Introduction

Zinc is an essential metal ion involved in numerous biological functions, including as a regulatory component to various proteins such as enzymes, transcription factors, and ion channels (Fayyazuddin et al., 2000; Gao et al., 2017; Maret, 2013; Sun et al., 2007; Vallee & Falchuk, 1993; Zheng et al., 2001). In the reproductive system, spermatozoa is a type of cell that is known to have high intracellular zinc content, which influences its production, maturation, and physiological functions (Chia et al., 2000; Fallah et al., 2018; Henkel et al., 1999; Riffo et al., 1992; Yamaguchi et al., 2009). Zinc is also present in the seminal fluid at high concentrations (∼1-1.5 mM) (Henkel et al., 1999; Riffo et al., 1992), and extensive studies have elucidated their role in regulating sperm physiology. For instance, extracellular zinc has been shown to influence human sperm motility, which negatively correlates with the concentration (Henkel et al., 1999, 2005). Another study also suggested that increased zinc concentration inhibits sperm motility (Riffo et al., 1992). Despite considerable evidence that sperm motility is regulated by extracellular zinc, intracellular zinc has also been implicated in playing a regulatory role. One study demonstrated that chelating intracellular zinc reduces sperm motility and velocity (Sørensen et al., 1999). However, the underlying mechanism by which zinc regulates sperm motility remains unclear.

During fertilization, spermatozoa undergo capacitation, a series of physiological processes that determine the success of fertilizing the egg within the female reproductive tract (Austin, 1951; Chang, 1951). This capacitation involves the changes of activities in various ion channels, which leads to diverse physiological changes, such as pH, calcium level, and membrane potential (Arnoult et al., 1999; Breitbart, 2002; Publicover et al., 2007; Vredenburgh-Wilberg & Parrish, 1995; Y. Zeng et al., 1995, 1996). Moreover, recent studies have shown that the activity of ion channels is regulated by zinc. Indeed, extracellular zinc has been shown to inhibit the flagellar voltage-gated proton channel Hv1, which is supposed to control intracellular pH in human sperm (Grahn et al., 2023; Lishko et al., 2010; Zhao et al., 2018). Thus, it is possible that the high levels of zinc in spermatozoa regulates their physiological properties.

In the present study, we find that intracellular zinc is exported during capacitation, indicating that zinc dynamics in spermatozoa play an important role in fertilization. Furthermore, zinc significantly inhibited the alkalinization-induced hyperpolarization in mouse sperm, which is mediated by Slo3 (K_Ca5.1), a sperm specific pH- and voltage-gated potassium channel (Schreiber et al., 1998; X. Zhang et al., 2006).

We further discovered that intracellular zinc directly inhibits mouse Slo3 (mSlo3) currents using Xenopus oocytes. Of note, this inhibition is pH-dependent, voltage-dependent and has a long-lasting inhibitory effect even after the washout. Coexpression of mSlo3 with the auxiliary subunit Lrrc52, to mimic the native environment (Yang et al., 2011; X. H. Zeng et al., 2015), consistently demonstrates zinc’s inhibitory effect. Additionally, we identify potential key amino acids in the mSlo3 channel that serve as the zinc binding site. Our findings highlight the importance of intracellular zinc dynamics and its regulatory role over ion channels during capacitation in spermatozoa.

Results

Intracellular zinc is exported from sperm during capacitation

To investigate the role of intracellular zinc during capacitation, we monitored the intracellular zinc concentration utilizing the zinc-sensitive fluorophore FluoZin3-AM. Fluorescence signals were successfully detected in both the head and flagellum of isolated mice spermatozoa (Fig. 1A). As expected, the head of the spermatozoa showed a stronger fluorescence signal compared to the flagellum, likely due to its higher zinc capacity. We also treated spermatozoa with Toyoda, Yokoyama, Hoshi (TYH) medium (Toyoda et al., 1971), which is well established capacitation-inducing medium in vitro. We observed that there was a gradual and significant decrease in fluorescence intensity in both regions (Fig. 1B), particularly prominent in the flagellum (Fig. 1C). This decline suggests the active release of intracellular zinc from sperm flagellum during capacitation.

Figure 1.

Figure 1—figure supplement 1.

Additionally, we examined the effect of intracellular zinc on sperm motility before and after capacitation (Fig. 1—figure supplement 1A-E). We incubated the isolated spermatozoa with cell permeable Zn²⁺ chelator N,N,N’,N’-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and measured the motility parameters before and after capacitation. We found that VAP (average path velocity), VCL (curvilinear velocity), and VSL (straight-line velocity) were influenced by the TPEN treatment only after the capacitation, as shown in Fig. 1—figure supplement 1. These results demonstrate that the dynamics of zinc levels during capacitation potentially contributes to sperm motility, highlighting the importance of zinc action in sperm physiology.

Intracellular zinc inhibits the alkalinization-induced hyperpolarization in sperm

It is already known that sperm capacitation is well associated with the increase of intracellular pH (Vredenburgh-Wilberg & Parrish, 1995; Y. Zeng et al., 1996), which leads to the hyperpolarization of the membrane (Arnoult et al., 1999; Y. Zeng et al., 1995) as well as the elevation of intracellular Ca²⁺ concentration level (Breitbart, 2002; Publicover et al., 2007) through diverse ion channel activities. To explore whether these pathways are influenced by intracellular zinc, we used perforated patch-clamp techniques. It has been reported that under the whole-cell current clamp of mouse epididymal spermatozoa, resting membrane potential (Vm) is hyperpolarized after intracellular alkalinization (Navarro et al., 2007). Therefore, we applied 10 mM NH₄Cl, which is routinely used to alkalize intracellular pH (Madshus, 1988), to spermatozoa. As expected, we observed that it hyperpolarized the membrane by −17.37 ± 2.89 mV; n=6 (Fig. 1E, G). Then, we applied 10 mM NH₄Cl with 100 µM zinc pyrithione (ZnPy), a zinc ionophore. In contrast to the control group, there was no remarkable hyperpolarization in the presence of ZnPy as the ΔVm was 0.99 ± 3.56 mV; n=3 (Fig. 1F, G). These findings indicate that either extracellular or intracellular zinc inhibits the alkalinization-induced hyperpolarization in mouse sperm.

