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J. Biol. Chem., Vol. 275, Issue 28, 21210-21217, July 14, 2000

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Ca_V2.2 and Ca_V2.3 (N- and R-type) Ca²⁺ Channels in Depolarization-evoked Entry of Ca²⁺ into Mouse Sperm^*

Gunther Wennemuth^§, Ruth E. Westenbroek^¶, Tao Xu, Bertil Hille, and Donner F. Babcock

From the Departments of  Physiology and Biophysics and ^¶ Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195-7290

Received for publication, March 13, 2000, and in revised form, April 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

As sperm prepare for fertilization, surface Ca²⁺ channels must open to initiate required, Ca²⁺-mediated events. However, the molecular identity and functional properties of sperm Ca²⁺ channels remain uncertain. Here, we use rapid local perfusion and single-cell photometry to examine the kinetics of calcium responses of mouse sperm to depolarizing stimuli. The linear rise of intracellular [Ca²⁺] evoked by ~10-s applications of an alkaline high [K⁺] medium directly reports activity of voltage-gated Ca²⁺ channels. Little response occurs if external Ca²⁺ is removed or if external or internal pH is elevated without depolarization. Responses are inhibited 30-40% by 30-100 µM Ni²⁺ and more completely by 100-300 µM Cd²⁺. They resist the dihydropyridines nitrendipine and PN200-110, but 1-10 µM mibefradil inhibits reversibly. They also resist the venom toxins calciseptine, -conotoxin MVIIC, and kurtoxin, but -conotoxin GVIA (5 µM) inhibits ~50%. GVIA also partially blocks transient, low voltage activated Ca²⁺ currents of patch-clamped spermatids. Differential sensitivity of sperm responses to Ni²⁺ and Cd²⁺ and partial blockade by GVIA indicate that depolarization opens at least two types of voltage-gated Ca²⁺ channels in epididymal sperm examined prior to capacitation. Involvement of a previously undetected Ca_V2.2 (N-type) channel, suggested by the action of GVIA, is substantiated by immunodetection of Ca²⁺ channel _1B subunits in sperm and sperm extracts. Resistance to dihydropyridines, calciseptine, MVIIC, and kurtoxin indicates that Ca_V1, Ca_V2.1, and Ca_V3 (L-, P/Q-, and T-type) channels contribute little to this evoked response. Partial sensitivity to 1 µM mibefradil and an enhanced sensitivity of the GVIA-resistant component of response to Ni²⁺ suggest participation of a Ca_V2.3 (R-type) channel specified by previously found _1E subunits. Our examination of depolarization-evoked Ca²⁺ entry indicates that mature sperm possess a larger palette of voltage-gated Ca²⁺ channels than previously thought. Such diversity may permit specific responses to multiple cues encountered on the path to fertilization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification and characterization of sperm Ca²⁺ entry channels and the mechanisms that modulate their function as sperm prepare to fertilize an egg remain major unsolved problems in reproductive biology (1-3). These challenges stand unmet largely because of the difficulty of applying patch-clamp methods and the tools of molecular biology to sperm. The poorly understood nature of the modifications to sperm that occur between mating and fertilization ("capacitation") presents an additional barrier to investigation.

Several indirect approaches have been informative. Probing of a germ line cell library for mRNA of Ca²⁺ channel ₁ subunits found predominant message for _1E (4). Whole cell recording from spermatocytes and spermatids found only transient, low voltage-activated (LVA)¹ Ca²⁺ currents (5-8). Finally, pharmacological sensitivities for spermatid Ca²⁺ currents showed some parallels with those for Ca²⁺-mediated responses of sperm (8, 9). On the basis of these results, it was proposed that a T-type Ca²⁺ channel is specified by _1E and is retained after spermiogenesis to provide the major route for depolarization-evoked entry of Ca²⁺ into sperm.

Newer work has shown that expression of _1E produces R-type rather than T-type channels (10) and has increased the number of Ca²⁺ channels found in the germ line. Message for the _1G and _1H subunits, now known to specify T-type channels (11-17), has been found in spermatogenic cells (18). Study by immunological methods showed that sperm contain _1E, _1A, and _1C channel proteins (19). If these proteins are functionally active, then P/Q- and L-type channels also might provide routes for entry of Ca²⁺ (see Table I).

