca2+ influx into eggs from the fruit fly, Drosophila melanogaster , to signal their activation (1). Eggs from insects including fruit flies, bees, and ants activate before fertilization when they are pushed out of the ovary. Independence of egg activation from fertilization in insect eggs allowed for the evolution of varied reproductive strategies ranging from parthenogenic reproduction, where females produce daughters with no male input, to sexual reproduction, where offspring are the result of maternal and paternal contributions (2). Other species also separate activation and fertilization. For example, eggs from zebrafish, an obligate sexual reproducer, activate as they are laid and transferred from the mother into the surrounding water (3). By contrast, egg activation is triggered by fertilization in organisms such as the mouse and human. Although the external signal that triggers egg activation differs among species, a wave of elevated intracellular Ca2+ is often the event … [↵][1]1Email: acarlson{at}pitt.edu. [1]: #xref-corresp-1-1

The hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are pacemaker channels whose currents contribute to rhythmic activity in the heart and brain. HCN channels open in response to hyperpolarizing voltages, and the binding of cAMP to their cyclic nucleotide-binding domain (CNBD) facilitates channel opening. Here, we report that, like cAMP, the flavonoid fisetin potentiates HCN2 channel gating. Fisetin sped HCN2 activation and shifted the conductance-voltage relationship to more depolarizing potentials with a half-maximal effective concentration (EC50) of 1.8 μM. When applied together, fisetin and cAMP regulated HCN2 gating in a nonadditive fashion. Fisetin did not potentiate HCN2 channels lacking their CNBD, and two independent fluorescence-based binding assays reported that fisetin bound to the purified CNBD. These data suggest that the CNBD mediates the fisetin potentiation of HCN2 channels. Moreover, binding assays suggest that fisetin and cAMP partially compete for binding to the CNBD. NMR experiments demonstrated that fisetin binds within the cAMP-binding pocket, interacting with some of the same residues as cAMP. Together, these data indicate that fisetin is a partial agonist for HCN2 channels.

The voltage-gated, K+-selective ether á go-go 1 (EAG1) channel is expressed throughout the brain where it is thought to regulate neuronal excitability. Besides its normal physiological role in the brain, EAG1 is abnormally expressed in several cancer cell types and promotes tumor progression. Like all other channels in the KCNH family, EAG1 channels have a large intracellular carboxy-terminal region that shares structural similarity with cyclic nucleotide–binding homology domains (CNBHDs). EAG1 channels, however, are not regulated by the direct binding of cyclic nucleotides and have no known endogenous ligands. In a screen of biological metabolites, we have now identified four flavonoids as potentiators of EAG1 channels: fisetin, quercetin, luteolin, and kaempferol. These four flavonoids shifted the voltage dependence of activation toward more hyperpolarizing potentials and slowed channel deactivation. All four flavonoids regulated channel gating with half-maximal concentrations of 2–8 µM. The potentiation of gating did not require the amino-terminal or post-CNBHD regions of EAG1 channels. However, in fluorescence resonance energy transfer and anisotropy-based binding assays, flavonoids bound to the purified CNBHD of EAG1 channels. The CNBHD of KCNH channels contains an intrinsic ligand, a conserved stretch of residues that occupy the cyclic nucleotide–binding pocket. Mutations of the intrinsic ligand in EAG1 (Y699A) potentiated gating similar to flavonoids, and flavonoids did not further potentiate EAG1-Y699A channels. Furthermore, the Y699A mutant CNBHD bound to flavonoids with higher affinity than wild-type CNBHD. These results suggest that the flavonoids identified here potentiated EAG1 channels by binding to the CNBHD, possibly by displacing their intrinsic ligand. EAG1 channels should be considered as a possible target for the physiological effects of flavonoids.

