ZP2 cleavage blocks polyspermy by modulating the architecture of the egg coat.

Following the fertilization of an egg by a single sperm, the egg coat or zona pellucida (ZP) hardens and polyspermy is irreversibly blocked. These events are associated with the cleavage of the N-terminal region (NTR) of glycoprotein ZP2, a major subunit of ZP filaments. ZP2 processing is thought to inactivate sperm binding to the ZP, but its molecular consequences and connection with ZP hardening are unknown. Biochemical and structural studies show that cleavage of ZP2 triggers its oligomerization. Moreover, the structure of a native vertebrate egg coat filament, combined with AlphaFold predictions of human ZP polymers, reveals that two protofilaments consisting of type I (ZP3) and type II (ZP1/ZP2/ZP4) components interlock into a left-handed double helix from which the NTRs of type II subunits protrude. Together, these data suggest that oligomerization of cleaved ZP2 NTRs extensively cross-links ZP filaments, rigidifying the egg coat and making it physically impenetrable to sperm.

Tiny Dancer: EFCAB9 Triggers Sperm Hyperactivation via CatSper.

The Ca2+-conducting ion channel, CatSper, is expressed exclusively on the sperm flagellum and regulates sperm motility. A new study (Hwang et al., Cell, 2019) reveals that the pH-sensing and Ca2+-binding protein, EFCAB9, is a subunit of the CatSper channel and has a key role in triggering mammalian sperm to change their swimming at fertilization.

Phosphate position is key in mediating transmembrane ion channel TMEM16A-phosphatidylinositol 4,5-bisphosphate interaction.

TransMEMbrane 16A (TMEM16A) is a Ca2+-activated Cl- channel that plays critical roles in regulating diverse physiologic processes, including vascular tone, sensory signal transduction, and mucosal secretion. In addition to Ca2+, TMEM16A activation requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, the structural determinants mediating this interaction are not clear. Here, we interrogated the parts of the PI(4,5)P2 head group that mediate its interaction with TMEM16A by using patch- and two-electrode voltage-clamp recordings on oocytes from the African clawed frog Xenopus laevis, which endogenously express TMEM16A channels. During continuous application of Ca2+ to excised inside-out patches, we found that TMEM16A-conducted currents decayed shortly after patch excision. Following this rundown, we show that the application of a synthetic PI(4,5)P2 analog produced current recovery. Furthermore, inducible dephosphorylation of PI(4,5)P2 reduces TMEM16A-conducted currents. Application of PIP2 analogs with different phosphate orientations yielded distinct amounts of current recovery, and only lipids that include a phosphate at the 4' position effectively recovered TMEM16A currents. Taken together, these findings improve our understanding of how PI(4,5)P2 binds to and potentiates TMEM16A channels.

Zinc protection of fertilized eggs is an ancient feature of sexual reproduction in animals.

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.

Optimized design of antisense oligomers for targeted rRNA depletion.

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 to <5% 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.

Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ are both required to open the Cl- channel TMEM16A.

Transmembrane member 16A (TMEM16A) is a widely expressed Ca2+-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 Ca2+-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 Ca2+, we found that TMEM16A-conducted currents decay shortly after patch excision. Such current rundown is common among channels regulated by phosphatidylinositol 4,5-bisphosphate (PIP2). Thus, we sought to investigate a possible role of PIP2 in TMEM16A gating. Consistently, synthetic PIP2 rescued the current after rundown, and the application of PIP2 modulating agents altered the speed kinetics of TMEM16A current rundown. First, two PIP2 sequestering agents, neomycin and anti-PIP2, 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 PIP2 using Mg-ATP or inhibiting dephosphorylation of PIP2 using ß-glycerophosphate slowed rundown by nearly 3-fold. Our results reveal that TMEM16A regulation is more complicated than it initially appeared; not only is Ca2+ necessary to signal TMEM16a opening, but PIP2 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.

Ion channels and signaling pathways used in the fast polyspermy block.

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.

The structural mechanism of KCNH-channel regulation by the eag domain.

The KCNH voltage-dependent potassium channels (ether-à-go-go, EAG; EAG-related gene, ERG; EAG-like channels, ELK) are important regulators of cellular excitability and have key roles in diseases such as cardiac long QT syndrome type 2 (LQT2), 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 is composed of a Per-Arnt-Sim (PAS) domain and a PAS-cap domain, whereas the carboxy-terminal region contains a cyclic nucleotide-binding homology domain (CNBHD), which is connected to the pore through a C-linker domain. Many disease-causing mutations localize to these specialized intracellular domains, which underlie the unique gating and regulation of KCNH channels. It has been suggested that the eag domain may regulate the channel by interacting with either the S4-S5 linker or the CNBHD. Here we present a 2 Å resolution crystal structure of the eag domain-CNBHD complex of the mouse EAG1 (also known as KCNH1) channel. It displays extensive interactions between the eag domain and the CNBHD, indicating that the regulatory mechanism of the eag domain primarily involves the CNBHD. Notably, the structure reveals that a number of LQT2 mutations at homologous positions in human ERG, in addition to cancer-associated mutations in EAG channels, localize to the eag domain-CNBHD interface. Furthermore, mutations at the interface produced marked effects on channel gating, demonstrating the important physiological role of the eag domain-CNBHD interaction. Our structure of the eag domain-CNBHD complex of mouse EAG1 provides unique insights into the physiological and pathophysiological mechanisms of KCNH channels.

Structure of the carboxy-terminal region of a KCNH channel.

