The role of Hv1 and CatSper channels in sperm activation

Abstract

Elevations of sperm intracellular pH and Ca²⁺ regulate sperm motility, chemotaxis, capacitation and the acrosome reaction, and play a vital role in the ability of the sperm cell to reach and fertilise the egg. In human spermatozoa, the flagellar voltage-gated proton channel Hv1 is the main H⁺ extrusion pathway that controls sperm intracellular pH, and the pH-dependent flagellar Ca²⁺ channel CatSper is the main pathway for Ca²⁺ entry as measured by the whole-cell patch clamp technique. Hv1 and CatSper channels are co-localized within the principal piece of the sperm flagellum. Hv1 is dedicated to proton extrusion from flagellum and is activated by membrane depolarisation, an alkaline extracellular environment, the endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. The CatSper channel is strongly potentiated by intracellular alkalinisation. Since Hv1 and CatSper channels are located in the same subcellular domain, proton extrusion via Hv1 channels should induce intraflagellar alkalinisation and activate CatSper ion channels. Therefore the combined action of Hv1 and CatSper channels in human spermatozoa can induce elevation of both intracellular pH and Ca²⁺ required for sperm activation in the female reproductive tract. Here, we discuss how Hv1 and CatSper channels regulate human sperm physiology and the differences in control of sperm intracellular pH and Ca²⁺ between species.

Yuriy Kirichok is an Assistant Professor of Physiology at the University of California San Francisco. He graduated from Moscow Institute of Physics and Technology, Russia and received his PhD in Biophysics from Bogomoletz Institute of Physiology, Kiev, Ukraine. He then moved to Boston, MA where he joined Dr David Clapham's Lab at Harvard Medical School as a postdoctoral fellow. He studies the role of ion channels in male fertility and ion channels that regulate mitochondria function. He adores the patch-clamp technique and can apply it to virtually any object no matter how small it is or how fast it moves. Polina Lishko graduated from Taras Shevchenko National University, Kiev, Ukraine and received her PhD in Biophysics from Bogomoletz Institute of Physiology, Kiev, Ukraine. In 2000 she joined Vadim Arshavsky's laboratory at Harvard Medical School to study mechanisms of vertebrate phototransduction. She continued her postdoctoral training to study the structure–function relationship of TRPV ion channels in Rachelle Gaudet's laboratory at Harvard University. She is currently a Specialist at the University of California San Francisco and together with Yuriy Kirichok, who happens to be her husband, studies regulation of sperm ion channels in humans. Her other interests include the history of science, cooking, and gardening.

When spermatozoa are introduced into the female reproductive tract they undergo several transformational steps to become able to fertilise an egg. Upon ejaculation, spermatozoa become motile for the first time and then become increasingly motile as they near the site of fertilisation in order to pass through the viscous luminal fluids of the female reproductive tract. Finally, they must successfully find the egg and penetrate its protective vestments to complete fertilisation. Several key steps in this process are controlled by sperm ion channels and transporters that set membrane potential and regulate intracellular pH and calcium levels, thus controlling motility patterns, chemotaxis, and the acrosome reaction.

Sperm intracellular pH is a key factor that controls sperm motility and fertilising ability. Morphologically mature mammalian spermatozoa stored in the caudal part of the epididymis and the vas deferens are essentially quiescent (Hammar, 1897) and are kept immotile primarily by an acidic intracellular and extracellular pH (Acott & Carr, 1984; Carr & Acott, 1989). Seminal plasma is more alkaline (pH > 7.0) and therefore provides a suitable environment for alkalinisation of the sperm cytoplasm (Hamamah & Gatti, 1998) and activation of sperm motility. As the sperm travel through the female reproductive tract, their intracellular pH gradually increases further, and this elevation is perceived as one of the key factors leading to functional activation (capacitation) of sperm in the oviduct.

The presence of a membrane transport mechanism for proton extrusion that results in alkalinisation of sperm cytoplasm was suggested in 1983 when Babcock et al. demonstrated elevation of bovine sperm intracellular pH upon membrane depolarisation (Babcock et al. 1983). This suggested the presence of either a voltage-gated proton channel or a voltage-dependent proton transporter. Since then it has been demonstrated that the Na⁺-dependent Cl⁻/HCO₃⁻ exchanger controls intracellular pH in mouse spermatozoa (Zeng et al. 1996). Additionally, a sperm-specific Na⁺/H⁺ exchanger (sNHE) was identified in mice, but its effect on sperm intracellular pH remains to be shown (Wang et al. 2003, 2007).

