NEW RESEARCH HORIZON Review
Rediscovering sperm ion channels
with the patch-clamp technique
Yuriy Kirichok* and Polina V. Lishko
Department of Physiology, University of California San Francisco UCSF Mail Code 2140, Genentech Hall Room N272F 600 16th Street,
San Francisco, CA 94158, USA
*Correspondence address. Tel: +1-415-476-6310; Fax: +1-415-502-8644; E-mail: email@example.com
Submitted on March 21, 2011; resubmitted on May 12, 2011; accepted on May 21, 2011
abstract: Upon ejaculation, mammalian spermatozoa have to undergo a sequence of physiological transformations within the female
reproductive tract that will allow them to reach and fertilize the egg. These include initiation of motility, hyperactivation of motility and
perhaps chemotaxis toward the egg, and culminate in the acrosome reaction that permits sperm to penetrate the protective vestments
of the egg. These physiological responses are triggered through the activation of sperm ion channels that cause elevations of sperm intra-
cellular pH and Ca2+in response to certain cues within the female reproductive tract. Despite their key role in sperm physiology and their
absolute requirement for the process of fertilization, sperm ion channels remain poorly understood due to the extreme difficulty in appli-
cation of the patch-clamp technique to spermatozoa. This review covers the topic of sperm ion channels in the following order: first, we
discuss how the intracellular Ca2+and pH signaling mediated by sperm ion channels controls sperm behavior during the process of fertiliza-
tion. Then, we briefly cover the history of the methodology to study sperm ion channels, which culminated in the recent development of a
reproducible whole-cell patch-clamp technique for mouse and human cells. We further discuss the main approaches used to patch-clamp
mature mouse and human spermatozoa. Finally, we focus on the newly discovered sperm ion channels CatSper, KSper (Slo3) and HSper
(Hv1), identified by the sperm patch-clamp technique. We conclude that the patch-clamp technique has markedly improved and shifted
our understanding of the sperm ion channels, in addition to revealing significant species-specific differences in these channels. This
method is critical for identification of the molecular mechanisms that control sperm behavior within the female reproductive tract and
make fertilization possible.
Key words: sperm motility / patch clamp / Ca2+/ H+/ K+ion channels / hyperactivation / acrosome reaction
Intracellular Ca2+and intracellular H+are two key messengers with
opposite effects on sperm activity and fertilizing ability: increasing
intracellular [Ca2+] stimulates sperm motility and fertility (Darszon
et al., 2005; Publicover et al., 2007; Fraser, 2010), whereas increasing
intracellular [H+] inhibits them (Babcock et al., 1983; Carr and Acott,
1989; Florman et al., 1989, 1992; Hamamah and Gatti, 1998; Suarez,
2008). Together with intracellular cAMP (Morton et al., 1974; Tash
and Means, 1983; Okamura et al., 1985; Brokaw, 1987; Visconti
et al., 1995; Fraser, 2010), these two antagonistic intracellular messen-
gers control the most important aspects of sperm behavior in the
female reproductive tract: initiation of sperm motility upon ejaculation,
sperm capacitation, hyperactivation, chemotaxis and the acrosome
reaction. Sperm intracellular [Ca2+] and [H+] are primarily deter-
mined by Ca2+and H+channels and transporters of the sperm
plasma membrane (Darszon et al., 2006a); however, intracellular
Ca2+stores such as the acrosome vesicle and the redundant
nuclear envelope may also contribute to sperm intracellular Ca2+sig-
naling (Ho and Suarez, 2003; Costello et al., 2009). Unfortunately, the
lack of direct methods to study ion channels and transporters of the
sperm plasma membrane has hampered our understanding of the
molecular mechanisms that regulate sperm activity and male fertility.
The invention of the patch-clamp technique by Neher and Sakmann
by the beginning of the 1980s (Hamill et al., 1981) initiated a revolution
in our understanding of plasma membrane ion channels and transpor-
ters of somatic cells. However, a similar routine application of the
patch-clamp technique to spermatozoa was considered impossible
due to their small size, vigorous motility and tight association of the
plasma membrane with rigid intracellular structures. Only recently,
in 2006, was this technical problem resolved for mouse sperm cells
with the development of a method that allowed reproducible patch-
clamp recording from whole sperm plasma membrane (Kirichok
et al., 2006). A few years later, a similar approach was successfully
used to patch-clamp human spermatozoa, opening new opportunities
to study the molecular mechanisms of male fertility, specifically in
humans (Lishko et al., 2010).
After an overview of sperm Ca2+and H+signaling, this review will
summarize the main approaches used to study the ion channels
responsible for this signaling. We will specifically focus on the recently
& The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Molecular Human Reproduction, Vol.17, No.8 pp. 478–499, 2011
Advanced Access publication on June 4, 2011doi:10.1093/molehr/gar044
developed sperm patch-clamp technique and how it has changed our
understanding of sperm ion channels. Because the application of the
patch-clamp technique has demonstrated that properties of sperm
ion channels differ significantly between species, in this review, we
focused on the physiology of human spermatozoa. Physiological prop-
erties of sperm cells from other species are considered only when
they are likely to apply to human spermatozoa and no data on
human cells are available.
General principles of sperm
intracellular H1and Ca21
Resting intracellular concentrations of Ca2+and H+within the sperm
cell are primarily set by ion pumps that use the energy stored in ATP
(directly or indirectly) to establish concentration gradients for these
two ions across the plasma membrane. Thus, while the concentration
of Ca2+in the extracellular medium is ?1–2 mM, its resting concen-
tration in the sperm cytosol is only ?100–200 nM (Babcock and Pfeif-
fer, 1987; Linares-Hernandez et al., 1998; Wennemuth et al., 2000). In
contrast, the concentration of protons seems to be always higher
inside the sperm cell than outside with resting DpH ≈ 0.4–1.0
(Babcock and Pfeiffer, 1987; Carr and Acott, 1989; Florman et al.,
1989; Vredenburgh-Wilberg and Parrish, 1995; Hamamah et al.,
1996; Zeng et al., 1996; Hamamah and Gatti, 1998). While mamma-
lian spermatozoa are stored in the caudal epididymis before ejacula-
tion, pH of the extracellular fluid varies between 5.5 and 6.8
depending on species (Acott and Carr, 1984). Since sperm pHiis
always more acidic, it should be round 6.0 or lower ([H+]i.1 mM)
for the majority of mammalian species in this portion of the male
reproductive tract. These pH values are significantly lower than
those normally found within somatic cells (Roos and Boron, 1981),
and help in rendering spermatozoa quiescent before ejaculation.
Upon ejaculation spermatozoa are exposed to higher extracellular
pH (average pHo≈ 7.0 or higher), and their pHiis also elevated to
about 6.5 ([H+]i≈ 0.3 mM), as they become motile for the first
time (Babcock et al., 1983; Carr and Acott, 1989; Hamamah et al.,
1996; Zeng et al., 1996). During subsequent transit through the
female reproductive tract, the sperm pHifurther increases as the
result of sperm capacitation (Parrish et al., 1989; Vredenburgh-
Wilberg and Parrish, 1995; Zeng et al., 1996; Suarez, 2008), but it
remains lower than pHo. In the female reproductive tract, pHois alka-
line (except for vagina) and can reach up to 8.4 ([H+]o¼ 4 nM) in cer-
vical mucus and 7.8 ([H+]o¼ 16 nM) in the Fallopian tubes depending
on species and the phase of the female cycle (Acott and Carr, 1984).
The high resting [H+]iand low resting [Ca2+]iestablished by H+
and Ca2+pumps of the sperm plasma membrane suppress the activity
of the sperm cell. Activation of spermatozoa requires reduction of
[H+]iand elevation of [Ca2+]i, which is easy to achieve due to the
steep resting concentration gradients for H+and Ca2+across the
sperm plasma membrane. Indeed, merely opening the H+and Ca2+
ion channels of the sperm plasma membrane would be sufficient to
allow H+to exit and Ca2+to enter the sperm cytosol down their
respective concentration gradients. The opening of the sperm H+
and Ca2+channels must be controlled by specific cues of the
female reproductive tract to regulate the activity of the sperm cells
in accordance with their position within the tract and depending on
the phase of the menstrual cycle. Among the cues of the female repro-
ductive tract that can activate sperm ion channels are progesterone
released by ovaries and cumulus cells surrounding the egg, glyco-
proteins of the zona pellucida and the major protein of the oviductal
fluid, albumin (Darszon et al., 2005, 2006a; Publicover et al., 2007; Xia
and Ren, 2009a, b; Fraser, 2010; Ren and Xia, 2010). However, there
are probably more factors of the female reproductive tract yet to be
discovered that directly or indirectly control the activity of sperm ion
channels and help us to synchronize the arrival of the egg and the
sperm at the site of fertilization (the ampulla of the oviduct).
Both sperm ion pumps and ion channels that participate in control-
ling [H+]i and [Ca2+]i are required for the regulation of sperm
function. For example, sperm contain two flagellar Ca2+transport
proteins: a Ca2+ATPase (PMCA4) that pumps Ca2+out of the
sperm cell to support low resting intraflagellar [Ca2+]i (Okunade
et al., 2004; Schuh et al., 2004) and a Ca2+channel CatSper (Ren
et al., 2001; Kirichok et al., 2006; Qi et al., 2007) that allows extra-
cellular Ca2+to enter the sperm flagellum. The balance between
the activities of these two proteins sets the concentration of Ca2+
in the sperm flagellum, and male mice deficient in either PMCA4 or
CatSper are infertile and have impaired sperm motility (Ren et al.,
2001; Okunade et al., 2004; Schuh et al., 2004; Qi et al., 2007).
However, pumps transport ions much more slowly than channels
and their activity can be overwhelmed by ion channels that operate
orders of magnitude faster (Hille, 1992). Pumps also cause relatively
slow changes in the intracellular ion concentrations, whereas ion chan-
nels are primarily responsible for rapid signaling events.
It should also be noted that due to the significant length (usually
.50 mm) and miniscule cytoplasmic cross-section (much ,1 mm2
throughout) of the sperm cell, diffusion between different parts of
the sperm cell, such as the head and the principal piece, is limited.
An extremely small amount of sperm cytosol squeezed in between
the plasma membrane and the intracellular structures further contrib-
utes to the spatial separation of the different cellular compartments.
Moreover, it has been demonstrated that the expression of sperm
ion channels is highly compartmentalized and differs significantly
between the head and the principal piece (Ren et al., 2001; Navarro
et al., 2007; Qi et al., 2007; Lishko et al., 2010). Thus, intracellular
H+and Ca2+signaling within the sperm head and the flagellum are
controlled by different mechanisms and are spatially isolated from
each other to allow for the independent control of Ca2+and
pH-dependent physiological responses occurring in the head and the
flagellum, such as the acrosome reaction and hyperactivation.
Intracellular Ca21and H1as
regulators of sperm activity in
the female reproductive tract
The female reproductive tract presents multiple barriers for the sperm
cells on their route to the egg (Suarez and Pacey, 2006). Female
immune responses, viscous cervical mucus, the thin uterotubal junc-
tion, sticky oviductal epithelium covered with mucus, the maze of
the oviductal epithelial folds near the site of fertilization and egg’s pro-
tective vestments all markedly reduce a sperm’s chances to reach and
fertilize the egg (Suarez and Pacey, 2006). Although millions of
Patch-clamp technique and sperm ion channels
spermatozoa are introduced into the female reproductive tract, only a
few will eventually traverse the viscous environment of the female
reproductive tract to arrive at the site of fertilization, and only one
will penetrate through the egg’s cumulus oophorus (CO) and zona
pellucida to fertilize the egg. To reach and fertilize the egg, spermato-
zoa have to (Fig. 1): (1) become motile for the first time; (2) develop
hyperactivated motility to overcome the viscous mucus and sticky epi-
thelium of the Fallopian tube and the protective vestments of the egg;
(3) use chemotaxis to find the egg among the labyrinthine folds of the
oviductal epithelium and (4) undergo acrosomal exocytosis to release
the hydrolytic enzymes that cleave glycoproteins of the zona pellucida
to allow the penetration of the zona. Below we discuss these four pro-
cesses essential for successful fertilization and demonstrate that they
require elevation of sperm pHiand [Ca2+]i.
