The Role of Sperm Membrane Potential and Ion Channels in Regulating Sperm Function

During the last seventy years, studies on mammalian sperm cells have demonstrated the essential role of capacitation, hyperactivation and the acrosome reaction in the acquisition of fertilization ability. These studies revealed the important biochemical and physiological changes that sperm undergo in their travel throughout the female genital tract, including changes in membrane fluidity, the activation of soluble adenylate cyclase, increases in intracellular pH and Ca2+ and the development of motility. Sperm are highly polarized cells, with a resting membrane potential of about −40 mV, which must rapidly adapt to the ionic changes occurring through the sperm membrane. This review summarizes the current knowledge about the relationship between variations in the sperm potential membrane, including depolarization and hyperpolarization, and their correlation with changes in sperm motility and capacitation to further lead to the acrosome reaction, a calcium-dependent exocytosis process. We also review the functionality of different ion channels that are present in spermatozoa in order to understand their association with human infertility.

Infertility is defined as the inability to conceive after 12 months or more of regular unprotected intercourse [1]. Currently, this reproductive disease is considered a global health and social problem estimated to affect between 8% and 15% of couples of reproductive age worldwide [2]. It has been reported that the male factor is solely responsible for approximately 20% of infertile couples and contributes to another 30% of couples [3]. During the last seventy years, studies on sperm cells have allowed a great advance in our knowledge about the physiology of spermatozoa and a parallel development of assisted reproduction technology (ART) as well [4,5,6,7,8,9,10,11]. However, the diagnosis of male infertility remains mainly based on semen analysis, and semen parameters alone are insufficient to determine infertility/fertility status. Moreover, the cause of abnormal semen parameters cannot always be identified, and no treatment can be offered to those patients [12]. To improve current infertility diagnosis, it is necessary to have a deeper knowledge about the control of physiological functions of spermatozoa at a cellular and molecular level.

Sperm cells are immature and non-competent immediately after ejaculation [4,5]. In order to obtain fertilization potential, spermatozoa need to undergo capacitation, which implies profound physiological and biochemical changes that occur inside the female genital tract [4]. For this capacitation to take place, spermatozoa must send and receive specific signals from the environment, which must be properly decoded under a precise spatio-temporal regulation that is still not completely unraveled. During their travel, sperm cells suffer an important loss of cholesterol from their membrane, causing an enhancement of membrane fluidity [13]. The increase in extracellular pH, as well as the high bicarbonate concentration in the seminal plasma and in the female genital tract, enable HCO3− influx to the sperm cell [14,15,16] and HCO3− uptake stimulates the activity of soluble adenylate cyclase (sAC) and the production of cyclic adenosine monophosphate (cAMP), which, in turn, causes the activation of protein kinase A (PKA) [9,17,18,19,20]. PKA induces the phosphorylation of proteins in serine, threonine and, ultimately, in tyrosine (Tyr) residues, which constitutes a mark of sperm capacitation [9,17]. This is followed by a modification of the membrane composition that enables the acrosome reaction (AR) [15,16,21,22]. The control of sperm membrane potential (Em) is essential for all these processes to occur and its regulation is orchestrated by complex signaling pathways that involve activation of a great number of ion transporters, exchangers and ion channels present in the sperm membrane [8,14,15]. Between them, ion channels play a central role due to their rapid response and transport capability [10,11,14,15,23,24,25].

In this review, we analyze the relationships between changes in sperm membrane potential and the functionality of different ion channels that are present in human spermatozoa, in order to better understand their association with sperm function and, consequently, with human fertility.

Spermatozoa are highly polarized cells with two clearly differentiated parts, a head and a tail. As occurs in neurons, sperm cells rapidly respond to changes in the environment and the regulation of ion balance across their membrane has been shown to be essential for sperm motility and fertilization capacity. The main ions involved in the acquisition of sperm fertilization ability are Na+, K+, Ca2+, Cl− and H+ [26], similar to those which occur in neurons. In addition, the Na+/K+ ATPase plays an important role in the establishment of the resting Em and in the maintenance of the electrochemical gradients for Na+ and K+ across the sperm plasma membrane [15,27].