Moreover, recent application of the patch-clamp technique to human spermatozoa revealed the molecule controlling intracellular pH in human sperm cells (Lishko et al. 2010). Unlike mouse sperm cells, human spermatozoa possess a voltage-gated proton channel, Hv1, which is localised to the principal piece of the sperm flagellum.

Hv1 is a flagellar regulator of intracellular pH in human spermatozoa

The existence of a plasma membrane ion channel selective for protons was first demonstrated by Thomas & Meech (1982), but it took several decades for the molecule responsible for this phenomenon to be identified (Sasaki et al. 2006; Ramsey et al. 2006). The proton-selective channel Hv1 (gene name HVCN1 for human or VSOP for mouse) is a four-transmembrane domain protein homologous to the voltage-sensor domain of voltage-gated cation channels. The Hv1 channel exists as a dimer in the plasma membrane; however, a single Hv1 subunit can function independently as a voltage-gated proton channel (Koch et al. 2008; Lee et al. 2008; Tombola et al. 2008). What makes Hv1 unique is the absence of the classical ion pore, and proton permeation is likely to occur via a water wire spanning the voltage sensor domain (Ramsey et al. 2010). Hv1 is abundant in phagocytes where it acts with NADPH oxidase to generate high levels of bactericidal reactive oxygen species by compensating membrane depolarisation and intracellular acidification, the side effects of NADPH oxidase enzymatic activity (Okochi et al. 2009; Ramsey et al. 2009). Activation by membrane depolarisation and by outward transmembrane pH gradient, selective inhibition by low micromolar concentrations of zinc, and the ability to be potentiated by fatty acids represent the classical electrophysiological fingerprint of Hv1 (DeCoursey, 2008). All these properties are also characteristics of sperm Hv1, and sperm Hv1 also has been shown to be potently activated by endogenous cannabinoid anandamide (Lishko et al. 2010), which is synthesised and possibly secreted by the protective vestment of the oocyte – the cumulus oophorus (El-Talatini et al. 2009) (Fig. 1).

Figure 1. Proton regulation and CatSper function in mouse versus human spermatozoa.

In mouse spermatozoa, intracellular alkalinisation is achieved via activity of Na⁺-dependent (Cl⁻/HCO₃⁻) exchanger (NCB) and sperm-specific Na⁺/H⁺ exchanger (sNHE). NCB also helps to accumulate bicarbonate ions (HCO₃⁻) to stimulate bicarbonate-dependent atypical adenylyl cyclase (ADCY10), which leads to elevation of intracellular cAMP (Hess et al. 2005; Carlson et al. 2007) and possible stimulation of sNHE, which has a cAMP binding site (Wang et al. 2003). In human spermatozoa, the voltage-gated proton channel Hv1 is likely to constitute the main proton extrusion mechanism. Activation of Hv1 by anandamide (AEA), removal of extracellular zinc, and the alkaline environment of the female reproductive tract cause intracellular alkalisation. In both mouse and human sperm cells, intracellular alkalinisation activates the calcium channel CatSper that allows calcium ions to enter sperm flagellum. The rise in intracellular calcium causes hyperactivation and prepares spermatozoa to undergo the acrosome reaction.

Hv1 is likely to induce sperm intracellular alkalinisation by equalizing the acidic sperm intracellular pH_i (5.5–6.5) (Hamamah & Gatti, 1998) with the more alkaline extracellular medium (pH_o > 7 at the site of fertilisation). As spermatozoa ascend the female reproductive tract and approach the egg, their pH_i approaches neutral (Zeng et al. 1996), and fertilising potential increases.

The voltage activation threshold of Hv1 depends on the ΔpH across the sperm plasma membrane. At intracellular pH 5.5 and extracellular pH 7.4, the activation threshold of Hv1 is approximately −60 mV (Lishko et al. 2010). However, since sperm intracellular pH is normally higher then 5.5, under physiological conditions Hv1 activation threshold is more positive, and the channel should not be active at the human sperm resting membrane potential (−40 mV) (Linares-Hernandez et al. 1998). Under these conditions sperm Hv1 can be activated by either membrane depolarisation or by extracellular anandamide. Membrane depolarisation can be induced, for example, by yet unidentified ligand-gated cationic channels of sperm. It is also tempting to speculate that anandamide produced by cumulus cells activates Hv1 in the egg vicinity, thereby providing more efficient alkalinisation of sperm cytoplasm during sperm penetration through the cumulus oophorus. Since Hv1 extrudes protons and therefore carries positive charge outside the cell, Hv1 should produce membrane hyperpolarisation that will limit Hv1 activity unless this hyperpolarisation is compensated for by other electrogenic transport mechanisms.

