Author response: A K+-selective CNG channel orchestrates Ca2+ signalling in zebrafish sperm (2015)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Calcium in the flagellum controls sperm navigation. In sperm of marine invertebrates and mammals, Ca2+ signalling has been intensely studied, whereas for fish little is known. In sea urchin sperm, a cyclic nucleotide-gated K+ channel (CNGK) mediates a cGMP-induced hyperpolarization that evokes Ca2+ influx. Here, we identify in sperm of the freshwater fish Danio rerio a novel CNGK family member featuring non-canonical properties. It is located in the sperm head rather than the flagellum and is controlled by intracellular pH, but not cyclic nucleotides. Alkalization hyperpolarizes sperm and produces Ca2+ entry. Ca2+ induces spinning-like swimming, different from swimming of sperm from other species. The “spinning” mode probably guides sperm into the micropyle, a narrow entrance on the surface of fish eggs. A picture is emerging of sperm channel orthologues that employ different activation mechanisms and serve different functions. The channel inventories probably reflect adaptations to species-specific challenges during fertilization. https://doi.org/10.7554/eLife.07624.001 eLife digest Mammalian sperm cells fertilize egg cells inside the female’s body; while for fish and other marine animals it is common for fertilization to take place outside in the environment. In general, sperm cells become attracted to egg cells by various chemical or physical signals. Sperm detect these cues and generate electrical signals that control their own movements and eventually guide sperm to the egg. In 2009, researchers identified a potassium ion channel, called CNGK, that starts the electrical signal in the sperm cells of sea urchins. This channel is activated by signalling molecules inside cells, called ‘cyclic nucleotides’, and its activity ultimately leads to calcium ions flowing into the sperm cell’s tail. This influx of calcium ions in turn controls the beating of the tail and, thereby, steers the sperm cell towards the egg. It was previously thought that this CNGK channel is found only in animals without a backbone (i.e. in invertebrates). However, Fechner et al. – including some of the researchers involved in the 2009 work – now report that the CNGK channel also exists in the sperm cells of a freshwater fish, the zebrafish. Unexpectedly, the CNGK channel is located in the heads of this fish’s sperm cells rather than in the tails. Electrophysiological experiments then revealed that the fish version of CNGK is not activated by cyclic nucleotides, but is activated when the inside of the cell becomes more alkaline. In zebrafish sperm, a more alkaline pH inside the cell causes calcium ions to flow in and this influx of calcium ions triggers a unique spinning-like swimming movement that is different from the swimming of other sperm from other species. Fechner et al. suspect that this unusual swimming behaviour guides sperm through a small hole in the protective coating of fish eggs, which eventually leads to fertilization. These findings suggest that while channel proteins found in sperm cells from different species look similar and serve similar roles, they are activated in ways that can be very different. https://doi.org/10.7554/eLife.07624.002 Introduction Fertilization is a complex task that, for different species, happens in entirely different spatial compartments or ionic milieus. In aquatic habitats, gametes are released into the water where sperm acquire motility and navigate to the egg. By contrast, mammalian fertilization happens in confined compartments of the female oviduct. From invertebrates to mammals, sperm use various sensing mechanisms, including chemotaxis, rheotaxis, and thermotaxis, to gather physical or chemical cues to spot the egg. These sensory cues activate various cellular signalling pathways that ultimately control the intracellular Ca2+ concentration ([Ca2+]i) and, thereby, the flagellar beat and swimming behaviours (Alvarez et al., 2012; Darszon et al., 2008; Eisenbach and Giojalas, 2006; Florman et al., 