1. Systems Biology in Reproductive Medicine, 56:37–43, 2010
Copyright & Informa Healthcare USA, Inc.
ISSN: 1939-6368 print/1939-6376 online
DOI: 10.3109/19396360903497217
Research Communication
+
Tetraethylammonium-Sensitive K Current
in the Bovine Spermatozoa and its
Blocking by the Venom of the Chilean
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Latrodectus mactans
Jorge Parodi,
Patricia Navarrete,
The morphology and size of spermatozoa hinder the study of the functional
Marcelo Marconi,
´ ´ ´
Raul Sanchez Gutierrez, properties of the spermatozoa plasma membrane. However, some studies have
´ ´
Ataulfo Martınez-Torres, and revealed the presence of a number of ion channels in this cell. We set out to
Fernando Romero Mejıas ´ measure the endogenous currents and to study the effect of the venom of the
Centro de neurociencia y Chilean black widow spider (Latrodectus mactans). By patch-clamping bovine
For personal use only.
´
Biologia de peptidos-BIOREN, spermatozoa our results indicate the presence of an outwardly rectifying
Departamento de Ciencias
current, sensitive to changes in K + concentration (30–140 mM) and to tetra-
´
Preclınicas, Facultad de
Medicina, Universidad de la
ethylammonium (TEA, 10–100 mM). The application of the venom (7.5 g/ml)
Frontera, Chile blocks these K+ currents and then alters the passive properties of the plasma
membrane. This leads to the entry of Ca ++ , reflected by a change in basal
´ ´
Ataulfo Martınez-Torres
fluorescent units (572 at 35710 FAU). The Ca ++ influx follows a reduction in
Laboratorio Neurobiologıa ´
Molecular y Celular II, the membrane conductance (control 2272; venom 1071 pS), as calcium
Departamento de Neurobiologıa ´ channels open in accord with voltage dependence.
Molecular y Celular, Instituto de
´
Neurobiologıa, Campus KEYWORDS ionic currents, Latrodectus venom, membrane potential, spermatozoa
´
Juriquilla-Queretaro, UNAM,
´
Mexico
INTRODUCTION
Abbreviations: TEA: tetraethyl-
ammonium; FAU: fluorescent arbitrary There is strong experimental evidence that mature mammalian sperma-
units; pS: membrane conductance; tozoa have several ionic-conductors, including those driven by voltage
CCVD: calcium channel voltage
dependent; PBS: phosphate saline dependent K + channels [Darszon et al. 1996; Labarca et al. 1995; Nuccitelli
buffer solution. and Ferguson 1994]. Recent reports using whole cell patch-clamping to
Received 13 August 2009; study ion-conductance, have described the functional properties of Ca ++
accepted 19 September 2009. channels (CATsper), which are key components of capacitation [Darszon
Address correspondence to et al. 2005; Wennemuth et al. 2000].
Jorge Parodi, Centro de Neuro-
´ ´
ciencias y Biologıa de Peptidos, The mammalian spermatozoa acquire the functional capacity to fertilize
´ ´
Nucleo cientıfico BIOREN, an egg during their trajectory along the female genital tract [Boni et al. 2007].
Universidad de la Frontera,
Montevideo 0870 Temuco, Chile. In this process the plasma membrane potential is hyperpolarized by the
E-mail: jparodi@ufro.cl activation of pH-sensitive K + -channels. This leads to an increase in Ca ++
37

2. permeability [Kumar et al. 2000; Linares-Hernandez with sensicam, for time lapse assessment with fast
et al. 1998; Shi and Ma 1998]. However, this increase perfusion of either venom or a high concentration of
of intracellular Ca ++ is not the sole cause of K + (30 mM). Notice the rise in fluorescence and the
capacitation, it has also been suggested that the immediate decrease of the emission after a few s as
intracellular pH and transient plasma membrane measured by its change in the midpiece. The
depolarization play a role [Fraire-Zamora and fluorometric data is summarized in Figure 1D
Gonzalez-Martinez 2004; Neri-Vidaurri Pdel et al. showing that both venom and K + increased the
2006] similar to the series of events that occur in free Ca ++ up to 35 fluorescent arbitrary units (FAU)
somatic cells [Baker et al. 1973]. The detailed as compared to the uninduced control. Together this
mechanism that leads to spermatozoa capacitation data suggests that spermatozoa calcium increases
remains to be detailed. after depolarization or perfusion with venom.
