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• W2140030603 abstract "Ion channels participate in crucial sperm functions such as motility, capacitation and the acrosome reaction. Chloride, the main anion in physiological solutions, is deeply involved in sperm physiology. We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa to investigate their ion channels. This work presents the first evidence for the presence of calcium-dependent chloride channels (CaCCs) in human spermatozoa; they could be constituted by TMEM16. The CaCCs play an important role in the physiology of human spermatozoa and participate in the acrosome reaction. Abstract Motility, maturation and the acrosome reaction (AR) are fundamental functions of mammalian spermatozoa. While travelling through the female reproductive tract, spermatozoa must mature through a process named capacitation, so that they can reach the egg and undergo the AR, an exocytotic event necessary to fertilize the egg. Though Cl− is important for sperm capacitation and for the AR, not much is known about the molecular identity of the Cl− transporters involved in these processes. We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa. Our whole cell recordings revealed the presence of a Ca2+-dependent Cl− current. The biophysical characteristics of this current and its sensitivity to niflumic acid (NFA) and 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid (DIDIS) are consistent with those displayed by the Ca2+-dependent Cl− channel from the anoctamin family (TMEM16). Whole cell patch clamp recordings in the cytoplasmic droplet of human spermatozoa corroborated the presence of these currents, which were sensitive to NFA and to a small molecule TMEM16A inhibitor (TMEM16Ainh, an aminophenylthiazole). Importantly, the human sperm AR induced by a recombinant human glycoprotein from the zona pellucida, rhZP3, displayed a similar sensitivity to NFA, DIDS and TMEM16Ainh as the sperm Ca2+-dependent Cl− currents. Our findings indicate the presence of Ca2+-dependent Cl− currents in human spermatozoa, that TMEM16A may contribute to these currents and also that sperm Ca2+-dependent Cl− currents may participate in the rhZP3-induced AR. From their germinal niche till they reach and fertilize the egg, mammalian spermatozoa must travel a long and winding road. Upon ejaculation and during their transit through the female reproductive tract, spermatozoa acquire progressive motility and undergo molecular, biochemical and physiological changes referred to as capacitation that enable them to reach and fertilize the egg (Bailey, 2010). To accomplish fertilization, spermatozoa must carry out the acrosome reaction (AR) (reviewed in Darszon et al. 2011). This exocytotic reaction enables spermatozoa to penetrate the ZP matrix and fuse with the egg plasma membrane, generating a zygote. Though for many years it has been believed that the zona pellucida (ZP), a glycoproteinaceous matrix that surrounds the mammalian oocyte, is the physiological inducer of the AR, how and where this reaction occurs has been re-examined recently (Ganguly et al. 2010; Inoue et al. 2011; Jin et al. 2011). The human ZP matrix is composed of four glycoproteins designated as ZP1 to ZP4; ZP3 is believed to be the main AR inducer (Conner et al. 2005; Caballero-Campo et al. 2006; Litscher et al. 2009). The AR is a calcium-dependent process and it is inhibited by several ion channel blockers, evidencing their predominant role in this process (Espinosa et al. 1998; Mayorga et al. 2007). It is well established that motility, capacitation and the AR require diverse ions (Ca2+, HCO3−, Na+, K+ and Cl−) (Visconti et al. 1995; Salicioni et al. 2007; Darszon et al. 2011). In mouse spermatozoa, the absence of external Cl− does not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the ZP-induced AR and finally fertilization are abolished or significantly reduced (Wertheimer et al. 2008; Chen et al. 2009). Similar results have been found in human sperm (Yeung & Cooper, 2008). As in other cells, Cl− is the principal anion that among other important functions is implicated in sperm volume regulation and protection from osmotic stress (Furst et al. 2002; Yeung et al. 2005; Cooper & Yeung, 2007). Mammalian spermatozoa confront drastic osmotic changes