The C at S per channel controls chemosensation in sea urchin sperm
2014; Springer Nature; Volume: 34; Issue: 3 Linguagem: Inglês
10.15252/embj.201489376
ISSN1460-2075
AutoresReinhard Seifert, Melanie Flick, Wolfgang Bönigk, Luis Álvarez, Christian Trötschel, Ansgar Poetsch, Astrid M. Müller, Normann Goodwin, Patric Pelzer, Nachiket D. Kashikar, Elisabeth Kremmer, Jan F. Jikeli, Bernd Timmermann, Heiner Kuhl, Dmitry Fridman, Florian Windler, U. Benjamin Kaupp, Timo Strünker,
Tópico(s)Reproductive biology and impacts on aquatic species
ResumoArticle22 December 2014free access Source Data The CatSper channel controls chemosensation in sea urchin sperm Reinhard Seifert Reinhard Seifert Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Melanie Flick Melanie Flick Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Search for more papers by this author Wolfgang Bönigk Wolfgang Bönigk Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Search for more papers by this author Luis Alvarez Luis Alvarez Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Search for more papers by this author Christian Trötschel Christian Trötschel Ruhr-Universität Bochum, Lehrstuhl Biochemie der Pflanzen, Bochum, Germany Search for more papers by this author Ansgar Poetsch Ansgar Poetsch Ruhr-Universität Bochum, Lehrstuhl Biochemie der Pflanzen, Bochum, Germany Search for more papers by this author Astrid Müller Astrid Müller Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Search for more papers by this author Normann Goodwin Normann Goodwin Marine Biological Laboratory, Woods Hole, MA, USA Laboratory of Molecular Signalling, Babraham Institute, Cambridge, UK Search for more papers by this author Patric Pelzer Patric Pelzer Marine Biological Laboratory, Woods Hole, MA, USA Institut für Anatomie und Zellbiologie, Abteilung für Funktionelle Neuroanatomie, Universität Heidelberg, Heidelberg, Germany Search for more papers by this author Nachiket D Kashikar Nachiket D Kashikar Marine Biological Laboratory, Woods Hole, MA, USA Sussex Neuroscience, School of Life Sciences, University of Sussex, Falmer, Brighton, UK Search for more papers by this author Elisabeth Kremmer Elisabeth Kremmer Helmholtz-Zentrum München, Institut für Molekulare Immunologie, München, Germany Search for more papers by this author Jan Jikeli Jan Jikeli Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Search for more papers by this author Bernd Timmermann Bernd Timmermann Max-Planck-Institut für Molekulare Genetik, Berlin, Germany Search for more papers by this author Heiner Kuhl Heiner Kuhl Max-Planck-Institut für Molekulare Genetik, Berlin, Germany Search for more papers by this author Dmitry Fridman Dmitry Fridman Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Florian Windler Florian Windler Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author U Benjamin Kaupp Corresponding Author U Benjamin Kaupp [email protected] Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Timo Strünker Corresponding Author Timo Strünker [email protected] Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Reinhard Seifert Reinhard Seifert Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Melanie Flick Melanie Flick Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Search for more papers by this author Wolfgang Bönigk Wolfgang Bönigk Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Search for more papers by this author Luis Alvarez Luis Alvarez Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Search for more papers by this author Christian Trötschel Christian Trötschel Ruhr-Universität Bochum, Lehrstuhl Biochemie der Pflanzen, Bochum, Germany Search for more papers by this author Ansgar Poetsch Ansgar Poetsch Ruhr-Universität Bochum, Lehrstuhl Biochemie der Pflanzen, Bochum, Germany Search for more papers by this author Astrid Müller Astrid Müller Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Search for more papers by this author Normann Goodwin Normann Goodwin Marine Biological