Here, we use the kinetics of depolarization-evoked increases in the intracellular free [Ca²⁺] (Ca_i) of epididymal sperm to monitor the activity of voltage-gated Ca²⁺ channels and examine their pharmacological sensitivity. The results indicate that a portion of the observed activity requires opening of N-type Ca²⁺ channels, specified by _1B Ca²⁺ channel proteins that are detected here for the first time in sperm and sperm extracts. The properties of the residual component of response are consistent with involvement of R-type channels, presumably specified by the _1E proteins found in past work. Contrary to expectations, blockers of L-, P/Q-, and T-type channels were ineffective. It remains possible that functional forms of these channels are present but of low abundance or not opened by the stimulus protocols used. These observations are summarized in Table I.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Indo-1 AM and Pluronic 147 were from Molecular Probes (Eugene, OR), calciseptine was from Calbiochem (La Jolla, CA), -conotoxins was GVIA from RBI (Natick, MA), and MVIIC was from RBI and Bachem (Torrance, CA). Kurtoxin (Dr. K. J. Swartz, NIH), mibefradil (Hoffman-LaRoche, Basel, Switzerland), and PN200-110 (Sandoz, Hanover, NJ) were gifts from the indicated sources. All other chemicals were from Sigma.

Sperm Preparation and Media-- Male mice (Swiss Webster, retired breeders) were euthanized by CO₂ asphyxiation. Caudae epididymides and vasa deferentia were excised and then rinsed with medium HS (135 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 30 mM HEPES, 10 mM glucose, 10 mM lactic acid, and 1 mM pyruvic acid) adjusted to pH 7.4 with NaOH. After transfer to 1 ml of medium HS containing 5 mg/ml bovine serum albumin and 15 mM NaHCO₃, semen was allowed to exude (15 min at 37° C, 5% CO₂) from three to five small incisions. All subsequent operations were at room temperature (22-25° C) in medium HS, unless otherwise noted. Cells were diluted to 4 ml and collected twice by sedimentation (400 × g; 5 min). Washed sperm were dispersed and stored at 1-2 × 10⁷ cells/ml.

Potassium-evoked responses were produced with medium K8.6 (135 mM KCl, 5 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 30 mM TAPS, 10 mM glucose, 10 mM lactic acid, 1 mM pyruvic acid) adjusted to pH 8.6 with NaOH, in accordance with past work indicating that an elevated external pH is required for depolarization-induced entry of Ca²⁺ into sperm from other species (20, 21). Other test solutions included media K8.6B and HSB (media K8.6 and HS fortified with 15 mM NaHCO₃), and medium Na8.6 (medium HS in which 30 mM TAPS, pH 8.6, replaced HEPES).

Dye Loading and Photometry-- Indo-1 AM was dispensed from 2 mM stocks in Me₂SO, dispersed in 10% Pluronic 147, diluted to 20 µM in 0.25 ml medium HS, and then immediately mixed with an equal volume of the sperm suspension. After 15-20 min, medium HS (1 ml) was added, and the cells were sedimented. After resuspension in 0.25 ml of fresh medium, incubation continued for 1-5 h before use.

Incubation chambers were constructed from 35-mm tissue culture dishes and #0 glass coverslips. An uncoated, ~5-mm square #00 coverslip was placed in the dish, and 10 µl of cell suspension was added. After ~5 min, the chamber was gently flooded with medium and transferred to the microscope stage. Test solutions were applied either by a solenoid-controlled, gravity-fed, multibarreled, local perfusion device or by bath perfusion (estimated exchange times of <0.2 and ~20 s, respectively). Photometric measurements were as described (22). The background-corrected ratiometric signal R (F₄₀₅/F₅₀₀) was calibrated with the constants R_min (1.372), R_max (9.784), and K* (3603 nM) determined empirically from cells equilibrated in solutions fortified with ionomycin (10 µM) that contained 50 mM EGTA, or 15 mM CaCl₂, or 20 mM EGTA with 15 mM CaCl₂ (calculated free [Ca²⁺] of 251 nM). Automated correction, calibration, and kinetic analysis of digital photometric records were performed in Igor (Wavemetrics, Lake Oswego, OR). Statistical analyses were performed in Excel (Microsoft, Redmond, WA). All results are presented as the means ± S.E.