Before ejaculation, morphologically mature, densely packed mam-
malian spermatozoa are stored in a quiescent state in the caudal
portion of the epididymis and vas deferens (Acott and Carr, 1984;
Carr and Acott, 1984; Hamamah and Gatti, 1998). This quiescence
is likely to help transcriptionally silent spermatozoa preserve their mol-
ecular and functional integrity for a longer time. Upon ejaculation,
spermatozoa are mixed with the seminal plasma and initiate their
motility for the first time. Sperm motility at this time is characterized
by relatively low-amplitude, symmetrical tail bending when compared
with the high-amplitude, asymmetrical tail bending characteristic of
hyperactivation and observed close to the site of fertilization (Suarez
and Pacey, 2006; Suarez, 2008). Elevations of sperm intracellular pH
and cAMP in response to a new extracellular environment have
been directly implicated in the initiation of sperm motility.
Although our understanding of sperm quiescence is limited, in many
species the acidic environment of cauda epididymis keeps spermato-
zoa immotile by lowering their intracellular pH (Acott and Carr,
1984; Carr and Acott, 1984; Hamamah and Gatti, 1998). High vis-
cosity of the cauda epididymal fluid may also help us to immobilize
spermatozoa (Usselman and Cone, 1983; Carr et al., 1985). The
exact mechanism by which low extracellular pH within the cauda epi-
didymis causes low sperm intracellular pH remains unclear, but lactate
and other weak acids that can shuttle protons across the cell mem-
brane have been implicated (Acott and Carr, 1984; Carr and Acott,
1984; Hamamah and Gatti, 1998). In some species such as humans,
lactate may not be essential, since the plasma membrane seems to
have a significant passive H+conductance (Hamamah et al., 1996;
Hamamah and Gatti, 1998). Finally, the ion pumps of the sperm
plasma membrane keep intracellular pH lower than the extracellular
pH (see earlier). During ejaculation, spermatozoa are diluted in the
seminal plasma that has a higher pH. Intracellular pH is thought to
follow the increase in the extracellular pH, and sperm motility is
initiated due to the direct stimulation of axonemal proteins by
Another factor important for the activation of sperm motility, intra-
cellular cAMP, stimulates the axoneme indirectly: it activates protein
kinase A (PKA) which is thought to cause phosphorylation of the
axoneme (Harrison, 2004; Nolan et al., 2004). Upon ejaculation,
sperm intracellular cAMP is elevated due to the activation of sperm
soluble adenylyl cyclase (sACY) by the high bicarbonate concentration
in the seminal plasma and the female reproductive tract when com-
pared with the epididymal fluid (Okamura et al., 1985; Chen et al.,
2000). Male mice deficient for sperm sACY (Esposito et al., 2004;
Hess et al., 2005, Xie et al., 2006) and PKA (Nolan et al., 2004) are
infertile and have impaired sperm motility. However, it should be
noted that PKA and sACY do not seem to be required for the
initiation of sperm motility, but rather increase the frequency of
sperm tail beating to improve progressive motility (Wennemuth
et al., 2003; Nolan et al., 2004; Xie et al., 2006). Interestingly, recom-
binant SACY or SACY in particulate fractions of sperm is also activated
by Ca2+(Jaiswal and Conti, 2003), and the presence of Ca2+in the
extracellular medium is required for sACY-dependent increase in
the frequency of flagellar beat triggered by bicarbonate (Carlson
et al., 2007).
Normal sperm motility is characterized by low-amplitude symmetri-
cal tail beating. This type of motility allows spermatozoa to traverse
the watery cervical mucus and uterus; however, it should be noted
that contractile activity of uterine muscle may facilitate transport of
spermatozoa through the cervix and uterus (Suarez and Pacey,
Figure 1 The milestones of sperm journey toward the egg. The
approximate locations where normal sperm motility (1), hyperacti-
vated sperm motility (2), chemotaxis (3) and the acrosome reaction
(4) occur within the female reproductive tract are shown by the cor-
responding numbers. Normal motility (1) is triggered upon ejaculation
in the anterior vagina and is characterized by the low amplitude, sym-
metrical tail bending. Hyperactive motility (2) occurs after spermato-
zoa enter Fallopian tubes. It characterized by the high-amplitude
asymmetrical tail bending and develops much higher thrust to pene-
trate through viscous environment of the female reproductive tract.
Sperm chemotaxis (3) is believed to occur close to the site of fertiliza-
tion in the ampulla of Fallopian tubes. Chemoattractant(s) (showed by
the red dots) released by the cumulus cells surrounding the egg (or by
the egg itself) help spermatozoa to find the egg among the epithelial
folds of the Fallopian tube. The acrosome reaction (4) occurs either
within the CO or upon the contact with zona pellucida. The hydro-
lytic enzymes released from the sperm head as the result of the acro-
some reaction help spermatozoa to penetrate these egg’s protective
Kirichok and Lishko
2006). After sperm entry into the Fallopian tubes, normal motility is
unlikely to provide sufficient thrust to overcome sticky oviductal
epithelium covered with dense mucus and, eventually, egg’s protective
vestments. To overcome these barriers, spermatozoa have to develop
hyperactivation, a special type of motility characterized by high-
amplitude, asymmetrical tail bending that develops a significantly
higher swimming force than the normal motility (Suarez and Pacey,
2006; Suarez, 2008).
After the activation of motility, all further milestones of the sperm’s
journey toward the egg (including hyperactivation, chemotaxis and the
acrosome reaction) require capacitation. Capacitation is defined as the
functional maturation of sperm cells within the female reproductive
tract (Austin, 1951; Chang, 1951; Yanagimachi, 1994). Since capaci-
tated spermatozoa acquire hyperactive motility and become able to
undergo chemotaxis and the acrosome reaction, they can now
reach and fertilize the egg. Capacitation is completed in the Fallopian
tubes, after spermatozoa have spent several hours in the female
reproductive tract. As assessed in vitro, extracellular Ca2+, bicarbonate
and albumin in the oviductal fluid are considered essential for capaci-
tation (Visconti et al., 2002). Capacitation is a complex and poorly
understood process accompanied by a wave of protein phosphoryl-
ation caused by cAMP-dependent PKA directly and through recruit-
ment of tyrosine kinases (Visconti et al., 1995, 2002; Harrison,
2004; Harrison and Gadella, 2005) as well as by the elevation of
basal levels of sperm pHiand [Ca2+]i(Parrish et al., 1989; Yanagima-
chi, 1994; Baldi et al., 1996; Zeng et al., 1996). These higher basal
levels of pHiand [Ca2+]iare likely to be sufficient to cause the hyper-
activation of sperm motility (Suarez, 2008). Chemotactic navigation
toward the egg and the acrosome reaction occur later with already
capacitated spermatozoa (Eisenbach and Giojalas, 2006; Florman
et al., 2008) and require further elevations of pHiand [Ca2+]iabove
the new basal levels achieved during capacitation. We will now
further consider hyperactivation, chemotaxis and the acrosome
reaction in detail.
As mentionedearlier, hyperactivation
large-amplitude, whip-like asymmetrical tail beating that results in
significantly stronger swimming force than in normal sperm motility
(Suarez, 2008). Hyperactivation not only allows spermatozoa to
move along the sticky oviductal epithelium covered with mucus, but
is also essential for the penetration of protective vestments of the
egg, the CO and zona pellucida (Suarez and Pacey, 2006; Suarez,
2008). Hyperactivation requires both elevation of intracellular pH
and elevation of intracellular Ca2+(Suarez, 2008), which are achieved
as a result of sperm capacitation. Elevated intracellular pH and Ca2+
directly affect the axoneme and, in combination, cause the high-
amplitude, asymmetrical flagellar beating, characteristic of hyperactiva-
tion (Ho et al., 2002). It has been demonstrated that the flagellar Ca2+
channel CatSper is required for hyperactivation, certainly in mice and
probably humans (Carlson et al., 2003, 2005; Qi et al., 2007). The
mechanism for H+extrusion is less certain and may differ between
species, even within mammals. In human spermatozoa, the dominant
H+conductor of the plasma membrane is the flagellar H+channel Hv1
(Lishko and Kirichok, 2010; Lishko et al., 2010). Activity of both
human CatSper and Hv1 is enhanced by capacitation (Lishko et al.,
2010, 2011). Since CatSper can be further potentiated by progester-
one (Lishko et al., 2011; Stru ¨nker et al., 2011) and Hv1—by ananda-
mide (Lishko and Kirichok, 2010), both released by cumulus cells
surrounding the egg, human spermatozoa may achieve an even
higher degree of hyperactivation during penetration through the CO
and zona pellucida.
As spermatozoa approach the site of fertilization, they have to find
the egg among the labyrinthine folds of the Fallopian tubes (Suarez and
Pacey, 2006). Several studies have reported that progesterone
released by the cumulus cells surrounding the egg is likely to serve
as the principal sperm chemoattractant in humans; however, other
possible chemoattractants have been proposed, and the very exist-
ence of sperm chemotaxis in vertebrates remains controversial (Eisen-
bach and Giojalas, 2006; Kaupp et al., 2008). In some marine
invertebrates, sperm chemotaxis has been convincingly demonstrated.
For the sea urchin Arbacia punctulata, spermatozoa perform chemo-
tactic turns that result in swimming trajectories directed toward a
source of the resact peptide that is released from the egg (Suzuki
and Garbers, 1984). Each turn is caused by a brief asymmetrical tail
motion that is triggered by a transient elevation of Ca2+in the
sperm flagellum (Kaupp et al., 2008; Guerrero et al., 2010). The
Ca2+required for chemotactic turns in A. punctulata comes from
the extracellular medium via an unidentified flagellar Ca2+channel
directly or indirectly activated by resact.
Although the chemotaxis of human sperm toward progesterone is
still being debated, human spermatozoa do possess a flagellar Ca2+
channel activated by progesterone: the CatSper channel (Lishko
et al., 2011; Stru ¨nker et al., 2011). As will be discussed later, the
CatSper channel is potently activated by low nanomolar concen-
trations of progesterone, and the CatSper-mediated Ca2+influx can
induce asymmetrical tail beating (Lishko et al., 2011; Stru ¨nker et al.,
2011). Thus, Ca2+influx through the CatSper channel appears to
be essential for both sperm hyperactivation and chemotaxis.
Finally, when the sperm cell finds the egg, it has to overcome its
protective vestments, the CO and the zona pellucida. The CO, the
outermost vestment of the egg, consists of the cumulus cells (follicular
cells retained around the oocyte) suspended in a viscoelastic matrix
formed primarily by hyaluronic acid secreted by the same cumulus
cells (Dandekar et al., 1992; Suarez, 2008). The dense layer of zona
pellucida lies under the CO and is formed by glycoproteins ZP1,
ZP2, ZP3 and ZP4 (ZP1–ZP3 in mice) secreted by the egg itself
(Wassarman and Litscher, 2008; Gupta et al., 2009). To penetrate
through the protective vestments of the egg, the sperm cells have
to be hyperactivated (Suarez, 2008). It should be noted that in the
CO, the concentration of progesterone released by the cumulus
cells is in the micromolar range (Osman et al., 1989). Since sperm
cells are completely buried in the CO during the penetration
through the protective vestments of the egg and subjected to high
concentrations of progesterone, their motility may reach the highest
degree of hyperactivation.
However, the matrix of the zona pellucida is so dense that hyper-
activation alone does not seem to be sufficient to get the sperm
through this layer. To penetrate the zona, spermatozoa complement
the mechanical force of hyperactivation with enzymatic disruption of
the zona’s matrix by hydrolytic enzymes released from the acrosome
vesicle located at the tip of the sperm head. The release of hydrolytic
enzymes, called the acrosome reaction, occurs upon contact of the
sperm head with the zona pellucida and is caused by exocytotic
fusion of the acrosomal vesicle with the sperm plasma membrane
(Yanagimachi, 1994). The acrosome reaction is thought to be
Patch-clamp technique and sperm ion channels
triggered by elevation of sperm [Ca2+]iand pHiupon binding of the
yet unidentified sperm head receptor to glycoproteins of the zona pel-
lucida (ZP3 in mice and ZP1, ZP3, ZP4 in humans) (Dean, 2007;
Florman et al., 2008; Wassarman and Litscher, 2008; Gupta et al.,
2009). However, the sperm receptor for the glycoproteins of the
zona pellucida as well as the identity of the Ca2+and H+transport
mechanisms associated with this receptor remains elusive.
