A K+-selective CNG channel orchestrates Ca2+ signalling in zebrafish sperm

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. DOI: http://dx.doi.org/10.7554/eLife.07624.001


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 Ca 2+ concentration ([Ca 2+ ] i ) and, thereby, the flagellar beat and swimming behaviours 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 Ca 2+ channel, called CatSper. Signalling events open CatSper by shifting its voltage-dependence to permissive, more negative V m 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 (Bö nigk et al., 2009;Strünker et al., 2006); the hyperpolarization activates a sperm-specific Na + /H + exchanger (sNHE) (Lee, 1984(Lee, , 1985Lee and Garbers, 1986) resulting in a long-lasting alkalization that shifts the voltage dependence of CatSper and leads to a Ca 2+ influx 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).
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 mM and 200 mM, respectively) compared to the orders-ofmagnitude higher concentrations in seawater or the oviduct (Alavi and Cosson, 2006;Hugentobler et al., 2007). Furthermore, [Ca 2+ ] 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 Ca 2+ 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 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.   (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). Figure 1 continued on next page 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 Ca 2+ 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 Ca 2+ 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.

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 Na v and Ca v 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 (Bö nigk et al., 2009).
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 (M w ) (174 ± 4 kDa (n = 13) and 175 ± 4 kDa (n = 3), respectively) ( Figure 2A). The M w is smaller than the predicted M w 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 (Kö rschen et al., 1995;1999).
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 thẽ 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 chemoattractantinduced hyperpolarization (Bö nigk 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 cAMP or cGMP (100 mM) in the pipette solution ( Figure 3A). We used also caged cyclic nucleotides to study the K + current in the absence and presence of cyclic nucleotides in the same sperm cell . Photorelease of cAMP or cGMP from caged precursors did not affect K + currents, suggesting that cyclic nucleotides do not modulate DrCNGK ( Figure 3B). In contrast, the photo-release of cAMP or cGMP induced a rapid current increase in heterologously expressed cyclic nucleotide-gated channels ApCNGK from sea urchin ( Membrane-permeant caged cyclic nucleotides have successfully been used to study sperm motility in sea urchin (Bö hmer et al., 2005;Kashikar et al., 2012;Wood et al., 2005) and humans (Gakamsky et al., 2009). We studied D. rerio sperm motility before and after photo-release of

Rectification of CNGK channels in zebrafish and sea urchin sperm is different
We noticed a much stronger rectification of currents carried by sea urchin ApCNGK compared to zebrafish DrCNGK ( Figure 4A) and, therefore, investigated the origin of this pronounced difference. In classical K + channels, block by intracellular Mg 2+ (Matsuda et al., 1987) or spermine (Fakler et al., 1995) produces inward rectification. However, neither Mg 2+ nor spermine affected ApCNGK rectification ( Figure 4B). Instead, intracellular Na + blocked outward currents in a strong voltage-and dose-dependent fashion ( Figure 4B,D). In the absence of Na + , the IV relation of ApCNGK and DrCNGK channels converged ( Figure 4A,B). We searched the pore regions of CNGK channels for clues regarding the molecular basis of the Na + block. In three of the four ApCNGK pore motifs, we identified a Thr residue that in most K + channels is replaced by a Val or Ile residue ( Figure 4C). When these Thr residues were changed to Val, the strong rectification of the mutant ApCNGK channel was lost and the IV relation became similar to that of the DrCNGK channel ( Figure 4E). We also tested the reverse construct, introducing Thr residues into the pore motif of DrCNGK channels. For unknown reasons, the mutants did not form functional channels.
Of note, the Thr residues are absent in CNGKs of freshwater organisms yet present in seawater organisms except for the sponge AqCNGK ( Figure 4C), suggesting that the different CNGK pores represent adaptations to vastly different ionic milieus.