2008; Guerrero et al., 2010; Ho and Suarez, 2001; Kaupp et al., 2008; Publicover et al., 2008). In species as phylogenetically distant as sea urchin and mammals, these pathways target a sperm-specific, voltage-dependent Ca2+ channel, called CatSper. Signalling events open CatSper by shifting its voltage-dependence to permissive, more negative Vm values. This shift is achieved by different means. In sea urchin sperm, opening of a K+-selective cyclic nucleotide-gated channel (CNGK) causes a transient hyperpolarization (Bönigk et al., 2009; Strünker et al., 2006); the hyperpolarization activates a sperm-specific Na+/H+ exchanger (sNHE) (Lee, 1984, 1985; Lee and Garbers, 1986) resulting in a long-lasting alkalization that shifts the voltage dependence of CatSper and leads to a Ca2+ influx (Kaupp et al., 2003; Seifert et al., 2015). By contrast, in human sperm, the shift is achieved by direct stimulation of CatSper with prostaglandins and progesterone in the seminal fluid or the oviduct (Brenker et al., 2012; Lishko et al., 2011; Smith et al., 2013; Strünker et al., 2011). Navigation of fish sperm and the underlying signalling pathways must be arguably different. First, teleost fish are lacking CatSper channels (Cai and Clapham, 2008), although activation of sperm motility requires Ca2+ influx (Alavi and Cosson, 2006; Billard, 1986; Cosson et al., 2008; Morisawa, 2008; Takai and Morisawa, 1995) stimulated by hyper- or hypoosmotic shock after spawning into seawater or freshwater, respectively (Alavi and Cosson, 2006; Cherr et al., 2008; Krasznai et al., 2000; Morisawa, 2008; Vines et al., 2002). Therefore, Ca2+ signalling in fish sperm must involve molecules different from those in marine invertebrates and mammals. Second, the ionic milieu seriously constrains ion channel function. Sperm of freshwater fish, marine invertebrates, and mammals are facing entirely different ionic milieus. K+ and Na+ concentrations in freshwater are extremely low (70 μM and 200 µM, respectively) compared to the orders-of-magnitude higher concentrations in seawater or the oviduct (Alavi and Cosson, 2006; Hugentobler et al., 2007). Furthermore, [Ca2+] in seawater is high (10 mM), whereas in freshwater it is low (< 1 mM). The low salt concentrations in freshwater probably require distinctively different ion channels. In fact, none of the ion channels controlling electrical excitation and Ca2+ signalling of fish sperm are known. Finally, fish sperm are not actively attracted to the whole egg from afar by chemical or physical cues, i.e. chemotaxis, thermotaxis, or rheotaxis (Cosson et al., 2008; Morisawa, 2008; Yanagimachi et al., 2013). Instead, many fishes deposit sperm directly onto the eggs. For fertilization, sperm must search for the narrow entrance to a cone-shaped funnel in the egg coat – the micropyle – that provides access to the egg membrane. Sperm reach the micropyle probably by haptic interactions with tethered molecules that line the egg surface and the opening or interior of the micropyle (Iwamatsu et al., 1997; Ohta and Iwamatsu, 1983; Yanagimachi et al., 2013). At surfaces, sperm swim with their flagellum slightly inclined, which pushes the head against the wall and stabilizes sperm at the surface (Denissenko et al., 2012; Elgeti et al., 2010). Thus, fish sperm motility might be governed by specific hydrodynamic and haptic interactions with the egg surface and the micropyle. Although the principal targets of a CNGK-mediated hyperpolarization – the Na+/H+ exchanger and CatSper – are absent in fish, vertebrate orthologues of the sperm CNGK channel are present in various fish genomes (Figure 1A). Here, we study the function of the CNGK channel in sperm of the freshwater fish Danio rerio (DrCNGK). The DrCNGK channel constitutes the principal K+ channel in D. rerio sperm. Unexpectedly, cyclic nucleotides neither regulate CNGK channel activity nor sperm motility; instead, intracellular alkalization, a key mechanism to control sperm function in many species, strongly activates CNGK and, thereby, triggers a Ca2+ signal and a motility response. Although its mechanism of activation is entirely