Although the importance of intracellular Ca2+and H+signaling in
sperm physiology has been recognized for a long time, until very
recently the Ca2+and H+ion channels of the plasma membrane
that cause elevations of pHiand [Ca2+]ihave remained unknown
despite significant efforts in this direction. In the following section,
we briefly summarize the methods that have been used to study
sperm intracellular pH and Ca2+signaling and to identify the
sperm ion channels involved. To explain why the identities of
sperm Ca2+and H+channels remained elusive until very recently,
we consider the relative effectiveness of every method discussed
in identification and characterization of the ion channels of mature
Overview of experimental
approaches to study sperm
intracellular Ca21and pH
The first evidence that Ca2+and H+are important for sperm physi-
ology came early from the observation that changing the level of
these two ions in extracellular medium strongly affects sperm motility,
chemotaxis and acrosome reaction. Extracellular Ca2+was demon-
strated to be essential for sperm capacitation, hyperactivation, chemo-
taxis and the acrosome reaction (Iwamatsu and Chang, 1971;
Yanagimachi, 1994; Eisenbach and Giojalas, 2006; Florman et al.,
2008; Suarez, 2008). It has also been convincingly demonstrated in
demembranated spermatozoa that Ca2+and pH directly affect the
activity of axoneme (Lindemann et al., 1987; Goltz et al., 1988; Ho
et al., 2002). Acidic pH suppressed the motility of demembranated
bull sperm, and flagellum beating was activated only when pH of the
incubation medium was 7.0 or higher (Ho et al., 2002). Hyperactiva-
tion required an even more alkaline pH of 7.9–8.5 (Ho et al., 2002).
Hyperactivation of demembranated sperm also required the elevation
of [Ca2+] in the incubation medium from 50 nM to 400 nM (Ho et al.,
2002). Unfortunately, the next logical step, identification of the mem-
brane transport mechanisms that control sperm pHi and [Ca2+]i,
turned out to be extremely challenging.
Development of ion-sensitive fluorescent probes for Ca2+and pH
provided a tool to approach this problem for the first time. These
probes allowed the observation of changes in [Ca2+]iand [H+]iin
live, motile spermatozoa (Babcock, 1983; Babcock et al., 1983;
Irvine and Aitken, 1986; Babcock and Pfeiffer, 1987) as they under-
went crucial physiological responses such as motility initiation, hyper-
activation, chemotaxis and acrosome reaction. These studies
demonstrated that when spermatozoa were incubated for several
hours in vitro under conditions similar to those found within the
female reproductive tract and acquired fertilizing competence
(in vitro capacitation), their pHi and [Ca2+]i rose (Parrish et al.,
1989; White and Aitken, 1989; Baldi et al., 1991, 1996; Zeng et al.,
1996). Comparison of hamster spermatozoa exhibiting normal or
hyperactivated motility demonstrated that [Ca2+]i is elevated in
hyperactivated sperm (Suarez et al., 1993). Replication of the acro-
some reaction in vitro by treating bovine spermatozoa with solubilized
zona pellucida induced the elevation of both [Ca2+]i and [H+]i
(Florman et al., 1989, 2008). Furthermore, in sea urchin,
(the peptide-chemoattractant released by the egg) induced elevation
of intracellular pH and Ca2+(Lee and Garbers, 1986; Schackmann
and Chock, 1986; Cook et al., 1994). In a resact gradient, freely swim-
ming A. punctulata spermatozoa generated flagellar Ca2+spikes that
triggered chemotactic turns to guide spermatozoa toward higher con-
centrations of the chemoattractant (Bohmer et al., 2005; Wood et al.,
2007). The likely chemoattractant of human spermatozoa, progester-
one, also induced massive Ca2+influx into human sperm cells
(Thomas and Meizel, 1989; Blackmore et al., 1990). When human
spermatozoa were exposed to a gradient of progesterone, they gen-
erated a series of Ca2+spikes similar to those observed during
chemotaxis of A. punctulata sperm (Harper et al., 2004). No
progesterone-induced elevation of intracellular pH has been reported
in human spermatozoa.
Although optical methods for measuring intracellular [Ca2+]iand
pHidemonstrated the existence of sperm intracellular Ca2+and pH
signaling for the first time, they failed to provide functional and mol-
ecular characterization of the Ca2+and H+transport mechanisms
of the sperm plasma membrane responsible for these signaling
events. The principal reason for this was that the technique did not
provide reliable control of the membrane voltage and the composition
of the intracellular medium and ionic gradients across the sperm
plasma membrane. Since these are essential determinants of the ion
channel activity, the interpretation of experimental results was difficult,
and there was no guarantee that only the single ion channel/transpor-
ter that is being studied contributes to the observed changes in
[Ca2+]i and [H+]i. Another substantial experimental difficulty was
associated with the fact that sperm cells have an extremely low cyto-
solic volume, and the level of fluorescent signals originating from a
single spermatozoon was weak. For this reason, the majority of
optical experiments with sperm cells were performed by measuring
fluorescent signals from sperm suspensions (Darszon et al., 2005).
This method detected signals averaged over large populations of
cells, which reduced the already low time resolution of the optical
methods and might mask fast signaling events. Although single-cell
measurements have sometimes been performed, primarily in recent
years, the fluorescent signal from the principal piece was often too
weak to be detected, and signals from the head and midpiece (that
probably have larger amounts of cytosol than the principal piece)
were primarily measured (Darszon et al., 2005). Unfortunately, even
measurements of [Ca2+]iand [H+]iin sperm suspensions did not
solve the problem of weak fluorescence from the principal piece,
and about 85% of the population signal originated from the head
and midpiece (Darszon et al., 2005). Thus, the optical methods pro-
vided little information on [Ca2+]i and [H+]i within the principal
piece, where the regulation of sperm motility occurs. A few recent
optical studies that succeeded in measuring [Ca2+]ifrom the sperm
flagellum have demonstrated a complex pattern of Ca2+transients
in this cellular domain (Bohmer et al., 2005; Wood et al., 2007;
Xia et al., 2007).
Kirichok and Lishko
The low volume of sperm cytoplasm can cause another problem for
optical measurements of sperm [Ca2+]iand [H+]i. Even a minute
plasma membrane disruption that results in only a few Ca2+or H+
ions entering the cell will lead to a significant change in [Ca2+]iand
[H+]i. The probability of such membrane disruption during the exper-
iment is especially high when motile sperm cells are attached to the
bottom of the recording chamber or when lipophilic compounds
such as progesterone are applied in relatively high concentrations. If
overlooked, this problem could lead to substantial experimental arti-
facts and incorrect conclusions regarding the mechanisms of sperm
[Ca2+]iand [H+]isignaling. In contrast, unspecific membrane leak is
instantly recognized during the patch-clamp experiment, since its
current–voltage relationship and ion selectivity is different from
those of Ca2+or H+currents.
In the beginning of the 1980s, concurrent with the introduction of
ion-sensitive fluorescent probes to study sperm intracellular Ca2+
and pH signaling, the patch-clamp technique was revealed as the
cutting-edge method to study plasma membrane ion channels
(Hamill et al., 1981). Using glass micropipettes that can form a very
tight seal with lipid membranes, the patch-clamp technique made
possible the direct measurement of ion currents across the whole
plasma membrane (whole-cell configuration) and even detection of
activity of a single ion channels (cell-attached and inside-out configur-
ations) (Fig. 2). The whole-cell configuration proved to be the most
useful, although single-channel patch-clamp measurements were also
often employed to obtain additional mechanistic insight into the
For ion channel research, the patch-clamp technique provided three
important advantages over the optical methods. First, it directly
measured ion channel activity—the transmembrane ion current—
rather than the result—the change in the intracellular concentration
of an ion. Second, it provided superior time resolution of ,1 ms.
Third, it allowed for very precise control of the conditions that
affect ion channel activity, namely the membrane voltage, which was
directly controlled by the patch-clamp amplifier, and the composition
of the solution on both sides of the plasma membrane, including the
concentration of the permeable ion and that of potential ion
channel regulators such as ATP, neurotransmitters and cAMP. The
latter provided the ability to adjust the compositions of the bath
and intracellular recording solutions through manipulation with ions,
channel activators and inhibitors so that the current through a single
type of ion channel was recorded and studied in isolation from
other ion channels present in the same membrane.
Due to these advantages, the patch-clamp technique produced a
marked advance in our understanding of the ion channels of somatic
cells, especially cells that depend heavily on electrical signaling such
as neurons or cardiomyocytes. Since spermatozoa apparently rely
on ion channels as heavily as neurons or cardiomyocytes, numerous
Figure 2 The modes of the patch-clamp technique. To form the cell-attached mode of the patch-clamp technique, a portion of the plasma mem-
brane must be gently driven into the tip of the patch pipette by light suction so that it forms an V-shaped invagination within the tip of the pipette and
establishes a tight seal with the internal walls of the tip. Since the cell-attached configuration allows only for a limited control of the membrane potential
and the recording solutions, it is rarely used for electrophysiological measurements. After formation of the cell-attached mode, the membrane patch
under the patch pipette can be destroyed by high-amplitude voltage pulses (up to 1 V) and/or suction to form the whole-mitoplast mode of the
patch-clamp technique that allows the recording from the whole-cell plasma membrane. Alternatively, the patch pipette can be withdrawn from
the cell to form the inside-out mode of the patch-clamp technique to record single channels. Two electrodes, one in the pipette and one in the
bath solution, are connected to the patch-clamp amplifier that controls potential across the cell membrane (V) and measures transmembrane currents
(I). The amplifier is shown only for the whole-cell mode.
Patch-clamp technique and sperm ion channels
attempts were made to apply the patch-clamp technique to sperm
cells from different species. Unfortunately, all early attempts to
achieve sperm patch-clamp recording were frustrated by the
obvious inability to form a tight seal between the glass patch pipette
and the sperm plasma membrane, the essential prerequisite of patch-
clamp recording. The special composition of the sperm plasma mem-
brane and the small size of sperm cells were usually given as the
primary reasons for the inability to achieve the seal.
Nevertheless, the formation of tight high-resistance seals between
the patch pipette and the sperm head, albeit with a very low prob-
ability, was reported (Guerrero et al., 1987). It was found that two
techniques improved the probability of seal formation: a perpendicular
approach between the pipette and the surface of the sperm head
(Espinosa et al., 1998; Gorelik et al., 2002; Gu et al., 2004; Jimenez-
Gonzalez et al., 2007), and swelling of the sperm cells in hypotonic
medium (Babcock et al., 1992; Sanchez et al., 2001). Unfortunately,
successful break-in into sperm cells to transition into the whole-cell
mode has never been achieved with these techniques and the
authors only reported recording of single-channel activity in the
cell-attached configuration, which has very limited application. These
experiments certainly whet the appetite for studying sperm ion chan-
nels, but the low success rate and inability to record in the whole-cell
mode made these approaches impractical.
To circumvent the problems associated with patch-clamping sperm
cells, attention was directed toward recording from spermatogenic
cells. The fact that all ion channels of transcriptionally and translation-
ally silent mature spermatozoa are synthesized during spermatogenesis
seemed to indicate that at least some of these channels might be func-
tional in spermatogenic cells. Spermatogenic cells for the patch-clamp
experiments were obtained from mouse testes by mechanical and
enzymatic dissociation of the seminiferous tubes (Hagiwara and
Kawa, 1984; Arnoult et al., 1996; Lievano et al., 1996; Santi et al.,
1996). Three cell types were most commonly present in such prep-
arations and were primarily used in electrophysiological recordings:
pachytene spermatocytes, round spermatids and condensing sperma-
tids (Darszon et al., 2005). The morphology of these cells is quite dis-
tinct from that of mature spermatozoa, as they have a mostly round
body and a significant amount of cytoplasm. Although this markedly
different morphology indicated a potential difference in ion channel
expression, it also simplified the application of the patch-clamp tech-
nique since the formation of the tight seal between the patch
pipette and the spermatogenic cell and breaking-in to record whole-
cell currents were straightforward.