along their journey to find the egg (Chen et al. 2010); for example, the acrosome swelling that occurs after binding to ZP leads to AR (Zanetti & Mayorga, 2009). Therefore, it is likely that Cl− plays a relevant role in sperm physiology. However, not much is known about the proteins that transport it across the membrane of this fundamental cell. Many different cell types in which cell volume control and secretion are critical (i.e. epithelial cells in exocrine glands and trachea, airway, vascular smooth muscle cells, reproductive tract smooth muscle cells, oviduct and ductus epididymis cells, and mouse spermatids) express Ca2+-dependent Cl− channels (CaCCs), exhibiting similar biophysical, pharmacological and molecular features (Hartzell et al. 2005; Huang et al. 2009; Kunzelmann et al. 2011). Interestingly, niflumic acid (NFA) and 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS), two CaCC blockers, inhibit the ZP-induced mouse spermatozoa AR in a similar dose-dependent manner as that with which they block CaCCs, indicating their involvement in this exocytotic event (Espinosa et al. 1998). The long journey of spermatozoa is accompanied by dynamic changes in the concentration of intracellular Ca2+ ([Ca2+]i) that trigger myriad signalling events which could include the modulation of CaCCs, as was demonstrated in other cells (Arreola et al. 1996). Though currents mediated by CaCCs, initially documented in Xenopus oocytes (Miledi, 1982), have now been recorded in many cells types, only recently has the transmembrane protein TMEM16A been identified as one of the major molecular counterparts of the CaCCs (Caputo et al. 2008; Schroeder et al. 2008; Yang et al. 2008). The development of small molecule inhibitors to investigate the contribution of TMEM16A to CaCC conductance in human tissues, airway, intestinal epithelial and salivary gland cells allows the pharmacological dissection of TMEM16A/CaCC function (Namkung et al. 2010). In spite of the evidence suggesting a role for CaCCs in human sperm physiology, they have never been recorded directly in these cells. In this work, we used a modified perforated patch-clamp technique to obtain whole cell recordings sealing on the head of mature human spermatozoa. Our findings show that these sperm possess CaCCs with biophysical and pharmacological properties resembling those reported in other native cells (Hartzell et al. 2005) and in CaCC expression cloning experiments in amphibian and mammalian systems (Caputo et al. 2008; Schroeder et al. 2008; Yang et al. 2008). We corroborate this by showing that these currents can be recorded sealing in the cytoplasmic droplet, as has been done for CatSper, Hv and Slo3 (Kirichok et al. 2006; Lishko et al. 2010; Santi et al. 2010). Our results are also consistent with a significant contribution of TMEM16A like channels to the CaCC currents obtained by whole cell recordings from heads of human spermatozoa. In addition, our observations suggest that these channels are involved in the human sperm rhZP3-mediated AR. This study was approved by the Bioethics Committee at the Biotechnology Institute from the National Autonomous University of Mexico. All semen donors gave written informed consent. Ejaculates were obtained from healthy donors by masturbation after at least 48 h of sexual abstinence. Only semen samples that fulfilled the World Health Organization guidelines were selected for experiments (WHO, 2010). Highly motile sperm were recovered after a swim-up separation for 1 h in Ham's F-10 medium at 37°C in a humidified atmosphere of 5% CO2–95% air. For electrophysiological experiments the cells were stored in physiological salt solutions immediately after swim-up separation until used. For AR determinations the swim-up procedure was carried out supplementing the F-10 medium with 5 mg ml−1 of BSA and 2 mm CaCl2, pH 7.4. Cell concentration was then adjusted to 107 sperm ml−1 with the capacitating Ham's F-10 medium, and incubation was continued for 5 h. After capacitation, sperm were preincubated with CaCC inhibitors (NFA, DIDS or TMEM16Ainh, 15 min in all cases) and the AR was induced in capacitated cells incubated for 30 min with 10 ng μl−1 of recombinant human ZP3 (rhZP3), purified as reported previously (Jose et al. 2010). Staining with fluorescein