Introduction

Calcium influx through voltage-sensitive channels controls a variety of cellular processes, including secretion and muscular contraction. Current flow through these channels is regulated primarily by membrane potential, but is also subject to facilitatory and inhibitory modulation that determine the resulting intracellular Ca²⁺ signal. Modulation has been attributed to alterations in the phosphorylation state of channel subunit proteins (Sculptoreanu et al., 1993b), to the direct interaction of G protein subunits with ion channel constituents (Bean, 1989; Hille, 1994) and to voltage-dependent processes (Artalejo et al., 1991). The mechanisms of modulation have been examined primarily in high voltage-activated Ca²⁺ channels. For example, L-type currents are positively modulated by protein kinases A and C, as well as by G_s, and are suppressed by G_o (reviewed by Hille, 1994). Similar mechanisms can regulate other high voltage-activated Ca²⁺ channels (Mintz and Bean, 1993; Hille, 1994).

The understanding of protein kinase-mediated channel regulation has progressed by the assignment of specific phosphorylation sites on recombinant channel subunits with selective aspects of modulation. In contrast, relatively little is known about the mechanism of voltage-dependent modulation. The potentiation, or facilitation, of Ca²⁺ currents by frequent or strong depolarizations was first described in chromaffin cells (Fenwick et al., 1982) and subsequently found in cardiac myocytes, neurons and a number of other cellular systems (reviewed by Dolphin, 1996). Ca²⁺ channel facilitation increases catecholamine secretion during stress (Artalejo et al., 1994), increases muscle contractile force (Sculptoreanu et al., 1993b) and generally is believed to either maintain or enhance Ca²⁺ signals. Several mechanisms have been proposed to explain voltage-dependent facilitation, including a voltage-dependent alteration of channel protein conformation, with the resultant exposure of new phosphorylation sites (Artalejo et al., 1992; Sculptoreanu et al., 1993a,b; Bourinet et al., 1994), the relief of a G protein-mediated inhibition of channel function (Grassi and Lux, 1989; Roche et al., 1995) or an alteration in the voltage dependence of gating (Feldmeyer et al., 1992). These studies focused on the high voltage-activated L- and N-type Ca²⁺ currents, whereas relatively little is known about the voltage-dependent facilitation of T-type channels (Alvarez et al., 1996; Ganitkevich and Isenberg, 1996).

A functional low voltage-activated, T-type Ca²⁺ channel is expressed during the meiotic and post-meiotic stages of mammalian spermatogenesis (Hagiwara and Kawa, 1984; Arnoult et al., 1996; Lievano et al., 1996). Recently, we have shown that this channel is retained in mature mouse sperm and is activated during fertilization by sperm contact with the egg's extracellular matrix, or zona pellucida (Arnoult et al., 1996). Ca²⁺ influx through the sperm T channel is required for the acrosome reaction, an exocytotic event that must be completed prior to sperm–egg fusion.

T channel regulation can be observed readily in mouse spermatogenic cells, which lack detectable levels of other voltage-sensitive Ca²⁺ currents (Hagiwara and Kawa, 1984; Arnoult et al., 1996; Lievano et al., 1996). Here, we report the voltage-dependent facilitation of the spermatogenic cell T current. Pharmacological and biophysical analyses suggest that a protein tyrosine kinase activity is required for inhibitory modulation of these channels, whereas a tyrosine phosphatase activity reverses this effect during strong or high frequency depolarizations.

Results

Mouse spermatogenic cell Ca²⁺ currents were recorded from dissociated cells using the whole-cell configuration of the patch–clamp. The biophysical and pharmacological characteristics of this current indicate that it is mediated by T-type Ca²⁺ channels (Arnoult et al., 1996). This T current is present during meiotic and post-meiotic stages of spermatogenesis, as are the regulatory processes described in these experiments.