To examine the possibility that intracellular, but not extracellular, zinc is responsible for inhibiting the alkalinization-induced hyperpolarization, we measured membrane potential of sperm using the patch-clamp technique in conventional whole-cell configuration with pH_i = 6.0 (Fig. 1H). In the test group, the patch pipette contained 100 µM ZnCl₂ (Fig. 1J), while the control group did not (Fig. 1I). In consistent with the perforated patch-clamp results, we observed alkalinization-induced hyperpolarization upon applying NH₄Cl in the control (ΔVm= −24.12 ± 5.885 mV; n=5; Fig. 1I, K). In contrast, no alkalinization-induced hyperpolarization was observed in the 100 µM ZnCl₂ group (ΔVm= −6.80 ± 1.18 mV; n=4; Fig. 1J, K). These results confirmed that intracellular zinc indeed inhibits alkalinization-induced hyperpolarization in mouse sperm.

Zinc inhibits mouse Slo3 (mSlo3) current

Our findings suggest that intracellular zinc inhibits a key process in sperm capacitation, specifically the alkalinization-induced hyperpolarization. Previous studies have identified the pH-and voltage-dependent potassium channel Slo3 is responsible for the principal K⁺ current (I_KSper) in mice spermatozoa (Navarro et al., 2007; Santi et al., 2010; Schreiber et al., 1998; X. H. Zeng et al., 2011). Given this context, we next investigated whether intracellular zinc acts directly on the Slo3 channel.

We expressed the mSlo3 channel in the Xenopus oocyte expression system and conducted inside-out patch-clamp recordings. The bath solution was initially set to pH 8.0, and recordings were performed using a high-speed perfusion system. A ramp pulse from −100 mV to + 100 mV with a holding potential of −60 mV was applied. We observed a robust pH- and voltage-dependent mSlo3 current at pH 8.0 (Fig. 2A, B), consistent with previous reports (Leonetti et al., 2012; Schreiber et al., 1998; X. Zhang et al., 2006). The application of 100 µM of ZnCl₂ resulted in the inhibition of 36.62 ± 4.76 % (n=6) of mSlo3 current (Fig. 2A, B). Of note, the inhibited mSlo3 current by zinc was never restored after the wash out of zinc even for more than 30 seconds. However, when pH 6.0 was applied to completely abolish the mSlo3 current, followed by the application of pH 8.0, the mSlo3 current recovered (Fig. 2A, B). We also determined the IC₅₀ of zinc inhibition by applying zinc at concentrations 0.1, 1, 10, 100, and 1000 µM for 30 seconds. The dose-response curve, fitted to the Hill equation with a standard slope (Hill slope of −1.0), yielded an IC₅₀ = 145.2 µM (Fig. 2C).

Figure 2.

Figure 2—figure supplement 1.

We also tested the effect of zinc at a lower pH of 7.5. We observed the pH- and voltage- dependent mSlo3 current at this pH. However, the 100 µM of ZnCl₂ application did not affect the mSlo3 current (Fig. 2D-F). To rule out instrumental and human error, we also tested the effect of pH 6.0 which suppresses Slo3 activity. As expected, the application of pH 6.0 completely inhibited the mSlo3 current, and the subsequent application of pH 7.5 restored the current (Fig. 2D-F).

We also conducted step pulse protocols from −100 mV to +180 mV with a holding potential of −60 mV to assess whether zinc inhibition is voltage-dependent. At pH 8.0, we observed a robust mSlo3 current, which was inhibited by 100 µM ZnCl₂ (Fig. 2G, H). The current-voltage (I-V) relationship indicated voltage-dependent zinc inhibition (Fig. 2H). The ratio of I_zinc to I_control plotted against membrane potential also demonstrated that zinc inhibition of mSlo3 current is voltage-dependent (Fig. 2—figure supplement 1A).

In addition, we plotted the conductance-voltage (G-V) relationship by calculating the conductance from the maximum current amplitude at steady-state, as the tail current recordings of mSlo3 were unsuccessful. We noted that V₅₀ of mSlo3 current shifted to more hyperpolarized potentials in the presence of zinc (V₅₀ control = 80.67 ± 2.32; V₅₀ zinc = 49.68 ± 1.38; n=13; Fig. 2—figure supplement 1B).

As we observed the long-lasting inhibitory effect of zinc on mSlo3 current, we examined whether a zinc chelator could restore the mSlo3 current to the baseline level without applying acidic pH. We used 5 mM EGTA, a divalent cation chelator which strongly chelates zinc (Kd_(Zn) ∼1 nM) (Hu et al., 2009). Zinc-inhibited mSlo3 current could not be recovered by pH 8.0 alone (Fig. 2I). However, significant current recovery was observed when 5 mM EGTA was included in the pH 8.0 bath solution (Fig. 2J). Specifically, 5 mM EGTA recovered 49.22 ± 16.98 % of the mSlo3 current (n=5), compared to pH 8.0 alone, which showed no significant recovery (5.62 ± 9.22 %; n= 7), as shown in Fig. 2K.

Additionally, we investigated the presence of endogenous zinc in Slo3 by perfusing Ph 8.0 followed by 5 mM EGTA at the beginning of the recording to detect any changes in the baseline current. The baseline current showed a slight increase upon perfusion with 5 mM EGTA, indicating the presence of endogenous zinc affecting the baseline current in Slo3 (Fig. 2—figure supplement 1C, D).

Taken together, these results showed that zinc has a long-lasting inhibitory effect on mSlo3 current that cannot be reversed by pH 8.0 alone. The inhibitory effect is pH-dependent, as complete inhibition at pH 6.0 followed by recovery at pH 8.0 was observed. In addition, zinc inhibition is voltage-dependent, and the application of 5 mM EGTA, which strongly chelates zinc, can partially recover mSlo3 current.