isothiocyanate-labelled Pisum sativum agglutinin (FITC-PSA) was performed for evaluation of the AR. Sperm were washed with phosphate-buffered saline (PBS) and aliquots of the cell suspensions were smeared onto AR glass slides and then air dried. To permeabilize the plasma and outer acrosomal membranes, the slides were fixed with cold methanol for 30 s. Samples were incubated with FITC-PSA (25 μg ml−1 in PBS, pH 7.4) for 30 min at room temperature in a moisture chamber. The excess of FITC-PSA was washed out with distilled water, slides were dried again and finally the AR assays were evaluated with a fluorescence microscope (Zeiss Axioskop, excitation filter 450–490 nm, emission filter 520 nm). The AR patterns (bright fluorescence: acrosome intact; no fluorescence or only fluorescence of the equatorial segment: acrosome reacted) were evaluated in at least 200 cells per condition. Negative (no stimulation) and positive (A-23187 10 μm) controls were included in all experiments. For each experiment, AR indexes (ARIs) were calculated by subtracting the percentage of spontaneously reacted spermatozoa in the negative control (value of 1.8 ± 0.4%, n= 3), from the raw mean values for all experimental conditions, expressing the results as a percentage of the AR observed in the positive controls (value of 29.3 ± 0.6%, n= 3). Data processing was done using the KyPlot 2.0 program (KyensLab Inc, Tokyo, Japan). In all cases, differences between raw experimental and control data were tested by unpaired Student's t tests. Differences were considered significant when P < 0.05. We have applied the perforated-cell patch-clamp technique to record ionic currents in the whole cell mode in mature human spermatozoa plated on poly-lysine-coated coverslips. The patch pipette was applied at a 90 deg angle to the sperm cell head plasma membrane using a three-axis piezo nano-positioning with servo-control (PZ 62E, Physik Instrumente, Germany) as represented in the online supplemental material Fig. S1. We used a mixture of saponin/β-escin (100 μg ml−1/5 μm) in the pipette tip (∼1 μl) to induce controlled patch permeabilization, and were able to record stable whole-cell currents after patch perforation from dialysed spermatozoa. This strategy has been used to record ion currents from cardiac myocytes and neurons (Fan & Palade, 1998; Sarantopoulos et al. 2004). It consists in perforating cell attached patches to obtain whole cell records, using a mixture of detergents in the pipette tip. The detergents were loaded through the back of the pipette after filling it with solution. The success rate to perforate the patch and record stable whole cell ionic currents for an adequate amount of time was about 50%. To visually confirm electrical and diffusion access to all sperm compartments (head, middle and principal pieces; Fig. 1A), using the adapted perforated patch technique; we dialysed the cell with internal solution containing fluorescein (1 μm) and recorded the fluorescence in an inverted fluorescence microscope (Olympus IX71) with a CCD camera. Glass pipettes (1BBL, World Precision Instruments, Inc., Sarasota, FL, USA) were designed to have 5–7 MΩ pipette resistance, when filled with internal solution. The junction potential between internal and external solutions, measured with the offset correction of the patch amplifier, was usually about 4–6 mV. Membrane potential was always corrected for the liquid junction potential (∼4 mV). The chloride concentration was maintained at least at 5 mm to insure the proper operation of the Ag–AgCl electrodes. Changing the chloride concentration did have an effect on the liquid junction potential as we had to correct the pipette potential differentially for each concentration. The seal resistance was typically 3–5 GΩ. After sealing and achieving the whole cell configuration, capacitive transients were obtained and used to measure sperm capacitance and series resistance (Fig. S2) to monitor the suitability of the recording conditions with this method. Such transients are shown in Fig. S2A for the onset and offset. High temporal resolution (>20 kHz) recordings allowed estimates of clamp quality and time constants. Such a record is shown in Fig. S2B. A double exponential of the form I=Io+I1× (1 − exp(−t/τ1)) +I2× (1 − exp(−t/τ2)) was fitted