In neurons, action potentials play a central role in cell-to-cell communication, assisting the propagation of signals along the neuronal axon toward synaptic boutons situated at the ends of the axon. Changes in Em occur due to the opening of voltage-dependent ion channels present in the cell plasma membrane, which change the polarity of transmembrane potential and generates an action potential [28]. It starts with a temporal Na+ permeability increase, producing an influx of this cation and an umbral membrane depolarization (~−55 mV) [29,30]. When Na+ is entering the cell (+30 mV), K+ permeability increases, but the opening and closing kinetics of K+ channels are slower than those of Na+ channels. For this reason, when K+ gets out of the cell to recover the resting Em, it takes more time to close the K+ channels and the K+ efflux produces a membrane hyperpolarization (−90 mV). At the time the K+ channels are closed, the Na+/K+ ATPase restores Em [28]. The membrane depolarization induces the activation of Ca2+ channels, which are essential for synaptic transmission, causing an increase in Ca2+ concentration at the presynaptic membrane. Consequently, neurotransmitters are released to the synaptic cleft and exocytotic vesicles fusion with the postsynaptic membrane to transmit the information to the next neuron [31,32].

As happens in neurons, changes in the ion balance across the membrane generate Em adaptations in spermatozoa [33]. During the journey from the testis to the fertilization site, sperm cells encounter different ion concentrations that modulate their ion channel activity, and therefore their Em [15]. External [K+] may change from ~39 to 5–8 mM; [Cl−] varies from ~27 to 130 mM, and external [Na+] goes from 38 to 140 mM between the cauda epididymis and the oviduct [33]. Non-capacitated human spermatozoa maintain a resting Em of around −40 mV [23,34] and ion channels are in a putative inactive state. Once in the female reproductive tract, human sperm are exposed to an increase in [Na+], leading to a Na+ influx that induces membrane depolarization [35,36], which promote linear sperm motility [36]. In parallel, membrane conductance has lower selectivity for K+ than for Na+ [37], proving that membrane depolarization prepares spermatozoa for a successful further capacitation [38]. In many species, including humans, sperm capacitation is accompanied by sperm plasma membrane hyperpolarization, with an increase in intracellular net negative charge to ~−60/−70 mV [33,39]. Membrane hyperpolarization, mainly induced by H+ and K+ currents, is necessary to achieve a successful capacitation in human sperm [23,35,40] and it is a previous event to prepare sperm for the AR [15,40,41]. For these processes to occur, an increase in intracellular [Ca2+] is needed, which can be induced by a calcium influx through calcium channels and be accompanied by Ca2+ release from intracellular reservoirs [15,34]. As in neurons and other cell types, calcium is needed to induce the exocytosis process known as acrosome reaction, an essential prerequisite for fusion with the oocyte and, therefore, fertilization [34,42].

Sperm maturation and the acquisition of motility initiates in the male genital tract [16,43]. The completion of cell modeling occurs in the epididymis where spermatozoa are coated with many different proteins and other secretory products that induce profound changes in their functional capability, metabolism and biochemistry [44,45,46]. Swimming capacity is also acquired in the epididymis, although due to the acid pH and low HCO3− concentration, they do not move actively until their transport to the female genital tract [16,43,46,47]. Prior to ejaculation, sperm cells are mixed with the seminal plasma, a HCO3−-rich medium formed by secretions from the testis, the epididymis, the prostate, the bulbourethral and the periurethral glands, with a major contribution of the seminal vesicles [43,45,46].

Seminal plasma, which constitutes approximately 95% of semen, provides the optimal environment that ensures sperm motility and fertilization ability and is considered as one of the main sources of factors necessary for capacitation [44,45,46]. At the same time, seminal plasma is rich in free cholesterol and Zn2+, which act as decapacitation factors and prevent a premature capacitation [15,43,44,45,46]. After ejaculation, seminal plasma proteins form the gelatinous coagulum that inhibits temporarily the initiation of swimming and due to its buffering properties (pH~7.3–8.4) protect spermatozoa from the acidic vaginal milieu (pH~5) [16,43,44,46]. Once in the female genital tract, a small number of spermatozoa are able to enter the cervix after liquefaction of the seminal coagulum, and they continue the travel throughout the female tract, where they are exposed to changes in the ionic environment that enable their motility.