The DrCNGK channel is controlled by intracellular pH Intracellular pH (pH i ) is an important factor controlling sperm motility in marine invertebrates and mammals (Alavi and Cosson, 2005;Dziewulska and Domagala, 2013;Hirohashi et al., 2013;Nishigaki et al., 2014;Santi et al., 1998;Seifert et al., 2015). Moreover, in mouse sperm, the Slo3 channels and the CatSper channels are exquisitely pH-sensitive (Kirichok et al., 2006;Schreiber et al., 1998;Zeng et al., 2011;Zhang et al., 2006a;2006b). Therefore, we examined whether DrCNGK is controlled by pH i . At pH i 6.4, almost no CNGK current was recorded from D. rerio sperm ( Figure 5A, left panel, B,C). To rule out that an increase of Ca 2+ (from 91 pM to 7.7 nM, see Materials and methods) due to the reduced buffering capacity of EGTA at pH 6.4 is responsible for current inhibition, we recorded sperm currents at pH 7.4 with a free Ca 2+ concentration of 1 mM ( Figure 5-figure supplement 2). Under these conditions, the K + current in sperm was still large, indicating that protons and not Ca 2+ , at least for the concentrations tested, are responsible for current inhibition. Exposing sperm to 10 mM NH 4 Cl rapidly elevates pH i ( Figure 5G), because NH 4 Cl overcomes the buffer capacity of HEPES   at pH 6.4 (Boron and De Weer, 1976;Seifert et al., 2015;Strünker et al., 2011). Alkaline pH i strongly enhanced CNGK currents ( Figure 5A, middle panel, B,C). Subsequent superfusion with 10 mM propionic acid, which lowers pH i , completely reversed the NH 4 Cl-induced CNGK currents ( Figure 5A, right panel, B and C). The NH 4 Cl action was very pronounced: 1 mM activated approximately 40% of the CNGK current; at 10 mM, the current was maximal ( Figure 5D, triangles). To quantitatively determine the pH dependence, we recorded sperm K + currents at different intracellular pH i values ( Figure 5C,D circles). The current is half-maximally activated at pH 7.08. The pH dependence allows to calibrate the NH 4 Cl action by superposing the data of the two different experimental conditions ( Figure 5D): For example, at an NH 4 Cl concentration of 1.5 mM, pH i in sperm will increase from 6.4 to 7.08 ( Figure 5D). Under currentclamp conditions, alkalization of D. rerio sperm with 10 mM NH 4 Cl evoked a rapid and reversible hyperpolarization from -49 ± 7 mV to -71 ± 4 mV ( Figure 5E, n = 10). We also studied the pH regulation of the DrCNGK channel expressed in Xenopus oocytes. Oocytes were first perfused with a K + bicarbonate solution, followed by a K + gluconate-based solution including 1 mM NH 4 Cl ( Figure 5-figure supplement 1). 1 mM NH 4 Cl reversibly increased DrCNGK currents by approximately 71%, demonstrating regulation by pH i ( Figure 5F). Higher concentrations of NH 4 Cl further increased the DrCNGK current; however, these conditions also elicited significant currents in control oocytes, thus precluding quantitative analysis. Furthermore, we tested, whether DrCNGK channels in oocytes are activated by hypoosmotic conditions. Reducing the osmolarity by~50% does not significantly change DrCNGK currents in oocytes ( Figure 5-figure supplement 3, n = 4). In sea urchin sperm, CNGK-mediated hyperpolarization leads to an increase of intracellular Ca 2+ . Therefore, we tested, whether the NH 4 Cl-induced hyperpolarization ( Figure 5E) and alkalization ( Figure 5G) evokes a Ca 2+ response. In fact, mixing of D. rerio sperm with 10 or 30 mM NH 4 Cl gave rise to a rapid Ca 2+ signal ( Figure 5H, n = 4). The time course of the pH i -and Ca 2+ signal was similar, suggesting that the CNGK-mediated hyperpolarization triggers a Ca 2+ influx. We conclude that DrCNGK represents a pH-sensitive channel that is strongly activated at alkaline pH i ; the ensuing hyperpolarization, like in sea urchin sperm, produces a Ca 2+ signal.