to the capacitive transient decay. Typical values of the best fit parameters were −52, 38 and 14 pA for Io, I1 and I2, respectively, whereas the time constants were 23 and 625 μS for the fast and slow components, respectively. The amplitude of the fast component was about 2–3 times greater than that of the slow one, suggesting that the fast component may be related to clamp conditions in the head and surrounding regions through the neck, whereas the slow one may be related to the clamp conditions in the tail of the sperm. Hence, although the whole cell procedure reports currents from all over the sperm, the recording conditions are better and faster in the head and surrounding areas than in the tail. The area vs. time, charge (Q), under the capacitive transients, needed to estimate membrane capacitance (Cm), sperm area and series resistance is shown in Fig. S2. The average charge measured under these conditions was 24 ± 9 fC (n= 5) and the mean membrane capacitance of the whole sperm cell membrane was 1.3 ± 0.1 pF (n= 96). This measurement was quite similar to that reported by the capacitance compensation from the Axopatch 200B. The human sperm area determined by electron micrographs using a stereological analysis method is approximately 106 μm2 (Curry et al. 1996). The capacitance values found by us are consistent with this value, assuming the specific Cm to be 1 μF cm−2. For 1.3 pF average capacitance, the estimated sperm area under these assumptions is approximately 130 μm2. Recordings were started after at least 4 min dialysis to allow equilibration of the cytosolic content with the pipette solution. Whole-cell patch-clamp recordings from mature human spermatozoa A, fluorescence image sequences of a spermatozoon after achieving the whole-cell configuration in the perforated-patch mode. Fluorescein (1 μm) diffused from the pipette throughout the interior of the spermatozoon in ∼15 s. The pipette fluorescence is not seen because the approach of the pipette is perpendicular to the sperm head (see supplemental Fig. S1). B, whole-cell currents elicited by voltage steps from a holding potential of 0 mV (inset: step protocol) in physiological salt solution conditions (see Methods) without and with NFA (10 μm). C, current–voltage relationships (I–V curve) of results as in B in the absence and presence of 10 μm NFA, showing inward and outward rectification at hyperpolarized and depolarized membrane potentials. Note that the macroscopic current family and I–V plot show a NFA-sensitive current component. For C, data represent means ± SEM with n= 4. Whole-cell macroscopic currents were acquired at 10 kHz using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) interfaced with a PC equipped with a DigiData 1300A (Molecular Devices) and filtered at 1–2 kHz (4-pole Bessel filter). Unless indicated otherwise in the figure, membrane potential was changed from −120 to 100 mV in 10 mV steps by delivering square pulses of 1 s duration every 5 s from a holding potential of 0 mV, followed by a 700 ms pulse to –120 mV. This protocol inactivates voltage dependent Ca2+ and K+ currents. The leak of the seal, calculated applying Ohm's law to current values measured at the middle of the capacitive transient, was very low under these circumstances (∼10–20 GΩ), suggesting that this technique did not alter the basic properties of the membrane. During the course of this study Lishko et al. (2010) applied classical whole-cell patch clamp techniques for human sperm sealing at the cytoplasmic droplet. Using this strategy we were able to record CatSper currents stimulated by progesterone (Fig. 6A) and Ca2+-dependent Cl− currents inhibited by NFA and TMEM16Ainh (Fig. 6B and C). Human spermatozoa patched at the cytoplasmic droplet display progesterone activated CatSper currents and CaCC currents inhibited by NFA and TMEM16Ainh A, a representative monovalent whole-cell ICatSper recorded from a human spermatozoon in HS solution (baseline trace, black) and in divalent-free solution (DVF) in the absence (dark grey), and presence (grey) of 500 nm of progesterone (n= 4). B, whole-cell CaCC currents recorded from a human spermatozoon dialysed with pipette solution containing 250 nm free Ca2+ and exposed to a bath TEA-Cl standard solution. Currents were obtained at a holding