Fast inactivating T-type voltage-gated Ca2+channels were identified
as the main Ca2+conductance of spermatogenic cells (Hagiwara and
Kawa, 1984; Arnoult et al., 1996; Lievano et al., 1996; Santi et al.,
1996), and it was postulated that they also play a key role in Ca2+sig-
naling in mature spermatozoa (Florman et al., 1998; Darszon et al.,
1999, 2006b). The notion that voltage-gated Ca2+channels (Cav)
are the primary Ca2+conductance of sperm was also supported
by optical methods thatseemingly
depolarization-induced Ca2+influx into the sperm cells in response
to application of extracellular medium containing a high concentration
of K+(elevation of extracellular K+causes membrane depolarization)
(Wennemuth et al., 2000). Although this putative voltage-gated Ca2+
influx also required a simultaneous marked elevation of the extracellu-
lar pH (to about 8.6) and thus did not behave exactly like regular Cav
channels (Wennemuth et al., 2000), the substantial agreement
between electrophysiological experiments on spermatogenic cells
and Ca2+imaging in mature sperm cells convinced the field that Cav
channels are the principal Ca2+entry pathway into the sperm cell.
Various Cavsubunits were also detected in mature spermatozoa by
immunocytochemistry (Carlson et al., 2003; Wennemuth et al.,
2000; Darszon et al., 2006b).
In spite of this conviction that Cavchannels play a key role in sperm
physiology, genetic evidence that Cavchannels (or any other type of
ion channel) are required for male fertility was lacking. In 2001, pro-
gress in this field advanced markedly with the identification of
CatSper (Cationic Channel of Sperm)—a putative cationic ion
channel specifically expressed in the principal piece of the sperm flagel-
lum (Ren et al., 2001). Male CatSper-deficient mice were completely
infertile while being otherwise normal, and their spermatozoa seemed
to have reduced motility (Ren et al., 2001).
CatSper (later renamed CatSper1 after identification of other
subunits of the same channel) belongsto the voltage-gated cation chan-
nels superfamily and has six predicted transmembrane domains (6TM),
similar to transient receptor potential (TRP) channels or voltage-gated
K+channels (Ren et al., 2001) (Fig. 3A). However, the predicted pore
region and overall homology of CatSper1 is closest to that of a single
6TM repeat of Cavchannels consisting of four 6TM repeats (24 trans-
membrane domains total). Surprisingly, stimulation of CatSper1-
deficient mouse spermatozoa with the classic high-K+high-pH extra-
cellular medium to induce the Ca2+influx mediated by putative
sperm Cavchannels gave no elevation of [Ca2+]ias measured by
optical methods (Carlson et al., 2003). Moreover, CatSper1 null sper-
matozoa were not able to develop hyperactivation after incubation
under capacitating conditions (Carlson et al., 2003). These obser-
vations seemed to indicate that CatSper1 forms a voltage-gated
Ca2+channel that provides Ca2+essential for hyperactivation.
However, since conclusions about the nature of the CatSper1
channel were based on indirect optical methods, there was still
doubt regarding the ion selectivity of the CatSper channel and its
mode of activation. It was not even clear whether CatSper1 could
form a functional channel, or just served as a regulatory subunit.
Thus, although the discovery of CatSper1 provided the first genetic evi-
dence for the importance of ion channels in sperm physiology, it sim-
ultaneously emphasized the need for a method for application of the
whole-cell patch-clamp technique to mature spermatozoa.
Soon after the discovery of CatSper1, in 2006, such a method was
finally developed for mouse spermatozoa (Kirichok et al., 2006). A
few years later, in 2010, it was reported that a similar approach
can be used to patch-clamp human spermatozoa (Lishko et al.,
2010). As expected, the whole-cell patch-clamp technique for
sperm brought about a quantum leap in our understanding of
sperm ion channels. First, it allowed comprehensive characterization
of the CatSper channel, the principal Ca2+conductance of the
sperm plasma membrane, which resulted in a better understanding
of the molecular mechanism of sperm hyperactivation (Kirichok
et al., 2006). Second, it shed light on the non-genomic mechanism
by which the female hormone progesterone induces massive Ca2+
influx into human spermatozoa and stimulates their activity (Lishko
et al., 2011; Stru ¨nker et al., 2011). Third, the principal H+conduc-
tance of the human sperm plasma membrane was identified as the
voltage-gated H+channel Hv1 (Lishko et al., 2010). Fourth, the
Kirichok and Lishko
principal K+conductance of the sperm plasma membrane that con-
trols membrane potential and thus should regulate the activity of
CatSper and Hv1 was identified and characterized (Navarro et al.,
2007; Santi et al., 2010; Zeng et al., 2011). Finally, significant differ-
ences in the Ca2+and H+ion channels were identified between
mouse and human spermatozoa (Lishko et al., 2010, 2011). This
stressed the importance of studying ion channel signaling specifically
in human spermatozoa if we want to understand the molecular
mechanisms of male infertility and develop new methods of contra-
ception. As a result of these discoveries, a coherent mechanistic
picture of regulation of sperm activity in the female reproductive
tract is taking shape. The sperm patch-clamp technique and the
discoveries it brought about are discussed in more detail in the
Figure 3 Molecular architecture of sperm ion channels characterized with the patch-clamp technique. (A) Predicted membrane topology of the
pore-forming CatSper1 subunit of CatSper channel. Note the classic six transmembrane helix structure (S1–S6) with the positively charged
voltage sensor helix S4 and the pore region (P) between transmembrane helices S5 and S6. The putative pH sensor is located in the histidine-rich
N-terminal domain of CatSper1. Other pore-forming subunits of the CatSper channel (CatSpers2–4) have similar membrane topology, but
contain less charge in the S4 transmembrane helix and lack the putative pH-sensor in the N-terminal domain. (B) The molecular composition of
CatSper channel complex. The Ca2+-selective pore is formed by the four different CatSpers1–4 subunits. The auxiliary CatSper subunits have
one (CatSperg and CatSperd) or two (CatSperb) transmembrane helices and large extracellular domains that may be involved in regulation of the
CatSper channel by cues of the female reproductive tract. (C) Predicted membrane topology of the Hv1 channel. Hv1 consists of four-transmembrane
helices (S1–S4) homologous to the voltage-sensor domain S1–S4 of voltage-gated ion channels, but it lacks the pore forming S5–S6 segment of these
channels. The remaining voltage-sensor domain only S1–S4 structure mediates transmembrane proton transport (Ramsey et al., 2010). (D) Predicted
membrane topology of the Slo3 channel. It has seven predicted transmembrane helices S0–S6, with S1–S6 helices homologous to classic voltage-
gated ion channels. The large intracellular C-terminal domain is likely to be involved in the pH-sensitivity of Slo3 (Xia et al., 2004).
Patch-clamp technique and sperm ion channels
Sperm patch-clamp recording:
the current approach
A prerequisite for successful transition into the whole-cell mode of the
patch-clamp technique is the formation of a reliable seal between the
patch-pipette and the cell plasma membrane (Hamill et al., 1981). If
the strength of the seal is not sufficient, the seal will be disrupted
during the break-in (which is performed by the application of negative
pressure into the pipette or by application of high transmembrane
voltage, or both) and the cell will be lost. Unfortunately, formation
of a tight seal (.10 GV, when the seal strength is expressed as elec-
trical resistance) was considered impossible in the case of sperm cells.
Moreover, if the seal was formed, as in the case of the vertical
approach to the sperm head (Gu et al., 2004; Jimenez-Gonzalez
et al., 2007), its strength was probably not sufficient to allow successful
When we first attempted patch-clamping of mouse epididymal
spermatozoa to provide electrophysiological characterization of the
CatSper channel (Kirichok et al., 2006), after numerous unsuccessful
attempts to form a GV seal we were completely convinced that the
plasma membrane of intact spermatozoa is extremely rigid and that
the formation of the seal is impossible. However, after swelling
mouse spermatozoa in a hypotonic solution (half normal tonicity) at
378C for 45 min, during which some cells turned into a membrane
‘pouch’ with the axoneme wound up inside and loosely attached to
the plasma membrane, we were able to form GV seals almost spon-
taneously (unpublished data). Furthermore, it was possible to break-in
into swollen spermatozoa and record whole-cell currents. After this
observation, we surmized that the apparent rigidity of the sperm
plasma membrane that prevented seal formation was not a native
property, but was caused by the tight association of the membrane
with the underlying intracellular structures. For the formation of the
GV seal, a portion of the plasma membrane must be gently driven
into the tip of the patch pipette by light suction so that it forms an
V-shaped invagination within the tip of the pipette and establishes a
tight seal with the internal walls of the tip (Fig. 2). Only pre-swollen
spermatozoa were able to ‘give’ a portion of the plasma membrane
for the formation of the GV seal; the intact spermatozoa had no
‘spare’ plasma membrane.
Unfortunately, swelling spermatozoa in hypotonic medium at 378C
could lead to a release of hydrolytic enzymes and degradation of
membrane proteins. Furthermore, disruption of the natural associ-
ation between the plasma membrane and the underlying structures
could alter the properties of sperm ion channels. Therefore, we
decided to identify potential regions of the sperm plasma membrane
loosely attached to the intracellular structures in intact spermatozoa.
After studying electronic microphotographs of mouse sperm cells,
we identified the cytoplasmic droplet as the only region of the
plasma membrane loosely associated with intracellular structures
During the process of spermatogenesis in the testis, the ‘nurturing’
Sertoli cell shapes the spermatozoon into its final slim flagellated form
by phagocytosing the residual cytosol of the initially round spermato-
genic cell. This phagocytosis continues until the spermatozoon is left
with a small (normally 1–3 mM in diameter) droplet of the cytoplasm
in the neck region (Cooper, 2011). In the majority of species, including
mice, during the transit of sperm through the epididymis, the cyto-
plasmic droplet migrates down the midpiece and eventually reaches
the connection between the midpiece and the principal piece (the
annulus) in the cauda epididymis (Cooper, 2011) (Fig. 4B). The
exact function of the cytoplasmic droplet is unknown, but it likely
helps in preventing damage to the sperm plasma membrane caused
by the sudden decrease in the osmolarity of the extracellular
medium and the associated increase in the volume of the sperm
cytosol during ejaculation (Cooper, 2011). Upon ejaculation, the
droplet is normally shed without disrupting the integrity of the
plasma membrane, and its presence on the spermatozoa beyond
this point may be associated with infertility at least in male mice,
bulls and boars (Cooper, 2011). Surprisingly, the only species in
which the cytoplasmic droplet is preserved upon ejaculation is
humans. Human spermatozoa preserve the droplet within the
female reproductive tract, and it does not interfere with the process
of fertilization (Cooper, 2011). Another difference in humans is that
the cytoplasmic droplet does not migrate down the midpiece
toward the annulus during the epididymal maturation, but always
stays in the neck region (Cooper, 2011) (Fig. 4C).
As mentioned earlier, the cytoplasmic droplet is the only region of
the sperm plasma membrane that is not tightly associated with the
rigid intracellular structures and can be driven into the tip of the
patch pipette by gentle suction to form a reliable GV seal (Kirichok
et al., 2006). To study electrophysiological properties of mouse sper-
matozoa, completely morphologically mature cells isolated from cauda
epididymis should ideally be used (Lishko et al., 2010, 2011).
However, patch-clamp recording from mouse spermatozoa isolated
from the corpus epididymis is easier, since the droplets are less
fragile (Kirichok et al., 2006; Navarro et al., 2007; Qi et al., 2007).
Unfortunately, since spermatozoa from the corpus epididymis are
not quite mature and cannot fertilize the egg, it is important to
ensure that the properties of the currents recorded match those
from the cauda epididymis sperm.
Human epididymal spermatozoa are much harder to obtain, but the
fact that human spermatozoa preserve the droplets after ejaculation
opens the door to study the molecular mechanisms of fertilization in
humans. In human sperm, the cytoplasmic droplet is located in the
neck region (Cooper, 2011). To the inexperienced eye it may seem
less conspicuous than the cytoplasmic droplet of mice, since it can
be treated as a continuation of the head (Fig. 4C). However, even
though the cytoplasmic droplet of a human spermatozoon is small
and barely visible, the placement of the patch pipette in the neck
region will often lead to the formation of the GV seal (Lishko et al.,
After the formation of seal with the cytoplasmic droplet of a mouse
or human spermatozoon, it is possible to disrupt the small portion of
the sperm plasma membrane just under the pipette (break-in) and
attain electrical access into the cell (Fig. 2). Breaking-in is best achieved
by simultaneous application of high voltage (?0.6–1 V) and negative
pressure to the pipette. After breaking-in, electrical access to all por-
tions of the sperm plasma is gained, as demonstrated by rapid distri-
bution of the fluorescent dye Lucifer Yellow through the whole
interior of the sperm cell (Kirichok et al., 2006; Lishko et al., 2010).