Similar to CNG and HCN channels, EAG and ERG channels contain a cyclic nucleotide binding domain (CNBD) in their C terminus. While cyclic nucleotides have been shown to facilitate opening of CNG and HCN channels, their effect on EAG and ERG channels is less clear. Here we explored cyclic nucleotide binding and modulation of mEAG1 and hERG1 channels with fluorescence and electrophysiology. Binding of cyclic nucleotides to the isolated CNBD of mEAG1 and hERG1 channels was examined with two independent fluorescence-based methods: changes in tryptophan fluorescence and fluorescence of an analog of cAMP, 8-NBD-cAMP. As a positive control for cyclic nucleotide binding we used changes in the fluorescence of the isolated CNBD of mHCN2 channels. Our results indicated that cyclic nucleotides do not bind to the isolated CNBD domain of mEAG1 channels and bind with low affinity (K(d)or = 51 microm) to the isolated CNBD of hERG1 channels. Consistent with the results on the isolated CNBD, application of cyclic nucleotides to inside-out patches did not affect currents recorded from mEAG1 channels. Surprisingly, despite its low affinity binding to the isolated CNBD, cAMP also had no effect on currents from hERG1 channels even at high concentrations. Our results indicate that cyclic nucleotides do not directly modulate mEAG1 and hERG1 channels. Further studies are necessary to determine if the CNBD in the EAG family of K(+) channels might harbor a binding site for a ligand yet to be uncovered.

Capacitation of mammalian sperm, including alterations in flagellar motility, is presumably modulated by chemical signals encountered in the female reproductive tract. This work investigates signaling pathways for adenosine and catecholamine agonists that stimulate sperm kinetic activity. We show that 2-chloro-2'-deoxyadenosine and isoproterenol robustly accelerate flagellar beat frequency with EC50s near 10 and 0.05 microM, respectively. The several-fold acceleration is maximal by 60 sec. Although extracellular Ca2+ is required for agonist action on the flagellar beat, agonist treatment does not elevate sperm cytosolic [Ca2+] but does increase cAMP content. Acceleration does not require the conventional transmembrane adenylyl cyclase ADCY3, since it persists in sperm of ADCY3 knockout mice and in wild-type sperm in the presence of the inhibitors of conventional adenylyl cyclases SQ-22536, MDL-12330A, or 2', 5'-dideoxyadenosine. In contrast, the acceleration by these agents is absent in sperm that lack the predominant atypical adenylyl cyclase, SACY. Responses to these agonists are also absent in sperm from mice lacking the sperm-specific Calpha2 catalytic subunit of protein kinase A (PRKACA). Agonist responses also are strongly suppressed in wild-type sperm by the protein kinase inhibitor H-89. These results show that adenosine and catecholamine analogs activate sperm motility by mechanisms that require extracellular Ca2+, the atypical sperm adenylyl cyclase, cAMP, and protein kinase A.

CatSper family proteins are putative ion channels expressed exclusively in membranes of the sperm flagellum and required for male fertility. Here, we show that mouse CatSper1 is essential for depolarization-evoked Ca 2+ entry and for hyperactivated movement, a key flagellar function. CatSper1 is not needed for other developmental landmarks, including regional distributions of Ca V 1.2, Ca V 2.2, and Ca V 2.3 ion channel proteins, the cAMP-mediated activation of motility by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} , and the protein phosphorylation cascade of sperm capacitation. We propose that CatSper1 functions as a voltage-gated Ca 2+ channel that controls Ca 2+ entry to mediate the hyperactivated motility needed late in the preparation of sperm for fertilization.