In a previous report we described that the venom
of the Chilean black widow spider Latrodectus
mactans increased the concentration of intra- Patch-Clamping Reveals a TEA-
cellular Ca ++ in spermatozoa [Romero et al. 2007]. Sensitive K+ Current
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Furthermore, this venom is known to block the TEA-
Spermatozoa exhibits varied permeability to ions
sensitive K + currents in neurons [Parodi and Romero
reflecting several channels, including the voltage-
2008] as well as endogenous K + currents of Xenopus
sensitive K + and ÀCa ++ [Darszon et al. 2006]. As
laevis oocytes [Parodi et al. 2008]. To assess the effect
illustrated in Figure 2A, whole-cell patch-clamping
of this venom on the plasma membrane potential
of spermatozoa showed inward and outward cur-
of bovine spermatozoa we used the nystatine-
rents. P pulses that ranged between À100 mV to
perforated patch-clamp technique. The results that
+ 100 mV were more sensitive to 50 mM TEA. These
begin to dissect the molecular components of
currents are plotted as a current/ voltage curve (I/V)
capacitation are reported below.
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(Fig. 2B). The TEA-sensitivity of the outward current
is consistent with the presence of a voltage depen-
dent potassium channel (Kv channel; IUPHAR
classification). These channels are crucial towards
RESULTS
maintaining the plasma membrane potential
Exposure to Venom Increases Free [Gutman et al. 2003]. Increasing the concentration
Intracellular Calcium of extracellular K + shifted the equilibrium potential
to more negative values (Fig. 2C). This is in accord
It has been shown by spectrophotometry that
with that predicted by the Nerst’s equation (Fig. 2D
exposing spermatozoa to the venom of the Chilean
and Table 1) indicating that the current was due to
L. mactans leads to an increase in pH and free
K + TEA-sensitive channels.
intracellular Ca ++ [Romero et al. 2007]. To extend
these results we assessed the changes in the
intracellular Ca ++ by fluorometry and patch-clamp.
Venom Alters the TEA-Sensitive
The spermatozoa loaded with Fluo-3 is shown in
Figure 1A. As expected, the basal level of free
K+ Current
intracellular Ca ++ is low, with most of the cells Outwardly-rectifying currents were efficiently and
emitting some faint fluorescence. In contrast, reversibly blocked when the spermatozoa were
30 min after the addition of the venom, the number exposed to the venom (Fig. 3A). The changes in
of cells exhibiting fluorescence increased consider- membrane conductance are summarized in Figure 3B.
ably (Fig. 1B). This was consistently observed Venom reduces the membrane conductance from
even after 3 repetitions suggesting that sudden 2271 pS to approximately 1072 pS (Fig. 3B). In
depolarization of the plasma membrane may induce contrast upon exposure to venom, the membrane
similar effects. resistance changed from 200 mO712 to 800 mO723,
Figure 1C shows typical fluorescence traces of the suggesting an important change in the passive mem-
spermatozoa midpiece loaded with Fluo-3 acquired brane properties.
38 J. Parodi et al.

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FIGURE 1 Venom-induced rise of intracellular Ca + + . Sample images of Fluo-3 AM loaded spermatozoa in the absence (A), or presence
(B) of total venom (7.5 mg/ml). (C), fluorometric records. (D), fluorescent arbitrary units (FAU) of spermatozoa in high K + (30 mM) or
venom (7.5 mg/ml); the bars represent means7 SD of three different experiments. Calibration bar ¼ 5 mm.