different compared to sea urchin sperm, the principal CNGK function, namely to provide a hyperpolarization that triggers a Ca2+ signal, is conserved. Our results show that sperm signalling among aquatic species shows unique variations that probably represent adaptations to vastly different ionic milieus and fertilization habits. Figure 1 with 3 supplements see all Download asset Open asset Identification of DrCNGK channel homologues and of a K+ channel in D. rerio sperm. (A) Phylogenetic tree (Page, 1996) of various ion channel families. The CNGK channel family exists in protozoa (dark blue), marine invertebrates and fish (medium blue), and freshwater fish (light blue). The HCN, CNG, and KCNH channel families are highlighted in green; voltage-gated Nav and Cav channels are highlighted in yellow; and voltage-gated Kv channels are highlighted in red. The following ion channel sequences were used: CNGK channels from zebrafish (DrCNGK), rainbow trout (OmCNGK), spotted gar (LoCNGK), West Indian Ocean coelacanth (LcCNGK), sea urchin (ApCNGK), acorn worm (SkCNGK), amphioxus (BfCNGK), starlet sea anemone (NvCNGK), vasa tunicate (CiCNGK), sponge (AqCNGK), choanoflagellate (SrCNGK); murine HCN channel subunits 1 (mHCN1), 2 (mHCN2), 3 (mHCN3), 4 (mHCN4), and the HCN channel from sea urchin (SpHCN1); rat CNGA subunits A1 (rCNGA1), A2 (rCNGA2), A3 (rCNGA3), and A4 (rCNGA4); the KCNH channels from fruit fly (DmEAG) and human (hERG); murine voltage-gated Nav (mNav 1.1 and mNav 1.6) and Cav channels (mCav1.1, mCav2.3 and mCav3.1) and voltage-gated Kv channels from fruit fly (DmShaker) and mouse (mKv3.1). Full-length Latin names and accession numbers are given in experimental procedures. Scale bar represents 0.1 substitutions per site. (B) Pseudo-tetrameric structure of CNGK channels. Numbers 1 to 4, homologous repeats; S1 to S6, transmembrane segments; yellow cylinders, cyclic nucleotide-binding domain CNBD; asterisks, epitopes recognized by antibodies anti-repeat1 of DrCNGK (polyclonal) and anti-repeat3 of DrCNGK (YENT1E2, monoclonal). (C) Whole-cell recordings from zebrafish sperm at low (left upper panel) and high (middle panel) extracellular K+ concentrations. Left lower panel: Voltage step protocol. Right panel: corresponding IV relations. (D) Whole-cell recordings from an isolated sperm head. Description see part C. (E) Whole-cell recording from zebrafish sperm (upper panel) and an isolated head (lower panel). (F) IV relation of recordings from part E. (G) Pooled IV relations ( ± sd) of currents from zebrafish sperm (filled circle, n = 23) and sperm heads (open squares, n = 6). https://doi.org/10.7554/eLife.07624.003 Results In several genomes, we identified genes encoding putative cyclic nucleotide-gated K+ channels (CNGK) (Figure 1A). CNGK channels are primarily present in marine invertebrates, yet absent in the genomes of vertebrates such as birds, amphibians, and mammals, except freshwater fish and coelacanths. The CNGKs of freshwater fish appear to form a phylogenetic sub-group on their own (Figure 1A). Moreover, the CNGK channel exists in the unicellular choanoflagellate (Salpingocea rosetta), the closest living relative of animals (Levin and King, 2013; Umen and Heitman, 2013). CNGK channels feature a chimeric structure. Their overall four-repeat pseudotetrameric architecture is reminiscent of voltage-dependent Nav and Cav channels, whereas the pore carries the canonical GYG or GFG motif of K+-selective channels (Figure 1B and Figure 1—figure supplement 1). Furthermore, CNGK channels are phylogenetic cousins of cyclic nucleotide-gated (CNG) channels and of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels (Figure 1A); each of the four repeats harbours a cyclic nucleotide-binding domain (CNBD) (Figure 1B). In fact, the CNGK channel of sea urchin sperm is activated at nanomolar cGMP concentrations (Bönigk et al., 2009). Identification of a K+ current in sperm of D. rerio We recorded currents from whole D. rerio sperm (Figure 1—figure supplement 2, left panel) in the whole-cell patch-clamp configuration. Voltage steps from a holding potential of -65 mV evoked slightly