After the formation of the whole-cell mode of the patch-clamp
technique, recording from sperm cells is performed exactly as in any
Kirichok and Lishko
other cell type (Fig. 5). Despite the long flagellum, we did not notice
significant problems with spatial voltage clamp (about the same voltage
was applied to all portions of the sperm flagellum) as judged by the
lack of error in reversal potentials of whole-cell currents. However,
precautions should be taken to ensure that concentrations of the per-
meable ions are well controlled within the flagellum. Although after
breaking-in, the sperm cell is perfused well with the pipette solution
and the intraflagellar ion concentrations reach the same values as
within the pipette, ion currents across the flagellar plasma membrane
can significantly change the concentration of the permeable ion in the
flagellum (either decrease or increase, depending on the direction of
the current). This is demonstrated well by the recording of Ba2+
currents through the flagellar CatSper channel. Although the pipette
solution contains no Ba2+, the Ba2+accumulates inside the flagellum
during the negative part of the voltage ramp protocol and causes an
outward Ba2+current through the CatSper channel during the positive
part of the voltage ramp (Fig. 6). The limited ability to clamp the intra-
flagellar concentration of permeable ions is the only peculiarity of the
sperm whole-cell patch-clamp technique. This problem can be
resolved by holding the cell at membrane potentials that cause
minimal transmembrane current (close to the reversal potential for
the permeable ion), and measuring the current amplitude at the
very beginning of the test voltage step while the intraflagellar concen-
tration of the permeable ion is still unaltered.
Patch-clamp recording can be performed not only from the whole
spermatozoon, but also from its fragments such as the flagellum to
study subcellular localization of ion channels. Upon treatment with
trypsin and gentle trituration, mouse sperm cells separate into two
parts either at the connection between the head and the midpiece
(neck) or at the connection between the midpiece and the principal
piece (annulus) (Kirichok et al., 2006; Navarro et al., 2007)
(Fig. 4D). Often, such separation does not result in the loss of integrity
of the plasma membrane, which closes at the site of separation. Since
the two resultant fragments, the ‘head plus midpiece’ fragment
(H+M) and the ‘principal piece plus midpiece’ fragment (P+M),
both contain the cytoplasmic droplet, it is possible to characterize
ion channels located on these fragments using the whole-cell patch-
clamp technique. Such recordings led to the conclusion that the
Figure 4 The sperm cytoplasmic droplet: the only gateway for sperm patch-clamp. (A) Electron microphotograph of the cytoplasmic droplet of a
ram spermatozoon isolated from cauda epididymis. Note the loose association of the plasma membrane with the underlying mitochondria and the
axoneme. The most anterior part of the principal piece is indicated with ‘P’ Reproduced from (Bloom and Nicander 1961; Figure 3) with kind per-
mission of Springer Science + Business Media. (B) Mouse spermatozoon isolated from corpus epididymis. Cytoplasmic droplet is indicated with the
red arrow. Annulus (connection between the principal piece and the midpiece is indicated with the blue arrow. (C) Ejaculated human spermatozoa.
Cytoplasmic droplet is indicated with the red arrow. Annulus (connection between the principal piece and the midpiece is indicated with the blue
arrow. (D) Diagram demonstrating fractionation of the spermatozoon for patch-clamp recording.
Patch-clamp technique and sperm ion channels
CatSper current originated only from the principal piece of the sperm
flagellum since it could be recorded from the P + M fragment, and not
from the H + M fragment (Kirichok et al., 2006).
A similar separation technique can be applied to human spermato-
zoa. Although trypsin does not seem to help us to fragment human
spermatozoa, simple trituration with a micropipette leads to the sep-
aration of a few spermatozoa into head and M + P fragments at the
neck region (Lishko et al., 2011). The P + M fragment contains the
cytoplasmic droplet and can be used for whole-cell recording. Due
to the resistance of human spermatozoa to trypsin treatment, we
failed to separate human spermatozoa at the connection between
the midpiece and the principal piece (annulus) to obtain H + M frag-
ments that contained the cytoplasmic droplet and were therefore suit-
able for patch-clamp recording. Although this complicated the
electrophysiological analysis of ion channel distribution in human sper-
matozoa, it was still possible to compare currents recorded from the
whole human spermatozoon with those from the P + M fragment.
This approach led us to conclude that the current activated by
flagellum (midpiece plus principal piece) and not from the head
(Lishko et al., 2011).
Although the whole-cell patch-clamp technique is the principal
patch-clamp mode used for characterization of ion channels, single-
channel recording is sometimes desirable in cell-attached and
especially the inside-out configuration that allows easy access to the
cytoplasmic face of the plasma membrane. However, the fact that
reproducible formation of the GV seal is only possible at the cyto-
plasmic droplet makes it impossible to record single-channel activity
from the domains most interesting in terms of Ca2+and H+signaling:
the head and the principal piece. This problem can potentially be
resolved by a perpendicular approach between the pipette and the
surface of the sperm head (Gu et al., 2004; Jimenez-Gonzalez et al.,
2007), but the formation of GV seal with the principal piece of the
intact spermatozoa is impossible due to its extremely small cross-
section and tight association of the plasma membrane with the intra-
cellular structures. The only possible approach to single-channel
recording from the principal piece seems to be pre-swelling of sper-
matozoa in a hypotonic solution to disrupt this association and
convert the flagellar plasma membrane into a ‘pouch’ with the
axoneme wound up inside. Formation of the GV seal with this
pouch is easy, and excision of the patch to form the outside-out
mode could be possible. Unfortunately, as mentioned earlier, swelling
and disruption of the plasma membrane-axoneme connections might
alter the properties of the flagellar ion channels.
in humanspermatozoaoriginates fromthe
Figure 5 Different effects of progesterone on mouse and human
CatSper channels. (A) Representative monovalent (Cs+) whole-cell
CatSper currents recorded from a human spermatozoon in the
absence (blue) and presence (red) of 500 nM progesterone. Note
that the human CatSper current is small (especially at the negative
membrane potentials) but increases dramatically after addition of pro-
gesterone to the bath solution. Currents were recorded in the
absence of divalent ions to allow Cs+permeation through normally
Ca2+-selective CatSper channel. Voltage protocol is shown above.
Right: a human spermatozoon attached to the recording pipette.
(B) Representative monovalent (Cs+) whole-cell CatSper currents
recorded from a mouse spermatozoon in the absence (blue) and
presence (red) of 500 nM P. Mouse CatSper current is overall
larger than human and is not affected by progesterone. All conditions
are the same as in (A). Right: mouse spermatozoon attached to the
recording pipette. Reproduced with permission from (Lishko et al.,
Figure 6 Sperm patch-clamp technique provides only limited
control of the concentration of the permeable ion in the sperm flagel-
lum. Representative whole-cell Ba2+CatSper current recorded from
a mouse spermatozoon isolated from corpus epididymis (red trace).
The recording solutions contained no ions that can permeate through
ion channels, except for 50 mM Ba2+in the bath solution. In spite of
the absence of Ba2+in the pipette solution, an outward Ba2+current
was observed at positive transmembrane potentials (indicated with
the blue arrow). This outward current is due to Ba2+accumulation
inside the flagellum during the negative part of the voltage-ramp pro-
tocol and subsequent efflux of the accumulated Ba2+through the
CatSper channel during the positive part of the voltage ramp. The
voltage protocol is shown above. The baseline current was recorded
in 50 mM of Mg2+in the bath solution.
Kirichok and Lishko
In conclusion, the sperm whole-cell patch-clamp technique rep-
resents a powerful tool for characterization of sperm ion channels.
Its application will further advance our knowledge of molecular mech-
anisms that control sperm activity and male fertility. Below we discuss
sperm ion channels that have been identified with the patch-clamp
technique and their physiological relevance.
CatSper channel: the principal
Ca21channel of sperm
By 2001, when the first pore-forming subunit of the CatSper (Cationic
Channel of Sperm) channel was identified (Ren et al., 2001), the
general agreement in the field was that voltage-gated Ca2+channels
(Cav) constitute the principal Ca2+conductance of sperm (Florman
et al., 1998; Darszon et al., 1999; Publicover and Barratt, 1999).
This notion was supported by electrophysiological identification of
Cavchannels in spermatogenic cells using the patch-clamp technique
(Hagiwara and Kawa, 1984; Arnoult et al., 1996; Lievano et al.,
1996; Santi et al., 1996) and by the observation of a putative voltage-
gated Ca2+influx into mature sperm cells in response to application of
high K+/high-pH extracellular medium (Wennemuth et al., 2000) (see
earlier). The only unresolved issue seemed to be the molecular iden-
tity of the sperm Cavchannels. There was little doubt that T-type
Ca2+channels are involved (Florman et al., 1998; Darszon et al.,
1999; Jagannathan et al., 2002a, b), but N-and R-type voltage-gated
channels also appeared to be implicated (Wennemuth et al., 2000).
The discovery of CatSper1, a putative Ca2+ion channel required
for male fertility (Ren et al., 2001) (Fig. 3A), provided a strong candi-
date molecule to explain the putative voltage-gated Ca2+influx into
sperm cells. Among all known channel proteins, CatSper1 had
highest homology to Cav channels and its S1–S4 voltage-sensor
domain contained a sufficient amount of positively charged lysine/argi-
nine residues in the S4 transmembrane helix to impart strong voltage
sensitivity (Ren et al., 2001). Indeed, it was soon demonstrated that
the elevation of [Ca2+]iin response to high K+/high-pH extracellular
medium that was initially assigned to the sperm Cavchannels was
completely abolished in CatSper1(2/2) spermatozoa (Carlson
et al., 2003). The tempting interpretation of this experiment was
that CatSper1 is the long-sought sperm voltage-gated Ca2+channel.
However, since these indirect experiments potentially allowed mul-
tiple interpretations (not only regarding the mode of activation of
CatSper1, but also its ion selectivity), the whole-cell patch-clamp tech-
nique was applied to mouse spermatozoa to provide direct electro-
physiological characterization of CatSper1 (Kirichok et al., 2006).
Comparisonof ion currents recorded from wild
CatSper1(2/2) spermatozoa confirmed that CatSper1 is indeed
required for a highly selective Ca2+current. By recording from frag-
ments of mouse spermatozoa, it was also established that this
current originated from the principal piece of the sperm flagellum
where CatSper1 protein is located. However, the voltage-dependence
of this current was very weak: the slope factor of the voltage activation
curve was 30, compared with four in strongly voltage-activated chan-
nels. Thus, the CatSper1-dependent channel was not voltage-gated.
Instead, it was potently activated by intracellular alkalinization: the
current was potentiated ?7-fold when intracellular pH was increased
from 6.0 to 7.0, the range of pHiconsidered the most physiologically
relevant for spermatozoa (Kirichok et al., 2006). Notably, the amino
terminus of CatSper1 is highly histidine-rich (83 histidines in the
446-residue N-terminus), suggesting that the N-terminus plays a
role in sensing intracellular pH (Ren et al., 2001) (Fig. 3A).
The pH sensitivity of CatSper can potentially explain why activation
of this Ca2+channel in optical experiments required extracellular alka-
linization. We can hypothesize (although this would not be the only
possible explanation) that the membrane depolarization induced by
the elevation of extracellular K+causes an increase in the permeability
of sperm plasma membrane for protons. Thus, the high extracellular
pH helps us to achieve intracellular alkalinization sufficient for the
activation of CatSper1 channel. Indeed, a very small H+conductance
activated by membrane depolarization was detected in mouse
spermatozoa (Lishko et al., 2010).