Immnunocytochemistry and Immunoblot Analysis-- Sperm and sperm extracts were probed with the affinity-purified antibody CNB-1 (23, 24), using methods for confocal immunomicroscopy and Western immunoblotting as described (19). Briefly, fixed and permeabilized mouse sperm were washed, blocked, and rinsed before incubation with diluted (1:15) antibody. Samples were rinsed again, treated with biotinylated anti-rabbit IgG (1:300), rinsed, treated with avidin D fluorescein (1:300), rinsed, and mounted for examination with the Bio-Rad MRC 600 confocal microscope at the Keck Imaging Facility of the University of Washington. Controls for nonspecific staining used antibody blocked by preliminary incubation with the peptide antigen. For immunoblot analysis, rat sperm were prepared in the presence of a protease inhibitor mixture. SDS extracts were clarified by sedimentation, reduced, separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, blocked, incubated with antibodies (10-20 µg/ml), rinsed, and developed with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).

Germ Line Cell Patch Clamp Procedures-- An excised mouse testis was bathed in a low [Ca²⁺] (0.1 mM) Ringer buffer at 20-23° C, the tunica was removed, and seminiferous tubules were teased apart with small forceps. Released cells were collected by sedimentation and then resuspended in 1 ml of a standard external buffer (140 mM NaCl, 2.5 mM KCl, 2 mM MgCl₂, 25 mM HEPES, 1.3 mM CaCl₂, 10 mM glucose, pH 7.4) with NaOH. The suspension was transferred to a 35-mm plastic recording chamber. Voltage clamp was performed with the whole cell configuration of the patch-clamp technique using an EPC 9 patch clamp amplifier and Pulse software (Heka, Lambrecht, Germany). The standard voltage protocol was 0.1 Hz application of 100-ms steps to 20 or 10 mV from a holding potential of 70 mV. Records were low pass filtered at 2 kHz (4-pole Bessel filter). Leak current subtraction used the P/n protocol (n = 4) implemented in the Pulse software. Typical current density was 7.1 ± 0.5 pA/picofarad (n = 6). Patch pipettes (2-4 M) were prepared from borosilicate glass. The pipette solution was 130 mM CsCl, 20 mM HEPES, 10 mM NaF, 4 mM MgATP, 2 mM EGTA, pH 7.4 with CsOH. The external solution for measurement of Ca²⁺ currents was 130 mM TEACl, 1 mM MgCl₂, 10 mM CaCl₂, 10 mM HEPES, 10 mM glucose, pH 7.4, with TEAOH.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the work here, indo-1 emission-ratio photometry reports spatially averaged Ca_i signals with an acceptable noise level from single sperm, sampled for 50 ms at 20 Hz for up to 60 s. Routinely, we examine small groups of 3-6 cells at a reduced excitation intensity to permit recordings lasting several minutes. Indo-1 photometry also allows continuous illumination with >600-nm light to provide bright field video images of the individual cells, which are motile but nonprogressive, oscillating about a point of attachment at the tip or base of the head. Cell viability is thus ensured. The photometric recording apparatus is synchronized with a local perfusion device that provides rapid application and removal of various test solutions and thus allows examination of reversibility and the kinetics of response and recovery.

Elevation and Recovery of Ca_i Evoked by Brief Perfusion with a Depolarizing Medium-- Fig. 1 shows representative photometric Ca_i records and their analysis. In Fig. 1A, sperm received five depolarizing stimuli by ~10-s perfusions with the alkaline, K⁺-substituted medium K8.6. In the first trial, Ca_i rose rapidly from a resting value of ~100 to ~500 nM and then recovered more slowly when the control medium was restored. The four subsequent stimuli produced even larger transient responses, indicating that brief elevations of Ca_i to 500 or 1000 nM do not irreversibly inactivate Ca²⁺ entry or the extrusion of Ca²⁺ that leads to recovery.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Repeated transient elevation and recovery of Cai evoked by brief perfusion with depolarizing medium. Fluorescence was recorded from sperm loaded with Indo-1 as described under "Experimental Procedures." A, calibrated Cai responses from representative cells perfused with HSB medium that contained 15 mM bicarbonate (15 BC) except during five brief (~10 s) stimuli with an alkaline, K+-substituted medium (K8.6). B, Cai responses from A () aligned to the initiation of stimulus and shown on an expanded time scale. C, rates of rise from regression analysis of the linear segments of the Cai responses in B were normalized to the largest of the five responses. Normalized responses from B and 15 similar experiments then were averaged. Boxes indicate the range of normalized data. D, averages of the rates of decay obtained by single exponential fits to the recovery phases in these same experiments. E, calibrated Cai responses from representative cells perfused with HS medium that lacked bicarbonate (0 BC), with HSB medium that contained 15 mM bicarbonate (15 BC), or with alkaline, K+-substituted versions of the same (K8.6 and K8.6B) to produce the indicated perfusate compositions. Symbols identify stimuli applied before (), during (), and after () exposure to HCO3. F, average rates of rise from regression analysis of the linear segments of the Cai responses in E and eight similar experiments.