inwardly rectifying currents (Figure 1C). Two pieces of evidence established that currents are carried by K+ channels and not by Cl- channels. The reversal potential (Vrev) shifted from -77 ± 3 mV (n = 18) at 5.4 mM extracellular [K+]o to -7 ± 1 mV (n = 7) at 140 mM [K+]o (△Vrev = 51 ± 3 mV/log [K+], n = 7) (Figure 1C, Figure 1—figure supplement 3C). Changing the intracellular [Cl-] did not affect Vrev (Figure 1—figure supplement 3A,B). These results demonstrate that the current is predominantly carried by K+ ions. To localize the underlying K+ channel, we recorded currents from isolated sperm heads (Figure 1D-G, Figure 1—figure supplement 2, middle panel). Head and whole-sperm currents displayed a similar K+ dependence (△Vrev = 52 ± 2 mV/log [K+], n = 6) (Figure 1D), rectification (Figure 1F,G), and amplitude (Figure 1F,G), suggesting that the underlying K+ channel is primarily located in the head. This result is unexpected, as ion channels involved in sperm signalling are usually localized to the flagellum. To test whether the DrCNGK channel is also localized to the head, we used Western blot analysis and immunocytochemistry. To this end, the DrCNGK protein was first characterized by heterologous expression in mammalian cell lines. DrCNGK constructs with a C-terminal HA-tag or with two tags, a C-terminal HA-tag and an N-terminal flag-tag, were expressed in CHOK1 cells (Figure 2A). In Western blots, the anti-HA-tag and the anti-flag-tag antibody labelled proteins of the same apparent molecular mass (Mw) (174 ± 4 kDa (n = 13) and 175 ± 4 kDa (n = 3), respectively) (Figure 2A). The Mw is smaller than the predicted Mw of 244.4 kDa. Because flag-tag and HA-tag antibodies recognized the N- and C-terminal end of the CNGK protein, respectively, we conclude that the 175-kDa band represents the full-length protein that, however, displays an abnormal electrophoretic mobility similar to other CNG channels (Körschen et al., 1995; 1999). Figure 2 Download asset Open asset Localization of the DrCNGK channel. (A) Western blot of membrane proteins (15 µg) from CHOK1 cells transfected with cDNA encoding either DrCNGK with a C-terminal HA-tag alone (lane C) or with both, a C-terminal HA-tag and an N-terminal flag-tag (N/C). Apparent molecular weight Mw is indicated on the left. (B) Characterization of anti-DrCNGK antibodies. Left: Western blot of membrane proteins (10 µg) from HEK293 cells transfected with cDNA encoding DrCNGK (Tr) and wild-type cells (wt). Right: Western blot of membrane proteins (15 µg) from zebrafish testis. (C) Western blot of membrane proteins (15 µg) from different zebrafish tissues. (D) Upper panel: Scheme of a testis cross-section. GC, germinal compartment; IC, intertubular compartment; SER, Sertoli cells; SGA, primary spermatogonia; SGB, secondary spermatogonia; SC, spermatocytes; ST, spermatids; scheme according to (Nobrega et al., 2009). Lower panel: Staining with anti-repeat1 antibody (red, left) and superposition (right) of the immunohistochemical image with a bright-field image of an in situ hybridization using an anti-DrCNGK-specific RNA probe (arrows). Bar represents 50 µm. (E) Staining of zebrafish sperm with anti-repeat1 (upper left panel) and anti-repeat3 antibody (lower left panel). Bars represent 10 µm. The respective bright-field images are shown (upper and lower right panels). (F) Western blot of equal amounts of total membrane proteins (15 µg) from purified heads and purified flagella. https://doi.org/10.7554/eLife.07624.007 Figure 2—source data 1 Indicators of merit for the mass spectrometric results of a testis preparation. a m indicates oxidized methionine, b△M [ppm] relative mass error, c XCorr, △Score, and △Cn indicate results based on searches with the sequest algorithm in Proteome Discoverer; d PEP (Posterior Error Probability) describes the probability that the observed hit is a chance event. https://doi.org/10.7554/eLife.07624.008 Download elife-07624-fig2-data1-v2.xlsx Figure 2—source data 2 Indicators of merit for the mass spectrometric results of different sperm preparations: whole sperm, head and flagella. a m indicates oxidized