The fluctuations in ion concentration through the female genital tract induce changes in ion channel permeabilities, which cause Em variations. Remarkably, it has been shown that Na+ changes have a great influence on the sperm Em [48,49,50,51]. The importance of this cation is further supported by the essential role played by the Na+/K+ ATPase, the electrogenic pump which maintains Na+ and K+ gradients across the cell membrane, in the regulation of sperm function. The Na+/K+ ATPase is a heteromeric membrane protein composed of two major polypeptides, an α and a β subunit, making a complex that associates with a γ subunit of the FXYD family [27,52]. The α subunit, formed by 10 transmembrane (TM) segments, constitutes the catalytic subunit and contains the binding sites for the cations and ATP, as well as for ouabain, which is a potent inhibitor of the pump activity, while the single membrane-spanning β subunit is necessary for localization of the ATPase to the plasma membrane [52,53]. Four different isoforms of the α subunit (named α1, α2, α3 and α4) and four different β subunits (named β1, β2, β3 and β4) have been cloned and each of them exhibit unique tissue expression profiles [52]. Between them, the α4 subunit is of particular interest, as it has been only detected in the testes and sperm of various mammalian species, being abundantly expressed in the sperm flagellum [27,49,54,55]. In mouse and rat sperm, blockade of the α4 subunit with ouabain impairs sperm motility and hyperactivation, causes sustained membrane depolarization, a decrease in pHi and an increase in [Na+]i and [Ca2+]i [27,49]. The essential role of the α4 subunit in sperm has been demonstrated by studies in α4 subunit-null mice, which show functional alterations similar to those observed after ouabain treatment and are infertile [27].

Human spermatozoa are exposed to an increase in [Na+] in the female reproductive tract, which induces a membrane depolarization caused by Na+ influx [35] and promotes linear sperm motility [36]. Sperm membrane depolarization, therefore, can be mediated by activation of Na+ channels and, among them, the presence of ENaC channels and of voltage-gated Na+ channels (VGSC) has been detected in human spermatozoa (Table 1).

The addition of external Na+ produces a depolarization in sperm cells that is potently inhibited by amiloride and its analog EIPA (5-(N-ethyl-N-isopropyl)-amiloride) [15]. ENaC are heteromultimeric Na+ selective channels made up of 4 subunits: α, β, γ and δ [63]. They are regulated by intracellular pH and Ca2+, extracellular Na+, Cl− and phosphorylation and inhibited by amiloride and EIPA [58,64]. These channels play a central role in the electrogenic Na+ transport in a variety of tissues and different studies have shown that they are essential for regulation of Em in spermatozoa [15,51,56,57,58]. The presence of three ENaC subunits, α, β and δ have been demonstrated in sperm from different mammalian species including humans, specifically in the flagellar midpiece and principal piece of the sperm (Table 1) [51,56,58,59], and an amiloride-sensitive inward Na+ current has been recorded in mouse spermatogenic cells [51]. This suggests a role for ENaC channels in sperm motility, which is confirmed by the fact that blockade of these channels increases sperm motility in asthenozoospermic patients [56]. Na+ permeability is involved in the establishment of the sperm resting Em, and it is thought to be reduced during sperm capacitation [50,51]. These channels, therefore, contribute to the regulation of Em in human sperm [58].

Veratridine, a highly selective VGSC activator, causes membrane depolarization and increases intracellular Na+ in human sperm cells, leading to a concentration-dependent increase in progressive sperm motility [61,65]. Voltage-gated sodium channels (Nav channels) belong to the voltage-gated ion channel (VGC) superfamily and are complex proteins composed of an α and one or more auxiliary β subunits [29,66,67]. The α subunit is a large protein that consist of four homologous domains, each with six transmembrane (TM) segments, and forms the ion-conducting aqueous pore. Nine different Nav α subunits (Nav1.1–Nav1.9) and a tenth, related, voltage-insensitive atypical isoform (Nax) have been cloned in mammals, each of them encoded by a different gene. Four different β subunits, named β1, β2, β3 and β4, are currently known and each of them consist of a single membrane-spanning segment with a large extracellular N-terminal domain and a smaller intracellular tail [25,29,68]. The mRNA of all VGSC, both α and β subunits, are highly expressed in the human testis, at a level comparable to that found in brain tissues [36,60] and, with the exception of the auxiliary subunit β2, they are also present in sperm (Table 1) [36,69]. Immunofluorescence studies have shown that Nav channel proteins are present in human sperm cells and display specific and different sites of localization, with Nav1.2, Nav1.6, Nav1.8 and Nax being predominantly localized in the flagellum, and Nav1.4, Nav1.7 and Nav1.9 in the connecting piece [36,61]. Nav1.8 has also been detected in bull [70] and ovine sperm [71] and its expression in the ovine sperm transcriptome is strongly downregulated under conditions of heat stress [71].