Laboratory, Woods Hole, MA, USA Laboratory of Molecular Signalling, Babraham Institute, Cambridge, UK Search for more papers by this author Patric Pelzer Patric Pelzer Marine Biological Laboratory, Woods Hole, MA, USA Institut für Anatomie und Zellbiologie, Abteilung für Funktionelle Neuroanatomie, Universität Heidelberg, Heidelberg, Germany Search for more papers by this author Nachiket D Kashikar Nachiket D Kashikar Marine Biological Laboratory, Woods Hole, MA, USA Sussex Neuroscience, School of Life Sciences, University of Sussex, Falmer, Brighton, UK Search for more papers by this author Elisabeth Kremmer Elisabeth Kremmer Helmholtz-Zentrum München, Institut für Molekulare Immunologie, München, Germany Search for more papers by this author Jan Jikeli Jan Jikeli Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Search for more papers by this author Bernd Timmermann Bernd Timmermann Max-Planck-Institut für Molekulare Genetik, Berlin, Germany Search for more papers by this author Heiner Kuhl Heiner Kuhl Max-Planck-Institut für Molekulare Genetik, Berlin, Germany Search for more papers by this author Dmitry Fridman Dmitry Fridman Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Florian Windler Florian Windler Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author U Benjamin Kaupp Corresponding Author U Benjamin Kaupp [email protected] Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Timo Strünker Corresponding Author Timo Strünker [email protected] Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Author Information Reinhard Seifert1,2,‡, Melanie Flick1,‡, Wolfgang Bönigk1, Luis Alvarez1, Christian Trötschel3, Ansgar Poetsch3, Astrid Müller1, Normann Goodwin2,4, Patric Pelzer2,5, Nachiket D Kashikar2,6, Elisabeth Kremmer7, Jan Jikeli1, Bernd Timmermann8, Heiner Kuhl8, Dmitry Fridman1,2, Florian Windler1,2, U Benjamin Kaupp *,1,2 and Timo Strünker *,1,2 1Center of Advanced European Studies and Research (Caesar), Abteilung Molekulare Neurosensorik, Bonn, Germany 2Marine Biological Laboratory, Woods Hole, MA, USA 3Ruhr-Universität Bochum, Lehrstuhl Biochemie der Pflanzen, Bochum, Germany 4Laboratory of Molecular Signalling, Babraham Institute, Cambridge, UK 5Institut für Anatomie und Zellbiologie, Abteilung für Funktionelle Neuroanatomie, Universität Heidelberg, Heidelberg, Germany 6Sussex Neuroscience, School of Life Sciences, University of Sussex, Falmer, Brighton, UK 7Helmholtz-Zentrum München, Institut für Molekulare Immunologie, München, Germany 8Max-Planck-Institut für Molekulare Genetik, Berlin, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 228 9656 162; Fax: +49 228 9656 9162; E-mail: [email protected] *Corresponding author. Tel: +49 228 9656 100; Fax: +49 228 9656 9100; E-mail: [email protected] The EMBO Journal (2015)34:379-392https://doi.org/10.15252/embj.201489376 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Sperm guidance is controlled by chemical and physical cues. In many species, Ca2+ bursts in the flagellum govern navigation to the egg. In Arbacia punctulata, a model system of sperm chemotaxis, a cGMP signaling pathway controls these Ca2+ bursts. The underlying Ca2+ channel and its mechanisms of activation are unknown. Here, we identify CatSper Ca2+ channels in the flagellum of A. punctulata sperm. We show that CatSper mediates the chemoattractant-evoked Ca2+ influx and controls chemotactic steering; a concomitant alkalization serves as a highly cooperative mechanism that enables CatSper to transduce periodic voltage changes into Ca2+ bursts. Our results reveal intriguing phylogenetic commonalities but also variations between marine invertebrates and mammals regarding the function and control of CatSper. The variations probably reflect functional and mechanistic adaptations that evolved during the transition from external to internal fertilization. Synopsis The identification of CatSper as the long-sought Ca2+ channel driving chemotaxis in sea urchin sperm reveals similarities and