Zinc more efficiently inhibits mSlo3 current when coexpressed with the auxiliary subunit Lrrc52

In its native environment of spermatozoa, Slo3 is expressed with its auxiliary subunit Lrrc52 (Yang et al., 2011; X. H. Zeng et al., 2015). Lrrc52 shifts the Slo3 gating to more negative potentials, making the channel easier to open (Yang et al., 2011). We coexpressed mSlo3 with mouse Lrrc52 (mLrrc52) in the Xenopus expression system to perform inside-out patch clamp recordings.

The mSlo3+mLrrc52 current was observed upon the application of pH 8.0. A ramp pulse protocol from −100 mV to +100 mV was applied with a holding potential of 0 mV. Similar to mSlo3 alone, in the presence of mLrrc52, mSlo3 current was also inhibited by 100 µM ZnCl₂ (Fig. 3A, B). In addition, zinc inhibition was not recovered after washing with the zinc-free solution (Fig. 3B). A significant leftward shift in IC₅₀ was observed when the dose-response curve was plotted for coexpression with mLrrc52 (Fig. 3C; IC₅₀ = 15.2 µM; n= 3-8) compared to mSlo3 alone (Fig. 2C, Fig. 3C).

Figure 3.

Figure 3—figure supplement 1.

Additionally, when pH 7.5 was applied to depolarized potentials, mSlo3+mLrrc52 current was activated and in contrast with mSlo3 alone (Fig. 2D-F), 100 µM ZnCl₂ already inhibited mSlo3+mLrrc52 current at pH 7.5 (Fig. 3D, E). Alkalinization increased the percentage of current inhibition by 100 µM ZnCl₂ (Fig. 3F; pH 7.5 = 32.51 ± 4.98 %; n=3 and pH 8.0 = 52.61 ± 2.91 %; n=8).

We next conducted recordings using a step pulse protocol from −100 mV to +180 mV with a holding potential of −60 mV. Upon applying pH 8.0, we observed a significant mSlo3+mLrrc52 current, which was inhibited by 100 µM ZnCl₂ (Fig. 3—figure supplement 1A, B). Again, the I-V relationship confirmed that zinc inhibition was voltage-dependent (Fig. 3—figure supplement 1B). V₅₀ of mSlo3+mLrrc52 current was also shifted into more hyperpolarized potentials in the presence of zinc (V₅₀ control = 72.93 ± 2.06 V₅₀ zinc = 56.27 ± 2.75; n=13; Fig. 3—figure supplement 1C).

Taken together, when co-expressed with mLrrc52, mSlo3 current was more efficiently inhibited by zinc.

E169 and E205 located within VSD are important for the sustained zinc inhibition of mSlo3 current

To elucidate the mechanism of zinc inhibition on the mSlo3 channel, we aimed to identify the structural determinants required for this inhibition using a range of computational tools and mutagenesis. First, we used the AlphaFold3 structure prediction webserver (Abramson et al., 2024) to predict the zinc binding site on the mSlo3 channel. Notably, the prediction has shown that the majority of the zinc binding sites are located in the channel’s intracellular regulator of K⁺ conductance (RCK) domain, which consists of RCK1 and RCK2, with very few zinc sites in the transmembrane domain (Fig. 4—figure supplement 1A). The RCK domain is also recognized as crucial for zinc binding in Slo channel families (Hou et al., 2010; J. Zhang et al., 2023).

Figure 4.

Figure 4—figure supplement 1.

To validate the site within the intracellular region, we conducted site-directed mutagenesis on intracellular domain focusing on RCK1, RCK1/RCK2 linker, and RCK2 (H354R, H383R, H423R, H516R, H564R, H606A, H695R, and H720R) to assess changes in channel sensitivity to zinc. However, we did not observe any significant decrease in the percentage of current inhibition (Fig. 4—figure supplement 1B, C). We also generated a chimera where RCK2 of mSlo3 was replaced with RCK2 from mSlo1, but this chimera exhibited slightly increased sensitivity to zinc (Fig. 4—figure supplement 1B, C). Taken together, this concludes that, unlike Slo1, the zinc binding site is unlikely to be located in the RCK or the intracellular domain of the channel.

We then diverge our focus to the intracellular site of the channel. Given that zinc has been shown to shift the G-V relationship of the mSlo3 channel and its complex with Lrrc52 (Fig. 2—figure supplement 1B, Fig. 3—figure supplement 1C), we suspect that the binding site is more likely to be located in the transmembrane (TM) region. To enhance the prediction within the transmembrane segment, we computationally truncated the intracellular domain, allowing a key binding site to be found near E169 and E205 within the voltage-sensing domain (VSD) with the AlphaFold3 structure prediction webserver (Fig. 4A). Next, we conducted triplicates of 200 ns molecular dynamics simulations of the AlphaFold3 full-length Slo3 tetrameric structure to validate this binding site where the Slo3 channel is solvated in 0.15 M ZnCl₂ and 0.15 M KCl (Fig. 4B). Here, we show that using MD simulations, a similar zinc site is also observed near E169 and E205, validating the prediction results by AlphaFold3 (Fig. 4C). In addition, we also observed another zinc binding near the pore at E310 and E313 (Fig. 5—figure supplement 1A).

Figure 5.

Figure 5—figure supplement 1.

To test the stability of zinc within E169 and E205, we removed all the ZnCl₂ in the simulation box, leaving just 4 zinc ions near E169 residues and conducted three separate simulations for 200 ns, leading to 12 data points. Our simulations show that zinc remains stable, with the low root mean square deviation (RMSD) within its binding site in the transmembrane region, validating the stability within its binding site (Fig. 4D).