Functional studies have shown that veratridine causes time- and concentration-dependent increases in progressive sperm motility that are reduced in the presence of tetrodotoxin (TTX) or A-803467, a specific Nav1.8 antagonist, suggesting that the effects of veratridine on motility involve activation of different, TTX-sensitive and insensitive Nav channels [61,62,65]. In addition, veratridine does not induce hyperactivation or AR by itself, but is able to inhibit the progesterone-induced AR [61,65]. Using fluorimetry, it was shown that veratridine causes membrane depolarization and increases intracellular Na+, but only induces a minor increase in the intracellular Ca2+ concentration in capacitated spermatozoa, which is not produced in non-capacitated cells [65]. All these data demonstrate that veratridine does not act by activating Ca2+ channels and suggest that VGSC might be involved in the regulation of the basal, linear progressive sperm motility and acquisition of capacitation, avoiding a premature hyperactivation and AR in an inadequate place.

Capacitation implies profound physiological and biochemical changes that initiates in the male genital tract and culminates inside the female genital tract [4,5]. At a molecular level, capacitation is associated to: loss of membrane cholesterol [72,73] and modification of other membrane lipids [74]; activation of a cAMP/PKA pathway [9,20]; increase in protein tyrosine phosphorylation and in intracellular pH (pHi) [9,75]. The increase in extracellular pH, as well as the high [HCO3−] in semen and in the female genital tract [14,16,47] enable a rapid HCO3− influx to the sperm cell and induces sperm capacitation [15]. HCO3− uptake leads to an increase in flagellar beat frequency by stimulating the activity of the soluble adenylyl cyclase ADCY10 (sAC) and the production of cAMP which, in turn, caused a quick activation of protein kinase A (PKA). This kinase induces the phosphorylation of proteins in serine, threonine and, ultimately, in tyrosine residues [9,17,18,76]. The increase in HCO3− regulated sAC activity is necessary for sperm intracellular alkalinization and membrane hyperpolarization [9,58,77] and modulates the activation of K+ (KSper) and Ca2+ (CatSper) channels, events that are all required for sperm capacitation and sperm motility hyperactivation, respectively [20,47,58]. PKA also regulates the activity of the Na+/K+ ATPase and causes, depending on the cell type, an activation or an inhibition of the pump [52], although the precise effects on human sperm remain unknown. The importance of sAC in male fertility is demonstrated by the observation that men with homozygous mutations in the gene encoding ADCY10 are infertile [78].

In this context, it has recently been shown that the blockade of sAC with the selective inhibitor TDI-10229 reduces the bicarbonate-induced increase in motility (without affecting basal motility) and prevents PKA activity, Tyr phosphorylation, intracellular alkalinization and the AR in mouse and human sperm, further confirming the key role played by sAC in the acquisition of sperm fertilization ability [20].

All this process is orchestrated by complex signaling pathways, with an important participation of Em hyperpolarization, which is regulated by different ion fluxes [23,35,40,51,79]. Specifically, there are two ion mechanisms that mainly contribute to sperm Em hyperpolarization: (1) the reduction in Na+ permeability and (2) the increase in K+ permeability. PKA inhibitors are able to block the capacitation-induced hyperpolarization, proving that, downstream cAMP signaling, hyperpolarization is necessary to prepare spermatozoa for AR [40,41,80]. The increase in cAMP/PKA causes the activation of K+ channels and indirectly inhibits ENaCs through a mechanism that involves the activation of the Cystic Fibrosis Transmembrane Conductance Regulator channel (CFRT) [51,58].