Ca 2+ controls swimming behaviour of D. rerio sperm
We studied the role of Ca 2+ for motility of D. rerio sperm using photo-release of Ca 2+ from caged Ca 2+ (NP-EGTA). Sperm motility was activated by hypoosmotic dilution (1:20 into 70 mM Na + ES, 167 mOsm x L -1 ) and was followed under a dark-field microscope ( Figure 6, Video 1). Unstimulated sperm swam on curvilinear trajectories of low curvature ( Figure 6A-F, green segment, Video 1). A UV flash almost instantaneously increased path curvature in NP-EGTA-loaded sperm ( Figure 6D-F,I), but not in control sperm ( Figure 6A-C,I); sperm swam on much narrower arcs ( Figure 6D-F, red segment, Video 1). The increase of path curvature was even more pronounced after a second UV flash ( Figure 6 D-F, cyan segment, I, Video 1). Many cells were pushing against the wall of the observation chamber and performed a 'spinning' or 'drilling' behaviour, as if to penetrate the wall ( Figure 6G). The asymmetry of the flagellar beat increased with each consecutive photo-release of Ca 2+ and eventually the flagellum pointed away from the glass surface ( Figure 6H, Video 1). This swimming behaviour likely represents a strategy followed by sperm on its search for the micropyle, a small opening (outer diameter about 8 mm in diameter) (Hart and Danovan, 1983) on the surface of the much larger egg (about 0.75 mm in diameter) (Selman et al., 1993). A similar swimming behaviour during fertilization has been reported for sperm of herring and black flounder (Cherr et al., 2008;Yanagimachi et al., 1992;. We propose that the spinning or drilling movements observed after Ca 2+ release reflect the swimming behaviour in vivo down the narrow micropyle. Video 1. Behavioural response of zebrafish sperm to successive Ca 2+ release. Representative recording of zebrafish sperm loaded with NP-EGTA (40 mM). Upon release of Ca 2+ , the swimming path curvature increases and, eventually, sperm spin against the wall of the recording chamber with their flagellum pointing away from the wall. Video recorded using dark-field microscopy at 30 frames per second using a 20x magnification objective. The field of view corresponds to 410 mm. The Video is shown in real time. DOI: 10.7554/eLife.07624.022

Discussion
A growing body of evidence reveals unexpected commonalities, but also notable differences among sperm from different species (for review (Darszon et al., 2006;Kaupp et al., 2008;Yoshida and Yoshida, 2011). Organisms, as phylogenetically distant as sea urchins and humans, share the CatSper channel as a common site of Ca 2+ entry into the sperm flagellum. By the same token, Slo3 K + channel orthologues in mouse and human sperm evolved different selectivity for intracellular ligands and might serve different functions (Brenker et al., 2014;Chavez et al., 2013;Lishko et al., 2012;Santi et al., 2009;Zeng et al., 2013). Here, we characterize a novel variant of CNGK channels in zebrafish sperm, whose key features depart from those of CNGK channels of marine invertebrates (Figure 7).
First, although the D. rerio CNGK carries four canonical CNBDs, it is gated by pH i rather than cyclic nucleotides, indicating that the CNBDs have lost their genuine ligand selectivity. The related ApCNGK channel from sea urchin sperm is also unique in that it displays an unusual cGMP dependence: Unlike "classic" cooperative CNG channels, it is gated by binding of a single cGMP molecule to the third CNBD, implying that the other CNBDs are non-functional (Bö nigk et al., 2009). Because the ApCNGK channel is activated through binding of cGMP to the third repeat, we searched for sequence alterations in the third repeat of the DrCNGK channel. Strikingly, in the C-linker region of the third repeat, we identified an insert of 42 amino-acid residues (Figure 1-figure supplement 1, blue) that is absent in other cyclic nucleotide-regulated channels. The insert shows no sequence similarity to any known functional domain of ion channels. One hypothesis is that this insert prevents the transmission of the binding signal to the channel pore. Another sequence peculiarity is identified in Figure 7. Models of signalling pathways in sea urchin and zebrafish sperm. Sea urchin (upper panel): Binding of the chemoattractant resact to a receptor guanylyl cyclase (GC) activates cGMP synthesis. Cyclic GMP opens K + -selective CNG channels (CNGK), thereby, causing a hyperpolarization, which in turn activates a sperm-specific Na + /H + exchanger (sNHE) that alkalizes the cell. Alkalization and subsequent depolarization by hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels lead to the opening of sperm-specific CatSper channels. Zebrafish (lower panel): Upon spawning, K + efflux through CNGK hyperpolarizes sperm. An unknown mechanism of alkalization (dashed lines) modulates the open probability of CNGK channels; the ensuing hyperpolarization opens voltage-gated Ca 2+ channels (Ca v ). DOI: 10.7554/eLife.07624.023 the second repeat (Figure 3-figure supplement 2). At amino-acid position 934, an Ala residue replaces a highly conserved Arg residue that is crucial for cyclic-nucleotide binding (Kaupp and Seifert, 2002). The CNBDs of repeat 1 and 4 do not show obvious sequence abnormalities and could represent bona fide CNBDs (Figure 3-figure supplement 2). In recent years, many structures of CNBDs have been solved (Clayton et al., 2004;Kesters et al., 2015;Kim et al., 2007;Rehmann et al., 2003;Schünke et al., 2011;Schünke et al., 2009;Zagotta et al., 2003). Can we learn from these structures something about the DrCNGK channel? Most of the CNBD structures feature a similar fold and are able to bind both cAMP and cGMP. However, for some CNG channels, ligands are full agonists, like cAMP and cGMP in the CNGA2 channel (Dhallan et al., 1990), only partial agonists, like cAMP in the CNGA1 channel (Altenhofen et al., 1991), or competitive antagonists, like cGMP in the bacterial SthK channel (Brams et al., 2014). Finally, in HCN channels, CNBDs interact and form a so-called gating ring (Zagotta et al., 2003), whereas in MloK1 channel, CNBDs do not interact at all (Cukkemane et al., 2007;Schünke et al., 2009;. In conclusion, at present, no sequence features can be identified that unequivocally explain the lack of cyclic-nucleotide regulation of the DrCNGK channel. The insensitivity of the DrCNGK channel to cyclic nucleotides is, however, reminiscent of EAG and hERG channels that carry classic CNBDs, yet are not gated by cyclic nucleotides (Brelidze et al., 2009;. Instead, small molecules such as flavonoids have been suggested as ligands that bind to the CNBD and modulate channel activity (Brelidze et al., 2010;Carlson et al., 2013). Moreover, in the C-terminus of these CNBDs, a conserved segment of residues was identified that occupies the CNBD and serves as an intrinsic "ligand" (Brelidze et al., 2012;Carlson et al., 2013). We can only speculate, that, in addition to protons, as yet unidentified ligands might bind to and regulate the DrCNGK channel. The apparent pK a value for channel activation by pH was approximately 7, suggesting that a His residue controls channel opening. There are a number of His residues in the C-linker of the four repeats that might serve as candidate sites. Future work is necessary to identify the site of pH regulation of the DrCNGK channel.
To take on a new ligand selectivity or activation mechanism is also reminiscent of orthologues of the sperm-specific K + channel Slo3. Whereas the mouse Slo3 channel is exclusively controlled by pH i (Brenker et al., 2014;Schreiber et al., 1998;Yang et al., 2011;Zeng et al., 2011;Zhang et al., 2006a), the human Slo3 is primarily regulated by Ca 2+ (Brenker et al., 2014). In conclusion, the zebrafish CNGK is a striking example for a channel featuring a CNBD that is not gated by cyclic nucleotides. In general, CNBDs might represent sensor domains that can relay information on ligands other than cyclic nucleotides.
Second, signalling pathways that control sperm motility are located to the flagellum: The GC receptor for chemoattractant binding in sea urchin (Bö nigk et al., 2009;Pichlo et al., 2014), the CatSper channel in humans, mice, and sea urchin (Chung et al., 2014;Kirichok et al., 2006;Seifert et al., 2015), the Slo3 K + channel in mice and humans (Brenker et al., 2014;Navarro et al., 2007), and the HCN and the CNGK channel in sea urchin (Bö nigk et al., 2009;Gauss et al., 1998). In contrast, the DrCNGK channel is located in the head rather than the flagellum. What might be the functional significance of such a peculiar location? The CNGK channel probably serves two related functions.
In seminal fluid, sperm of freshwater fish are immotile due to a high [K + ] and high osmolarity. Upon release into hypoosmotic freshwater, sperm become motile for a few minutes (Morisawa et al., 1983;Takai and Morisawa, 1995;Wilson-Leedy et al., 2009). The osmolarityinduced activation hyperpolarizes sperm and induces a Ca 2+ signal (Krasznai et al., 2000). We propose that the CNGK triggers Ca 2+ signalling events upon spawning: In the high-K + seminal fluid, partially open CNGK channels keep sperm depolarized. When exposed to low-K + hypoosmolar conditions, sperm hyperpolarize and, ultimately, Ca 2+ is entering the cell and activates general motility (Figure 7).
Moreover, during the search for the micropyle on the egg surface, the sense of direction might be provided by haptic interaction with tethered molecules that line the opening or the funnel of the micropyle (Iwamatsu et al., 1997;Ohta and Iwamatsu, 1983;Yanagimachi et al., 2013). The haptic interactions could directly control CNGK activity in the head. For example, near or inside the micropyle, the CNGK might become further activated by alkaline pH and initiate the Ca 2+ -dependent 'drilling' behaviour.