At mating, mammalian sperm are diluted in the male and female reproductive fluids, which brings contact with HCO(3)(-) and initiates several cellular responses. We have identified and studied two of the most rapid of these responses. Stop-motion imaging and flagellar waveform analysis show that for mouse epididymal sperm in vitro, the resting flagellar beat frequency is 2-3 Hz at 22-25 degrees C. Local perfusion with HCO(3)(-) produces a robust, reversible acceleration to 7 Hz or more. At 15 mM the action of HCO(3)(-) begins within 5 seconds and is near-maximal by 30 seconds. The half-times of response are 8.8+/-0.2 seconds at 15 mM HCO(3)(-) and 17.5+/-0.4 seconds at 1 mM HCO(3)(-). Removal of external HCO(3)(-) allows a slow return to basal beat frequency over approximately 10 minutes. Increases in beat symmetry accompany the accelerating action of HCO(3)(-). As in our past work, HCO(3)(-) also facilitates opening of voltagegated Ca(2+) channels, increasing the depolarization-evoked rate of rise of intracellular Ca(2+) concentration by more than fivefold. This action also is detectable at 1 mM HCO(3)(-) and occurs with an apparent halftime of approximately 60 seconds at 15 mM HCO(3)(-). The dual actions of HCO(3)(-) respond similarly to pharmacological intervention. Thus, the phosphodiesterase inhibitor IBMX promotes the actions of HCO(3)(-) on flagellar and channel function, and the protein kinase A inhibitor H89 blocks these actions. In addition, a 30 minute incubation with 60 micro M cAMP acetoxylmethyl ester increases flagellar beat frequency to nearly 7 Hz and increases the evoked rates of rise of intracellular Ca(2+) concentration from 17+/-4 to 41+/-6 nM second(-1). However, treatment with several other analogs of cAMP produces only scant evidence of the expected mimicry or blockade of the actions of HCO(3)(-), perhaps as a consequence of limited permeation. Our findings indicate a requirement for cAMP-mediated protein phosphorylation in the enhancement of flagellar and channel functions that HCO(3)(-) produces during sperm activation.

Cyclic nucleotide-gated (CNG) channels have been shown to be blocked by diltiazem, tetracaine, polyamines, toxins, divalent cations, and other compounds. Dequalinium is an organic divalent cation which suppresses the rat small conductance Ca(2+)-activated K(+) channel 2 (rSK2) and the activity of protein kinase C. In this study, we have tested the ability of dequalinium to block CNGA1 channels and heteromeric CNGA1+CNGB1 channels. When applied to the intracellular side of inside-out excised patches from Xenopus oocytes, dequalinium blocks CNGA1 channels with a K(1/2) approximately 190 nM and CNGA1+CNGB1 channels with a K(1/2) approximately 385 nM, at 0 mV. This block occurs in a state-independent fashion, and is voltage dependent with a zdelta approximately 1. Our data also demonstrate that dequalinium interacts with the permeant ion probably because it occupies a binding site in the ion conducting pathway. Dequalinium applied to the extracellular surface also produced block, but with a voltage dependence that suggests it crosses the membrane to block from the inside. We also show that at the single-channel level, dequalinium is a slow blocker that does not change the unitary conductance of CNGA1 channels. Thus, dequalinium should be a useful tool for studying permeation and gating properties of CNG channels.

Ca2+-activated Cl- channels (CaCCs) mediate some of the most important physiologic processes, such as the regulation of neuronal and cardiac excitability, electrolyte balances in secretory epithelia, and olfactory transduction. However, it wasn’t until 2008 that transmembrane proteins with unknown function 16 (TMEM16a) was identified as a bona fide CaCC. One of the cell types that TMEM16a was originally identified in was Xenopus laevis oocytes. Although TMEM16a is present in these oocytes, we do not know if it is maintained during oogenesis and functional in the fertilization-competent egg. During fertilization in X. laevis, a Ca2+-activated Cl- current is evoked in a pathway known as the fast polyspermy block, which inhibits sperm from entering an already fertilized egg. Here, we sought to uncover the molecular identity of the Cl- channel mediating this polyspermy block. We tested whether the rate of the fertilization-evoked depolarization was altered in eggs inseminated in the presence of TMEM16a-targeting small molecule inhibitors including: MONNA, Ani9, CaCCinh-A01, and T16ainh-A01. We reasoned that the rate of depolarization is proportional to the number of channels opened and thus looked for changes in the depolarization rate. We found that the depolarization of the fast block was significantly slowed or completely prevented in eggs inseminated in the presence of these TMEM16a inhibitors. To verify that the TMEM16a-targeted inhibitors block the X. laevis version of the channel, we recorded the Ca2+ activated Cl- current evoked by uncaging IP3 to evoke Ca2+ release from the endoplasmic reticulum, in X. laevis oocytes using two electrode voltage clamp. We found that these inhibitors indeed were effective on the X. laevis TMEM16a channels. Taken together, these results suggest that TMEM16a is conserved through the oogenesis process and that TMEM16a mediates the fast block to polyspermy in X. laevis.