CFTR is an ATP-gated anion channel that conducts Cl− and HCO3−. It is expressed at the equatorial segment of the human sperm head [77] and the flagellar midpiece [51] and mutations in the CFTR gene are responsible for infertility and Cystic Fibrosis disease. The activation of CFRT, which are localized with ENaC in the midpiece of sperm flagella [15,51,56], is coupled to inhibition of ENaC, resulting in membrane hyperpolarization. Consistent with this activity, it has been shown that a reduction in [Na+]e or blockade of ENaC by amiloride causes membrane hyperpolarization and increases sperm motility in asthenozoospermic patients [51,56,58]. Similarly, inhibition of CFRT significantly reduced capacitation and HCO3−-associated events [77] and a specific CFRT inhibitor was shown to prevent ZP3-induced AR and sperm–oocyte fusion in humans [81].

On the other hand, K+ channels participate in the modulation of the intracellular K+ concentration and have a major role in determining Em, being particularly important in sperm hyperpolarization [25,82,83]. They are the most diverse class of ion channels and show a great structural diversity (Table 2). Different types of K+ channels, including Ca2+-activated K+ channels of the SLO subfamilies [84,85], voltage-gated K+ channels (Kv channels) [86], inwardly rectifying K+ channels (Kir channels) [82,87] and two-pore domain K+ channels (K2P) [88,89] have been detected in human sperm.

SLO channels, also known as KSper, have a major role in regulating capacitation- induced hyperpolarization in the sperm cells [23,24,83,84,91]. SLO channels belong to the family of Ca2+-activated K+ channels (KCa) and are composed of a pore-forming α subunit, and three types of auxiliary subunits [25,85,112,113,114] (Table 2). The four known α subunits are named SLO1, SLO2.1, SLO2.2 and SLO3, and among them, SLO1 and SLO3 are abundantly expressed in mammalian spermatozoa [15,83,84,85,90]. Although SLO are members of the 6 TM K+ channels, SLO1 and SLO3 have an additional TM domain, named S0, which play an important role in the interaction with auxiliary subunits. They are, therefore, unique K+ channels with 7 TM. The functional protein is an homotetramer, formed by the association of four α subunits, leading to a 7 × 4 TM basic structure [85,112,113,115] (Table 2). Three types of auxiliary subunits have been described for SLO channels: β subunits (β1–4), γ subunits (γ1–4), which belong to the family of extracellular leucine-rich-repeat-containing proteins (LRRC), and the recently described LINGO subunits (LINGO 1–4) that belong to the extracellular leucine-rich repeat and immunoglobulin-like (Ig) domains (LRRIG) protein family [85,92,95,98,114]. The functional properties of SLO channels are dramatically altered by the co-assembled auxiliary subunits, their identity and the number of them. Thus, the α subunit tetramer can be surrounded by 0–4 β subunits, 0–4 γ subunits and a still undetermined number of LINGO subunits [85,113,114].

SLO1, also known as KCa1.1, BK or Maxi-K channel, is highly expressed in the flagellum of human spermatozoa [40,90] (Table 2). The SLO1 current is activated by two independent physiological stimuli that act synergistically, [Ca2+]i and membrane depolarization, but it is insensitive to intracellular alkalinization [85,90,97,112,113]. This large conductance K+ channel is regulated by cholesterol and 17β-estradiol (E2), activated by Mg2+ and inhibited by progesterone [90,113,114]. On the other hand, the sperm-specific SLO3 or KCa5.1 channel, an evolutionary duplication of SLO1 in mammals, is also abundant in the human sperm flagella. This channel is sensitive to intracellular alkalinization and membrane depolarization and it is inhibited by progesterone [23,40,84,91,114,116]. In mice, the SLO3 channel is responsible for KSper, and deletion of Kcnu1, the gene that encode SLO3, inhibited the alkalinization-induced K+ current, and caused infertility [23,83,92,93]. Sperm from these mice are unable to swim progressively, to hyperpolarize and to undergo the acrosome reaction. Remarkably, these sperm cannot acrosome react even when exposed to the Ca2+ ionophore A23187. These results show the importance of SLO3 channel activation in the capacitation-associated processes necessary for fertilization [92,93].