potential of 0 mV with the indicated voltage step protocol (top panel) in the absence or presence of 10 μm NFA. Note that 10 μm NFA inhibited nearly 50% of the current indicating that it is a similar current to that observed in Fig. 4. C, family of CaCC currents recorded from another spermatozoon before and after exposure to 10 μm TMEM16Ainh. D, current–voltage relationship (I–V) of currents normalized with respect to maximal current in control conditions measurements at the end of each voltage pulse of recordings as in B and C. E, dose dependent blockade of CaCC currents by TMEM16Ainh. Current amplitudes were measured at +100 mV by averaging seven to nine original current traces and normalizing with respect to the maximal blocked fraction. For E and D data represent the mean ± SEM with n= 6. Pulse protocols, and data capture and analysis were performed with pCLAMP software (Molecular Devices), SigmaPlot 9.0 (Systat Software Inc., San Jose, CA, USA) and Origin7.5 (OriginLab Corp., Northampton, MA, USA). Total whole-cell ionic currents were initially recorded using normal physiological salt solutions (HS) with the following composition; external solution (in mm): 135 NaCl, 5 KCl, 1.8 CaCl2, 1 MgSO4, 5 dextrose, 10 lactic acid, 1 sodium pyruvate, 10 Hepes, pH 7.35; and internal solution (in mm): 10 NaCl, 140 KCl, 1 MgSO4, 10 EGTA, 5 CaCl2, 5 ATP-Mg, 10 Hepes, pH 7.25. The standard external solution for recording Cl− currents had the following composition (in mm): 135 TEA-Cl, 0.5 CaCl2, 45 d-mannitol, 20 TES, pH 7.35 and 330 mosmol kg−1. The solutions with different Cl− concentrations are shown in Table 1. The tonicity of the internal solutions was 40 mosmol kg−1 more hypotonic than the external solutions, to get rid of swelling-activated Cl− channels possibly present in these cells (Yeung et al. 2005). The Ca2+-buffered pipette solutions containing free [Ca2+] near 0 (1 nm) and up to 1 μm were prepared as in Arreola et al. (1996). EGTA and N-(2-hydroxyethyl)ethylenedinitrilo-N,N′,N′-triacetic acid (HEDTA) were selected as high and low affinity Ca2+ buffers (Kd values of 95 nm and 3.5 μm, respectively), to prepare Ca2+ buffers in the nano- and micromolar range, respectively (Table 2). The pH of the pipette solutions was set to 7.25 with N-tris(hydroxymethyl)methyl-2-aminoethanesulphonic acid (TES). High concentrations of Ca2+ and pH buffers assured control of both Ca2+ and pH during the course of these experiments. To determine the free Ca2+ concentrations we used the Maxchelator (v.2.1.) software program (written by Chris Patton, Hopkins Marine Station, Stanford University). Monovalent cations were substituted by tetraethylammonium (TEA) in both the internal and external solutions to avoid contamination of the Cl− currents by K+ channels such as Slo3 or cationic currents through non-selective channels (Navarro et al. 2007; Martinez-Lopez et al. 2009; Santi et al. 2009). For recording monovalent CatSper currents, seals were formed in HS solution and pipettes were filled with (in mm): 135 caesium methanesulphonate (CsMeSO3), 5 CsCl, 10 EGTA, 5 Na-ATP, 0.5 Na-GTP, 10 Hepes, pH 7.3 adjusted with CsOH. Bath divalent-free solution (DVF) for recording monovalent CatSper currents contained the following (in mm): 140 CsMeSO3, 1 EDTA, 20 Hepes, pH 7.4 adjusted with CsOH. HS solution was used to record the baseline current which was then exchanged by DVF for measuring monovalent CatSper currents. Bovine serum albumin (BSA), CaCl2, A23187, Ham's F-10 medium, fluorescein isothiocyanate–Pisum sativum agglutinin (FITC-PSA), poly-lysine, niflumic acid, DIDS, saponin, β-escin, ionomycin, dimethyl sulfoxide (DMSO) and other inorganic salts were acquired from Sigma-Aldrich Chemical Co. (St Louis, MO, USA), and methanol was from J. T. Baker (Phillipsburg, NJ, USA). Stock solutions in DMSO were prepared for each compound and aliquots were stored at −20°C. Numeric results are expressed as means ± standard error mean (SEM); n, mean number of individuals, tested and analysed with Student's t test. Two-tailed P values <0.05 were considered statistically significant. To investigate the mature human spermatozoa plasma membrane ionic currents, we applied the perforated-cell patch-clamp technique (see Methods) by attaching a patch pipette to the sperm head plasma