Voltage-dependent facilitation of the T-type Ca²⁺ current

Maximal inward T current ('basal' current) is obtained when cells are depolarized from a holding potential of -90 mV to a test potential of -20 mV. Current amplitude is not affected by successive depolarizing steps to this potential at a frequency of <0.1 Hz (Figure 1A), but is enhanced by increasing the stimulation rate above 0.2 Hz (data not shown) or by providing a depolarizing prepulse to more positive membrane potential before the test depolarization (Figure 1B). The additional current recruited by such prepulses is referred to as 'facilitated' current. Voltage-dependent facilitation does not alter significantly the activation and inactivation kinetics of the current. This is demonstrated in Figure 1C, where basal and facilitated current traces superimpose after normalization to the same peak amplitude. The activation and inactivation time constants, as determined on 20 cells, are 3.6 1.2 and 16.8 3.6 ms respectively, for basal current, while comparable values for the facilitated current are 3.4 1.2 and 14.8 2.7 ms, respectively (mean SD). Similar results were obtained when Ba²⁺ replaced Ca²⁺ as a charge carrier (data not shown). These kinetic data suggest that faciltation reflects the regulation of a single population, rather than the activation of a different class of channels.

The voltage and time dependence of the facilitating prepulse were determined (Figure 2A). When cells are depolarized from a holding potential of -90 mV, the prepulse necessary for facilitation has the following characteristics: (i) a voltage threshold of -30 mV and a maximal response following depolarizations to +60 mV (Figure 2B); (ii) a temporal threshold of 5 ms producing a maximal effect following prepulses of 100 ms duration (Figure 2C); and (iii) a transitory effect, as a prepulse–test pulse interval of 0.5–1 s produced a maximal response and facilitation decreased as this interval was lengthened (loss of facilitation, t_1/2 4.8 s; Figure 2D). The degree of facilitation also decreased as the prepulse–test pulse interval was shortened, most likely due to incomplete recovery of T channels from inactivation during the prepulse. Optimal facilitation occurs when cells that are held at -90 mV are subjected to a 100 ms prepulse to +60 mV, returned to holding potential for 500 ms, and then subjected to test depolarizations.

The extent of facilitation was also reciprocally dependent upon the basal current density. Spermatogenic cells were subjected to the optimal facilitation protocol described above. Cells with a basal current density of 2–5.5 mA/cm² exhibit a large facilitation (166 43% of basal current, n = 27). In contrast, cells with a larger basal current density of 6–10 mA/cm² exhibit a much lower increase (108 9% of initial current, n = 12). The mean degree of facilitation was 148 45% increase (n = 39) and regression analysis of these pooled data indicate a maximal current of 9.2 mA/cm².

Role of protein phosphorylation in the voltage-dependent facilitation of T-type calcium current

As shown in Figure 2D, the potentiation of spermatogenic cell T currents by a facilitating prepulse decays slowly. Sustained facilitation was also reported for the L-type channels of chromaffin cells (Artalejo et al., 1992) and skeletal muscle (Sculptoreanu et al., 1993a,b), where the operation of a voltage-dependent protein kinase has been proposed. We therefore determined the role of the protein phosphorylation state in T channel facilitation using modulators of protein kinase and phosphatase activities.

Figure 3 illustrates the effects of tyrphostins A47 and A25, specific membrane-permeant inhibitors of protein tyrosine kinases, on the spermatogenic cell T current. The values of the basal and facilitated currents obtained after a depolarizing prepulse were relatively stable during sequential recording at a frequency of 0.1 Hz (Figure 3B–D). However, addition of A47 or A25 enhanced the initial current amplitude to a level equal to that of the prepulse-facilitated current in the absence of tyrphostin. This enhancement of basal currents occurs in the absence of any associated alterations in the activation and inactivation kinetics, thus producing a larger net conductance. This effect was produced by 2–50 M tyrphostin A47, although higher concentrations acted more rapidly (t_1/2 at 2 and 10 M of 2.5 min and <0.5 min, respectively). These effects are readily reversible when tyrphostin A47 or A25 are removed by perfusion (Figure 3D). In contrast, no effects on T currents were observed when spermatogenic cells were treated with tyrphostin A1 (Figure 3D), which is not a potent inhibitor of protein tyrosine kinase activity, or with AG1478 (data not shown), which is a highly selective antagonist of the epidermal growth factor receptor tyrosine kinase (Levitzki and Gazit, 1995).

Several additional features of the tyrphostin-dependent enhancement of Ca²⁺ current are notable. (i) The increase in current produced by tyrphostin A47 and A25 is equal to, but never exceeds that produced by a strong depolarizing prepulse. (ii) The amplitude of the prepulse-facilitated current obtained after a depolarizing prepulse is not increased by tyrphostins A47 or A25 (Figure 3). (iii) Tyrphostins A47 and A25 had no stimulatory effect on those cells with large basal currents, in which depolarizing prepulses also failed to produce any enhancement.