On a final note, the study of zebrafish sperm provides insight into adaptive mechanisms of sperm evolution. Boundary conditions might constrain sperm to develop different signalling strategies for similar functions. One obvious constraint is the ionic milieu, which strongly affects ion channel function. In freshwater, ion concentrations are low and opening of Na + -, K + -, and non-selective cation channels would hyperpolarize rather than depolarize cells. We speculate that, in freshwater fish, a depolarization-activated Ca 2+ channel like CatSper may not work and has been replaced by another Ca v channel.
Furthermore, the Thr/Val difference in ApCNGK versus DrCNGK, which determines Na + blockage, probably represents an adaptation to the respective ionic milieu. Na + blockage of sea urchin CNGK resists hyperpolarization in seawater and, thereby, facilitates the opening of depolarizationactivated CatSper channels. The observation that CNGK channels from seawater organisms carry this Thr residue indicates a specific evolutionary pressure on this pore residue. Why is this Thr residue lost in CNGK channels of freshwater organisms? We speculate that Na + blockage disappeared along with the loss of CatSper genes and that Ca 2+ ions enter fish sperm through a Ca 2+ channel that is activated by hyperpolarization rather than depolarization. Future work needs to identify this Ca 2+ channel, its mechanism of activation, and its role for fertilization of teleost fish.
In summary, we identify a zebrafish CNGK channel that is activated at alkaline pH, and is set apart from its cousins of sea urchins that are activated by cGMP. Orthologues of CNGK also exist in the choanoflagellate S. rosetta, suggesting that this channel sub-family is phylogenetically ancient. Interestingly, this protozoon has a sexual life cycle: during anisogamous mating, small flagellated cells fuse with large cells (Levin and King, 2013). This mating behaviour represents the ancestor of sexual reproduction in animals (Levin and King, 2013;Umen and Heitman, 2013). The role of the S. rosetta CNGK channel for sexual reproduction without sperm will be interesting to study.

Cloning of the DrCNGK gene
We identified two putative annotated sequences in the database: Eb934551 and XM_0013354. Eb934551 contained the putative N-terminal region and XM_0013354 contained the putative repeat 3, repeat 4, and parts of repeat 2. Using four sets of primer pairs, we did nested PCR reactions on testis cDNA to obtain the full-length sequence: the primer pairs #4811/#4812 and #4813/#4814 were used for XM_0013354 and the primer pairs C0274/C0275 and C0276/C0277 for Eb934551. The primer sequences were: TATTTCAAGTAGCTGTTACCG (#4811), ACATTCCCTTATAATAAT-GTCC (#4812), AAAAAAGCTAAGCTTTTCAGAAACACAG (#4813), AAAATCTGACAGGTACCCTG-CAGAATGC (#4814), CATACAGGATGCATGACCCC (C0274), CCAGGAATGTATGTGTAGGTC (C0275), GAGGAATTCATGCATGACCCCAGAGAAATGAAG (C0276) and CTCGGATCCGTATGTGT-AGGTCTTTAATTTCAGGG (C0277). Due to failure of expression, we used a codon-optimized version (human codon usage) of the DrCNGK gene separated into three modules (Eurofins Genomics). Each module was flanked by restriction sites. The first module contained bases 1 to 2,108 and was flanked on the 5' end with BamHI and on the 3' end with XbaI; the second module contained bases 2,109 to 4,655 and was flanked on the 5' end with XbaI and on the 3' end with EcoRI; the third module contained bases 4,656 to 6,360 and was flanked on the 5' end with EcoRI and on the 3' end with NotI. At the 3' end, the coding sequence for the hemagglutinin tag (HA-tag) was added. The construct was cloned into the pcDNA3.1 vector (Life technologies) (DrCNGK). To enhance expression levels, we added a QBI SP163 sequence (Stein et al., 1998) in front of the start codon (QBI-DrCNGK).