The KCNH family of ion channels, comprising ether-à-go-go (EAG), EAG-related gene (ERG), and EAG-like (ELK) K(+)-channel subfamilies, is crucial for repolarization of the cardiac action potential, regulation of neuronal excitability and proliferation of tumour cells. The carboxy-terminal region of KCNH channels contains a cyclic-nucleotide-binding homology domain (CNBHD) and C-linker that couples the CNBHD to the pore. The C-linker/CNBHD is essential for proper function and trafficking of ion channels in the KCNH family. However, despite the importance of the C-linker/CNBHD for the function of KCNH channels, the structural basis of ion-channel regulation by the C-linker/CNBHD is unknown. Here we report the crystal structure of the C-linker/CNBHD of zebrafish ELK channels at 2.2-Å resolution. Although the overall structure of the C-linker/CNBHD of ELK channels is similar to the cyclic-nucleotide-binding domain (CNBD) structure of the related hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channels, there are marked differences. Unlike the CNBD of HCN, the CNBHD of ELK displays a negatively charged electrostatic profile that explains the lack of binding and regulation of KCNH channels by cyclic nucleotides. Instead of cyclic nucleotide, the binding pocket is occupied by a short ß-strand. Mutations of the ß-strand shift the voltage dependence of activation to more depolarized voltages, implicating the ß-strand as an intrinsic ligand for the CNBHD of ELK channels. In both ELK and HCN channels the C-linker is the site of virtually all of the intersubunit interactions in the C-terminal region. However, in the zebrafish ELK structure there is a reorientation in the C-linker so that the subunits form dimers instead of tetramers, as observed in HCN channels. These results provide a structural framework for understanding the regulation of ion channels in the KCNH family by the C-linker/CNBHD and may guide the design of specific drugs.

Flavonoid regulation of HCN2 channels.

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 SARS coronavirus accessory protein ORF3a rescues potassium conductance in yeast.

ORF3a is an accessory protein expressed by all human pathogen coronaviruses and is the only accessory protein that strongly affects viral fitness. Its deletion reduces severity in both alpha- and beta-coronaviruses, demonstrating a conserved function across the superfamily. Initially regarded as a non-selective cation channel, ORF3a's function is now disputed. Here, we show that ORF3a from SARS, but not SARS-CoV-2, promotes potassium conductance in a yeast model system commonly used to study potassium channels. ORF3a-mediated potassium conductance is also sensitive to inhibitors, including emodin, carbamazepine, and nifedipine. This model may be used in future studies on ORF3a and related proteins.

Characterization of hyperactive mutations in the renal potassium channel ROMK uncovers unique effects on channel biogenesis and ion conductance.

Hypertension affects one billion people worldwide and is the most common risk factor for cardiovascular disease, yet a comprehensive picture of its underlying genetic factors is incomplete. Amongst regulators of blood pressure is the renal outer medullary potassium (ROMK) channel. While select ROMK mutants are prone to premature degradation and lead to disease, heterozygous carriers of some of these same alleles are protected from hypertension. Therefore, we hypothesized that gain-of-function (GoF) ROMK variants which increase potassium flux may predispose people to hypertension. To begin to test this hypothesis, we employed genetic screens and a candidate-based approach to identify six GoF variants in yeast. Subsequent functional assays in higher cells revealed two variant classes. The first group exhibited greater stability in the endoplasmic reticulum, enhanced channel assembly, and/or increased protein at the cell surface. The second group of variants resided in the PIP2-binding pocket, and computational modeling coupled with patch-clamp studies demonstrated lower free energy for channel opening and slowed current rundown, consistent with an acquired PIP2-activated state. Together, these findings advance our understanding of ROMK structure-function, suggest the existence of hyperactive ROMK alleles in humans, and establish a system to facilitate the development of ROMK-targeted antihypertensives.

Xenopus laevis lack the critical sperm factor PLCζ.

Fertilization of eggs from the African clawed frog Xenopus laevis is 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 from X. laevis are injected into mouse eggs, suggesting that mammals and X. laevis share a sperm factor. However, X. laevis lacks an annotated PLCZ1 gene, which encodes the PLCζ enzyme. In this study, we attempted to determine whether sperm from X. laevis express an unannotated PLCZ1 ortholog. We identified PLCZ1 orthologs in 11 amphibian species, including 5 that had not been previously characterized, but did not find any in either X. laevis or the closely related Xenopus tropicalis. Additionally, we performed RNA sequencing on testes obtained from adult X. laevis males 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 the Xenopus lineage and raise the question of how fertilization triggers calcium release and egg activation in these species.

Hybridization led to a rewired pluripotency network in the allotetraploid Xenopus laevis.

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 predicted 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.

TMEM16A activation for the fast block to polyspermy in the African clawed frog does not require conventional activation of egg PLCs.

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 phospholipase C, which generates IP3 to signal a Ca2+ release from the ER. Currently, the signaling pathway by which fertilization activates PLC during the fast block 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 PLCß. 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 PLCß. These data suggest that the PLC that signals the fast block depolarization in X. laevis is activated by a novel mechanism.

Actin polymerization is not required for the fast block to polyspermy in the African clawed frog, Xenopus laevis.

Fertilization of an egg by multiple sperm presents one of the earliest and most prevalent obstacles to successful reproduction. Eggs employ multiple mechanisms to prevent sperm entry into the nascent zygote. The fast block to polyspermy uses a depolarization to inhibit sperm entry. For some external fertilizers, fertilization and the fast block require actin polymerization. Here we explored whether the fast block to polyspermy in the external fertilizer, Xenopus laevis, requires actin polymerization. Inseminating in the presence of inhibitor cytochalasin B, here we demonstrate that actin polymerization is not required for the fast block to polyspermy in X. laevis.

Absence of direct cyclic nucleotide modulation of mEAG1 and hERG1 channels revealed with fluorescence and electrophysiological methods.

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.