Background The necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy. Methodology/principal finding Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+. Conclusions/Significance Taken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu.

Preventing polyspermy is essential for the normal embryonic development of most animals. Polyspermy, or the fertilization of an egg by more than one sperm, causes severe chromosomal defects and embryonic mortality. The eggs of nearly all externally fertilizing species employ a fast block to polyspermy in the form of a fertilization signaled depolarization. In the African clawed frog, Xenopus laevis, the fast block requires an increase in intracellular calcium that activates a Ca2+-activated Cl- channel; an efflux of chloride from the egg then depolarizes the membrane. Although increased Ca2+ is crucial for the fast block, the source of this Ca2+ remains unknown. Here we tested the hypothesis that Ca2+ enters the egg to signal the fast block in X. laevis using whole cell recordings on eggs inseminated in the nominal absence of Ca2+or in the presence of 10 μM Gd3+. We recorded normal depolarizations, as well as normal embryonic development, from eggs fertilized in both treatments thereby suggesting that Ca2+ is not entering from the extracellular environment. To test the importance Ca2+ released from the endoplasmic reticulum (ER) in the fast block, the membrane potential was recorded from eggs that had inhibited inositol trisphophate (IP3) synthesis or IP3 receptor binding. Our data show that inseminated eggs under both of these conditions failed to depolarize. Taken together, these data suggest that the primary source of calcium needed for the fast block to polyspermy is the ER.

Background: Ether-a`-go-go (EAG) channels are expressed throughout the central nervous system and are also crucial regulators of cell cycle and tumor progression. The large intracellular amino- and carboxy- terminal domains of EAG1 each share similarity with known ligand binding motifs in other proteins, yet EAG1 channels have no known regulatory ligands. Methodology/Principal Findings: Here we screened a library of small biologically relevant molecules against EAG1 channels with a novel two-pronged screen to identify channel regulators. In one arm of the screen we used electrophysiology to assess the functional effects of the library compounds on full-length EAG1 channels. In an orthogonal arm, we used tryptophan fluorescence to screen for binding of the library compounds to the isolated C-terminal region. Conclusions/Significance: Several compounds from the flavonoid, indole and benzofuran chemical families emerged as binding partners and/or regulators of EAG1 channels. The two-prong screen can aid ligand and drug discovery for ligandbinding domains of other ion channels.

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca(2+) entry evoked by alkaline depolarization. In the absence of external Ca(2+), Na(+) carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na(+)](i)-reporting probe SBFI in populations of intact sperm. Removal of external Ca(2+) increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na(+)](i) is greater for sperm alkalinized with NH(4)Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na(+)](i) rise is slowed by candidate CatSper blocker HC-056456 (IC(50) approximately 3 microM). HC-056456 similarly slows the rise of [Ca(2+)](i) that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO(3) (-) but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca(2+) entry through the CatSper channel is terminated by removal of external Ca(2+). Thus, open CatSper channels and entry of external Ca(2+) through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC(-/-) spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC(-/-) spermatozoa and are undetectable after sperm purification. HCO(3)(-) is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca(2+) entry into sAC(-/-) spermatozoa. Moreover, the delayed HCO(3)(-)-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC(-/-) spermatozoa. However, sAC(-/-) sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC(-/-) sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC(-/-) spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.

Capacitation of mammalian sperm, including alterations in flagellar motility, is presumably modulated by chemical signals encountered in the female reproductive tract. This work investigates signaling pathways for adenosine and catecholamine agonists that stimulate sperm kinetic activity. We show that 2-chloro-2'-deoxyadenosine and isoproterenol robustly accelerate flagellar beat frequency with EC50s near 10 and 0.05 microM, respectively. The several-fold acceleration is maximal by 60 sec. Although extracellular Ca2+ is required for agonist action on the flagellar beat, agonist treatment does not elevate sperm cytosolic [Ca2+] but does increase cAMP content. Acceleration does not require the conventional transmembrane adenylyl cyclase ADCY3, since it persists in sperm of ADCY3 knockout mice and in wild-type sperm in the presence of the inhibitors of conventional adenylyl cyclases SQ-22536, MDL-12330A, or 2', 5'-dideoxyadenosine. In contrast, the acceleration by these agents is absent in sperm that lack the predominant atypical adenylyl cyclase, SACY. Responses to these agonists are also absent in sperm from mice lacking the sperm-specific Calpha2 catalytic subunit of protein kinase A (PRKACA). Agonist responses also are strongly suppressed in wild-type sperm by the protein kinase inhibitor H-89. These results show that adenosine and catecholamine analogs activate sperm motility by mechanisms that require extracellular Ca2+, the atypical sperm adenylyl cyclase, cAMP, and protein kinase A.