Hv1 sensitivity to zinc is likely to play a significant role in sperm physiology, since zinc has long been known to regulate male fertility. Seminal plasma has the highest concentration of zinc in the human body (3 mm) (Saaranen et al. 1987). Thus, when spermatozoa are mixed with seminal plasma, zinc inhibits Hv1 and may prevent premature sperm activation. During their travel through the female reproductive pathways, this zinc inhibition should be halted by albumin chelation and absorption by uterine and oviductal epithelium (Gunn & Gould, 1958; Ehrenwald et al. 1990; Lu et al. 2008), so that, upon arrival at the fertilisation site within the fallopian tube, spermatozoa should be essentially free from inhibition by extracellular zinc.

Fallopian tubes play an important role in sperm maturation and are the site of the capacitation process (Austin, 1951; Chang, 1951). It must be noted that freshly ejaculated spermatozoa are unable or poorly able to fertilise (Stauss et al. 1995) and must first undergo capacitation, a chain of intrinsic temperature-dependent reactions during which spermatozoa become hyperactivated (the amplitude of the flagellar bend increases, which results in more asymmetrical whip-like movements) and acquire the ability to undergo the acrosome reaction (Yanagimachi, 1969, 1970; Mahi & Yanagimachi, 1973). During capacitation the following processes occur: depletion of plasma membrane cholesterol, removal of adherent seminal plasma proteins, increase in cyclic AMP content, and increase in intracellular pH (Nixon & Aitken, 2009; Fraser, 2010). Interestingly, the activity of sperm Hv1 is greatly enhanced in capacitated sperm cells (Lishko et al. 2010). Hv1 current recorded from capacitated spermatozoa is characterized by a larger amplitude, faster activation kinetics, and a negative shift in the activation voltage (Lishko et al. 2010), which may be associated with channel phosphorylation – a well known mechanism for Hv1 up-regulation in somatic cells (DeCoursey, 2008). Since phosphorylation of multiple proteins is a crucial step in the process of sperm capacitation (Visconti et al. 1995a,b;), we believe that phosphorylation may enhance the ability of Hv1 to cause elevation of intracellular pH during capacitation.

Surprisingly, Hv1 current is absent in mouse spermatozoa (Lishko et al. 2010). This difference between mouse and human sperm cells indicates that the Na⁺-dependent Cl⁻/HCO₃⁻ exchanger (and maybe sNHE) should be solely responsible for pH regulation within flagella of murine spermatozoa (Zeng et al. 1996; Florman et al. 2010) (Fig. 1). Since Hv1 current is absent in mouse spermatozoa, it is not surprising that an Hv1 knockout mouse is fertile (Ramsey et al. 2009). Since the patch-clamp technique cannot detect certain transmembrane transport mechanisms such as non-electrogenic transporters, it is possible that such mechanisms may contribute to acid extrusion in the sperm of mice and humans.

Sperm pH-dependent CatSper channel controls intraflagellar calcium signalling

The intracellular alkalinisation caused by pH regulating molecules activates the pH-dependent sperm-specific calcium channel CatSper (Cation channel of Sperm), which is confined to the principal piece of the sperm flagellum similar to Hv1 (Ren et al. 2001; Kirichok et al. 2006). The role of the CatSper channel is to provide calcium influx into the sperm flagellum and therefore induce Ca²⁺-dependent hyperactivated motility (Carlson et al. 2003). Interestingly, spermatozoa from CatSper1- and CatSper2-deficient mice can be artificially hyperactivated by addition of thimerosal that releases calcium from internal stores (Marquez et al. 2007) or by procaine, which is likely to induce CatSper-independent Ca²⁺ entry (Carlson et al. 2005). Without hyperactivation of motility, spermatozoa cannot traverse the oviduct and penetrate the protective barriers on the egg's surface, the cumulus oophorus and zona pellucida (Avidan et al. 2003; Suarez & Ho, 2003; Suarez & Pacey, 2006. Therefore the CatSper channel is absolutely required for male fertility in both mice and humans (Ren, 2001; Suarez, 2008; Avenarius et al. 2009; Ho et al. 2009).