Soon after the discovery of CatSper1, three other related sperm-
specific 6TM proteins were identified based on their sequence hom-
ology to CatSper1: CatSper2, CatSper3 and CatSper4 (Quill et al.,
2001; Lobley et al., 2003; Qi et al., 2007). CatSper2-, CatSper3-
and CatSper4-deficient mice showed the same phenotype as
CatSper1(–/–) mice: the males were infertile and their spermatozoa
could not develop hyperactivation (Quill et al., 2003; Carlson et al.,
2005; Qi et al., 2007). Interestingly, whole-cell patch-clamp recording
from mouse spermatozoa demonstrated that CatSpers 2, 3 and 4
were required for the same flagellar pH-dependent Ca2+channel as
CatSper1 since this channel disappeared on spermatozoa deficient
for any one of the CatSper proteins (Qi et al., 2007). Moreover,
CatSper2, CatSper3 and CatSper4 could be co-immunoprecipitated
with CatSper1 from mouse testes lysate and after co-expression in
a heterologous system, demonstrating that CatSper2, CatSper3 and
CatSper4 form molecular complexes with CatSper1 (Qi et al.,
2007). From these experiments, and based on the classic architecture
of other ion channels such as Kvand TRP (in which four 6TM subunits
are required to form a single pore) (Yu et al., 2005), it was concluded
that the pore of CatSper channel is formed by four different CatSper
subunits: CatSpers 1, 2, 3 and 4 (Qi et al., 2007; Navarro et al., 2008).
In contrast to CatSper1, other pore-forming CatSper subunits have a
lower number of positively charged residues in the S4 transmembrane
domain, which probably explains the low voltage sensitivity of the
CatSper channel (Qi et al., 2007; Navarro et al., 2008).
Although the CatSper channel has only weak voltage-dependence, it
is still important for CatSper function and is directly connected to the
channel’s pH-sensitivity (Kirichok et al., 2006). At low intracellular pH
(? 6.0–6.5), the CatSper voltage dependence is such that it holds the
channel virtually closed at sperm physiological membrane potentials
(from about 270 to 0 mV). However, an increase in intracellular
pH significantly shifts CatSper voltage dependence toward the negative
membrane potentials (Kirichok et al., 2006). This new voltage depen-
dence now allows the channel to be opened by lower potentials in the
The discovery that the CatSper channel is activated by the elevation
of intracellular pH helped us to explain how capacitation can cause
hyperactivation. One of the hallmarks of capacitation is elevation of
sperm pHi (Parrish et al., 1989; Zeng et al., 1996; Suarez, 2008).
This intracellular alkalinization should not only directly stimulate moti-
lity of the axoneme, but, by activating CatSper channel, will also
provide the elevation of [Ca2+]i required for hyperactivation.
Indeed, it was later demonstrated that intracellular alkalinization
Patch-clamp technique and sperm ion channels
caused by extracellular application of NH4Cl is sufficient for the induc-
tion of sperm hyperactivation (Marquez and Suarez, 2007).
Using optical methods for measuring sperm [Ca2+]i, it was found
that in intact mouse spermatozoa membrane-permeable analogs of
cAMP and cGMP activated CatSper-dependent Ca2+entry (Ren
et al., 2001, Xia et al., 2007). Thus, it was originally proposed that
CatSper channel can be directly or indirectly activated by cyclic
nucleotides (Ren et al., 2001). However, other groups failed to
demonstrate [Ca2+]ielevation in response to membrane-permeable
analogs of cAMP, to photolysis of caged cAMP or in response
to bicarbonate-induced elevation of cAMP (Carlson et al., 2003;
Wennemuth et al., 2003; Stru ¨nker et al., 2011). It was, however,
demonstrated that CatSper-dependent Ca2+elevation induced by
high K+/high-pH extracellular medium was strongly facilitated by
bicarbonate that causes sACY-dependent elevation of intracellular
cAMP (Carlson et al., 2003; Wennemuth et al., 2003; Xie et al.,
2006). It is important to stress out that in these experiments, the
bicarbonate-induced elevation of sperm cAMP did not cause Ca2+
entry itself but simply facilitated the activation of CatSper by high
K+/high-pH extracellular medium (Carlson et al., 2003; Wennemuth
et al., 2003). In the patch-clamp experiments the CatSper current
was not affected by cyclic nucleotides (Kirichok et al., 2006). This
demonstrated that the facilitation of CatSper channel by cyclic nucleo-
tides is indirect and requires an intermediary signaling cascade that was
obviously disrupted during the patch-clamp recording. In this respect,
it has been proposed that cAMP facilitates CatSper-dependent Ca2+
entry via PKA-dependent phosphorylation (Wennemuth et al., 2003;
Nolan et al., 2004).
Since in intact cells the activity of CatSper channel can be facilitated
by cyclic nucleotides, the cyclic nucleotide-induced Ca2+entry into
mammalian spermatozoa that was originally assigned to the cyclic
nucleotide gated channels (Wiesner et al., 1998) can in fact be
mediated by the CatSper channel. This hypothesis is supported by
the fact that no ion channels directly gated by cyclic nucleotides
have been detected with the patch-clamp technique in mouse or
human spermatozoa (Kirichok et al., 2006; Navarro et al., 2007)
(and unpublished observations in human spermatozoa). However,
such channels are likely to be present in spermatozoa of marine invert-
ebrate species (Darszon et al., 1999, 2006a; Strunker et al., 2006;
Bonigk et al., 2009).
Albumin, the main protein of the oviductal fluid and an important
component of the in vitro capacitation media, has also been shown
to cause CatSper-dependent Ca2+influx into mouse spermatozoa
(Xia and Ren, 2009a). Finally, it has been demonstrated that the
Ca2+influx into mouse spermatozoa induced by the glycoproteins
of the egg’s zona pellucida requires CatSper channel (Xia and Ren,
2009b). In the past, the zona-induced Ca2+influx was assigned to
the putative sperm Cav channels (Florman et al., 1998; Darszon
et al., 1999). Although the mechanisms by which albumin and the gly-
coproteins of zona pellucida activate CatSper channel are yet to be
established, these discoveries may lead to a new and improved under-
standing of fundamental aspects of sperm physiology such as capacita-
tion and the acrosome reaction (Ren and Xia, 2010).
In addition to the four pore-forming subunits, three auxiliary
subunits of the CatSper channel have been discovered: CatSperb,
CatSperg and CatSperd (Liu et al., 2007; Wang et al., 2009; Chung
et al., 2011) (Fig. 3B). These are all sperm-specific proteins that
were identified as proteins that co-purify as a complex with
CatSper1 from mouse testis lysate. CatSperb, CatSperg and CatSperd
are located in the principal piece of the sperm flagellum, the region
where the pore-forming CatSper subunits reside. While CatSperb
has two predicted transmembrane helices with a large extracellular
loop between them, CatSperg and CatSperd have just one predicted
transmembrane helix and a large extracellular domain ( Liu et al., 2007;
Wang et al., 2009; Chung et al., 2011) (Fig. 3B). Although the inter-
action of CatSperb and CatSperg with the CatSper complex was
clearly demonstrated biochemically (Liu et al., 2007; Wang et al.,
2009; Ren and Xia, 2010; ), it is not clear whether they are required
for the functional CatSper channel. In contrast, CatSperd does not
only interact with the CatSper complex but has been shown to be
essential for the functional CatSper channel: the whole-cell CatSper
current disappears on CatSperd (2/2) mouse spermatozoa
(Chung et al., 2011). CatSperd (2/2) spermatozoa also have very
low levels of CatSper1 protein compared with wild-type cells
(Chung et al., 2011). Since all three auxiliary subunits of the CatSper
channel have large extracellular domains, any of them could potentially
interact with extracellular ligands to modulate the activity of CatSper
channel, similar to the auxiliary subunit of the voltage-gated Cav
channels. However, the exact functions of CatSperb, CatSperg and
CatSperd remain to be established.
CatSperb, CatSperg, CatSperd and CatSper2, are all undetectable
on CatSper1 (2/2) sperm (Carlson et al., 2005; Liu et al., 2007;
Wang et al., 2009; Chung et al., 2011). Conversely, CatSper1
protein disappears on CatSper2 (2/2) and CatSperd (2/2) sper-
matozoa (Carlson et al., 2005; Chung et al., 2011). These observations
suggest that if one of the proteins of the CatSper channel is lacking and
the whole channel complex cannot be formed, the remaining CatSper
proteins are degraded. At this time, the CatSper channel contains the
highest number of independent subunits among all known ion chan-
nels, and it is quite possible that more CatSper subunits will be discov-
ered in the future. It is likely that this complexity is the reason why all
attempts to express CatSper channel in heterologous expression
systems have failed. If, at some point, heterologous expression is
achieved, it will make studies of function–structure relationships
within CatSper channel possible and will further improve our under-
standing of how the regulation of sperm intracellular Ca2+occurs at
the molecular level.
The subunits of CatSper channel are present in the genome of all
mammals and some invertebrate species, such as the sea urchin and
sea squirt (Cai and Clapham, 2008). Since the discovery of CatSper
channel, several mutations and deletions in CatSper1 and CatSper2
subunits have been shown to be associated with cases of male inferti-
lity in humans (Hildebrand et al., 2010). Recent development of the
whole-cell patch-clamp technique for human spermatozoa provided
a tool for the identification and characterization of the current
mediated by human CatSper channel (Lishko et al., 2010). Under
the recording conditions used to identify the mouse CatSper current
and where it can be recorded in isolation from other sperm ion chan-
nels, we identified a highly Ca2+-selective flagellar channel in human
sperm (Lishko et al., 2010, 2011). Similar to mouse CatSper, it was
potently activated by intracellular alkalinization and was only weakly
voltage dependent. However, the voltage dependence of human
CatSper was slightly steeper (slope factor approximately 20 compared
with 30 in mice) and the half activation voltage was more positive
Kirichok and Lishko
(+85 mV in human versus +11 mV in mice at pHi¼ 7.5) than those
of mouse CatSper (Kirichok et al., 2006; Lishko et al., 2011). Slope
factors for strongly voltage-sensitive ion channels are ?4.
Although in general, human CatSper current was similar to mouse
CatSper current, the human CatSper had a steeper voltage depen-
dence that was also shifted toward more positive potentials. This
voltage dependence made human CatSper much less active at the
negative physiological potentials across the sperm plasma membrane
compared with mouse CatSper (Lishko et al., 2011) (Fig. 5). Interest-
ingly, we and others (Lishko et al., 2011; Stru ¨nker et al., 2011) were
able to identify a potent extracellular physiological activator of
human CatSper channel that, similar to intracellular pH, shifted the
voltage dependence of human CatSper channel toward more negative,
physiological potentials. This activator was progesterone, a major
female steroid hormone released by the ovaries and the cumulus
cells surrounding the egg in the Fallopian tubes (Lishko et al., 2011;
Stru ¨nker et al., 2011) (Fig. 5). Interestingly, mouse CatSper was not
activated by progesterone (Lishko et al., 2011)(Fig. 5B). When com-
bined with intracellular alkalinization, progesterone made human
CatSper as active at negative physiological potentials as mouse
CatSper (Lishko et al., 2011).
Progesterone is probably the most potent activator of human sper-
matozoa (Publicover et al., 2007). In nanomolar concentrations, it
induces robust Ca2+influx into human sperm cells (Thomas and
Meizel, 1989; Blackmore et al., 1990). Progesterone triggers sperm
hyperactivation and the acrosome reaction, and is probably also the
chemoattractant of human spermatozoa, although this role of pro-
gesterone is still debated (Uhler et al., 1992; Roldan et al., 1994;
Revelli et al., 1998; Eisenbach and Giojalas, 2006; Teves et al.,
2006). The progesterone receptor associated with human spermato-
zoa is probably the best-known example of a ‘non-genomic progester-
one receptor’ whose action does not depend on the regulation of
gene expression (Revelli et al., 1998; Losel and Wehling, 2003;
Luconi et al., 2004), in contrast to the classic nuclear progesterone
receptor (Evans, 1988). The effect of progesterone on human
sperm cells was much faster than that mediated by the nuclear pro-
gesterone receptor and certainly did not depend on regulation of tran-
scription since spermatozoa are transcriptionally silent. Although all
physiological effects of progesterone on human spermatozoa
depend on the progesterone-induced Ca2+influx, the putative
sperm progesterone receptor and the associated Ca2+ion channel
remained elusive for more than 20 years.