variations in the molecular makeup of chemosensory signaling pathways in mammals and marine invertebrates. CatSper mediates chemoattractant-induced Ca2+ bursts and controls chemotactic steering in sea urchin sperm. CatSper activation is controlled by an intimate interplay of pHi and Vm. CatSper activation requires that chemoattractant-induced pHi and Vm changes proceed in precise chronology. Allosteric regulation of CatSper by ΔpHi enables sperm to transduce periodic Vm changes into periodic Ca2+ signals, thereby controlling sperm navigation on periodic paths in a chemoattractant gradient. Introduction The intracellular Ca2+ concentration ([Ca2+]i) coordinates several sperm functions required for fertilization (Ho & Suarez, 2001; Eisenbach & Giojalas, 2006; Florman et al, 2008; Kaupp et al, 2008; Publicover et al, 2008). In particular, Ca2+ controls the beat of the flagellum and, thereby, the swimming behavior. In mice and humans, the sperm-specific Ca2+ channel CatSper (cation channel of sperm) represents the principal pathway for Ca2+ entry into the flagellum (Quill et al, 2001; Ren et al, 2001; Kirichok et al, 2006; Lishko et al, 2010). Targeted disruption of CatSper in mice impairs sperm motility (Qi et al, 2007), and CatSper−/− sperm fail to traverse the oviduct (Ho et al, 2009; Miki & Clapham, 2013; Chung et al, 2014) and to penetrate the egg coat (Ren et al, 2001)—deficits that cause male infertility (Quill et al, 2001; Ren et al, 2001; Qi et al, 2007). Similarly, mutations in human CatSper genes cause infertility in men (Avenarius et al, 2009; Hildebrand et al, 2010). CatSper has been proposed to serve as a polymodal sensor that integrates diverse chemical and physical cues (Brenker et al, 2012; Miki & Clapham, 2013; Tavares et al, 2013; Schiffer et al, 2014): In general, CatSper is activated at depolarized membrane potentials (Vm) and at alkaline intracellular pH (pHi) (Kirichok et al, 2006; Lishko et al, 2010, 2011; Strünker et al, 2011). However, the interplay between Vm and pHi to control CatSper during fertilization is unknown. In human sperm, hormones in the seminal fluid and the oviduct, i.e. prostaglandins and progesterone, directly activate CatSper (Lishko et al, 2011; Strünker et al, 2011; Brenker et al, 2012; Smith et al, 2013) and, thereby, affect sperm motility (Aitken & Kelly, 1985; Alasmari et al, 2013). Progesterone has been implicated in human sperm chemotaxis (Oren-Benaroya et al, 2008; Publicover et al, 2008; Teves et al, 2009); yet, in vivo, neither sperm chemotaxis nor the physiological role of these hormones during fertilization has been definitely established (Baldi et al, 2009). This is due to the demanding challenge to experimentally emulate the complex chemical, hydrodynamic, and topographical landscape of the female genital tract (Suarez & Pacey, 2006; Suarez, 2008; Kirkman-Brown & Smith, 2011; Miki & Clapham, 2013). In contrast, many aquatic species, in particular marine invertebrates, release their gametes into the ambient water; consequently, gametes of broadcast spawners can be studied under close to native conditions. For 100 years, sperm of marine invertebrates have served as a powerful model of fertilization research (Kaupp, 2012). It is well established that in the aquatic habitat, sperm are guided to the egg by chemotaxis. A case in point is sea urchin sperm. In sea urchin sperm, a cGMP signaling pathway generates Ca2+ bursts in the flagellum that coordinate chemotactic steering (Böhmer et al, 2005; Wood et al, 2005; Darszon et al, 2008; Kaupp et al, 2008; Guerrero et al, 2010a,b; Alvarez et al, 2012). Important components and cellular events of this signaling pathway have been identified (reviewed in Darszon et al, 2008; Kaupp et al, 2008; Alvarez et al, 2014). Briefly, the chemoattractant activates a receptor guanylyl cyclase (GC) and, thereby, stimulates rapid cGMP synthesis (Dangott & Garbers, 1984; Bentley et al, 1986, 1988; Shimomura & Garbers, 1986; Dangott et al, 1989; Kaupp et al, 