We explored whether venom also modulates expected the pre-pulse currents were approximately
spermatozoa K + currents by increasing the free 300% higher than the control. Interestingly, 10 mM
intracellular Ca ++ as known to occur in somatic cells TEA did not affect the current.
[Romero et al. 2007]. Briefly hyperpolarizing the
spermatozoa plasma membrane from À60 mV to
À80 mV activates a calcium-dependent K + current DISCUSSION
(Fig. 3C, insert). The tail-current derived from this A previous study has indicated that the venom
response in the presence of either venom, high K + , induced cellular effect is mediated by low molecular
or TEA is plotted in Figure 3C. In most cases at weight heat sensitive peptides present in the venom,
7.5 mg/ml venom with 30 mM potassium or 50 to [Parodi and Romero 2008]. As described above,
100 mM TEA the K + currents were blocked. As the venom of the Chilean L. mactans is effective
Effect of Venom on Mature Spermatozoa Currents 39

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FIGURE 2 A TEA-sensitive outwardly rectifying K + . (A), sample records of membrane currents obtained in the absence or the presence
of 5 mM TEA. (B), I/V relation from the sample currents (n ¼ 6). (C), I/V plot in increasing concentrations of K + .
TABLE 1 Calculated [I/pA ¼ f (Vm/mV)] and Experimental venom of L. mactans increases the free intracellular
Reversal Membrane Potentials. Ca ++ negatively impacting the TEA-sensitive K +
[K + ] Predicted I/V (mV) Experimental I/V (mV) spermatozoa current. All conductances are crucial to
spermatozoa capacitation.
30 mM À40 mV À43 7 7 mV
80 mM À14 mV À16 7 4 mV After several min of exposure of the spermatozoa to
140 mM 0 mV 57 2 mV the venom, free intracellular Ca ++ rises. As shown in
Figures 3A and B, the change in the membrane con-
ductance after exposure to venom suggests depolari-
even in the absence of a-latrotoxin, which is the zation and calcium channel voltage dependent
primary component described for this venom in (CCVD) activation in the spermatozoa cells. This
another species of spider [Ushkaryov et al. 2008]. may reflect the blockage of voltage-dependent K +
The results reported in this study suggest that the channels leading to the entrance of Ca ++ from the
40 J. Parodi et al.

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FIGURE 3 Venom blocks the TEA-sensitive current. (A), I/V relation in the absence or presence of 7.5 mg/ml venom. (B), the membrane
conductance is reduced in spermatozoa exposed to the venom. (C), comparative effect of venom, high K + , and increasing concentra-
tions of TEA (10 to 50 mM) on the prepulse current. The insert shows sample recordings of the prepulse in control cells and cells
exposed to venom. The bars represent means7SD of six different experiments.n
extracellular medium in accord with previous studies observed. Nevertheless the functional data reported
that suggested that the venom inhibits several K + here and elsewhere [Darszon et al. 2006; Marconi
conductances [Parodi and Romero 2008; Parodi et al. et al. 2008; Navarro et al. 2008] is consistent with
2008]. Like the oocyte Kv1.1 s [Parodi et al. 2008], this the view that Kv channels are integral to the
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channel is also present in the spermatozoa midpiece spermatozoa.
[Darszon et al. 1999]. Since Kv1.1 is integral to Other natural sources of spermicidal action have
membrane potential regulation [Gutman et al. 2003] been reported, including scorpion toxins and plant
we compared the effect with high K + added in the extracts [Harat et al. 2008; Lopez-Gonzalez et al.
medium. In both cases the concentration of free Ca ++ 2003]. Together with the previous studies of the
exhibited a transient rise and sharp fall as detected spermicidal properties of the venom of the Chilean
by spermatozoal midpiece fluorescence. However, L. mactans we were enabled, with a new set of
a rise in Ca ++ was observed throughout the cell pharmacological tools, to identify the active
after long periods of time. This is consistent with the compound(s) that mediate the modulation of ionic-
view that a CCVD was present [Darszon et al. 2005]. conductances. Perhaps they could provide a
A direct pharmacological study is required to specify resource for a new generation of contraceptives.
the Ca ++ and K + currents involved.