We established that progesterone activates human CatSper with an
EC50≈ 7.7 nM (Lishko et al., 2011). The progesterone-binding site
associated with CatSper channel is external and its pharmacology is
different from that of the nuclear progesterone receptor (Lishko
et al., 2011; Stru ¨nker et al., 2011). The effect of progesterone upon
CatSper is enhanced by sperm capacitation (Lishko et al., 2011; Stru ¨n-
ker et al., 2011). The action of progesterone is very rapid (latency
,36 ms) and does not depend on intracellular ATP, GDP, cyclic
nucleotide, Ca2+or other soluble intracellular messengers (Lishko
et al., 2011; Stru ¨nker et al., 2011). The simplest explanation of
these results is that receptors for progesterone are located within
the CatSper channel complex consisting of CatSpers1–4, CatSperb,
CatSperg or CatSperd. However, a different, currently unidentified,
protein associated with the CatSper channel may also serve as the
CatSper-associated progesterone receptor.
As measured by optical methods in human sperm, prostaglandin E1
(PGE1) causes intracellular Ca2+transients similar in amplitude and
waveform to those induced by progesterone (Aitken et al., 1986;
Schaefer et al., 1998; Shimizu et al., 1998). Interestingly, patch-clamp
experiments with human spermatozoa established that in addition
to progesterone, human CatSper channel is activated by nanomolar
concentrations of select PGs including PGE1(Lishko et al., 2011; Stru ¨n-
ker et al., 2011). However, the effect of PGs is additive to the effect of
progesterone and is likely mediated through a different receptor
(Lishko et al., 2011; Stru ¨nker et al., 2011). The relative potency of
the human CatSper activators identified in this work followed the
sequence Progesterone . PGF1a≈ PGE1. PGA1.PGE2≫PGD2
(Lishko et al., 2011). Although the physiological role of the effect of
PGs on human spermatozoa remains unclear, PGs are present in
large quantities in the seminal plasma (Mann and Lutwak-Mann,
1981) and are secreted by the oviduct and cumulus cells surrounding
the oocyte (Espey and Lipner, 1994). Millimolar concentrations of
Zn2+present in the seminal plasma (Saaranen et al., 1987) are likely
to block CatSper channel and prevent massive elevation of [Ca2+]
and hyperstimulation of spermatozoa in the seminal plasma.
However, since free concentration of Zn2+in the oviduct is very
low (total concentration ≈ 20 mM, nearly all of which is bound to
albumin), CatSper channel could be potentiated by PGs as spermato-
zoa approach the site of fertilization (Schaefer et al., 1998).
Considering all its direct (intracellular pH, progesterone and PGs)
and indirect (cAMP, albumin and zona pellucida glycoproteins) activa-
tors, the CatSper channel appears to be a channel with true polymo-
dal regulation. It should also be noted that CatSper channel is
potentiated as a result of capacitation, presumably due to phosphoryl-
ation (Lishko et al., 2011). The large number of subunits required to
form CatSper channel may provide the molecular basis for such poly-
modal regulation. Since orthologs of CatSper subunits present in
different species have low identity (50% or less) (Liu et al., 2007;
Ren and Xia, 2010), the regulation of main sperm Ca2+entry
pathway may differ significantly between species. Mouse CatSper
channel, for example, is not sensitive to the activators of human
CatSper, progesterone and PGs (Lishko et al., 2011). This difference
in CatSper regulation probably reflects different mechanisms of fertili-
zation adopted by different species.
The last question that should be discussed in relation to the CatSper
channel is the existence (or non-existence) of sperm Cavchannels.
The discovery that the putative voltage-gated Ca2+influx disappeared
on CatSper1 (2/2) and CatSper2 (2/2) knockout spermatozoa
has shaken the very foundation of the hypothesis about the existence
of sperm Cav channels (Carlson et al., 2003, 2005). Currently,
immunocytochemical reactivity for several different Cavsubunits in
mature spermatozoa remains the only evidence for the existence of
sperm Cavin mature sperm cells (rather than spermatocytes) (Wes-
tenbroek and Babcock, 1999; Wennemuth et al., 2000; Carlson
et al., 2003; Darszon et al., 2006b). On the other hand, the lack of
fertility phenotypes in Cav3.1 and 3.2 knockout mice lacking T-type
Cavchannels do not support the existence of functional sperm Cav
channels (Escoffier et al., 2007). Finally, all attempts to identify voltage-
gated Ca2+currents electrophysiologically in mature mouse or human
spermatozoa have failed. T-type Ca2+channels could be recorded
from mouse testicular sperm (Darszon et al., 2006b), but they
seemed to be silenced via an unknown mechanism and are
Patch-clamp technique and sperm ion channels
undetectable in the epididymal mouse sperm (Xia and Ren, 2009b).
Ejaculated human spermatozoa do not posses functional Cavchannels
as recorded with the patch-clamp technique (Lishko and Kirichok,
unpublished data). It is possible that the Cavchannels identified in
mature sperm cells by immunocytochemistry do exist, but remain
silent during optical and patch-clamp experiments and can only be
activated under certain conditions. However, this special type of acti-
vation would be very unusual for Cavchannels, which are simply gated
by membrane depolarization in somatic cells.
Voltage-gated proton channel
Hv1: the dominant proton
conductance of human sperm
Since sperm pHiis an important regulator of sperm motility and the
acrosome reaction, together with Ca2+it has been considered an
important regulator of sperm function. However, when the discovery
of pH-sensitive CatSper channel revealed that intracellular pHiis also
an important regulator of sperm intracellular Ca2+signaling, it became
obvious that pHiis likely to be the principal regulator of sperm activity
over Ca2+. Intercellular alkalinization appears to be essential for the
initiation of motility, capacitation, hyperactivation, chemotaxis and
the acrosome reaction, and is likely to lie well upstream in the
sequence of signaling events leading to sperm functional activation
within the female reproductive tract. Despite the important role
that intracellular alkalinization plays in sperm activation, the H+trans-
port mechanisms of the sperm plasma membrane that extrude
protons out of the sperm cell have long remained unknown.
Although intracellular pH-sensitive fluorescent probes were able to
detect changes in sperm intracellular pH associated with activation of
motility, capacitation, chemotaxis and the acrosome reaction, identifi-
cation of the H+transport mechanisms involved using this method
was very challenging. It was suggested that a Na+/H+exchanger
(NHE) (Bibring et al., 1984; Lee and Garbers, 1986; Schackmann
and Chock, 1986; Garcia and Meizel, 1999; Woo et al., 2002) and a
zation at least in some species (Tajima and Okamura, 1990; Zeng
et al., 1996), but the actual molecules controlling sperm intracellular
pH have never been identified.
The NHE seemed to be an especially attractive mechanism for
elevation of sperm intracellular pH. At the very least, it seems to be
ideally posed to cause the intracellular alkalinization required for
initiation of sperm motility. Upon ejaculation, mammalian spermato-
zoa experience significant elevation of extracellular [Na+] from
about 30 mM in cauda epididymis to 100–150 mM in the seminal
plasma and 130–150 mM in the oviduct (Mann, 1964; Borland
et al., 1980; Owen and Katz, 2005). This increase in extracellular
Na+could be used by the NHE to drive the export of protons and
raise sperm pHi. Therefore, the identification of a sperm-specific mol-
ecule that bore significant homology to known NHEs and was loca-
lized in the principal piece of sperm flagellum was especially exciting
(Wang et al., 2003). Male mice deficient in this putative NHE were
completely infertile, and their spermatozoa had greatly impaired moti-
lity (Wang et al., 2003). Although the newly identified molecule was
dubbed sNHE (sperm NHE), it has been difficult to demonstrate
that it actually functions as a NHE: similar to CatSper subunits, after
2exchanger are involved in sperm alkalini-
expression in heterologous systems, sNHE failed to be transported
to the plasma membrane and exhibit functional activity (Wang et al.,
2003). Functional data from the native system (spermatozoa) were
even less encouraging: sNHE seemed to have no effect on
sperm pHisince there was no significant difference in intracellular
pH between wild type and sNHE (–/–) spermatozoa (Wang
et al., 2003).
Although it was later demonstrated that a chimera between sNHE
and the ubiquitously expressed plasma membrane NHE isoform-1
(NHE1) is transported to the plasma membrane after heterologous
expression, the chimeric protein showed very modest H+transport
activity compared with NHE1 (Wang et al., 2007). Finally, it was dis-
covered that the expression level of the sACY is extremely low in
sNHE (2/2) spermatozoa, and that the sperm motility defect can
be rescued by the addition of membrane-permeable analogs of
cAMP (Wang et al., 2003, 2007). This certainly raised the question
of whether the infertility phenotype of male sNHE-deficient mice is
solely due to disruption of sACY, which has previously been shown
to be required for sperm motility and male fertility (Esposito et al.,
2004; Hess et al., 2005; Xie et al., 2006). In conclusion, it is still not
clear whether sNHE is a real NHE, and whether its putative NHE
activity is required for male fertility. Further experiments are certainly
needed to answer these important questions.
Development of the whole-cell patch-clamp technique for mouse
and human spermatozoa provided a tool to directly address the
problem of H+transport across the sperm plasma membrane. So
far, patch-clamp experiments with mouse spermatozoa have not
detected any significant proton currents (Lishko et al., 2010). Cer-
tainly, this result does not mean that protons are not transported
across the plasma membrane of mouse spermatozoa. Since the
NHE is electroneutral (does not transport a net charge), its activity
cannot be recorded with the patch-clamp technique. Furthermore,
there may be other H+transport mechanisms that require specific
intracellular or extracellular activators that were not present in our
In contrast to mouse spermatozoa, patch-clamping human sperma-
tozoa revealed a very large voltage-gated H+current (Lishko et al.,
2010). We called this current and the underlying H+channel of the
human sperm plasma membrane HSper (H+channel of sperm). The
HSper current was one of the highest native H+currents ever
recorded across the cell plasma membrane (Decoursey, 2003; 2008;
Lishko et al., 2010). Interestingly, HSper was a one-way channel: it
only allowed outward transport of H+and thus was specifically
designed for extrusion of protons, leading to intracellular alkalinization.
HSper was activated by membrane depolarization, but the transmem-
brane H+concentration gradient favoring H+extrusion also activated
HSper and helped the channel to open at much more negative mem-
brane potentials. Thus, effectively, HSper was opened by the com-
bined transmembrane electrochemical H+gradient favoring proton
extrusion. A similar gradient favoring H+entry into the human sper-
matozoa failed to induce any inward HSper current. HSper current
was potently inhibited by extracellular Zn2+: nanomolar concen-
trations of this ion significantly reduced the amplitude and activation
kinetics of HSper current. The combination of properties possessed
by HSper (high selectivity for protons, conducting only the outward
current, slow kinetics of activation and deactivation, gating by com-
bined electrochemical H+gradient and high sensitivity to Zn2+)
Kirichok and Lishko
(Lishko et al., 2010) is shared by only one other channel, the voltage-
gated proton channel Hv1 (Ramsey et al., 2006; Sasaki et al., 2006;
DeCoursey, 2010). Moreover, similar to Hv1 (Alabi et al., 2007),
HSper was potentiated by fatty acids and inhibited by crude venom
of Chilean rose tarantula Grammostola rosea that contained hanatoxin
and other homologous toxins (Lishko et al., 2010).
Hv1 is a four-transmembrane domain protein homologous to the
voltage-sensor domain of voltage-gated cation channels (Ramsey
et al., 2006; Sasaki et al., 2006) (Fig. 3C). A functional Hv1 channel
is a dimmer of two identical subunits, although each of the two sub-
units appears to function as an H+channel (Koch et al., 2008; Lee
et al., 2008; Tombola et al., 2008). Hv1 is highly expressed in phago-
cytic immune cells, where it helps the enzyme NADPH oxidase gen-
erate high levels of reactive oxygen species required to kill bacteria
(DeCoursey, 2010). More specifically, Hv1 compensates for the mem-
brane depolarization and intracellular acidification generated as the
result of NADPH oxidase enzymatic activity and helps it produce reac-
tive oxygen species at the highest possible rate (DeCoursey, 2010).