To validate the binding site further, we conducted a computational site-directed mutagenesis to further assess zinc stability after the mutation of the nearby acidic residue, E169A and E205A. Here, we show that the RMSD of the E169A mutant increases more than WT and E205A (Fig. 4E). Together, our simulation suggested that E169 and E205 are indeed important for the binding of zinc by Slo3.

We then examined zinc sensitivities of mSlo3 currents from oocytes expressing mutants by electrophysiology with mutation of zinc binding residues on the mSlo3 channel predicted by MD simulations, which is located in the S2-S3 linker (D162), S3 domain (E169), S4 domain (E205) and the intracellular linker between TM and RCK1 (E326) (Fig. 4C-E, Fig. 5A, Fig. 5—figure supplement 1B). All of these mutants exhibited similar current inhibition by zinc compared to wildtype (WT = 29.58 ± 4.47 %, n=7; D162S = 19.85 ± 6.71 %, n=3; E169A = 44.17 ± 10.14 %, n=4; E205 = 33.08 ± 5.14 %, n=4; E326 = 27.54 ± 0.89 %, n=2; Fig. 5B, C, Fig. 5—figure supplement 1C, D).

On the other hand, when we analyzed the percentage of current recovery of all the mutants, E169A and E205A showed significant current recovery upon the wash-out by pH 8.0 alone (WT = 5.62 ± 9.22 %, n=7; D162S = 0.86 ± 21.71 %, n = 3; E169A = 48.74 ± 15.81 %, n=4; E205A = 48.65 ± 7.19 %, n=4; E326 = −3.27 ± 1.01 %, n=2; Fig. 5B, D, Fig. 5—figure supplement 1C, E). Taken together, in consistent with MD simulation results, our electrophysiological recordings demonstrated that the long-lasting inhibitory effect of zinc was partly abolished by these mutations. Thus, our findings highlight the contribution of E169A in the bottom of S3 domain and E205A in the bottom of S4 domain in zinc-mediated inhibition of mSlo3 current.

Zinc alters the dynamics of VSD motion in mSlo3

Given the observed zinc-induced shift in the G-V relationship of both mSlo3 and mSlo3+mLrrc52, along with MD simulations and mutagenesis data, it is likely that VSD is involved in zinc inhibition of mSlo3. Then, we performed voltage-clamp fluorometry (VCF) experiments to track the motion of the VSD in the presence and absence of zinc. First, to identify suitable site for labeling with TMRM, we performed VCF scanning by introducing Cys mutations one at a time at the top of S3, S3-S4 linker, and the top of S4 of mSlo3 VCF construct (Fig. 6—figure supplement 1A). Two native, extracellular cysteines were mutated to avoid non-specific labeling (mSLo3 C19S/C131S) and the desired Cys mutations were then added on this double-mutant background. Initial scanning of VCF constructs L188C, K189C, S190C, and N191C (Fig. 6—figure supplement 1A-E) conjugated with TMRM resulted in no detectable fluorescence intensity (F) changes (ΔF). Subsequently, we coexpressed the mSlo3 VCF constructs with mLrrc52 to increase the level of mSlo3 expression in heterologous expression system (Leonetti et al., 2012). Out of three mutants coexpressed with mLrrc52 (L193C, G194C, and L195C; Fig. 6—figure supplement 1F-H), L193C, which is located in the top of S4 domain, produced fluorescence intensity changes in response to voltage steps (Fig. 6—figure supplement 1F) (ΔF/F= 1.15 ± 0.24 %; n=4).

Figure 6.

Figure 6—figure supplement 1.

We next performed VCF recordings in the absence and presence of zinc. Oocytes were held at −60 mV and the step pulses were applied from −160 mV to + 160 mV. Fluorescence change could be observed in response to voltage steps (ΔF/F_Control = 2.84 ± 0.47 % at +160 mV; n=7; Fig. 6A). A puff of 10 mM zinc solution was then manually injected into the same oocyte using a glass needle with positive pressure. 60 seconds post-injection, a second set of step pulses was applied to record the response under zinc application (ΔF/F_Zinc = 2.53 ± 0.52 % at +160 mV; n=7; Fig. 6A). In the presence of zinc, the V₅₀ of fluorescence-voltage (F-V) relationship was shifted into more depolarized potentials (V₅₀ control = −21.68 ± 3.04; V₅₀ zinc = 27.04 ± 1.05; Fig. 6B). This decrease in ΔF/F was more pronounced at certain voltages, such as 0 mV (Fig. 6E-F), and extended up to +80 mV. I-V relationship (Fig. 6C) showed that mSlo3 current was inhibited by zinc, consistent with what has been observed in the patch-clamp recordings (Fig. 2 and Fig. 3). In consistent with F-V relationship, the conductance-voltage (G-V) was also shifted into more depolarized potentials (V₅₀ control = 21.11 ± 4.8; V₅₀ zinc = 38.62 ± 28.8; Fig. 6D). Collectively, these results clearly indicate that the VSD motion of mSlo3 is influenced by the application of intracellular zinc.

Discussion

The present study aims to understand the roles of intracellular zinc in sperm physiology, focusing on sperm capacitation. Our results demonstrated that intracellular zinc is exported from spermatozoa during capacitation. We further discovered that intracellular zinc regulates alkalinization-induced hyperpolarization in mice spermatozoa, mediated by Slo3 channel. Detailed analysis of zinc’s regulatory effects mSlo3 in the Xenopus oocyte expression system revealed that intracellular zinc directly inhibits mSlo3 currents with exceptionally slow dissociation. By combining MD simulations and electrophysiology, we identified amino acid residues important for zinc-mediated inhibition of Slo3 activities.