2003). cGMP opens K+-selective cyclic nucleotide-gated (CNGK) channels (Strünker et al, 2006; Galindo et al, 2007; Bönigk et al, 2009). The ensuing hyperpolarization (Cook & Babcock, 1993; Reynaud et al, 1993; Beltrán et al, 1996; Strünker et al, 2006) activates a sperm-specific voltage-dependent Na+/H+ exchanger (sNHE) (Lee, 1984a; Lee, 1984b; Lee & Garbers, 1986), mediating a rapid rise of pHi (Nishigaki et al, 2001; Solzin et al, 2004) and, eventually, opens voltage-gated Ca2+ channels (Gonzáles-Martínez et al, 1992; Beltrán et al, 1996; Nishigaki et al, 2001; Kaupp et al, 2003; Strünker et al, 2006). However, to date, the molecular identity of the Ca2+ channel and its mechanism of activation have been elusive. CatSper genes exist in many metazoan genomes, including aquatic animals (Cai & Clapham, 2008), yet the expression and function of CatSper in non-mammalian species are unknown. Here, we show that CatSper represents the long-sought Ca2+ channel of the chemotactic signaling pathway in sperm of the sea urchin A. punctulata. CatSper mediates the chemoattractant-induced Ca2+ bursts and controls chemotactic steering. We unveil the intimate, allosteric relationship between pHi and Vm for CatSper activation: A minute chemoattractant-induced increase of pHi enables CatSper in a highly cooperative fashion to open during a subsequent depolarization. The pHi-induced shift of the voltage dependence of CatSper activation enables sperm to transduce periodic Vm changes into periodic Ca2+ bursts during sperm navigation on periodic paths in a chemoattractant gradient. We reveal intriguing commonalities and variations in the function and molecular makeup of chemosensory signaling pathways in sperm from mammals and marine invertebrates. Although different in design, these pathways share the CatSper channel as a key component of Ca2+ signaling. Results CatSper is expressed in the flagellum of Arbacia punctulata sperm From a cDNA library of A. punctulata testis, we cloned four cDNAs encoding pore-forming CatSper subunits (ApCatSper 1–4) (Fig 1A, Supplementary Fig S1). Each ApCatSper subunit harbors six transmembrane segments (S1–S6), a voltage sensor in S4, and a pore loop between S5 and S6 (Fig 1A, Supplementary Fig S1). The pore loops carry the signature sequence of Cav and CatSper channels (Fig 1B, upper panel) (Navarro et al, 2008). Similar to other voltage-gated channels, the S4 segments of all four ApCatSper subunits carry six to seven positively charged residues (Fig 1B, lower panel). The intracellular N- or C-termini of ApCatSper 1, 2, and 4, but not of ApCatSper 3, carry coiled-coil domains (Fig 1A, Supplementary Fig S1) that were proposed to mediate heterotetramerization in mammalian CatSper (Lobley et al, 2003). The overall homology of ApCatSper subunits with their mammalian CatSper ortholog is low (25–35%). Figure 1. Features of ApCatSper 1–4, β, γ, and δ and localization of CatSper in A. punctulata sperm Predicted membrane topology and predicted molecular weight of ApCatSper 1–4 cloned from A. punctulata testis, and of ApCatSper β, δ, and γ. A gene encoding ApCatSper δ was identified in the A. punctulata genome; transcripts encoding ApCatSper β and ApCatSper γ were identified in the A. punctulata transcriptome. S1 to S6, transmembrane segments; +, positively charged amino acids in S4; gray cylinder, coiled-coil domain. Upper panel, alignment of pore regions of ApCatSper 1–4. Amino acids of the Ca2+ selectivity-filter motif are highlighted. Lower panel, alignment of S4 voltage-sensor segments of ApCatSper 1–4 and of D. melanogaster ShakerB Kv channel. Positively charged amino acids are highlighted. Numbers indicate start and end position of amino acids of the selected region. Western blots of total protein of CHO cells transfected with ApCatSper 2 or 3, non-transfected control cells (-), and A. punctulata sperm (Sp). The Western blots were probed with anti-HA, anti-ApCatSper 2, or anti-ApCatSper 3 antibodies. Arrows indicate bands