ApCNGK pore mutants: for the pore mutant ApCNGK-4V, the following amino-acid substitutions in the ApCNGK wild-type channel (Bö nigk et al., 2009) were produced: T252V, T801V and T1986V. Three PCR reactions were required for each mutation: two with the primers containing the point mutation and one recombinant PCR reaction. For the amino-acid exchange T252V, the primer pairs #4433/C0531 and C0530/#4409 were used and for the recombinant PCR, the primer pair #4433/ #4409. The PCR product was cloned into the ApCNGK gene with restriction enzymes BamHI and XhoI. For the amino-acid exchange T802V, primer pairs #4436/C0533 and C0532/#4439 were used and for the recombinant PCR the primer pair #4436/#4439. The PCR product was cloned into the ApCNGK gene with restriction enzymes XhoI and XbaI. For the amino-acid exchange T1986V, the primer pairs #4412/C0535 and C0534/#4447 were used and for the recombinant PCR, the primer pair #4412/#4447. The PCR product was cloned into the ApCNGK gene with restriction enzymes For expression in X. laevis oocytes, the DrCNGK and QBI-DrCNGK constructs were cloned into a modified version of the expression vector pGEMHEnew (Liman et al., 1992); because cloning of DrCNGK was only possible using BamHI and NotI, a NotI restriction site present in pGEMHEnew was removed and a new one was introduced into the multiple-cloning site. This new vector has been named pGEMHEnew-NotI. In vitro transcription to generate cRNA was performed using the mMES-SAGEmMACHINEKit (Ambion); the plasmid was linearized with SpeI.

Preparation of testis, sperm, heads and flagella
Animals were sacrificed according to the "Guidelines for housing and care, transport, and euthanasia of laboratory fishes", ("Empfehlung fü r die Haltung, den Transport und das tierschutzgerechte Tö ten von Versuchsfischen", published by the Tierä rztliche Vereinigung fü r Tierschutz e.V. January 2010). To obtain intact sperm, zebrafish male were anesthetized with MS-222 (0.5 mM, 3 min). After a brief wash with fresh water, the head was quickly separated from the body. The body of the fish was ventrally opened and two testis strands were removed and transferred into ES buffer (see Electrophysiology) or phosphate-buffered saline (PBS) containing (in mM: NaCl 137, KCl 2.7, Na 2 HPO 4 6.5, KH 2 PO 4 1.5, pH 7.4), additionally containing 1.3 mM EDTA, mPIC protease inhibitory cocktail (Sigma-Aldrich), and 1 mM DTT. Sperm were collected with a pipette tip. After 15 min, 80% of the supernatant was transferred into a fresh reaction tube. To separate heads from flagella, the sperm suspension was sheared 30-40 times on ice with a 24 gauge needle (Braun, Bethlehem, PA). The sheared suspension was centrifuged for 10 min (800xg, 4˚C) to sediment intact sperm and sperm heads. This procedure was repeated twice. The purity of flagella preparations was assessed using dark-field microscopy ( Figure 1-figure supplement 2). For testis sections, males were ventrally sliced and kept overnight in 4% paraformaldehyde. After 24 hr, testis strands were removed and embedded in paraffin. Sections (8 mm) were made using a microtome (Leica, Wetzlar, Germany).
For in situ hybridization, tissue was permeabilized with protein kinase K (1 mg/ml in 0.1 M Tris/ HCl, pH 8.0) and hybridized using the DrCNGK-3 antisense probe. After washing, the antibody staining was performed using an anti-Digoxigenin antibody (1:500, Roche) conjugated with alkaline phosphatase. RNA was visualized with a mixture of nitro-blue tetrazolium chloride (500 mg) and 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt (188 mg, Roche). Cross sections were covered with a glass slip. For antibody staining of the in situ hybridization sections, cover slips were removed keeping the slides 5 min in xylene and briefly in PBS. This step was repeated; the fixative was removed from the sections. Afterwards, sections were stained as described for sperm immunocytochemistry. Proteins were probed with antibodies: anti-repeat1 (1:500) or anti-repeat3 (1:10) and visualized with Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories).