Although this is currently the best-established physiological role of
Hv1, the channel is also highly expressed in other cells such as baso-
phils, B lymphocytes, microglia and alveolar epithelium and may
have other functions independent of NADPH oxidase (Capasso
et al., 2011).
After demonstrating that the electrophysiological and pharmacologi-
cal properties of HSper are identical to those of Hv1, we further estab-
lished that human spermatozoa contain high amounts of Hv1 protein
and its corresponding mRNA (Lishko et al., 2010). Interestingly,
immunocytochemical analyses of human sperm revealed that Hv1
was specifically localized within the principal piece of the sperm flagel-
lum (Lishko et al., 2010). This correlated with electrophysiological
recordings from fractionated human spermatozoa showing that the
HSper current originated from the sperm flagellum (Lishko et al.,
These experiments demonstrated that Hv1 is the main H+conduc-
tance of the human spermatozoa, at least as recorded with the patch-
clamp technique. Since sperm Hv1 conducts H+only in the outward
direction and due to the large amplitude of this current (Lishko
et al., 2010), sperm Hv1 is likely to be the principal H+conductance
responsible for intracellular alkalinization and functional activation of
human sperm. Specific localization of Hv1 in the principal piece of
the sperm flagellum makes it ideally positioned to activate
pH-dependent proteins of the axoneme and the pH-dependent
CatSper channel, and thus to control sperm motility.
Similar to CatSper, sperm Hv1 appears to be controlled by multiple
cues of the male and female reproductive tracts. First, the female
reproductive tract has a higher pH (?7.4) than the acidic fluid of
the cauda epididymis, which can have a pH as low as 6.5. The
elevation of extracellular pH upon penetration of the human sperma-
tozoa into the female reproductive tract should increase the trans-
membrane electrochemical driving force favoring H+extrusion and
activate sperm Hv1 (Lishko et al., 2010). Hv1 can also be activated
by membrane depolarization (Lishko et al., 2010), but currently it is
not clear how such depolarization of the sperm plasma membrane
can be achieved. Based on indirect studies, several ligand-gated ion
channels were proposed to function in sperm cells, and some of
these channels can potentially provide membrane depolarization to
activate sperm Hv1 (Meizel, 2004).
Second, sperm Hv1 can be activated by removal of extracellular zinc
(Lishko et al., 2010). Human seminal plasma contains high levels of zinc
(total concentration 2.2+1.1 mM compared with 14+3 mM in
serum) (Saaranen et al., 1987). Upon ejaculation, seminal zinc diffuses
into the female reproductive tract and in rats, has been shown to
reach the Fallopian tubes (Gunn and Gould, 1958), where it is even-
tually absorbed by the oviductal epithelium and chelated by the domi-
nant protein in the oviductal fluid, albumin (Ehrenwald et al., 1990; Lu
et al., 2008). Thus, sperm Hv1 should be inhibited by seminal zinc up
to the moment when spermatozoa reach the Fallopian tubes, where
the inhibition will be relieved to allow sperm intracellular alkalinization,
activation of Ca2+influx through pH-sensitive CatSper channels and
sperm hyperactivation. Regulation of sperm Hv1 by zinc could
explain why sperm hyperactivation occurs only in the Fallopian
tubes close to the site of fertilization.
Third, low micromolar concentrations of endogenous cannabinoid
anandamide strongly potentiate sperm Hv1 (Lishko et al., 2010). A
related compound, arachidonic acid, required approximately 100
times higher concentration to achieve a similar potentiation (Lishko
et al., 2010). The effect of anadamide is not mediated by CB1 or
CB2 cannabinoid receptors and is likely due to a direct interaction
of anandamide with Hv1 (Lishko et al., 2010). Bulk concentrations of
anandamide in the fluids of the male and female reproductive tracts
are in the nanomolar range (Schuel and Burkman, 2005). However,
it is possible that spermatozoa experience much higher concentrations
in the direct proximity of sources of anandamide such as the cumulus
cells that surround the egg in the Fallopian tubes (El-Talatini et al.,
2009). If the concentration of anandamide in CO reaches low micro-
molar levels, anandamide can serve as an activator of sperm motility
and help spermatozoa to penetrate the egg’s protective vestments.
Fourth, sperm Hv1 is potentiated during in vitro capacitation (Lishko
et al., 2010). The mechanism of this potentiation remains unknown,
but since multiple proteins are phosphorylated during capacitation,
we can hypothesize that Hv1 also becomes phosphorylated at this
time. Notably, phosphorylation is the primary mechanism of Hv1 regu-
lation in other tissues (Decoursey, 2003; Musset et al., 2010).
Although capacitation is a poorly understood process, intracellular
alkalinization is considered a key factor in this process (Parrish et al.,
1989; Zeng et al., 1996; Darszon et al., 1999; Suarez, 2008). The
coincidence of capacitation and the enhancement of Hv1 activity
suggest a strong connection between these two events. Although it
is possible that potentiation of Hv1 is an important causative factor
in capacitation, we cannot exclude the possibility that Hv1 enhance-
ment occurs late in the process of capacitation and is a mere
consequence of it.
In conclusion, sperm Hv1 appears to play an important role in the
regulation of human sperm intracellular pH and the pH-dependent
CatSper channel. By doing so, it can potentially influence almost
every aspect of sperm behavior in the female reproductive tract,
including initiation of motility, capacitation, hyperactivation, chemo-
taxis and the acrosome reaction. However, the exact physiological
function of sperm Hv1 remains to be established. Since no functional
sperm Hv1 channel has been identified in mice (Lishko et al., 2010),
this convenient genetic model cannot be used to identify the
physiological role of human sperm Hv1. It is important to note that
Hv1-deficient mice do not exhibit any fertility phenotype (Ramsey
et al., 2009); however, low levels of sperm HVCN1 (Hv1) mRNA
Patch-clamp technique and sperm ion channels
are strongly correlated with male infertility in humans (Platts et al.,
2007). It is thus possible that studies of genetic infertility in humans
will help us understand the exact role of Hv1 in male fertility.
Slo3 (KSper): a K1channel that
sets sperm resting membrane
Since the sperm Hv1 channel and, to a lesser degree, CatSper channel
depend on the membrane potential, it is important to identify the ion
channels that control the sperm membrane potential. In many differ-
ent cell types, the resting membrane potential is primarily set by K+
channels, and is usually slightly more positive than the K+reversal
are no exception to this rule.
Whole-cell patch-clamp recording from mouse spermatozoa iso-
lated from corpus epididymis identified a constitutively active,
weakly voltage-dependent K+channel that was potentiated by
depolarization and was initially named KSper (K+channel of sperm)
(Navarro et al., 2007). Interestingly, similar to CatSper channel,
mouse KSper was strongly potentiated by intracellular alkalinization
(Navarro et al., 2007). Patch-clamp recording from fractionated sper-
matozoa showed that the KSper channel originated from the principal
piece of the sperm flagellum (Navarro et al., 2007). Current-clamp
recording in the whole-cell mode demonstrated that mouse sperm
membrane potential is strongly dependent on intracellular pH (it
becomes more negative at alkaline pH) and is affected by pharmaco-
logical modulators of KSper channel (Navarro et al., 2007). Based on
these experiments, it was concluded that KSper is the K+channel that
sets the sperm resting membrane potential (Navarro et al., 2007). It
was also suggested that the membrane hyperpolarization observed
as a result of sperm capacitation in mice is due to activation of
KSper by the capacitation-dependent intracellular alkalinization. The
most likely molecule mediating the KSper current was proposed to
be a sperm-specific channel Slo3 (Navarro et al., 2007) (Fig. 3D),
which—after expression in a heterologous system—gave rise to K+
and was activated by intracellular alkalinization (Schreiber et al., 1998).
A few years later, the generation of Slo3-deficient mice confirmed
that Slo3 is required for the mouse sperm K+channel potentiated
by intracellular alkalinization (Santi et al., 2010; Zeng et al., 2011).
Slo3-deficient male mice are infertile and their spermatozoa do not
undergo membrane depolarization in response to intracellular alkalini-
zation and do not exhibit the capacitation-dependent membrane
hyperpolarization (Santi et al., 2010; Zeng et al., 2011). Slo3 null sper-
matozoa also show markedly deficient progressive motility and an
impaired acrosome reaction induced by the calcium ionophore
A23187 (Santi et al., 2010, Zeng et al., 2011).
It has recently been demonstrated that mouse KSper (Slo3) channel
requires phosphatidylinositol 4,5-bisphosphate (PIP2) for its activity
and can be inhibited by sperm membrane receptors that cause PIP2
hydrolysis such as putative sperm epidermal growth factor (EGF)
receptor (Tang et al., 2010). Since stimulation of spermatozoa with
EGF triggers the acrosomal exocytosis, it was proposed that EGF
may mediate its action at least partially through inhibition of KSper
channel and membrane depolarization (Tang et al., 2010).
K≈ −80 mV, depending on the cell type). Sperm cells
These experiments seemed to identify Slo3 as the K+channel
responsible for setting the resting membrane potential of mouse sper-
matozoa. However, the K+channel performing this function in human
spermatozoa has not yet been identified. It is tempting to postulate
that the same alkalinization-activated K+channel is present on
human spermatozoa, but the significant differences in sperm ion chan-
nels between mice and humans, as well as an apparent lack of
suggest that such conclusions should be confirmed experimentally.
It is also important to mention that to understand the mechanism of
regulation of sperm membrane potential, it is not sufficient to merely
establish ion channels that control resting membrane potential (such
as Slo3). In somatic cells, potassium channels set the resting potential,
but the membrane depolarization required for the activation of
voltage-gated ion channels is normally induced by Na+-permeable
ion channels controlled by intracellular or extracellular cues such as
cAMP or neuromediators. Similar Na+-permeable channels controlled
by sperm intracellular second messengers or cues of the female repro-
ductive tract remain to be identified in spermatozoa. Identification of
these channels will help us to better understand the mechanisms of
regulation of other sperm ion channels that depend on the membrane
potential, such as Hv1 and possibly CatSper.
in human spermatozoa,
The sperm whole-cell patch-clamp technique, combined with
methods of molecular genetics and biochemistry, has resulted in a
quantum leap in our understanding of sperm ion channels. Identifi-
cation and functional characterization of important sperm ion channels
such as CatSper, HSper (Hv1) and KSper (Slo3) have paved the way
for a comprehensive and coherent model of electrical signaling in
Although characterization of sperm ion channels is not complete, it
is very unlikely that the 40 or so different ion channel proteins suppo-
sedly expressed in spermatozoa are important for sperm physiology in
any one particular species (Darszon et al., 2006a). First, although many
of these channels may be expressed as protein or as mRNA in sper-
matogenic cells or spermatozoa, they are not necessarily functionally
active in the mature sperm cells. This seems to be the case, for
example, with the voltage-gated Ca2+channels that are functionally
expressed in spermatogenic cells but become quiescent in mature
spermatozoa (Ren and Xia, 2010, Xia and Ren, 2009b). Second,
some of these channels may regulate sperm activity in one species
but be functionally absent in another. The dramatic difference in func-
tional expression of the Hv1 channel between mouse and human sper-
matozoa should serve as a good example of closely related species
with very different ion channel physiology (Lishko et al., 2010). In
any case, the patch-clamp technique should be used to prove that
channels that were proposed to be functionally present on spermato-
zoa (Darszon et al., 2006a) are indeed valid sperm channels. Three
decades after its invention, the patch-clamp technique remains the
most reliable method for functional identification and characterization
of ion channels.
There is no doubt that functional identification of sperm ion chan-
nels using the whole-cell patch-clamp technique and optical methods
for measuring intracellular Ca2+and pH in intact sperm cells will even-
tually allow us to identify a relatively small number of ion channels that
Kirichok and Lishko
control sperm activity and male fertility in any particular species. Since
properties of sperm ion channels seem to differ significantly from
species to species, successful completion of this work is especially
important in humans to help understand numerous unexplained
casesof male infertilityand
Y.K. and P.V.L. conceived and wrote the manuscript.
This work was supported by grant #R01HD068914 from the Eunice
Kennedy Shriver National Institute of Child Health & Human Develop-
ment. The content is solely the responsibility of the authors and does
not necessarily represent the official views of the Eunice Kennedy
Shriver National Institute of Child Health & Human Development or
the National Institutes of Health.
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