Zinc levels are dynamic during capacitation

Using FluoZin3-AM for zinc imaging, we confirmed the presence of intracellular zinc in sperm (Fig. 1A), which is consistent with previous findings (Henkel et al., 1999). Our observations revealed that treatment with capacitation medium induced a decrease in zinc fluorescence intensity (Fig. 1B, C), suggesting that zinc levels are dynamic during capacitation. Previous studies using boar, bull and human spermatozoa have identified four distinct patterns of zinc localization associated with the state of capacitation (Kerns et al., 2018). In non-capacitated sperm, high concentration of zinc is present throughout the whole sperm from the sperm head to the whole sperm-tail, and the subcellular zinc localization shifts to a medium level in both the sperm head and the sperm tail mid piece (Kerns et al., 2018). In congruent with this, our findings also demonstrated a decrease in fluorescence intensity in both the head and flagellum of mice spermatozoa during capacitation (Fig. 1B, C). We also observed that zinc chelator significantly affected the sperm motility only after, but not before, capacitation (Fig. 1—figure supplement 1). These findings indicate that zinc dynamics play a crucial role in controlling sperm motility during the capacitation process.

To maintain zinc homeostasis, two protein families facilitate the transport of Zn²⁺ across cellular and intracellular membranes in opposite directions: zinc transporter proteins (ZnT) and Zrt-, Irt-like proteins (ZIP) (Kambe et al., 2004). ZIP12 has been reported to be highly expressed in mouse testis (Zhu et al., 2022), as well as ZnT-1 (Elgazar et al., 2005). The functional significance of other ZnTs and ZIPs, particularly those related to capacitation, is still largely unclear, leaving the mechanisms of zinc transport in sperm during capacitation poorly understood.

Intracellular zinc directly inhibits mSlo3 channels

Our findings from inside-out patch-clamp recordings indicate that zinc directly inhibits mSlo3 channels in a dose-dependent manner (Fig. 2). Zinc inhibition in mSlo3 channels is dependent on pH (Fig. 2A-E), voltage (Fig. 2G-H; Fig.2—figure supplement 1A, B) and exhibits a long-lasting inhibitory effect (Fig. 2I, K). This sustained inhibitory effect of zinc can be partially restored by EGTA, a divalent ion chelator with high affinity for zinc (Fig. 2J, K; Fig.2—figure supplement 1C, D). Previous studies indicate that zinc also regulates the Slo channel family, albeit with varying effects among Slo3 channel subtypes: zinc activates Slo1 channels (Hou et al., 2010) while inhibiting Slo2.2 channels (Budelli et al., 2016; J. Zhang et al., 2023). Zinc may produce these opposite effects on Slo2.2 and Slo1 by binding to distinct, non-conserved sites among the subtypes (J. Zhang et al., 2023).

Zinc inhibition on mSlo3 channels is characterized by the slow dissociation of zinc from the channel (Fig. 2I-K). Similar slow dissociation has been observed in zinc-regulated ion channels and receptors, such as NMDA and glycine receptors (Paoletti et al., 1997; Trombley et al., 2011). In NMDA receptors, high zinc affinity results in slow dissociation, which has been suggested to have a physiological relevance in neurons (Paoletti et al., 1997). Glycine receptors show complex responses depending on the balance of agonist and antagonist concentrations and whether the receptors have been pre-incubated with zinc (Trombley et al., 2011).

Despite the relatively high IC₅₀ of zinc inhibition for mSlo3 channels (145.2 µM), the slow dissociation usually indicates high-affinity binding. This might be due to the ∼40-second zinc application time in our study, which may have been insufficient to fully observe the effects at lower concentrations (Fig. 2B, C). Extended exposure of intracellular zinc in sperm could potentially result in greater inhibition.

Our results also show that when mSlo3 is coexpressed with Lrrc52 the lower concentration of zinc was sufficient to inhibit mSlo3 channels (Fig. 3). Although we still did not identify the exact mechanism underlying it, Lrrc52 shifts the gating of mSlo3 to more hyperpolarized potentials and lower pH, making the channel easier to open (Leonetti et al., 2012; Yang et al., 2011; X. H. Zeng et al., 2015). Considering that the zinc inhibition of Slo3 depends on membrane potential and intracellular pH, it is understandable that the presence of this auxiliary subunit would lead to more efficient inhibition by zinc.

Structural determinants of zinc inhibition in the mSlo3 channel

We identified potential key amino acids in the mSlo3 channel that may serve as zinc binding sites using MD simulations and amino acid mutagenesis (Fig. 4, 5; Fig. 4—figure supplement 1, Fig. 5—figure supplement 1). Previous studies on the Slo channel family indicates that zinc binding sites are located in RCK1 domain for Slo1 (Hou et al., 2010) and RCK2 domain for Slo2.2 (J. Zhang et al., 2023). However, in mSlo3 channel, our results didn’t show that either RCK1 or RCK2 may serve as zinc binding site (Fig. 4— figure supplement 1). Our MD simulations focusing on transmembrane region of mSlo3 channel suggest two possible zinc binding locations: one located within the intracellular part of the VSD and another one close to the pore (Fig. 4B, C; Fig. 5—figure supplement 1A). Given that zinc affects the G-V relationship of mSlo3 and its complex with Lrrc52, we focused on the VSD site.

Our analysis of current recovery for mutants in the VSD region revealed that E169A and E205A showed significant recovery upon wash-out with pH 8.0. This result aligns with MD simulations showing faster zinc dissociation for both E169A and E205A, indicating their contributions in zinc-mediated inhibition. Thus, the cluster of negative residues within the VSD serve as the zinc binding site in the mSlo3 channel.

However, our single amino acid mutations in this site, which contains clustered negative residues, did not significantly change zinc-mediated current reduction compared to the wildtype. This may be due to mutating one single amino acid might not be sufficient to fully elucidate other contributing residues in the predicted mSlo3 zinc binding site. Thus, more extensive mutagenesis studies will be needed to fully understand zinc inhibition in mSlo3.

Physiological relevance of zinc inhibition of the mSlo3 channel in mouse sperm

Zinc is thought to be involved in capacitation and the acrosome reaction, suggesting its role in regulating sperm fertilizing ability in vitro (Aonuma et al., 1978). This finding is supported by studies indicating that seminal zinc inhibits sperm’s ability to undergo the acrosome reaction (Riffo et al., 1992). However, the specific function of intracellular zinc in relation to the capacitation process has not been thoroughly investigated. Previous research indicates that zinc inhibits sperm motility in ejaculation medium as well as acrosome reaction (Henkel et al., 1999; Riffo et al., 1992), leading to the hypothesis that zinc might influence capacitation.