membrane. Figure 1A shows the image of a spermatozoon after achieving the whole-cell configuration and dialysing with the fluorescent dye fluorescein (1 μm) through the patch pipette. Electrical access to all sperm compartments was corroborated by the fluorescence throughout the whole interior of the human spermatozoon. The average time necessary to accomplish cell dialysis after achieving a giga-ohm (GΩ) seal between the patch pipette and the plasma membrane of the sperm head was ∼15 s. Figure 1B shows whole-cell macroscopic currents obtained from the same spermatozoon using physiological salt solutions (HS). Under these recording conditions the macroscopic currents result from a mixture of cationic (K+, Ca2+ or Na+) and anionic (Cl−) conductances. As Cl− is essential for sperm physiology (pointed out in the Introduction) and its transport systems not well defined, we decided to explore the Cl− contribution to the sperm whole-cell macroscopic currents, obtained by this methodology. The application of NFA decreased the total current revealing the presence of a minor but significant and reproducible NFA-sensitive component possibly due to Cl− channels. The current–voltage relationship showed an inward and an outward rectifying component in the negative and positive membrane potential values; specifically, at negative (−120 mV) and positive (100 mV) voltages, 10 μm NFA reduced the conductance 40% and 17%, respectively (Fig. 1C). Since NFA is best known as a blocker of Ca2+ regulated Cl− channels, we examined the Cl− dependence of the macroscopic current recorded in human spermatozoa using a modified bath solution containing different TEA-Cl concentrations (see Table 1 and Fig. 2A). To examine if the currents could include a voltage-activated Ca2+ channel (CaV) current, we applied a pre-pulse to –120 mV after holding the cells at 0 mV. Under these conditions we never observed such Ca2+ currents. When the bath solution contained high Cl− concentrations, robust currents were recorded and they decreased as the external Cl− concentration was reduced in the external medium. Lowering extracellular Cl− from 147 to 12 mm strongly reduced the outward currents; see records in Fig. 2A and I–V curve in Fig. 2B. Different extracellular Cl− concentrations, at a given intracellular Cl− concentration, elicited a shift in the reversal potential (Erev) (Fig. 2C). Between the extreme extracellular Cl− concentrations (147 and 12 mm), we found a shift of 49 ± 4 mV in the reversal potential (Erev). The expected shift for an ideal Cl− selective channel is 64 mV. Such a difference may indicate that the underlying channel has a small, but significant, permeability to gluconate, as it has been reported for several CaCCs (Frings et al. 2000; Kim et al. 2003). In addition, as there is external Ca2+ in the medium, CatSper (Lishko & Kirichok, 2010) and TRP-like channels could contribute to the currents. The sperm macroscopic currents depend on the concentration of extracellular Cl− A, family of macroscopic currents obtained from a spermatozoon exposed to high and low extracellular Cl− concentrations ([Cl−]e) using TEA-Cl solutions (see Methods). Currents were elicited by voltage steps, indicted in the voltage step protocol, from a holding potential of 0 mV. Note that lowering of [Cl−]e from 147 to 12 mm strongly decreased outward currents. B, the I–V plot clearly shows the [Cl−]e dependence of the current and the reversal potential (Erev). Reducing [Cl−]e shifts Erev to the right, as expected for a Cl− selective channel. C, The relation between the measured Erev of the current and the calculated equilibrium potential for Cl− is very close. The dashed line is the fitting expected for an ideal Cl− channel. In all conditions [Ca2+]i= 250 nm. For C, data represent means ± SEM with n= 5. To determine if the Cl− currents recorded in mature human spermatozoa were due to CaCCs, we varied the Ca2+ concentrations of the pipette internal solutions and applied voltage-step protocols to avoid other ionic currents as described in Fig. 3A. The macroscopic currents were negligible when [Ca2+]i was 1 nm and very small with 25 nm[Ca2+]i. Increasing the [Ca2+]i to 250 or 1000 nm induced the appearance of a robust current with an instantaneous component followed by a fractional slower