In contrast, the human KSper current has mixed characteristics, as it shows weak sensitivity to alkalinization, high sensitivity to [Ca2+]i, and is inhibited by high concentrations of progesterone [84,90,91,117,118]. Thus, the precise identity of the channel mediating the [Ca2+]i-dependent K+ outward fluxes that hyperpolarize the human sperm membrane remains undefined. It has been suggested that human KSper can be mediated by SLO1 [90], by both SLO1 and SLO3 [40,83,91] or by a human-specific SLO3 variant [15,24,84,119]. In this context, a recent study described a human case of male infertility in which a homozygous mutation in SLO3 causes severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations [94]. Additionally, the differential characteristic of human KSper may be due to association with specific auxiliary subunits, which can produce profound changes in SLO channel gating properties [85,95,99]. For example, auxiliary β subunits alter channel gating in the presence of elevated [Ca2+]i while γ subunits affect SLO gating even at low [Ca2+]i [114]. Yang et al. (2009) found that all β subunits can be coexpressed with SLO3 in Xenopus oocytes, although only β4, both from human and mice, can modify the channel activation kinetics and surface expression [95]. These authors showed that all β subunits mRNAs are present in mouse sperm [95]. It has also been shown that SLO3 binds strongly to γ1 (LRRC26) and γ2 (LRRC52) subunits but interact only weakly with γ3 (LRRC55) and γ4 (LRRC38) [98]. In mice, the γ2 subunit controls the physiological activation of KSper current and, as shown in Lrrc52 null mice, is critical for fertility [83,99]. Further studies are needed to determine the identity, stoichiometry and influence of the auxiliary subunits that coassemble with SLO subunits in human sperm.

Kv channels constitute the largest family of K+ channels, with 40 members, and belong to the VGC superfamily. The Kv family is formed by 12 subfamilies (Kv1–Kv12) that are widely distributed in a great variety of tissues [113,120]. The pore-forming α subunit is formed by 6 TM, and the functional channel is a homo- or heterotetramer, composed of 4 α subunits, leading to the classical VGC structure of 24 TM segments [113]. Kv channels may also contain auxiliary β subunits (β1–β3), which regulates channel localization and gating properties [113]. Mammalian Kv channels can be modulated by phosphorylation, being activated by Protein Kinase C (PKC) [121] and downregulated by Tyrosine Kinase (TK) activity [122]. Jacob et al. (2000) described the presence of Kv1.3 mRNA in rat testis and showed that it can be modulated in a similar way [86]. Felix et al. (2002) detected a tetraethyl ammonium (TEA)-sensitive current in mouse spermatogenic cells which was attributed to activation of Kv channels, and Kv1.1, Kv1.2 and Kv1.3 were found to be expressed in mouse spermatozoa [100] (Table 2). The presence of Kv1.1 has also been described in bull spermatozoa [101]. Additionally, male mice with a targeted deletion of the Kv6.4 subunit have immotile spermatozoa and, as a consequence, are infertile [103]. Different studies have shown the presence of other Kv channels in human sperm such as Kv1.5 [89,102] and Kv7.1 [104] (Table 2). Kv7.1, also known as KCNQ1 or KvLQT1, is present in the sperm head and flagellum, and its inhibition reduced sperm motility and AR but had no effect on hyperactivation [104]. The auxiliary subunit KCNE1 is also expressed in human sperm, being mainly localized in the tail and neck regions, and co-localize partially with Kv7.1 [89,104].

In humans, Kv inhibitors, such as Pb2+, are able to block the progesterone-induced acrosome reaction, supporting a role for these channels in AR [123,124]. These authors provided indirect evidence probing the presence of Kv channels in human sperm, using a biotinylated charybdotoxin probe, and show that these channels are distributed over the sperm head and colocalize with progesterone receptors [86,123,124]. Several Kv isoforms are sensitive to Pb2+ and an inverse relationship between fertilization rates and Pb2+ concentration in blood and seminal plasma has also been described [123,124]. Therefore, it has been suggested that Kv channels are responsible for the metal ion-related male infertility, in which AR is prevented [123].

However, despite the demonstration of the presence of different Kv channels, recent electrophysiological studies have shown that the K+ current recorded in sperm cells is mainly if not solely mediated by activation of SLO channels [23,24,92,93] and the precise role of Kv in sperm function remains to be determined.