Slo3 plays a critical role in sperm fertility, since mice lacking Slo3 are infertile despite their normal sperm morphology (Santi et al., 2010). Furthermore, our previous work showed that VSP-deficient sperm exhibited enhanced Slo3 channel activity, which indirectly increases Ca²⁺ influx and results in abnormal sperm motility (Kawai et al., 2019). Taken together, our findings suggest that intracellular zinc is potentially a key regulator of sperm capacitation, functioning through inhibition of the Slo3 channel. Therefore, this study highlights the critical role of intracellular zinc in the regulation of sperm capacitation.

Materials and Methods

Ethical approval

All animal experiments were approved by the Animal Care and Use Committee of the Osaka University Graduate School of Medicine and performed in accordance with its guidelines.

Drug and Reagent

Zinc pyrithione was purchased from Fujifilm Wako (Osaka, Japan). TPEN was obtained from Cayman (Ann Arbor, MI, USA). Gramicidin was obtained from Merck (Kenilworth, NJ).

Patch Clamp Recordings from Spermatozoa

Patch clamp recordings from spermatozoa were conducted as previously described (Kawai et al., 2019; Kirichok et al., 2006). Briefly, sperm were harvested from the mouse corpus epididymis using an HS-based solution containing (in mM): 135 NaCl, 5 KCl, 2 CaCl₂, 1 MgSO₄, 20 HEPES, 5 glucose, 10 lactic acid, and 1 sodium pyruvate (pH 7.4). After 10 minutes, the supernatant was collected, centrifuged, washed twice with the HS-based solution, resuspended, and placed on untreated glass coverslips.

For conventional whole-cell recordings, the intracellular solution contained (in mM): 120 KCl, 3 MgCl₂, 40 MES, and 0.3 EGTA (pH 6.0), with the osmolality adjusted to approximately 300 mOsm/kg using sucrose. The same HS-based solution was used as the bath solution.

For perforated patch recordings, the intracellular solution included (in mM): 120 KCl, 3 MgCl₂, 40 HEPES, 0.3 EGTA (pH 7.0), and 0.05 mg/ml gramicidin. Once pores formed, the access resistance ranged between 50 and 100 MΩ.

Recording pipettes were made with borosilicate glass (BF-150-86-10; Sutter Instruments, CA, USA) using a puller (P-97; Sutter Instruments). The recordings were conducted using an Axopatch 200B amplifier (Molecular Devices, CA, USA) and sampled at 5 kHz using a Digidata 1550A system (Molecular Devices) with pCLAMP 10.5 software (Molecular Devices). Low pass filter of 20 Hz was applied to increase S/N ratio.

Zinc imaging from Spermatozoa

Sperm were isolated from the cauda epididymis of mice and transferred to HS medium. The sperm were then loaded with 10 μM FluoZin3-AM (Invitrogen) and 0.05% Pluronic F-127 in HS medium, followed by a 30-minutes incubation at 37°C. After incubation, the sperm were washed with HS medium and incubated on Cell-Tak (Corning, Bedford, MA)-coated round cover glasses for 10 minutes. The cover glass was mounted on a perfusion chamber for imaging experiments.

Fluorescence signals were detected using a filter set (Ex 495/10; DM 515; Em 535/25) with an IX-81 upright microscope (Olympus, Tokyo, Japan) equipped with a C9100 EMCCD camera (Hamamatsu Photonics, Shizuoka, Japan) and Metamorph imaging software (Molecular Devices). Images were captured every 30 seconds.

For capacitation, TYH medium (Toyoda et al., 1971) was used, consisting of: 120 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 5.6 mM glucose, 1.0 mM sodium pyruvate, 1.7 mM CaCl₂, 1.2 mM MgSO₄, 25 mM NaHCO₃, 4.0 g/L ALBMAX I (Thermo Fisher Scientific), and penicillin (50 units/ml)-streptomycin (50 μg/ml).

The experiment focused on the head and tail regions of spermatozoa. For analysis, cells in which both the head and tail were visible throughout the entire experiment were selected.

Sperm motility analysis

Sperm velocity was analyzed as previously described (Miyata et al., 2021). Briefly, spermatozoa isolated from the cauda epididymis were suspended in TYH medium. The TYH medium contained: 120 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 5.6 mM glucose, 1.0 mM sodium pyruvate, 1.7 mM CaCl₂, 1.2 mM MgSO₄, 25 mM NaHCO₃, 4.0 g/L ALBMAX I (Thermo Fisher Scientific), penicillin (50 units/ml) - streptomycin (50 μg/ml), and 0.6% Phenol Red.

Average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), straightness (STR), and linearity (LIN) was measured using the CEROS II sperm analysis system (Hamilton Thorne Biosciences, MA, USA) at 10 minutes and 2 hours after incubation. In some experiments, 20 μM TPEN was directly added to the TYH medium.

Molecular Biology

All Slo3 mutants were constructed from WT mus musculus Slo3 (mSlo3) in PSD64TF. Single amino acid mutations were introduced using PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc.). Chimeras were generated using In-Fusion HD Cloning Kit (Takara Bio Inc.). The introduced mutations and generated chimeras were confirmed by DNA sequencing. The mMESSAGE SP6 RNA transcription kit (Thermo Fisher Scientific) was used to transcribe WT, mutants, and chimeras of mSlo3 cRNAs from plasmid cDNA linearized by Xba1 restriction enzyme (Takara Bio Inc.).