The molecular identity of the K + channel
modulated by the venom was indirectly assessed
by the activation kinetics and TEA-sensitivity of the MATERIALS AND METHODS
currents. At 5 mM TEA-blocks the outward-rectifying
K + current and likely represents a Kv1-like current
Spider Retrieval
[Marconi et al. 2008], although mouse brain large- Female adult L. mactans were captured in Chile
conductance (mSlo) has also been identified in during the summer months (December 2006 and
spermatozoa [Navarro et al. 2008]. The currents January 2007) from the area of ‘‘Alto Bio Bio’’ in the
blocked by the spider venom appear similar to those Bio-Bio state (721160 5100 W, 71450 2400 S) as previously
found in frog oocytes and neurons in culture [Parodi described [Romero et al. 2003]. Care was taken not to
and Romero 2008; Parodi et al. 2008] although damage the breeding zones. The specimens were
differences in the expression profiles of Kv channels separately maintained in individual jars. To stimulate
[Darszon et al. 2006] among species have been the production and concentration of venom in the
Effect of Venom on Mature Spermatozoa Currents 41

6. glands, the spiders were raised without food and and Marty 1988] at room temperature, using a patch-
only given water for 30 days. clamp amplifier (Warner PC-501A). For whole-cell
recordings, the external solution contained 150 mM
NaCl, 5.4 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2,
Venom Retrieval 10 mM glucose buffered with 10 mM HEPES, pH 7.4
The spiders were immersed in liquid nitrogen and buffer. The internal solution contained 120 mM KCl,
after 1 min transferred to a phosphate saline buffer 2.0 mM MgCl2, 2 mM ATP-Na2-ATP, 10 mM BAPTA,
solution (PBS: 0.1 M NaH2PO4, 0.01 M Na2HPO4, 0.5 mM GTP buffered with 10 mM HEPES, pH 7.4
1.35 M NaCl, pH 7.4) at 41C. The glands were buffer. For perforated patch-clamp 10 mg/ml nystatine
removed and the membrane that binds them to the was added [Ermishkin et al. 1976]. For some experi-
base was sectioned. Each gland, was placed into ments, the external solution included either 7.5 g/ml
a tube containing PBS (25 pairs of glands for 100 ml of venom or 5–100 mM TEA. Spermatozoa were
of PBS) and homogenized. The homogenate was clamped at a holding potential of À60 mV. The ionic
immediately centrifuged at 1,000 Â g for 15 min. The currents were captured online and digitized at a
supernatant was subsequently aliquoted, and frozen sampling rate of 1.2 kHz using pClamp 9 (Molecular
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at À 201C. The protein content of the venom was Devices, Axon) after being filtered (internal 4 Bessel;
determined by a modification of the Bradford frequency cutoff ¼ 3 kHz). Pulse protocols, data
method (BioRad Protein Assay). capture, and recording analysis were performed using
pClampfit 9.0 software (Molecular Devices, Axon).
Spermatozoa
Bovine spermatozoa were obtained from samples
Fluo-3 AM Loading
cryopreserved in liquid nitrogen, which were Bovine spermatozoa were loaded with 5 mM Fluo-3
thawed at 351C for 1 min then suspended in human AM in pluronic acid/DMSO, 10% w/v (Molecular
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tubal fluid (HTF) medium supplemented with 3% Probes, Eugene, OR, USA) and mounted in a fast-
BSA. Spermatozoa were selected by the swim-up switching flow perfusion chamber adapted on the
technique (recommendation of [World Health stage of an inverted fluorescent microscope (Eclipse
Organization 1999]) and motile spermatozoa were TE, Nikon) equipped with a xenon lamp and a 40 Â
placed in a recording chamber coated with poly-L- objective (22–241C). The cells were briefly illumi-
lysine (0.2 mg/mL). The recording chamber was then nated (200 ms) for fast record real time analysis, using
washed with a standard external solution that a computer-controlled Lambda 10-2 filter wheel
contained 150 mM NaCl, 5.4 mM KCl, 2.0 mM CaCl2, (Sutter Instruments). Regions of interest (ROI) were
1.0 mM MgCl2, 10 mM glucose buffered with 10 mM simultaneously selected on midpiece containing
HEPES, pH 7.4 buffer. Fluo-3 fluorescence (excitation 480 nm, emission
510 nm). Images were collected at 2–5 s intervals
during a continuous 5-min period or at 0 or 30 min.