Among several candidate Ca(2+) entry channels in sperm, only CatSper1 and CatSper2 are known to have required roles in male fertility. Past work with CatSper1 null sperm indicates that a critical lesion in hyperactivated motility underlies the infertility phenotype and is associated with an absence of depolarization-evoked Ca(2+)entry. Here we show that failure of hyperactivation of CatSper2 null sperm similarly correlates with an absence of depolarization evoked Ca(2+) entry. Additional shared aspects of the phenotypes of CatSper1 and -2 null sperm include unperturbed regional distributions of conventional voltage-gated Ca(2+) channel proteins and robust acceleration of the flagellar beat by bicarbonate. Further study reveals that treatment of both wild-type and CatSper2 null sperm with procaine increases beat asymmetry, a characteristic of the flagellar waveform of hyperactivation. This partial rescue of the loss-of-hyperactivation phenotype suggests that an absence of CatSper2 precludes hyperactivation by preventing delivery of needed Ca(2+) messenger rather than by preventing flagellar responses to Ca(2+). CatSper2 null sperm also have an increased basal cAMP content and beat frequency. Protein kinase A inhibitor H89 lowers beat frequency to that of wild-type sperm, suggesting that CatSper2 is required for protein kinase A-mediated, tonic control of resting cAMP content. Relative to wild-type testis, CatSper1 and -2 null testes contain normal amounts of CatSper2 and -1 transcripts, respectively. However, CatSper1 null sperm lack CatSper2 protein and CatSper2 null sperm lack CatSper1 protein. Hence, stable expression of CatSper1 protein requires CatSper2 and vice versa. This co-dependent expression dictates identical loss-of-function sperm phenotypes for CatSper1 and -2 null mutants.

Ether-a-go-go (Eag) family channels, which include hErg1, are voltage-gated K+ channels that are important in cardiac and neural function and have a well-documented role in disease. Eag1 channels in particular are found throughout the central nervous system and are crucial regulators of cell cycle and tumor progression. Wild-type Eag1 channels exhibit voltage-gated activation and deactivation, but no apparent inactivation. Here we find that deletion of the entire intracellular amino-terminal domain uncovers an inactivated channel state at depolarizing potentials. We characterized this new inactivated state in inside-out patches excised from Xenopus oocytes expressing the mutant Eag1 channels. Intriguingly, we find that application of the membrane-impermeable cysteine modifying reagent MTSES to the intracellular side of the amino-terminal deletion mutant abolishes this inactivation. We have localized this cysteine-modifying effect to one or more of three cysteine residues in the intracellular carboxy-terminal domain. One of these residues resides in a domain that shares sequence similarity with the cyclic nucleotide binding domain (CNBD) of other channels and enzymes, and the other two reside in the C-linker which connects the CNBD-like region to the S6 transmembrane domain. As the CNBD-like region in does not bind to, and these channels are not regulated by cyclic nucleotides, the role of domain in channel gating is unknown. Our results suggest that the entire amino-terminus, or a fraction thereof, may act as a ligand that binds to the CNBD-like region to prevent inactivation in the wild-type channel.