Preparation of Xenopus laevis oocytes

As an anesthetic agent, 0.15% tricaine (Sigma-Aldrich / Tokyo Chemical Industry) was used for Xenopus laevis before surgical operation for isolation of oocytes. Follicular membranes were removed from isolated oocytes by collagenase treatment (2 mg ml−1; type 1; Sigma-Aldrich or Collagenase-P from Roche) for 3 hr. Oocytes were then rinsed and stored in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), pH 7.5, with NaOH) supplemented with 0.1 mg/mL gentamycin (FUJIFILM Wako Pure Chemical Corporation or Nacalai Tesque, Inc.) and 5 mM sodium pyruvate. The incubation temperature was 18 °C.

Expression of mSlo3 channel

Xenopus oocytes injected with 15 ng of WT mSlo3 cRNA and incubated for 2 days at 18°C. For mSlo3 mutants, oocytes were injected with 50 ng–60 ng of cRNA and incubated for 1–3 days, depending on the desired expression level for patch-clamp and voltage-clamp fluorometry (VCF) experiments. cRNA for mLrrc52 was also injected for data shown in Fig. 3, Fig. 3—figure supplement 1, Fig. 6, and Fig. 6—figure supplement 1F-H.

Oocyte patch-clamp recording of mSlo3

Patch-clamp recordings of Xenopus oocytes were performed using an Axopatch-200B amplifier (Molecular Devices) connected through an AD/DA converter (Digidata 1440A: Molecular Devices) and operated with pClamp software. Current traces were filtered at 5 kHz and sampled at 100 kHz. Vitelline membrane was manually removed using forceps after incubation for 1 to 5-minute in a hypertonic solution containing 192 mM NMDG, 4 mM KCl, 3.6 mM CaCl₂, 2 mM MgCl₂, and 10 mM HEPES, pH 7.4, with HCl. Leak currents were subtracted by using a P/-8 protocol for step pulses recordings only. Patch pipettes, with resistance ranging from 1 to 3 MΩ were pulled from borosilicate glass (Drummond Scientific Company). The pipette solution contains 0.5 mM potassium gluconate, 0.5 mM KCl, 1.1 mM KOH, 10 mM HEPES, 159 mM sodium gluconate, and 2 mM MgCl₂, pH 7.1, adjusted with methanesulfonic acid.

Patches were perfused with: (1) bath solution containing 184 mM potassium gluconate, 10 mM KOH and 10 mM HEPES in pH 6.0, 7.5, and 8.0; (2) zinc-containing bath solution at pH 7.5 and pH 8.0, with zinc concentrations of 0.1, 1, 10, 100, and 1000 µM. (3) bath solution containing 5 mM EGTA in pH 8.0. All pH values were adjusted using methanesulfonic acid. Slo3 macroscopic currents were evoked by an alkaline pH and by a depolarizing ramp pulse (from −100 mV to +100 mV with the holding potential of either - 60 mV or 0 mV) or step pulses (from −100 mV to +180 mV with the holding potential at - 60 mV). All bath solutions were perfused using a high-speed perfusion system ALA-VM8 OCTAFLOW (ALA Scientific Instruments Inc.)

Voltage-clamp fluorometry (VCF) recording of mSlo3

In mSlo3 VCF construct, the target labeling site (in S3-S4 linker) was substituted with cysteine residue. Two native, extracellular cysteines were mutated to avoid non-specific labeling (mSLo3 C19S/C131S). Oocytes for VCF recordings were labeled with Tetramethylrhodamine-5-maleimide (TMRM) (Invitrogen). In prior to VCF recordings, oocytes were incubated in a labeling solutions (120 mM K-methanesulfonate (MES), 2 mM CaCl₂, and 10 mM HEPES pH 7.4) containing 10 µM TMRM (Savalli et al., 2006) on ice for 1 hour. Labeled oocytes were then rinsed in TMRM-free ND96 solution four times and kept in the dark until and throughout the recording.

Oocytes were placed in a recording chamber with the animal pole facing upward and held at −60 mV. For screening experiments, step pulses ranging from −80 mV to +160 mV were applied, while pulses from −160 mV to +160 mV were used for direct zinc injection experiments. For the VCF recording in the absence and presence of zinc, voltage steps were first applied to record the initial response. Subsequently, a puff of 10 mM zinc solution was manually injected into the same oocyte using a glass needle and positive pressure. Sixty seconds post-injection, a second set of step pulses was applied to record the response under zinc application. In VCF recording, borosilicate glass capillaries (Harvard Apparatus) were used with a resistance of 0.2–0.4 MΩ when filled with 3 M KOAc and 10 mM KCl. Bath solution contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.4-7.5, with NaOH was used for recordings.

Fluorometric recordings were performed with an upright fluorescence microscope (Olympus BX50WI) equipped with a water immersion objective lens (Olympus XLUMPLAN FL 20x/1.00) to collect the emission light from the voltage-clamped oocytes. The light from LED source (MCWHL8 Thorlabs) was applied through a band-pass excitation filter (531 nm/40 nm BrightLine: Semrock). Emitted light was passed through band pass emission filter of 593nm/40nm (BrightLine, Semrock). The emission signals were detected by a photomultiplier (H10722-20; Hamamatsu Photonics). The detected emission intensities were acquired by a Digidata 1440A (Axon Instruments) and Clampex 10.3 software (Molecular Devices) at 25 kHz for VCF mutants screening and 50 kHz for VCF recording in the absence and presence of zinc. To improve the signal-to-noise ratio, VCF recording was repeated 3 times. Averaged data were used for data presentation and analysis.

Three-dimensional structural modeling of mouse Slo3 and prediction of zinc binding site

The amino acid sequence of mouse Slo3 (mSlo3) (O54982-1) was obtained from the UniProt database (Fig. 4) and was retrieved from NCBI NP_032458.3 (Fig. 5). The sequence (only the transmembrane region from residue M1 to G330, or the full-length protein) was input to the AlphaFold3 (Abramson et al., 2024) structural modelling web platform to generate an initial structure with (Fig. 4A) or without (Fig. 5A) 50 zinc ions. All the structural data presented in this study were generated using PyMOL molecular graphics system ver. 2.5.0 (Schrodinger LLC).