These observations suggest a role for protein tyrosine kinase activity in the control of T channel activity. In order to explore this regulatory role further, we examined the effects of tyrosine phosphatase modulators on Ca²⁺ currents. As shown in Figure 4, current amplitude was relatively stable during repetitive depolarization at low frequency (<0.1 Hz) and voltage-dependent facilitation was observed following a depolarizing prepulse (point '1').

When 10 M phenylarsine oxide, a specific and membrane-permeant protein tyrosine phosphatase inhibitor, was added to the external solution, there was a marked decline in the amplitude of both the basal current and the facilitated current produced by a depolarizing prepulse (Figure 4). However, the loss of facilitated current is always more rapid, and within 100–200 s there is no longer additional Ca²⁺ current as a result of a depolarizing prepulse (Figure 4, point '2').

Similar results were obtained when 1 mM Na⁺-orthovanadate is added to the pipet solution. This compound is a membrane-impermeant inhibitor of a wide range of phosphatases, including protein tyrosine phosphatases. The additional current produced by a facilitating prepulse is reduced by 50% during 600 s of recording in the presence of Na⁺-orthovanadate, whereas only a 15% reduction was observed in parallel experiments in the absence of this antagonist (n = 5). The requirement for intracellular diffusion of Na⁺-orthovanadate from the pipet to its site of action most likely contributes to its relatively slow time course and low potency. These data support the hypothesis that the spermatogenic cell T current is regulated by its tyrosine phosphorylation state.

In contrast, modulators of serine/threonine phosphorylation, such as H-7 and H-9, do not increase the basal Ca²⁺ currents (200 mM; n = 9 for each). Furthermore, H-7 does not affect the relative amplitude of the facilitated current obtained following depolarizing prepulses (data not shown). Okadaic acid (1 M), an inhibitor of the protein phosphatase 2A, has no effect on the amplitude of the basal or the facilitated currents (data not shown). Finally, neither the basal nor the facilitated Ca²⁺ currents of spermatogenic cell are affected by the addition of 500 M guanine nucleotides (GDP, GTP) or of their thiophosphate derivatives (GDPS, GTPS) to the pipet solution (data not shown).

Discussion

We have demonstrated that the T-type Ca²⁺ current is enhanced when mouse spermatogenic cells are subjected to either a depolarizing prepulse or to inhibition of protein tyrosine phosphorylation. Three types of evidence indicate that these two treatment protocols produce the same facilitated state. (i) The extent of the effects of these treatments are similar. For example, the basal current is enhanced selectively by tyrphostin A25 and A47 to the level of the facilitated current, but these inhibitors of protein tyrosine kinase activity never produce currents that exceed the facilitated level. Similarly, protein tyrosine phosphatase inhibitors selectively suppress the facilitated current to the level of the standard current, but never produce currents that are smaller than the affected basal current. (ii) The effects of these protocols are not additive. For example, tyrphostin treatment precludes further enhancement of current by a depolarizing prepulse. (iii) The effects of a depolarizing prepulse and of an altered protein tyrosine phosphorylation state on the biophysical characteristics of the T current are similar: both treatments alter the current amplitude without affecting either the activation or the inactivation kinetics.

The indistinguishable biophysical characteristics of the basal and facilitated currents suggest that facilitation is due to an increase in the T current rather than the recruitment of a class of different cryptic channels, such as may occur in chromaffin cells (Artalejo et al., 1992). The enhancement of spermatogenic cell T currents can be due either to an increase in the number of functional channels or to alteration in the conductance characteristics of individual T channels. Taken together with the conclusion that a single facilitated state is produced by either experimental treatment, these observations suggest that spermatogenic cell T-type Ca²⁺ channels are present in two functional states: a low conductance, or even silent, basal state and a higher conductance facilitated state. Transitions between these functional states are promoted either by voltage stimulation or by modulation of protein tyrosine phosphorylation. Single channel recordings will be required to assess this model.

Voltage-dependent facilitation has been reported previously for Ca²⁺ channels, as well as a number of other voltage-sensitive ion channels (Fenwick et al., 1982). This stimulation has been attributed to several mechanisms, including phosphorylation of channel subunits through either Ca²⁺- or voltage-dependent protein kinases as well as the voltage-dependent relief of a G protein-mediated inhibition (Hille, 1994; Dolphin, 1996). However, two observations indicate that a Ca²⁺-regulated process is unlikely to mediate the voltage-dependent facilitation of the spermatogenic cell T channel: (i) the effects of a depolarizing prepulse are observed when Ba²⁺ replaces Ca²⁺ as a charge carrier; and (ii) internal Ca²⁺ is buffered in all recordings by EGTA or by BAPTA.