Micropipette Preparation The imaging was carried out with a 12 bit cooled
Borosilicate glass capillary tubes 1.5 mm of internal SensiCam camera (PCO, Germany), using the imag-
diameter (Sutter Instrument Co., CA, USA) were ing software Axon Workbench 2.2 (Axon Instru-
placed in a vertical puller (Narishige PP 830 m ments). The spermatozoa were exposed to 7.5 g/ml
Barishige, Tokyo, Japon) and then treated with a of total venom () or 30 mM potassium () by bath
micropolisher (Narishige MF 900) to obtain a micro- perfusion, and changes in the emission of midpiece
pipette tip with electrical resistances ranging from fluorescence, measured in FAU and scored.
10 to 12 MO.
ACKNOWLEDGMENTS
Electrophysiology Fernando Romero, Raul Sanchez, and Jorge Parodi
Electrical currents were recorded by using the were supported by the Fondef-Conicyt Chile No
perforated whole-cell patch-clamp technique [Horn ´
DO5I10416. Ataulfo Martinez-Torres was supported
42 J. Parodi et al.

7. by CONACYT 55025 and UNAM-PAPIIT 204806. Jorge Labarca, P., Zapata, O., Beltran, C. and Darszon, A. (1995) Ion channels
from the mouse sperm plasma membrane in planar lipid bilayers.
Parodi has postdoctoral fellow from CTIC-UNAM Zygote 3:199–206.
and from MIDEPLAN-Chile. Patricia Navarrete has Linares-Hernandez, L., Guzman-Grenfell, A. M., Hicks-Gomez, J. J. and
CONICYT grant. We are in debt with Dr. Luis Aguayo Gonzalez-Martinez, M. T. (1998) Voltage-dependent calcium influx in
human sperm assessed by simultaneous optical detection of intracellular
for help with measurement equipment for calcium calcium and membrane potential. Biochim Biophys Acta 1372:1–12.
experiments. Lopez-Gonzalez, I., Olamendi-Portugal, T., De la Vega-Beltran, J. L.,
Van der Walt, J., Dyason, K., Possani, L. D., Felix, R. and Darszon, A.
(2003) Scorpion toxins that block T-type Ca2+ channels in
spermatogenic cells inhibit the sperm acrosome reaction. Biochem
REFERENCES Biophys Res Commun 300:408–414.
Marconi, M., Sanchez, R., Ulrich, H. and Romero, F. (2008) Potassium
Baker, P. F., Meves, H. and Ridgway, E. B. (1973) Calcium entry in current in mature bovine spermatozoa. Syst Biol Reprod Med
response to maintained depolarization of squid axons. J Physiol 54:231–239.
231:527–548. Navarro, B., Kirichok, Y., Chung, J. J. and Clapham, D. E. (2008)
Boni, R., Gualtieri, R., Talevi, R. and Tosti, E. (2007) Calcium and other ion Ion channels that control fertility in mammalian spermatozoa. Int J
dynamics during gamete maturation and fertilization. Theriogenology Dev Biol 52:607–613.
68 Suppl 1:S156–164. Neri-Vidaurri Pdel, C., Torres-Flores, V. and Gonzalez-Martinez, M. T.
Darszon, A., Acevedo, J. J., Galindo, B. E., Hernandez-Gonzalez, E. O., (2006) A remarkable increase in the pHi sensitivity of voltage-
Syst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona
Nishigaki, T., Trevino, C. L., Wood, C. and Beltran, C. (2006) Sperm dependent calcium channels occurs in human sperm incubated
channel diversity and functional multiplicity. Reproduction 131:977–988. in capacitating conditions. Biochem Biophys Res Commun
Darszon, A., Labarca, P., Nishigaki, T. and Espinosa, F. (1999) Ion channels 343:105–109.
in sperm physiology. Physiol Rev 79:481–510. Nuccitelli, R. and Ferguson, J. E. (1994) Fertilization and ion channels.