Inwardly rectifying K+ Channels (Kir channels) have also been described in mammalian spermatozoa [87]. Contrary to Kv channels, Kir channels do not activate by depolarization. Kir comprises a variety of K+ channels classified in seven different subfamilies which are activated by intracellular mediators [113,120]. These channels are classified, from a functional point of view, in four different groups: (1) K+ transport channels, including Kir1.1, Kir4.1, Kir4.2, Kir5.1, and Kir7.1; (2) Classical Kir channels, comprising Kir2.1–Kir2.4 and Kir2.6 channels; (3) G-protein-gated Kir channels (GIRK), including Kir3.1–Kir3.4, and (4) ATP-sensitive K+ channels (KATP), comprising Kir6.1 and Kir6.2 [125]. The pore-forming α subunit is composed of two hydrophobic TM domains and the functional channel derives from the association of four subunits, forming homo- or heterotetramers [113,126].

Kir channels play an important role in the maintenance of resting Em [108,113,127,128] and their conductance is strictly regulated by intracellular Mg2+ and endogenous polyamines, such as spermine, spermidine and putrescine [108,125]. At membrane potentials positive to the equilibrium potential of K+ (EK), intracellular Mg2+ and polyamines block the channel, allowing a small outward current, while at potentials negative to EK, Mg2+ and polyamines flow into the cell, and the channel unlocking permits a large inward K+ current [125]. Kir channels can also be regulated by intracellular pH, Na+ or ATP and their conductance augments at higher extracellular K+ concentrations [87,107,113,125,129]. Additionally, the component of the plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) plays an essential role in Kir channel activation, with each Kir group showing a differential sensitivity to this signaling phospholipid [125].

The presence of Kir channels in mammalian sperm has been poorly studied. Kir3.1 and Kir3.2d (a splice variant that is expressed only in the testis) have been detected in the rat and mouse testis and mouse sperm [100,105,106,107,125], Kir4.1 in the mouse testis [107], Kir7.1 in the rat testis [126] and Kir5.1 has been identified in the rat testis [108,109] and in the head and body of rat and mouse sperm [107,108]. Recent findings suggest that Kir5.1 could participate in the regulation of pHi changes that occur during sperm capacitation [107] (Table 2). Moreover, the deletion of Kcnj16 in mice, the gene encoding Kir5.1, increases the percentage of sperm with abnormal flagellar morphology and causes subfertility in an age-dependent manner, providing a role for this channel in male infertility [107]. The detection of Kir4.1 in the mouse testis further support a participation of Kir5.1, as this is only functional as a heteromer with Kir4.1 and/or Kir4.2 [125].

Within the Kir family, ATP-sensitive potassium channels (KATP) are a group with differential properties from other members of the family which include Kir6.1 and Kir6.2. These channels have a hetero-octameric structure, composed of four preforming KATP subunits (Kir6.1 and/or Kir6.2, each of them with 2 TM) and a combination of four regulatory sulphonylurea receptor (SUR) subunits (SUR1, SUR2A and/or SUR2B) [82,113,130]. These channels are weak inwardly rectifiers and are regulated by adenine nucleotides, thus coupling cellular metabolism with membrane excitability and Em [82,125]. KATP are inactivated by intracellular ATP, causing a membrane depolarization, while intracellular ADP activates the channel through interaction with SUR in a Mg2+-dependent manner, leading to membrane hyperpolarization [125]. The channels are also regulated by intracellular acidification, which causes activation, and by PIP2 and other phosphatidylinositol phosphates that antagonize the ATP inhibition with the consequent opening of KATP. The activity of KATP is also controlled by PKC and PKA phosphorylation [82,125].

The KATP subunits Kir6.2 and SUR2 have been detected using RT-PCR, Western blot, immunohistochemistry and immunofluorescence in epididymal epithelial cells and epididymal spermatozoa from several mammalian species including rats, mice, dogs, cats, cattle and humans, with strong co-localization [111]. Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B are also present in rat and mouse spermatogenic cells and mature sperm [82,110] and there is a co-localization of Kir6.2 with SUR2B in the acrosome of rat spermatids [110] and of Kir6.2 with SUR1 in the post-acrosomal region and flagellar midpiece of mouse spermatozoa [82]. In mouse sperm, KATP function has been linked to sperm hyperpolarization and AR during capacitation [82]. More studies are needed to clarify the role of KATP, and in general, of Kir channels in human sperm.