Electrophysiology
We electrically recorded from intact zebrafish sperm and from isolated sperm heads using the patch-clamp technique in the whole-cell configuration. Recordings were accomplished within 4 hr after preparation. Seals between pipette and sperm were formed at the neck region in standard extracellular solution (ES). The following pipette solutions were used: standard intracellular solution (IS) (in mM): NaCl 10, K + aspartate 130, MgCl 2 2, EGTA 1, Na 2 ATP 2, and Hepes 10 at pH 8.4, 7.9, 7.4, 6.9, or 6.4 adjusted with KOH; Cl --based IS (in mM): NaCl 10, KCl 130, MgCl 2 2, EGTA 1, Na 2 ATP 2, and Hepes 10 at pH 7.4 adjusted with KOH. The following bath solutions were used: standard ES (in mM): NaCl 140, KCl 5.4, MgCl 2 1, CaCl 2 1.8, glucose 10, and Hepes 5 at pH 7.4 adjusted with NaOH; for K + -based ES solutions, the equivalent amount of Na + was replaced by K + (concentrations are indicated in the Figure legends). Calculations of the free Ca 2+ concentrations were carried out using the Maxchelator program (http://maxchelator.stanford.edu/webmaxc/webmaxcE.htm) assuming a residual Ca 2+ concentration in water of 1 mM. At pH 6.4, [Ca 2+ ] i was 7.7 nM and at pH 7.4, it was 91 pM. To obtain an intracellular solution with 1 mM free Ca 2+ , 1 mM CaCl 2 was added to the IS solution at pH 7.4.
Caged compounds (100 mM BCMACM-caged cAMP or 100 mM BCMACM-caged cGMP) were added to the IS. The final concentration of DMSO was 0.1%. The compounds were photolyzed by ã 1 ms flash of ultraviolet light from a Xenon flash lamp (JML-C2; Rapp OptoElectronic, Wedel, Germany). The flash was passed through a BP295-395 nm filter (Rapp OptoElectronic) and delivered to the patch-clamp chamber in the microscope by a liquid light guide. Pipette resistance in IS/ES was between 11.5 and 15.0 MW. Voltages were corrected for liquid junction potentials.
We recorded ApCNGK and mutant ApCNGK currents from transfected (Lipofectamine 2000, Life technologies) HEK293 cells with the patch-clamp technique in the whole-cell configuration. A HEK293 cell line stably expressing the ApCNGK channel was used for inside-out recordings. The pipette solution for whole-cell recordings was standard IS. Channels were activated with 100 mM cGMP. The pipette solution for inside-out recordings was standard ES. The following bath solutions were used for inside-out recordings: IS-30 NMDG-0Na + solution (in mM): NaCl 0, NMDG 30, KCl 110, EGTA 0.1, Hepes 10 at pH 7.4 adjusted with KOH; IS-NMDG-30Na + solution (in mM): NaCl 30, KCl 110, EGTA 0.1, Hepes 10 at pH 7.4 adjusted with KOH. 30 NMDG-0Na + and 0 NMDG-30Na + solutions were mixed to obtain the desired Na + concentrations. 100 mM Na + -cGMP was added to the bath solution. For the solution with 0 mM Na + , we used 100 mM Na + -free cGMP. Pipette resistance in IS/ES was between 4.0 and 7.0 MW.

Measurement of changes in intracellular Ca 2+ concentration and pH
We measured changes in [Ca 2+ ] i , and pH i in a rapid-mixing device (SFM-4000; BioLogic, Claix, France) in the stopped-flow mode using the Ca 2+ indicator Cal-520-AM (AAT Bioquest, Sunnyvale, CA) or the pH indicator BCECF-AM (Life Technologies). All sperm from a zebrafish male were diluted into 100 ml of ES solution and incubated with either 10 mM Cal-520-AM and 0.5% Pluronic for 120-180 min or 10 mM BCECF-AM for 10 min. Sperm were washed once, diluted 1:20 into ES solution, and loaded into the stopped-flow device. The sperm suspension was rapidly mixed 1:1 (vol/vol) with control ES solution or with ES solution containing NH 4 Cl to obtain final concentrations of 10 mM and 30 mM after mixing. Fluorescence was excited by a SpectraX Light Engine (Lumencor, Beaverton, OR). Cal-520 was excited with a 494/20 nm (Semrock, Rochester, NY), BCECF with a 452/45 nm (Semrock) excitation filter. Emission was recorded by photomultiplier modules (H9656-20; Hamamatsu Photonics). Fluorescence of Cal-520 was recorded using a 536/40 nm (Semrock) emission filter and normalized (without background subtraction) to the value before stimulation. BCECF fluorescence was recorded in the dual emission mode using a 494/20 nm (Semrock) and a 549/15 nm (Semrock) emission filter. The pH i signals represent the ratio of F494/549 and were normalized (without background subtraction) to the value before stimulation. All stopped-flow traces represent the average of 3-6 recordings. The signals were normalized to the first 5-10 data points before the onset of the signal to yield 4F/F and 4R/R, respectively.