It is also unlikely that the voltage-dependent facilitation of the spermatogenic T-type current is due to the reversal of G protein-dependent inhibition. In a number of other cellular systems, external signals suppress Ca²⁺ currents through a G protein-mediated mechanism, and voltage-dependent facilitation is due to the reversal of this inhibition (Doupnik and Pun, 1994; Roche et al., 1995; Dolphin, 1996). The voltage-dependent reversal of G protein-mediated inhibition has been studied extensively in the high voltage-activated class of Ca²⁺ channels.

However, several observations are inconsistent with the presence of a similar G protein-mediated pathway in the facilitation of spermatogenic cell T currents. First, facilitation by either a depolarizing prepulse or by the application of tyrosine kinase inhibitors is not affected by the intracellular addition of G protein inhibitors (GDPS or absence of GTP) or activators (GTPS or GTP). For example, while GDPS inhibits either the voltage-dependent facilitation or the receptor-mediated inhibition of Ca²⁺ currents in several other preparations (Grassi and Lux, 1989; Buisson et al., 1992, 1995; Doupnik and Pun, 1994; Roche et al., 1995; Alvarez et al., 1996), we were able to record stable facilitation for long periods (10–15 min) in the presence of this G protein blocker. Second, the duration of the voltage-dependent facilitation of the spermatogenic cell T current is not consistent with the presence of a G protein-mediated pathway. We observed that a depolarizing prepulse produces a relatively long-lived facilitation that decays with a time constant of 4.8 s. In contrast, facilitation is transitory in those examples in which it is due to removal of G protein-mediated inhibition and decays with a time constant of 30 ms (Tsunoo et al., 1986; Golard et al., 1993). Finally, a characteristic feature of voltage-dependent relief of G protein inhibition is an increased rate of current inactivation (Hille, 1994; Roche et al., 1995; Alvarez et al., 1996), whereas the facilitated current of spermatogenic cells does not exhibit any change in kinetics (Figure 1C).

The most plausible mechanism for the control of this T current is by the regulation of tyrosine phosphorylation. The pharmacological experiments presented here suggest a model in which dephosphorylation of key but unidentified tyrosine residues accounts for the transition of the channel to the facilitated state, whereas the phosphorylation of these residues produces the lower conductance basal state. It is now well accepted that tyrosine phosphorylation alters the function of the nicotinic acetylcholine receptor (reviewed by Huganir, 1991) and of a number of voltage-gated ion channels (reviewed by Siegelbaum, 1994; Jonas and Kaczmarek, 1996). In addition, it has been suggested that tyrosine phosphorylation also modulates Ca²⁺ channel function, including a possible role in the angiotensin II-dependent inhibition of the T-type current of NG108-15 cells (Buisson et al., 1992, 1995). The consensus of these previous studies is that Ca²⁺ currents are increased by tyrosine phosphorylation. In contrast, the T channel of mouse spermatogenic cells represents, to the best of our knowledge, the first example of a Ca²⁺ current that is potentiated by a voltage-dependent dephosphorylation process.

The inhibitory and facilitatory modulation of T currents by protein tyrosine kinases and tyrosine phosphatases, respectively, may control sperm function during mammalian fertilization. We have shown recently that T channels are retained on the mature sperm cell following the completion of spermatogenesis (Arnoult et al., 1996). Prior to penetrating the egg's zona pellucida and fusing with the egg plasma membrane, sperm must complete the acrosome reaction, a Ca²⁺-dependent exocytotic event. Zona pellucida contact activates sperm T channels, and the resultant Ca²⁺ entry is essential for the acrosome reaction (Arnoult et al., 1996). Sperm have only a single secretory vesicle, and available evidence indicates that cells that complete the acrosome reaction prior to contact with eggs are infertile (Ward and Kopf, 1993; Yanagimachi, 1994). Thus, a key feature of sperm physiology is the suppression of spontaneous exocytosis until the activation of specific signal transducing pathways by the zona pellucida glycoprotein, ZP3 (Ward and Storey, 1984; Florman and First, 1988; Florman and Babcock, 1990).