The HCO3(-) anion activates sperm motility, an important early step in capacitation, by increasing flagellar beat frequency through a pathway that requires the atypical adenylyl cyclase SACY and the sperm-specific C alpha2 catalytic subunit of PKA. Here we show that the accelerating action of HCO3(-) also requires the continued presence of external Ca2+ (EC50 approximately 0.5 mM), and find that Ca2+ can be replaced by Sr2+ but not by Mn2+. Ca2+ is required for HCO3(-) to elevate cAMP, but not for cAMP-AM to increase beat frequency, indicating that external Ca2+ acts before rather than after stimulation of SACY by HCO3(-). With external Ca2+ present, HCO3(-) does not alter cytosolic or near-membrane [Ca2+]. Removal of external Ca2+ initiates a slow decline in intracellular [Ca2+] and rapid block of the HCO3(-)-evoked acceleration that is not relieved upon increasing internal [Ca2+] by rapid photolysis of caged Ca2+. We also find that the rapid (t(1/2) approximately 10 s) accelerating action of HCO3(-) is slowed more than three-fold by the carbonic anhydrase inhibitor acetazolamide. It is unaltered by the broad spectrum anion transport inhibitor SITS, and is not accompanied by detectable changes in intracellular pH. We propose that external Ca2+ binds an unidentified extracellular protein that is required for HCO3(-) to engage cAMP-mediated activation of motility.

Ether-a-go-go (Eag) family channels, which include hErg1 channels, are voltage-gated K+ channels that are important in cardiac and neural function. From the amino acid sequence of Eag family channels, we identified two probable ligand-binding sites based on their similarities with well-characterized ligand-binding domains. The first putative ligand-binding domain is in the carboxy-terminal region and shares sequence similarity with the cyclic nucleotide-binding domain (CNBD) of proteins modulated by cyclic nucleotides. The second is a Per-Arnt-Sim (PAS) domain in the amino-terminal region. As no ligands have been reported for Eag family channels, we categorize these channels as orphan receptors. We reasoned that a chemical screen of metabolites will lead us to physiologically relevant channel-regulators. Using a novel, high through-put screen of the “Fragments of Life” chemical library of metabolites and metabolite-like compounds (deCODE Biostructures, Bainbridge Island WA) and inside-out patch-clamp recording, we have identified the flavonoid quercetin, and various structurally-related flavonoids, as regulators of Eag1 channels. Flavonoids are secondary plant metabolites found in all vascular plants that share a three-ring structure. Flavonoids have existed in nature for more than a billion years, and thus have interacted with evolving organisms for eons. Identified flavonoids slow channel deactivation and shift the conductance/voltage relationship toward more negative potentials in a concentration-dependent manner. For example, 10 uM quercetin shifted the Vhalf of the conductance/voltage relation an average of −13 mV, and slowed the tau of deactivation from 3 to 9 ms (in 5 separate experiments). Flavonoids retained their activity on Eag1 constructs lacking the PAS domain, the entire amino-terminus, or the post-CNBD of distal-carboxy terminal region. Further, identified flavonoids bind to the the isolated CNBD in a fluorescence-based binding assay. Thus, we predict that flavonoids activate Eag1 channels by binding to the CNBD of the carboxy-terminal region.

The KCNH voltage dependent potassium channels are key regulators of cellular excitability, involved in cardiac long QT syndrome type 2 (LQTS2), epilepsy, schizophrenia and cancer. The intracellular domains of KCNH channels are structurally distinct from other voltage-gated channels. The amino-terminal region contains an eag domain, which includes a Per-Arnt-Sim (PAS) module and a PAS-cap region, while the carboxy-terminal region encompasses a cyclic nucleotide-binding homology domain (CNBHD), connected to the pore domain through a C-linker domain. These specialized intracellular domains are the site of many disease-causing mutations and bestow unique gating and regulation on KCNH channels. It has been suggested that the eag domain may interact with either the S4-S5 linker or the CNBHD in human ERG (hERG) and EAG channels. We have used fluorescence approaches to determine that the eag domain and the CNBHD, from the mEAG1 channel, form a complex in solution, with an apparent affinity of 13.2 ± 2.3 μM. Moreover, an equimolar mixture of purified eag domain and CNBHD produced co-crystals of the complex, belonging to the P65 space group, which diffracted to 2.0 A resolution. The structure of the eag domain-CNBHD complex was solved using molecular replacement with mEAG1 CNBHD as a model. Harboring many LQTS2 and cancer-associated mutations, the eag domain-CNBHD interface involves three important regions: (i) the "intrinsic ligand" motif, a unique structural feature of the CNBHD; (ii) the post-CNBHD region, known to mediate EAG channels regulation by a variety of cellular signaling events; and finally, (iii) the PAS-cap region, which constitutes the first 25 amino acids of the eag domain, and forms a highly conserved amphipathic helix (αCAP). The structure of the EAG domain-CNBHD complex of mEAG1 provides a detailed physiological and pathophysiological description of the intracellular domain of the KCNH channels.