methionine, b△M [ppm] for all peptides is below ± 2.5, c XCorr indicates results based on searches with the sequest algorithm; Amanda indicates results based on searches with the MS Amanda algorithm, d PEP (Posterior Error Probability) describes the probability that the observed hit is a chance event; △Score and △Cn are not indicated separately since the scores for all peptides are 1 and 0, respectively. https://doi.org/10.7554/eLife.07624.009 Download elife-07624-fig2-data2-v2.xlsx We raised two antibodies against epitopes in repeat 1 and 3 of the DrCNGK protein (Figure 1B, asterisks). Both antibodies labelled membrane proteins of about 170 kDa in Western blots of DrCNGK-expressing CHOK1 cells, D. rerio testis, and sperm, but not of heart, brain, ovaries, and eyes (Figure 2B,C). To scrutinize the antibody specificity, we analyzed by mass spectrometry the ~170-kDa protein band from testis, mature whole sperm, isolated heads, and isolated flagella; 7, 23, 18, and 15 proteotypic DrCNGK peptides were identified, respectively (Figure 1—figure supplement 1, Figure 2—source data 1,2). Peptides covered almost the entire polypeptide sequence (Figure 1—figure supplement 1). The presence of DrCNGK in testis was confirmed by immunohistochemistry and in situ hybridization of D. rerio testis slices. The anti-repeat1 antibody labelled structures, most likely sperm, in the lumen of testicular compartments (Figure 2D, bottom left). An antisense RNA probe stained sperm precursor cells, in particular spermatocytes (Figure 2D, bottom right), but almost no primary or secondary spermatogonia. Finally, the anti-repeat1 and anti-repeat3 antibodies intensely labelled the head and, to a lesser extent, the flagellum of single sperm cells (Figure 2E). In Western blots of isolated heads and flagella, the DrCNGK was readily identified in head preparations, yet was barely detectable in flagella preparations (Figure 2F, n = 4). In summary, the CNGK channel is located primarily in the head of mature D. rerio sperm. The DrCNGK channel is not sensitive to cyclic nucleotides The sea urchin ApCNGK channel is opened by cyclic nucleotides and mediates the chemoattractant-induced hyperpolarization (Bönigk et al., 2009; Strünker et al., 2006). Unexpectedly, patch-clamp recordings of K+ currents from D. rerio sperm required no cyclic nucleotides in the pipette (Figure 1C-G). Therefore, we scrutinized the action of cyclic nucleotides on sperm K+ currents. Mean current amplitudes were similar in controls and in the presence of either or cGMP in the pipette (Figure We used also cyclic nucleotides to study the K+ current in the and presence of cyclic nucleotides in the same sperm cell (Kaupp et al., of or cGMP from did not affect K+ suggesting that cyclic nucleotides not DrCNGK (Figure In contrast, the of or cGMP a current in expressed cyclic nucleotide-gated channels ApCNGK from sea urchin (Figure supplement We also expressed the DrCNGK channel in The relation (Figure K+ dependence (Figure supplement and by (Figure supplement was similar to that of the K+ current recorded from D. rerio sperm. Moreover, currents in were also to the and (Figure whereas with currents in that the ApCNGK channel from sperm (Figure supplement Figure 3 with supplements see all Download asset Open asset nucleotides not activate K+ channels in sperm. (A) amplitude of whole-cell recordings from zebrafish sperm at mV in the or presence of or cGMP in the pipette ± (n = ± (n = ± (n = data and ± of experiments in (B) of cyclic nucleotides from inside sperm. Left panel: Whole-cell recordings at mV from sperm with (upper panel) or cGMP (lower panel). indicate the of the to cyclic nucleotides by Right panel: Mean current 3 and 3 after the of or as in part from sperm are indicated by of expressed DrCNGK channels in the or presence of of cyclic nucleotides. (C) Left: recordings from shown are in the (left and presence of 10 mM Voltage steps as shown in Figure supplement Right: IV relations of current recordings from the left (D) Pooled IV from DrCNGK and control recordings in the and presence of 10 mM (E) Left: recordings from DrCNGK shown are in the (left and presence of 10 mM Right: IV relations of current recordings from the left (F) Pooled IV from and control recordings in the and presence of 10 mM (G) and after of (left panel) or cGMP panel). The indicates the swimming of cyclic nucleotides was by the of of the (Figure supplement et al., and after of or Sperm were with or as in part cyclic nucleotides been used to study sperm motility in sea urchin et al., et al., 2012; et al., and et al., 2009). We D. rerio sperm motility and after of (Figure left) or cGMP (Figure from The was by the of of the (Figure supplement (Bönigk et al., 2009). i.e. (Figure and swimming (Figure supplement were not by that neither nor cGMP a in the control of sperm In we no action of cyclic nucleotides on the DrCNGK channel and on the swimming behaviour of D. rerio sperm. of CNGK channels in zebrafish and sea urchin sperm is different We a rectification of currents carried by sea urchin ApCNGK compared to zebrafish DrCNGK (Figure and, the of this In K+ channels, by intracellular et al., or et al., 1995) produces However, neither nor ApCNGK rectification (Figure Instead, intracellular Na+ currents in a and (Figure In the of the IV relation of ApCNGK and DrCNGK channels (Figure We the pore of CNGK channels for the molecular of the Na+ In of the four ApCNGK pore we identified a that in most K+ channels is by a or (Figure these were to the rectification of the ApCNGK channel was and the IV relation similar to that of the DrCNGK channel (Figure We also the into the pore motif of DrCNGK channels. For the did not form channels. Figure 4 Download asset Open asset of sperm K+ current with current from expressed ApCNGK channels. (A) IV relations of whole-cell recordings from zebrafish sperm and ApCNGK channels expressed in HEK293 were to at (B) IV relations current ± n = 6) of recordings from ApCNGK channels expressed in HEK293 with the indicated concentrations of and were to at (C) of pore from different CNGK channels. fishes are highlighted in and seawater species in The of the is given on the indicate the A key that is in repeats of the ApCNGK channel and other seawater species is highlighted in at this are indicated in (D) IV relations of recordings of ApCNGK channels expressed in HEK293 Na+ concentrations were to the (E) IV relations current ± sd) of whole-cell recordings from zebrafish sperm (n = 18) and from channels (n = 7) expressed in HEK293 were to at sequence of the pore of the ApCNGK channels were activated with the are absent in CNGKs of freshwater yet present in seawater except for the sponge (Figure suggesting that the different CNGK represent adaptations to vastly different ionic milieus. The DrCNGK channel is controlled by intracellular pH pH is an controlling sperm motility in marine invertebrates and mammals (Alavi and Cosson, and 2013; et al., 2013; Lishko et al., 2010; Lishko and 2010; et al., et al., Seifert et al., 2015). Moreover, in mouse sperm, the channels and the CatSper channels are et al., 2006; et al., et al., 2011; 2013; et al., Therefore, we whether DrCNGK is controlled by At almost no CNGK current was recorded from D. rerio sperm (Figure left To that an of Ca2+ to see Materials and to the of at pH is for current we recorded sperm currents at pH with a Ca2+ concentration of 1 (Figure supplement these the K+ current in sperm was that and not at for the concentrations are for current sperm to 10 mM (Figure the of at pH and Seifert et al., Strünker et al., 2011). strongly CNGK currents (Figure middle with 10 mM which the CNGK currents (Figure right and The action was very 1 mM activated of the CNGK at 10 the current was (Figure To the pH we recorded sperm K+ currents at different intracellular (Figure The current is activated at pH The pH dependence to the action by the data of the two different experimental (Figure For at an concentration of in sperm from to (Figure alkalization of D. rerio sperm with 10 mM evoked a and hyperpolarization from ± mV to ± 4 mV (Figure n = We also the pH of the DrCNGK channel expressed in were first with a K+ by a K+ including 1 mM (Figure supplement 1). 1 mM DrCNGK currents by by (Figure