A second characteristic of mammalian sperm is that fertility is only expressed during the final phase of functional maturation, or capacitation, that occurs within the female reproductive tract prior to sperm–egg contact (Yanagimachi, 1994). Recently, it was shown that one component of capacitation is the enhanced tyrosine phosphorylation of an array of sperm proteins (Visconti et al., 1995a,b). Tyrosine phosphorylation of the sperm T channel or associated proteins during capacitation would minimize Ca²⁺ influx through this pathway, thereby suppressing spontaneous exocytosis as sperm prepare for fertilization. A corollary of this model, which has not yet been demonstrated, is that a sperm tyrosine phosphatase is activated by ZP3 during sperm–zona pellucida contact, thus relieving T channel inhibition and permiting egg-induced acrosome reactions.

Sperm receive a variety of chemical signals in addition to that provided by ZP3 as they approach and contact eggs. Progesterone (Foresta et al., 1993) and extracellular ATP (Foresta et al., 1992, 1996) depolarize sperm membrane potential in vitro and may be present at the site of fertilization in vivo. It is plausible that these and other compounds act either through voltage- or receptor-dependent mechanisms to switch the sperm T channel from the low conductance state to a facilitated state, thereby enhancing the Ca²⁺ current triggered by ZP3.

In sum, the present study demonstrates that the T channel of the mouse male germ cell is modulated by its tyrosine phosphorylation state. In particular, the potentiation of this current by tyrosine dephosphorylation provides a new model for Ca²⁺ channel regulation. This type of modulation may regulate the development of fertility in mature sperm.

Materials and methods

Biological preparations

Seminiferous tubules were isolated from the testis of CD1 mice (16 weeks old; Charles River Laboratories) and incubated at 37°C for 30 min in 3 ml of a solution containing (mM): NaCl (150), KCl (5), CaCl₂ (2), MgCl₂ (1), NaH₂PO₄ (1), NaHCO₃ (12), glucose (11), pH 7.3 and collagenase type IA (1 mg/ml, Sigma). Tubules were rinsed twice in collagenase-free medium and cut into 2 mm sections. Spermatogenic cells were obtained by manual trituration and were attached to culture dishes coated with Cell-Tak (Collaborative Biomedical Products, Bedford, MA). The pachytene spermatocyte and the round spermatid are the prominent cell types obtained from the diploid meiotic and the haploid post-meiotic stages of spermatogenesis, respectively. These cells are readily distinguished based on cellular and nuclear size and morphology, as described previously (Romrell et al., 1976; Arnoult et al., 1996). These stages were used routinely for electrophysiological recordings.

Electrophysiological recordings

Ca²⁺ currents were recorded in the whole-cell configuration of the patch–clamp technique (Hamill et al., 1981) and analyzed using Biopatch (BioLogic, FR). Pipets were pulled from 7052 thin glass (Gardner Glass Co., CA), coated with Sylgard 184 (Dow Corning, MI), and fire-polished. Pipet resistance was 2–10 M. Currents were obtained with an Axopatch 1-D amplifier (Axon Inst., Burlingame, CA). All traces were corrected for leakage and capacitance currents, filtered at 3 kHz, and digitized every 250 msec. Other details of the voltage protocols used here are provided in the Results section.

The pipet solution, which was designed to eliminate K⁺ currents, contains (in mM): Cs-glutamate (120), TEA-Cl (20), MgCl₂ (5), D-glucose (5), MgATP (3), EGTA (10), HEPES (10), pH 7.0 (adjusted with CsOH). Spermatogenic cells were attached to culture dishes during 5 min incubations, following which the bath solution was changed to a standard recording solution, containing (mM): NaCl (100), KCl (5), CaCl₂ (10), MgCl₂ (1), TEA-Cl (26), Na-lactate (6), HEPES (10), D-glucose (3.3), pH 7.4 (adjusted with 1 M NaOH). The cells were isolated in a 1 ml chamber and perfused at a rate of 4–8 ml/min. Tyrphostin A1, A25, A47, AG1478 (Calbiochem, CA) and phenylarsine oxide (Sigma) were prepared as dimethylsulfoxide (DMSO) stocks. The DMSO concentration during recording was <0.01% (v/v). H-7 and H-9 (RBI, Natick, MA) were prepared in the standard recording solution. All experiments were preformed at room temperature (25°C).