representing ApCatSper 2 and 3. Immunocytochemical analysis of sperm stained with anti-ApCatSper 3, anti-GC, or anti-CNGK antibodies; superposition of images obtained by fluorescence and bright-field microscopy; scale bar = 10 μm. The DNA was stained with DAPI (blue). Western blot analysis of co-immunoprecipitation (IP) of A. punctulata sperm proteins. The input (I), flow through (FT), washes (W1-5), and the eluate (E) of the IP using the anti-ApCatSper 2 antibody were probed with the anti-ApCatSper 3 antibody (upper panel) and vice versa (lower panel). ApCatSper 3 and ApCatSper 2 were co-immunoprecipitated with the anti-ApCatSper 2 (upper panel) and anti-ApCatSper 3 antibody (lower panel), respectively. Analysis by mass spectrometry of immunoprecipitated proteins. ApCatSper 1-4, β, γ, and δ were identified in the immunoprecipitates obtained with both the anti-ApCatSper 2 and anti-ApCatSper 3 antibodies. The number of tryptic peptides identified and the respective sequence coverage are given. Source data are available online for this figure. Source Data for Figure 1C [embj201489376-SourceData-Fig1C.jpg] Source Data for Figure 1E [embj201489376-SourceData-Fig1E.jpg] Download figure Download PowerPoint To localize ApCatSper subunits in sperm, we raised monoclonal antibodies against ApCatSper 2 and 3 (Supplementary Fig S1). Hemagglutinin (HA)-tagged ApCatSper 2 and 3 subunits were heterologously expressed in Chinese hamster ovary (CHO) cells. In Western blots, an anti-HA antibody labeled polypeptides with apparent molecular weights (Mw) of 66.5 ± 3.1 kDa (ApCatSper 2, n = 24) and 41.6 ± 2.1 kDa (ApCatSper 3, n = 9). The same polypeptides were recognized by the monoclonal anti-ApCatSper 2 and 3 antibodies in transfected CHO cells and in sperm (Fig 1C), demonstrating that ApCatSper 2 and 3 are expressed in A. punctulata sperm. In immunocytochemistry, the anti-ApCatSper 3 antibody stained the flagellum (Fig 1D, left). The staining pattern of the receptor GC (Fig 1D, middle), the CNGK channel (Fig 1D, right), and ApCatSper 3 overlapped, showing that CatSper colocalizes with components of the chemotactic signaling pathway. Mass spectrometry confirmed the presence of ApCatSper 1–4 in the flagellum: In protein preparations from purified flagella, we identified proteotypic peptides for all four ApCatSper subunits (Supplementary Fig S1, Supplementary Table S1); the peptides covered 5–25% of the respective protein sequences (Supplementary Table S1). Moreover, in the A. punctulata genome and testis transcriptome (to be published), we identified a gene encoding the accessory subunit CatSper δ (Chung et al, 2011) and mRNAs encoding CatSper β (Liu et al, 2007) and CatSper γ (Wang et al, 2009) (Supplementary Fig S1, Fig 1A). In purified flagella, we identified proteotypic peptides of the predicted accessory subunits (Supplementary Table S1). We conclude that ApCatSper 1–4 and ApCatSper β, δ, and γ are expressed in sperm and are located in the flagellum. We immunoprecipitated ApCatSper 2 and ApCatSper 3, using the respective anti-ApCatSper antibodies. Analysis of the co-immunoprecipitates by Western blotting (Fig 1E) and mass spectrometry (Fig 1F, Supplementary Table S2) indicates that ApCatSper 1–4, β, δ, and γ interact to form a protein complex. Therefore, we propose that the architecture of the CatSper channel is similar in sea urchins and mammals. Unfortunately, like their mammalian counterparts (Ren et al, 2001), heterologously expressed ApCatSper subunits did not yield functional channels. Intracellular pH and membrane voltage control Ca2+ influx in sea urchin sperm Using a stopped-flow apparatus and fluorescent probes for Ca2+, Vm, and pH, we studied the role of CatSper in intact A. punctulata sperm. Ammonium chloride (NH4Cl) evoked a rapid and sustained intracellular alkalization (Supplementary Fig S2) that stimulated a Ca2+ increase (Fig 2A). At low NH4Cl concentrations (≤ 3 mM), Ca2+ signals slowly reached a plateau; at higher concentrations (≥ 10 mM), NH4Cl evoked rapid, oscillatory Ca2+ responses (Fig 2A). Mixing of sperm with both NH4Cl and EGTA, which lowers extracellular [Ca2+] to ≤ 400 nM, abolished the Ca2+, but not the pHi response (Supplementary Fig S2), demonstrating that alkalization stimulates Ca2+ influx. Figure 2. CatSper inhibitors abolish alkaline-evoked Ca2+ influx in A. punctulata sperm Alkaline-evoked Ca2+ signals in sperm mixed with NH4Cl; sperm were loaded with the Ca2+ indicator Fluo-4. ΔF/F (%) indicates the change in Fluo-4 fluorescence (ΔF) with respect to the basal fluorescence (F, mean of the first 3–5 data points). Ca2+ signals evoked by mixing of sperm simultaneously with NH4Cl (30 mM) and the CatSper inhibitor MDL12330A. Dose–response relation of inhibition of the Ca2+ signals shown in panel (B), Ki = 20 μM, amplitudes were determined at t = 4 s. Download figure Download PowerPoint Two distinct CatSper inhibitors, MDL12330A (MDL) (Brenker et al, 2012) and mibefradil (Strünker et al, 2011), suppressed the alkaline-evoked Ca2+ signal (Fig 2B and C, Supplementary Fig S2); the constants of half-maximal inhibition (Ki) were 15.6 ± 3.3 μM (MDL) and 20.7 ± 5.1 μM (mibefradil) (n = 4) (Fig 2B and C, Supplementary Fig S2). Sperm were mixed simultaneously with NH4Cl and the inhibitors, and the time course of inhibition probably reflects the time required for the drug to reach the blocking site; we did not test whether drug action reached steady state within the recording time. The drugs inhibit CatSper-mediated Ca2+ signals in human sperm with similar potency (Strünker et al, 2011; Brenker et al, 2012). We conclude that in sea urchin sperm, similar to mouse and human sperm, CatSper mediates alkaline-evoked Ca2+ influx. Because MDL and mibefradil are not selective for CatSper, we cannot exclude that the sperm might harbor additional, so far unknown Ca2+-permeable channels that are also activated at alkaline pHi and inhibited by both drugs. We determined the pHi sensitivity of the alkaline-induced Ca2+ influx using the "pHi pseudo-null-point" method (Eisner et al, 1989; Chow et al, 1996; Bond & Varley, 2005; Swietach et al, 2010) that allows clamping of pHi to fixed values and calibration of the pH indicator BCECF. Key is a set of pHi-clamp solutions composed of a weak acid (butyric acid, BA) and a weak base (trimethylamine, TMA) at different molar ratios (see 4). TMA and BA freely equilibrate across the membrane and, at sufficiently high concentrations (see 4), establish a defined pHi that is set by the acid/base ratio (Chow et al, 1996). Mixing of sperm with a pHi 7.2-clamp solution changed pHi only slightly, suggesting a resting pH (pHrest) of about 7.2 (Fig 3A). Mixing with pHi-clamp solutions < 7.2 and > 7.2 evoked acidification and alkalization, respectively, that was stable after 4–5 s and persisted for at least 14 s (Fig 3A). The changes in ΔR/R of BCECF fluorescence were linearly related to pHi-clamp values (Fig 3B); interpolation yielded a pHrest of 7.16 ± 0.04 (Fig 3B; n = 7). Similar pHrest values of sea urchin sperm were determined by other methods (Babcock et al, 1992; Guerrero et al, 1998). Moreover, the calibration allowed rescaling of the data in Fig 3A to absolute pHi values (inset in Fig 3B). Figure 3. Determination of the threshold pHi for alkaline-evoked Ca2+ influx Changes in pHi evoked by mixing with pHi-clamp solutions (see explanation in the text); sperm were loaded with the pHi indicator BCECF. ΔR/R (%) indicates the change in the BCECF fluorescence emission ratio (ΔR = F494/F540) with respect to the basal ratio (R, mean of the first 3–5 data points). Steady-state change (at t = 14 s) of BCECF fluorescence for the pHi signals shown in (A). The intercept of the fitted straight line with the x-axis yields the resting pHi; the slope of the straight line yields the ΔR/R (%) × ΔpH−1. Inset: calibrated changes in pHi evoked by various pHi-clamp solutions. Ca2+ signals evoked by mixing of sperm with pHi-clamp solutions. Dose–response