Flooding simulations

The full-length mSlo3 from the AlphaFold3 webserver was trimmed to remove long disordered regions (residue 1-18, 616-698 and 1050-1121), resulting in 12 separate chains. The mSlo3 complex was embedded into the POPC bilayer and solvated in 0.15 M NaCl and 0.15 ZnCl₂ using CHARMM-GUI Membrane Builder (Wu et al., 2014). The production run simulations were simulated with a 2 fs timestep using the CHARMM36m forcefield (Huang et al., 2016) in GROMACS 2023.3 (Abraham et al., 2023). The system was then energy minimized and equilibrated using the standard six steps CHARMM-GUI equilibration protocol. This includes the following set-up: The protein backbone was restrained at the force constant of 4000, 2000, 1000, 500, 200 and 50 kJmol^-1nm^-2, the protein side chains were restrained with the force constant of 2000, 1000, 500, 200, 50, and 0 kJmol^-1nm^-2, the lipids non-H atoms were restrained at the force constant of 1000, 400, 400, 200, 40 and 0 kJmol^-1nm^-2 and the dihedral restraint was set at the force constant of 1000, 400, 200, 200, 100 and 0 kJmol^-1rad^-2. The simulations were equilibrated with a 1 fs timestep for 125 ps for the first three steps, and then to a 2 fs timestep for 500 ps the next two, and 5 ns for the final step. The first two steps were conducted with the NVT ensemble, whereas the last four were conducted with the NPT ensemble. All equilibration runs were conducted at 310 K using Berendsen thermostat (Berendsen et al., 1984). In all NPT ensemble equilibration, the semi-isotropic pressure was maintained at 1 bar using Berendsen barostat. The production runs were conducted for 200 ns under 310 K using the v-rescale thermostat (Bussi et al., 2007). The semi-isotropic pressure of all systems was maintained at 1 bar using a C-rescale barostat (Bernetti & Bussi, 2020). All simulations were carried out in triplicates, where velocity was randomly generated at the beginning of every simulation. All simulation frames were saved every 0.1 ns.

Zn stability

Based on the flooding simulations, the binding pose for Zn²⁺ is adjusted so that the ion is 3-4 Å away from E169. This results in the system with 4 Zn²⁺ per system, one per subunit. The system was then energy minimized using the steepest descent algorithm and equilibrated with the Cα restraint at 1000 kJ kJmol^-1nm^-2 for 10 ns. The production run was conducted for 200 ns, with the same barostat and thermostat setting as the flooding simulation.

Data Analysis

Inside-out patch-clamp data were analyzed using Clampfit 11.3 software (Molecular Devices), Igor Pro 6.22A (Wavemetrics), and GraphPad Prism 10. Analysis of current-voltage (I-V) were obtained from the maximum current amplitude at steady-state. I-V relationship was fitted with Boltzmann equation:

where G is a scaling factor (maximum conductance or current), V₅₀ is the midpoint, and s is the slope value.

And then the I-V relationship in the presence of 100 µM of ZnCl₂ was fitted with Woodhull equation (Woodhull, 1973) to express the current as a function of voltage, incorporating both the voltage-dependent gating of the channel and the voltage-dependent blocking of the channel:

where V_block is the voltage at which blocking is half-maximal, Slope_block is the slope factor for the voltage dependence of the blocking effect, and δ is the fraction of the membrane potential that the blocking ion experiences.

V_rev is the reversal potential and was calculated with following equation as described in (Schreiber et al., 1998) using Goldman-Hodgkin-Katz equation:

where P is the Na⁺ to K⁺ permeability ratio (Schreiber et al., 1998).

Conductance-voltage (G-V) relationship of Slo3 were obtained from conductance calculation using the following equation:

V_rev was calculated using equation (4) and fitted to Boltzmann equation in (1), adjusted for G-V.

IC₅₀ (half maximal inhibitory concentration) was calculated using equation while constraining the Hill Slope to a constant value of −1.0:

where I_max is the maximal current when inhibition is minimal, I_min is the minimal current inhibition is maximal, and X is the inhibitor concentration.

Arithmetic operations were performed by Clampfit to compensate bleaching from the fluorescence traces:

where [time] is the value of the point given by Clampfit.

All the compensated traces were then normalized by setting each baseline level (F signal at −60 mV, the holding potential) to be 1, to calculate the % F change:

Conductance-voltage (G-V) relationship for VCF data were obtained from conductance calculation using equation (5). Fluorescence-voltage (F-V) relationship were obtained from the normalized ΔF/F and fitted with the equation (1).

Statistical analysis

Statistical analysis was performed by either one-way ANOVA, unpaired t-test or paired t-test. Following one-way ANOVA, Dunnett’s post-hoc test was applied. The data were expressed as mean± s.e.m. with n indicating the number of samples. Values of p were stated in each bar graph. All the statistical analysis and the bar graphs were performed and generated with GraphPad Prism 10.

Acknowledgements

The authors thank Prof. Fredrik Elinder (Linköping University, Sweden) for discussion and the guidance on analysis and experiments, Ms. Hikari Ginama and Ms. Megumi Kobayashi for technical support and Dr. Natsuki Mizutani for discussion and guidance of experiment. This work was supported by fundings provided by Grants-in-Aid from MEXT KAKENHI Grant Number JP20H05507 (T.K), JP20H05791 (Y.O), JP22K15074 (R.T.A), JSPS KAKENHI Grant Number JP17K15558, JP23K06334 (T.K), 19H03401, 22H02804 (Y.O), and JST FOREST Program Grant Number JPMJFR225Z (T.K). The research is supported by JSPS Postdoctoral Fellowship for Research in Japan (R.T.A) and Lee Kuan Yew Postdoctoral Fellowship from Nanyang Technological University, Singapore (T.P). Additionally, financial support was received from the Takeda Science Foundation and Mochida Memorial Foundation for Medical and Pharmaceutical Research, all of which contributed to T.K.‘s research.