Darszon, A., Lievano, A. and Beltran, C. (1996) Ion channels: key Science 263:988.
elements in gamete signaling. Curr Top Dev Biol 34:117–167. Parodi, J. and Romero, F. (2008) Synaptic effects of low molecular
Darszon, A., Nishigaki, T., Wood, C., Trevino, C. L., Felix, R. and Beltran, C. weight components from Chilean Black Widow spider venom.
(2005) Calcium channels and Ca2+ fluctuations in sperm physiology. Neurotoxicology 29:1121–1126.
Int Rev Cytol 243:79–172. Parodi, J., Romero, F., Miledi, R. and Martinez-Torres, A. (2008) Some
Ermishkin, L. N., Kasumov, K. M. and Potzeluyev, V. M. (1976) Single effects of the venom of the Chilean spider Latrodectus mactans on
ionic channels induced in lipid bilayers by polyene antibiotics endogenous ion-currents of Xenopus laevis oocytes. Biochem Biophys
amphotericin B and nystatine. Nature 262:698–699. Res Commun 375:571–575.
Fraire-Zamora, J. J. and Gonzalez-Martinez, M. T. (2004) Effect of Romero, F., Altieri, E., Urrutia, M. and Jara, J. (2003) Venom of
For personal use only.
intracellular pH on depolarization-evoked calcium influx in human Latrodectus mactans from Chile (Araneae, Theridiidae): effect on
sperm. Am J Physiol Cell Physiol 287:C1688–1696. smooth muscle. Rev Biol Trop 51:305–312.
Gutman, G. A., Chandy, K. G., Adelman, J. P., Aiyar, J., Bayliss, D. A., Romero, F., Cunha, M. A., Sanchez, R., Ferreira, A. T., Schor, N. and
Clapham, D. E., et al. (2003) International Union of Pharmacology. XLI. Oshiro, M. E. (2007) Effects of arachnotoxin on intracellular pH and
Compendium of voltage-gated ion channels: potassium channels. calcium in human spermatozoa. Fertil Steril 87:1345–1349.
Pharmacol Rev 55:583–586. Shi, Y. L. and Ma, X. H. (1998) Ion-channels reconstituted into lipid
Harat, Z. N., Sadeghi, M. R., Sadeghipour, H. R., Kamalinejad, M. and bilayer from human sperm plasma membrane. Mol Reprod Dev
Eshraghian, M. R. (2008) Immobilization effect of Ruta graveolens L. 50:354–360.
on human sperm: a new hope for male contraception. Ushkaryov, Y. A., Rohou, A. and Sugita, S. (2008) alpha-Latrotoxin and its
J Ethnopharmacol 115:36–41. receptors. Handb Exp Pharmacol 184:171–206.
Horn, R. and Marty, A. (1988) Muscarinic activation of ionic currents Wennemuth, G., Westenbroek, R. E., Xu, T., Hille, B. and Babcock, D. F.
measured by a new whole-cell recording method. J Gen Physiol (2000) CaV2.2 and CaV2.3 (N- and R-type) Ca2+ channels in
92:145–159. depolarization-evoked entry of Ca2+ into mouse sperm. J Biol Chem
Kumar, S., Ying, Y. K., Hong, P. and Maddaiah, V. T. (2000) Potassium 275:21210–21217.
increases intracellular calcium simulating progesterone action in World Health Organization (1999) Commentry: legal and ethical aspects
human sperm. Arch Androl 44:93–101. of sperm retrieval. J Law Med Ethics 27:359–361, 295.
Effect of Venom on Mature Spermatozoa Currents 43