After a short lag, Ca_i rose linearly for the duration of the applied stimuli (Fig. 1B). Rates of rise, from regression analysis of Ca_i responses in this and 15 similar experiments, showed a cell-to-cell variation of more than 2-fold, which depended in part upon the age of the preparation. Normalization to the fastest response of each experiment removed most of this variability and revealed that the average rate of rise increased progressively during the first four stimuli (Fig. 1C).

During the recovery from depolarization, the fall in Ca_i was well fitted by a single exponential (not shown). Rates of recovery showed relatively little cell-to-cell variability and did not change significantly for the series of stimuli (Fig. 1D). Average half-time for recovery was 30.3 ± 2.4 s.

A Persistent Stimulatory Action of Bicarbonate-- In sperm, bicarbonate rapidly stimulates cAMP production and motility (25) and is required for the slow development of sperm capacitation (26). To prevent capacitation, which might obscure rapid responses, bicarbonate and bovine serum albumin were intentionally deleted from the sperm storage medium (HS). Bicarbonate also was omitted from the depolarizing medium to allow inclusion of otherwise incompatible components, such as Ni²⁺ or Cd²⁺. However, 15 mM HCO₃ was included in the perfusing medium HSB to promote Ca_i responses to depolarizing stimuli. Fig. 1 (D and E) documents this rapidly developing enhancement, whose mechanism is still under investigation. The average rate of Ca_i rise evoked in the presence of HCO₃ was 3.5-fold that found for stimuli applied before exposure to HCO₃. Responses evoked immediately after removal of HCO₃ were slightly but not significantly (p > 0.2) faster than those observed in its continued presence. Experiments not shown indicate that the enhancing action of HCO₃ is complete within a few minutes and persists for a similar period after its removal.

Requirements for Evoked Elevation of Internal [Ca²⁺]-- In Fig. 2 and experiments to follow, a pair of depolarizing stimuli with medium K8.6 bracketed the test stimulus. First, we tested the need for extracellular Ca²⁺. In Fig. 2A, a brief perfusion with Ca²⁺-deficient medium HSB preceded the test stimulus with a nominally Ca²⁺-free version of medium K8.6. This ensured that Ca²⁺ was removed before membrane depolarization began, while precluding possible depletion of internal Ca²⁺ stores. The depolarization in the absence of external Ca²⁺ evoked a barely detectable response, smaller than the transient elevation of Ca_i produced upon subsequent restoration of external Ca²⁺. Average rates of rise for the test stimulus and the two control stimuli (Fig. 2B, left panel) show that responses to alkaline-depolarization require external Ca²⁺ and therefore represent Ca²⁺ entry. Fig. 2B (right panel), summarizing similar experiments in which the test stimulus was an alkaline version of medium HS (Na8.6), shows that elevation of external pH without depolarization does not evoke a substantial Ca_i response.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Negligible increases in Cai produced by elevation of internal or external pH or by depolarization in the nominal absence of external Ca2+. A, Cai responses from cells transferred to and perfused with medium HSB, stimulated with K8.6, and then perfused again with HSB. After recovery, a second stimulus with a Ca2+-free version of K8.6 was preceded and followed by ~5-s perfusion with a nominally Ca2+-free HSB. A dashed box marks the period of exposure to Ca2+-free conditions. Following recovery in HSB, a third stimulus with K8.6 was applied. B, average rates of rise from regression analysis of the linear segments of Cai responses. Left panel, from A and nine similar experiments; right panel, from 16 similar experiments in which initial and terminal stimuli with medium K8.6 bracketed a stimulus with Na8.6, an alkaline version of medium HS. C, Cai responses from cells transferred to and perfused with medium HSB, briefly (~5 s) with HS and then either with K8.6 or with HS containing 25 mM NH4Cl, as indicated. A dashed box marks the period of exposure to NH4+. D, average rates of rise from regression analysis of the linear segments of the responses. Left panel, from C and seven similar experiments; right panel, from eleven similar experiments using a test a stimulus with HS containing 15 mM NH4Cl.