The KCNH family of ion channels comprises Ether-a -go-go (EAG), EAG-related gene (ERG), and EAG-like (ELK) K+ channel subfamilies. KCNH channels are best known for their regulatory function in tumor progression, repolarization of the cardiac action potential and regulation of neuronal excitability. The C-terminus of KCNH channels contains a cyclic nucleotide-binding homology domain (CNBHD) coupled to the pore with the C-linker. Mutations in the C-linker/CNBHD affect KCNH channel function and, in ERG channels, can lead to Long-QT Syndrome. The CNBHD shares sequence similarity with the cyclic nucleotide-binding domain (CNBD) of hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels. However, unlike the HCN and CNG channels that are regulated by direct binding of cyclic nucleotides to the CNBDs, KCNH channels are not directly regulated by cyclic nucleotides and their CNBHDs do not bind cyclic nucleotides. The structural basis of the regulation of the KCNH channels by the C-linker/CNBHD and the failure of cyclic nucleotides to regulate the channels are not clear. Here we present the crystal structure of the C-linker/CNBHD of zebrafish ELK (zELK) channels at 2.2 A resolution. The structure revealed unique features of the KCNH channels including: 1) a negatively charged electrostatic profile that explains the lack of binding and regulation of ion channels in the KCNH family by cyclic nucleotides, 2) intersubunit interactions that produce a two-fold symmetry in the C-terminal region as opposed to the four-fold symmetry of HCN channels, and 3) a novel β-strand occupying the pocket where cyclic nucleotides bind to HCN channels. Mutations in the β-strand shifted the half-maximal activation voltage by ∼15 mV, implicating the β-strand as a regulatory element in KCNH channels. These findings provide a structural framework for understanding the regulation of ion channels in the KCNH family by the C-linker/CNBD.

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels are pace-making channels that contribute to rhythmic activity in the heart and brain. All HCN-family channels contain a large intracellular domain in their carboxy terminal region which includes a cyclic nucleotide binding domain (CNBD) and a C-linker that connects it to the pore. We recently reported that the flavonoids luteolin and myricetin regulate Eag1 channels by binding a domain in their carboxy-terminal region that shares sequence and structural similarity with the CNBD of HCN2. In a screen of flavonoids, we identified fisetin as another potent regulator of Eag1. Fisetin shifted the voltage-dependence of Eag1 channels toward more hyperpolarizing potentials and slowed channel deactivation. Here we demonstrate that fisetin also regulates the kinetic and steady state properties of HCN2 channels. We found that 30 μM fisetin sped activation at −140 mV, decreasing the time constant from 1.17 ± 0.10 s to 0.80 ± 0.8 s (N=10). Additionally, 30 μM fisetin shifted the Vhalf of the conductance-voltage relationship from −127.8 ± 0.7 mV, to −120.4 ± 0.7 mV (N=10). Fisetin shifted the voltage-dependence of Eag and HCN channels in opposite directions, indicating that it is not simply interacting with the voltage sensor. Furthermore we found that fisetin and cAMP had non-additive effects on HCN2 channel gating. Using a FRET-based binding assay, we showed that fisetin bound to the purified CNBD of HCN2. Additionally, fisetin did not speed activation or shift the voltage-dependence of HCN2 channels lacking their CNBD. Together these data indicate that the CNBD mediates fisetin regulation of HCN2 channels. Based on the similar regulation of Eag and HCN channels by fisetin we propose that the mechanisms underlying ligand-mediated gating is well-conserved amongst these channels.