Mass spectrometric identification of the DrCNGK channel
Proteins of whole sperm, isolated heads, or flagella were resuspended in an SDS sample buffer and loaded on a SDS gel; after proteins had migrated approximately 1 cm into the separation gel, the gel was stained with Coomassie. The single gel band was excised for every sample, and proteins were in-gel digested with trypsin (Promega, Sunnyvale, CA); peptides were separated by RP-LC (180 min gradient 2-85% acetonitrile, (Thermo Fisher Scientific)) using a nanoAcquity LC System (Waters, Milford, MA) equipped with a HSS T3 analytical column (1.8 mm particle, 75 mm x 150 mm) (Waters) and analyzed twice by ESI-LC-MS/MS, using an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) with a 300-2,000 m/z survey scan at 240,000 resolution, and parallel CID of the 20 most intense precursors from most to least intense (top20) and from least to most intense (bot-tom20) with 60 s dynamic exclusion. All database searches were performed using SEQUEST and MS Amanda algorithm (Dorfer et al., 2014), embedded in Proteome Discoverer (Rev. 1.4, Thermo Electron 2008-2011, Thermo Fisher Scientific), with both a NCBI (26,623 entries, accessed December 20th, 2010) and a Uniprot (40,895 entries, accessed April 24th, 2014) zebrafish sequence protein database, both supplemented with the DrCNGK protein sequence (Figure 1-figure supplement 2). Only peptides originating from protein cleavage after lysine and arginine with up to two missed cleavages were accepted. Oxidation of methionine was permitted as variable modification. The mass tolerance for precursor ions was set to 8 ppm; the mass tolerance for fragment ions was set to 0.6 amu. For filtering of search results and identification of DrCNGK, a peptide FDR threshold of 0.01 (q-value) according to Percolator (Käll et al., 2007) two unique peptides per protein and peptides with search result rank 1 were required.

Sperm motility
Sperm were loaded for 45 min at room temperature with either 30 mM DEACM-caged cAMP, 30 mM DEACM-caged cGMP, or 40 mM caged Ca 2+ (NP-EGTA, Life Technologies). A UV light-emitting diode (365-nm LED; M365L2-C, Thorlabs, Newton, NJ) was used for photolysis of caged compounds. Experiments using caged Ca 2+ and DEACM-caged nucleotides were carried out using a UV power of 25 and 22 mW, respectively. Flash duration was 300 ms. Pluronic (0.5%) was added to the incubation solution. Sperm were kept quiescent during incubation in ES solution (292 mOsm x L -1 ). Swimming was initiated by a hypoosmotic shock diluting sperm 1:20 in an activation solution containing (in mM): NaCl 70, KCl 5.4, MgCl 2 1, CaCl 2 1.8, glucose 10, and Hepes 5 at pH 7.4 adjusted with NaOH (167 mOsm x L -1 ). Swimming behaviour was observed with a dark-field condenser in an inverse microscope (IX71, Olympus, Tokio, Japan) with 20x magnification (UPLSAPO, NA 0.75). Movies were recorded at 30 Hz using a back-illuminated electron-multiplying charge-coupled device camera (DU-897D; Andor Technology, Belfast, United Kingdom). Sperm trajectories were tracked using custom-made software written in MATLAB (Mathworks). The software can be made available upon request. The average swimming path (ASP) was calculated by filtering the tracked coordinates with a second degree Savitzky-Golay filter with a 200 ms span. The curvature (k) of the swimming path was calculated using the formula:bbb , where x and y are the coordinates of the ASP.
To assess cAMP loading and release, we recorded the fluorescence increase due to DEACM-OH release after photolysis of DEACM-caged cAMP (Bö nigk et al. 2009). Release and fluorescence excitation was achieved simultaneously using the same UV LED (power 1.75 mW). Light was coupled to the microscope with a dichroic mirror (455DRLP, XF2034, Omega Optical, Brattleboro, VT) and fluorescence was long-pass filtered (460ALP; XF309; Omega Optical). Single cells were recorded at 50 Hz.
Data analysis: Statistical analysis and fitting of data were performed, unless otherwise stated, using Sigma Plot 11.0, GraphPadPrism 5, or Clampfit 10.2 (Molecular Devices). All data are given as mean ± standard deviation (number of experiments).
Note: All cell lines used in this study will be sent for STR profiling. Mycoplasma testing was performed using the Promokine Mycoplasma Test Kit 1/C (PromoCell GmbH, Heidelberg, Germany). Results of this test can be supplied upon request.