References

Alvarez JL, Rubio LS and Vassort G (1996) Facilitation of T-type calcium current in bullfrog atrial cells: voltage-dependent relief of a G protein inhibitory tone. J Physiol, 491, 321–334. | PubMed | ChemPort |

Arnoult C, Cardullo RA, Lemos JR and Florman HM (1996) Egg-activation of sperm T-type Ca²⁺ channels regulates acrosome reactions during mammalian fertilization. Proc Natl Acad Sci USA, 93, 13004–13009. | Article | PubMed | ChemPort |

Artalejo CR, Dahmer MK, Perlman RL and Fox AP (1991) Two types of Ca²⁺ currents are found in bovine chromaffin cells: facilitation is due to the recruitment of one type. J Physiol, 432, 681–707. | PubMed | ChemPort |

Artalejo CR, Rossie S, Perlman RL and Fox AP (1992) Voltage-dependent phosphorylation may recruit Ca²⁺ current facilitation in chromaffin cells. Nature, 358, 63–66. | Article | PubMed | ChemPort |

Artalejo CR, Adams ME and Fox AP (1994) Three types of Ca²⁺ channel trigger secretion with different efficacies in chromaffin cells. Nature, 367, 72–76. | Article | PubMed | ISI | ChemPort |

Bean BP (1989) Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence. Nature, 340, 153–156. | Article | PubMed | ISI | ChemPort |

Bourinet E, Charnet P, Tomlinson WJ, Stea A, Snutch TP and Nargeot J (1994) Voltage-dependent facilitation of a neuronal _1C L-type calcium channel. EMBO J, 13, 5032–5039. | PubMed | ChemPort |

Buisson B, Bottari SP, de Gasparo M, Gallo-Payet N and Payet MD (1992) The angiotensin AT2 receptor modulates T-type calcium channel current in non-differentiated NG108-15 cells. FEBS Lett, 309, 161–164. | Article | PubMed | ChemPort |

Buisson B, Laflamme L, Bottari SP, de Gasparo M, Gallo-Payet N and Payet MD (1995) A G protein is involved in the angiotensin AT₂ receptor inhibition of the T-type calcium current in non-differentiated NG108-15 cells. J Biol Chem, 270, 1670–1674. | Article | PubMed | ChemPort |

Dolphin AC (1996) Facilitation of Ca²⁺ current in excitable cells. Trends Neurosci, 19, 35–43. | Article | PubMed | ISI | ChemPort |

Doupnik CA and Pun RY (1994) G-protein activation mediates prepulse facilitation of Ca²⁺ channel currents in bovine chromaffin cells. J Membr Biol, 140, 47–56. | PubMed | ChemPort |

Feldmeyer D, Melzer W, Pohl B and Zollner P (1992) Modulation of calcium current gating in frog skeletal muscle by conditioning depolarization. J Physiol, 457, 639–653. | PubMed | ChemPort |

Fenwick EM, Marty A and Neher E (1982) A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine. J Physiol, 331, 577–597. | PubMed | ChemPort |

Florman HM and Babcock DF (1990) Progress towards understanding the molecular basis capacitation. In Wassarman,P.M. (ed.), Elements of Mammalian Fertilization. Vol I: Basic Concepts. CRC Press, Boca Raton, FL, pp. 105–132.

Florman HM and First NL (1988) The regulation of acrosomal exocytosis. I. Sperm capacitation is required for the induction of acrosome reactions by the bovine zona pellucida in vitro. Dev Biol, 128, 453–463. | PubMed | ChemPort |

Foresta C, Rossato M and Di Virgilio F (1992) Extracellular ATP is a trigger for the acrosome reaction in human spermatozoa. J Biol Chem, 267, 19443–19447. | PubMed | ChemPort |

Foresta C, Rossato M and Di Virgilio F (1993) Ion fluxes through the progesterone-activated channel of the sperm plasma membrane. Biochem J, 294, 279–283. | PubMed | ChemPort |

Foresta C, Rossato M, Chiozzi P and Di Virgilio F (1996) Mechanism of human sperm activation by extracellular ATP. Amer J Physiol, 270, C1709–C1714. | PubMed | ChemPort |

Ganitkevich VY and Isenberg G (1996) Stimulation-induced potentiation of T-type Ca²⁺ currents in myocytes from guinea-pig coronary artery. J Physiol, 443, 703–725.