relation for the Ca2+ signals shown in (C). Calibrated pHi increase (red) and respective Ca2+ response (black) evoked by mixing of sperm with a pHi 7.6-clamp solution; depicted are, on an extended time scale, the first 500 ms of the respective pHi increase and Ca2+ signal shown in (B, inset) and (C), respectively. The threshold pHi for CatSper activation was deduced from the latency of the Ca2+ signal. Threshold pHi and latency of Ca2+ signals evoked by various pHi-clamp solutions (mean ± SD; n ≥ 3). Download figure Download PowerPoint Figure 3C shows the time course of Ca2+ responses in sperm mixed with different pHi-clamp solutions. Plotting the amplitude of the Ca2+ signals versus the respective pHi-clamp values disclosed an exceptionally steep dose–response relation with a pH½ of 7.47 ± 0.01 and a Hill coefficient of 10.8 ± 2.2 (Fig 3D, n = 4). From the time course of the changes in pHi and Ca2+, we reconstructed the threshold pHi (pHthr) at which the Ca2+ influx commenced (Fig 3E). For example, using the pHi 7.6-clamp solution, the Ca2+ signal was observed after a latency of ≅ 200 ms (Fig 3E, dotted black line), at which the pHi of sperm had increased to ≅ 7.3 (Fig 3E, dotted red line), i.e. pHthr for Ca2+ influx. We determined pHthr for the entire range of pHi-clamp solutions (Fig 3F). The latency of the Ca2+ influx decreased with increasing pHi-clamp values (Fig 3F, black), because the alkalization proceeded on a faster time scale (Fig 3A and inset of Fig 3B). However, pHthr was largely independent of the rate and magnitude of the pHi increase (Fig 3F, red). The invariant pHthr for the alkaline-induced Ca2+ influx and its exceptionally steep, switch-like dose–response relation suggest that intracellular alkalization sensitizes CatSper to open during depolarization. We also wondered whether depolarization evokes a Ca2+ increase. In fact, rapid elevation of the extracellular K+ concentration ([K+]o) to ≥ 30 mM evoked a transient Ca2+ signal (Fig 4A), whose amplitude was graded with [K+]o. MDL inhibited Ca2+ signals evoked by 80 mM and 160 mM K+ with a Ki of 38.8 ± 7.5 μM and 29.2 ± 11.2 μM (n = 3), respectively (Fig 4B–D); the Ca2+ signals were also suppressed by mibefradil (Supplementary Fig S2). We conclude that CatSper also supports depolarization-evoked Ca2+ influx in sea urchin sperm. Figure 4. The threshold pHi for alkaline-evoked Ca2+ influx is controlled by Vm Depolarization-evoked Ca2+ signals in sperm mixed with ASW containing high KCl concentrations. Ca2+ signals evoked by mixing of sperm with 80 mM KCl and the CatSper inhibitor MDL12330A. Ca2+ signals evoked by mixing of sperm with 160 mM KCl and MDL12330A. Dose–response relation for the Ca2+ signals shown in (B, C) at t = 1–2 s. Threshold pHi for Ca2+ signals evoked by pHi-clamp solutions in sperm bathed in ASW containing low (3 mM), high (191 mM), and normal (9 mM) KCl (mean ± SD; n ≥ 3); data for 9 mM KCl are from Fig 3F. Resting pHi and resting Vm in sperm bathed in ASW containing low (3 mM), high (191 mM), and normal (9 mM) KCl (black) (mean ± SD; n ≥ 3). Mean threshold pHi for CatSper activation at different membrane potentials (red); mean threshold pHi was derived from data shown in (E). Download figure Download PowerPoint We examined the relationship between pHthr and Vm. To manipulate the resting potential (Vrest), sperm were incubated at different [K+]o. In standard artificial sea water (ASW, 9 mM [K+]o), Vrest was −51.9 ± 2 mV (Fig 4E, n = 6); sperm were hyperpolarized and depolarized to −54.9 ± 2.2 mV and −26.3 ± 4.2 mV, respectively, at low (3 mM) and high (191 mM) [K+]o (Fig 4F, n = 3); Vrest was determined by the [K+]o null-point method (Strünker et al, 2006; see also 4), assuming an intracellular K+ concentration of 423 mM. Probing cells with different pHi-clamp solutions and analyzing the time course of pHi and Ca2+ signals revealed pHthr for different Vrest values (Fig 4E, Supplementa
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