The CatSper ion channel is a sophisticated protein complex composed of at least six subunits, of which four α subunits (CatSper 1–4) form a calcium-selective pore (Lobley et al. 2003; Qi et al. 2007). The first member of the family, CatSper1, possesses a large N-terminus in which histidine residues are remarkably abundant. This His-rich domain may be involved in pH regulation of the CatSper current (Ren et al. 2001; Navarro et al. 2008). The complex includes two additional auxiliary subunits, CatSperβ and CatSperγ, which are transmembrane proteins with large extracellular domains and unknown functions (Liu et al. 2007; Wang et al. 2009). Studies with mice deficient in CatSper 1, 2, 3, and 4 revealed that all four subunits are required for stable expression of the heteromeric channel complex, and knockout of any one of them results in absence of the other subunits (Qi et al. 2007). To this day, heterologous expression of the functional CatSper channel complex remains a challenge, and study of the CatSper channel is limited to electrophysiological, optical and classical biochemical applications in the native system.

Similar to many other voltage-gated calcium channels, CatSper can conduct monovalent cations such as sodium and caesium in the absence of divalent cations. However, the sensitivity of CatSper to calcium ions is much greater than that of other calcium channels (Kirichok et al. 2006). CatSper is not permeable to magnesium ions (Kirichok et al. 2006).

Both mouse and human CatSper channels have weak voltage dependence (Kirichok et al. 2006; Lishko et al. 2010). The voltage-activation curves for strongly voltage-sensitive ion channels (slope factors of ∼4) are much steeper than for those for mouse CatSper (slope factor ∼30) (Kirichok et al. 2006). Although CatSper voltage dependence is weak, it plays a significant role in the regulation of the CatSper channel by pH. At acidic intracellular pH 6.0, CatSper voltage activation curve is shifted toward the positive potential so much (half-activation voltage +87 mV) that only a very small fraction of the channels are active at physiologically relevant negative membrane potentials (Kirichok et al. 2006). Elevation of pH_i to 7.5 produces a dramatic 76 mV negative shift of the CatSper voltage dependence and makes the channel active in the physiological range of potentials (Kirichok et al. 2006).

In many voltage-gated sodium channels and voltage-gated calcium channels the extracellular domains of auxiliary subunits modulate channel activity upon binding of extracellular ligands (Liu et al. 2007; Eroglu et al. 2009; Wang et al. 2009). Interestingly, CatSperγ has a large extracellular domain of 1066 amino acids and the very N-terminal part of this domain shares weak homology with the von Willebrand factor A-like (vWFA) domain of the α2δ2 subunit of the voltage-gated calcium channel. Recently, it was reported that this vWFA-like domain binds thrombospondin and simpler organic molecules such as gabapentin (Eroglu et al. 2009). It is possible that CatSperγ plays a similar role and can modulate CatSper ion channel function upon association with some signal molecule(s) present in the female reproductive tract.

Since the egg is normally fertilised by the spermatozoon that was most successful in overcoming the barriers encountered on the route towards the egg, the properties of the most successful sperm are immediately passed on to the future generation. Thus, in the essence of the ‘survival of the fittest’ concept, evolution of the sperm cell represents one of the most rapidly changing biological systems. Therefore, it should be of no surprise that sperm physiology and sperm ion channels differ between species. The CatSper channel is a good example of such variation. According to Cai & Clapham (2008), the CatSper complex disappeared several times during evolution: it is absent in roundworms, insects, teleost fish, amphibian and birds, yet it is preserved not only in mammals and reptiles, but also in tunicates, echinoderms and cnidarians (Cai & Clapham, 2008; Navarro et al. 2008). Thus, spermatozoa of sea squirts, sea urchins and sea anemones also express a functional CatSper complex. In species where the oocyte has a thin wall (such as birds and fish), the job of the spermatozoon is easier and the CatSper channel may be redundant. In fact, in these species sperm hyperactivation required for penetration through egg protective vestments is not observed (Cummins, 2009). In marine animals with external fertilisation such as sea urchins, spermatozoa need to find the egg in sea water. Sea urchin spermatozoa may use asymmetrical, hyperactive-like tail movements to make chemotactic turns that direct them toward the egg (Kaupp et al. 2008). Therefore, the presence of the CatSper channel in sea urchin spermatozoa might be essential for chemotaxis. In contrast, fish spermatozoa, which are delivered directly onto the egg, do not require chemotaxis and may have disposed of the CatSper channel during evolution.

In conclusion, the molecular mechanisms that control sperm intracellular Ca²⁺ and pH play key roles in the process of fertilisation. Future studies will shed more light on the molecular identities and physiological regulation of these mechanisms. These advances will help in the development of new tools to control sperm cell behaviour in the female reproductive tract and will help to increase or decrease male fertility. However, we predict that the mechanisms by which human spermatozoa control intracellular pH and calcium differ from those in other species. Since human fertilisation is of special concern, more efforts should be directed to studying the physiology of the human sperm cell on the molecular level.