Golard A, Role LW and Siegelbaum SA (1993) Protein kinase C blocks somatostatin-induced modulation of calcium current in chick sympathetic neurons. J Neurophysiol, 70, 1639–1643. | PubMed | ChemPort |

Grassi F and Lux HD (1989) Voltage-dependent GABA-induced modulation of calcium currents in chick sensory neurons. Neurosci Lett, 105, 113–119. | Article | PubMed | ChemPort |

Hagiwara S and Kawa K (1984) Calcium and potassium currents in spermatogenic cells dissociated from rat seminiferous tubules. J Physiol, 356, 135–149. | PubMed | ChemPort |

Hamill OP, Marty A, Neher E, Sakmann B and Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recordings from cell and cell-free membrane patches. Pflugers Arch, 391, 85–100. | PubMed | ISI | ChemPort |

Hille B (1994) Modulation of ion-channel function by G-protein-coupled receptors. Trends Pharmacol Sci, 17, 531–536. | Article | ChemPort |

Huganir RL (1991) Regulation of the nicotinic acetylcholine receptor by serine and tyrosine protein kinases. Adv Exp Med Biol, 287, 279–294. | PubMed | ChemPort |

Jonas EA and Kaczmarek LK (1996) Regulation of potassium channels by protein kinases. Curr Opin Neurobiol, 6, 318–323. | Article | PubMed | ISI | ChemPort |

Levitzki A and Gazit A (1995) Tyrosine kinase inhibition: an approach to drug development. Science, 267, 1782–1788. | PubMed | ISI | ChemPort |

Lievano A, Santi CM, Serrano J, Trevino CL, Bellve AR, Hernandez-Cruz A and Darszon A (1996) T-type Ca²⁺ channels and _1E expression in spermatogenic cells and their possible relevance to the sperm acrosome reaction. FEBS Lett, 388, 150–154. | Article | PubMed | ChemPort |

Mintz IM and Bean BP (1993) GABA_B receptor inhibition of P-type Ca²⁺ channels in central neurons. Neuron, 10, 889–898. | PubMed | ISI | ChemPort |

Roche JP, Anantharam V and Treistman SN (1995) Abolition of G protein inhibition of 1A and 1B calcium channels by coexpression of the 3 subunit. FEBS Lett, 371, 43–46. | Article | PubMed | ChemPort |

Romrell LJ, Bellve AR and Fawcett DW (1976) Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization. Dev Biol, 49, 119–131. | PubMed | ISI | ChemPort |

Sculptoreanu A, Rotman E, Takahashi M, Scheuer T and Catterall WA (1993a) Voltage-dependent potentiation of the activity of cardiac L-type calcium channel a₁ subunits due to phosphorylation by cAMP-dependent protein kinase. Proc Natl Acad Sci USA, 90, 10135–10139. | PubMed | ChemPort |

Sculptoreanu A, Scheuer T and Catterall WA (1993b) Voltage-dependent potentiation of L-type Ca²⁺ channels due to phosphorylation by cAMP-dependent protein kinases. Nature, 364, 240–243. | Article | PubMed | ISI | ChemPort |

Siegelbaum S (1994) Ion channel control by tyrosine phosphorylation. Curr Biol, 4, 242–245. | PubMed | ISI | ChemPort |

Tsunoo A, Yoshii M and Narahashi T (1986) Block of calcium channels by enkephalin and somatostatin in neuroblastoma–glioma hybrid NG108-15 cells. Proc Natl Acad Sci USA, 83, 9832–9836. | PubMed | ChemPort |

Visconti PE, Bailey JL, Moore GD, Pan D, Olds-Clarke P and Kopf GS (1995a) Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development, 121, 1129–1137. | ChemPort |

Visconti PE, Moore GD, Bailey JL, Leclerc P, Connors SA, Pan D, Olds-Clarke P and Kopf GS (1995b) Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development, 121, 1139–1150. | ChemPort |

Ward CR and Kopf GS (1993) Molecular events mediating gamete activation. Dev Biol, 158, 9–34. | Article | PubMed | ISI | ChemPort |

Ward CR and Storey BT (1984) Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev Biol, 104, 287–296. | PubMed | ISI | ChemPort |

Yanagimachi R (1994) Mammalian fertilization. In Knobil,E. and Neill,J.D. (eds), The Physiology of Reproduction. 2nd edn. Raven Press, New York, pp. 189–317.