component (Fig. 3A). This is clearly illustrated in the current–voltage (I–V) relationship at different [Ca2+]i concentrations (Fig. 3B) and especially at [Ca2+]i= 1000 nm where noticeable increments are recorded of the inward current at –120 mV and the outward-rectifying current at +100 mV. The currents at [Ca2+]i= 1000 nm are about 18- to 25-fold those at [Ca2+]i= 25 nm. Figure 3C shows steady-state average currents measured at −120 mV normalized against the current induced by 1000 nm of [Ca2+]i obtained from recordings in both the head, as well as by the now established protocol sealing at the cytoplasmic sperm droplet (Lishko et al. 2010, 2011). To provide a quantitative description of the Ca2+ dependence, we fitted the Hill equation to these data (continuous line) and obtained an apparent Kd of 236 nm and a Hill coefficient (nH) of 1.3. Figure 3D shows the relative current normalized against the current induced by 1000 nm of [Ca2+]i at +100 mV along with the fit of the Hill equation. The apparent Kd at +100 mV was 163 nm and nH of 1.9. The current at this potential was a very steep function of [Ca2+]i as indicated by the nH value. These results imply that the Ca2+ sensitivity of Cl− channels is a function of membrane potential as it has been reported for CaCCs in parotid acinar cells (Arreola et al. 1996). The Kd and nH values at negative and positive membrane potential are similar (nano-molar range) to those reported in rat parotid acinar cells (Kd= 300 nm and nH= 1.2 at –66 mV; Kd= 61 nm and nH= 2.7 at +74 mV) (Arreola et al. 1996). Consistently, they are also similar to those reported in heterologously expressed CaCCs (EC50= 400 nm at 60 mV) (Yang et al. 2008). Behaviour of the Cl− currents at different [Ca2+]i A, representative whole-cell currents recorded from one of four human spermatozoa dialysed with pipette solutions containing (from top to bottom) 1, 25, 250 and 1000 nm free Ca2+ with TEA-Cl standard solution in the bath. Currents were obtained from a holding potential of 0 mV with the voltage step protocol shown in the top panel. Note the strong [Ca2+]i dependence of the macroscopic current; voltage is not sufficient to activate the current in [Ca2+]i= 1 nm, and 25 nm activates only 4% of the current with respect to 1000 nm, the maximal concentration used. B, I–V plot of the currents obtained in A. C and D, illustrate concentration–response curves of the effect of different [Ca2+]i on the macroscopic inward and outward Cl− currents obtained from four independent experiments sealing on the sperm head and three on the cytoplasmic droplet (see Methods). Currents were normalized against the current obtained at 1000 nm[Ca2+]i recorded at –120 mV (C) and +100 mV (D). The continuous lines represent the data fitted to a Hill equation with the following parameters: Kd= 236 nm and nH= 1.3 at –120 mV and Kd= 163 nm and nH= 1.9 at +100 mV. Data represent means ± SEM with n= 7. To further characterize the channels we recorded, we analysed the dependence of the gating parameters of CaCC activation with increasing [Ca2+]i. Figure S3A shows typical records of CaCC obtained with the same pulse protocol as described before and [Ca2+]i= 500 nm. The gating for activation of the outward currents was well described by a double exponential (see continuous line superimposed to the current trace). In Fig. S3B, we plotted the activation constants of the CaCC currents versus[Ca2+]i. These results show that elevating [Ca2+]i levels increased not only the currents amplitude, as described in Fig. 3, but also accelerated the activation kinetics. To examine the pharmacological properties of the putative CaCC currents recorded in mature human spermatozoa, two well known blockers of this channel were tested. We found that both NFA and DIDS potently affected the Ca2+-dependent Cl− curr" @default.
• W2140030603 created "2016-06-24" @default.
• W2140030603 creator A5011954284 @default.
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• W2140030603 creator A5080274967 @default.
• W2140030603 date "2012-05-31" @default.
• W2140030603 modified "2023-09-30" @default.
• W2140030603 title "Human spermatozoa possess a calcium-dependent chloride channel that may participate in the acrosomal reaction" @default.
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