Previous studies of sperm from other species showed that alkaline depolarizing medium elevates both internal pH and [Ca²⁺] (20, 27), and recent work with mouse sperm (28) indicated that increased internal pH also can cause Ca_i to rise. Fig. 2 (C and D) compares responses to alkaline depolarization with responses evoked by elevation of internal pH with NH₄⁺. Control and test stimuli were applied soon after starting a brief perfusion with medium HS (to remove external HCO₃). Although NH₄⁺ evokes a slight increase in Ca_i, the mean rates of rise (Fig. 2D) in response to 25 (left panel) or 15 mM (right panel) NH₄⁺ are much smaller than in response to the alkaline depolarizing medium (K8.6).

Sensitivity of Evoked Responses to Inorganic and Organic Ca²⁺ Channel Blockers-- To determine the kind of Ca²⁺ channels involved, we examined responses to a test stimulus applied in the absence or presence of various blockers of voltage-gated Ca²⁺ channels. The protocols were similar to those of Fig. 2. Rates of response to the middle, test stimulus were normalized to the mean of the rates for the bracketing control stimuli. The first bar of Fig. 3A validates this normalization procedure. The average normalized response for test stimuli applied in the absence of blocker was 1.01 ± 0.04. Similar analysis of the first three responses in the experiments of Fig. 1C yielded an average value of 1.04 ± 0.06. Fig. 3A also reports relative dose-dependent sensitivities of evoked responses to Ni²⁺ and Cd²⁺. At 10 µM, Ni²⁺ was ineffective, at 30 µM it caused 31 ± 4% inhibition, and at 100 µM it produced only a marginally greater inhibition (41 ± 5%). Inhibition of a similar extent (39 ± 4%) was produced by 30 µM Cd²⁺, but inhibition by 100 µM Cd²⁺ (74 ± 3%) was nearly 2-fold greater than that by Ni²⁺. Blockade by 300 µM Cd²⁺ was nearly complete (87 ± 4%). Thus, evoked responses may use two types of channels. Moderate concentrations of Ni²⁺ apparently block one but not the other, and both are sensitive to Cd²⁺.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Inhibition of evoked responses by inorganic and organic blockers of voltage-gated Ca2+ channels. Rates of rise from regression analysis of Cai responses in experiments similar to those shown in Fig. 2. Responses to a middle test stimulus were first normalized to the mean of the rates for the bracketing control stimuli, then averaged. A, initial and terminal stimuli with medium K8.6 bracketed either a stimulus with unmodified K8.6 (None; n = 14) or with K8.6 containing the indicated concentrations of NiCl2 (n = 22, 25, and 15) or CdCl2 (n = 22, 15, and 25). B, the test stimulus was with K8.6 that variously contained the indicated concentrations of nitrendipine (Nitr; n = 17 and 10), PN200-110 (PN; n = 20), or mibefradil (Mib; n = 6, 13, and 12). The test stimulus was preceded by 30 s of perfusion with medium HSB that contained the same concentration of these agents as the K8.6 test solution.

Fig. 3B examines the effectiveness of two classes of organic blockers of voltage-gated Ca²⁺ channels. Sensitivity to dihydropyridines, such as nitrendipine and the more potent PN200-110, is a hallmark of L-type Ca²⁺ currents but also is found for T-type currents of some somatic cells (10). PN200-110 at 1 µM did not inhibit Ca_i responses of sperm to alkaline depolarization. This concentration is greater than the reported IC₅₀ for LVA Ca²⁺ currents of spermatids (8) and for exocytotic responses of sperm (21). Nitrendipine at 10 µM was similarly ineffective despite its reported action at this or lower concentrations on Ca²⁺ currents of germ line cells (6, 8) and exocytotic responses of sperm (21, 29). At 30 µM nitrendipine slightly enhanced Ca_i responses (18 ± 6%) but caused a rapid decline in sperm motility, indicating that this result should be viewed cautiously. Apparently, Ca²⁺ channels sensitive to dihydropyridines do not contribute substantially to the evoked responses studied here.

The nondihydropyridine, benzimidazoylyl-substituted tetraline antagonist mibefradil is a potent inhibitor of T-type currents (12, 30) and also is a less potent blocker of R-type and other currents (10, 31, 32). At 1 µM, near the IC₅₀ for inhibition of R- and T-type currents of somatic cells (10), mibefradil inhibited evoked Ca_i responses of sperm by ~30%. At 3 or 10 µM, mibefradil produced near complete, reversible blockade, without obvious effects on cell viability.

Sensitivity of Evoked Responses to Peptide Toxins-- Several venom peptide neurotoxins target selected ion channels. This selectivity is widely used in characterization of ion currents of somatic cells but has seen little application in studies of sperm and male germ line cells. Figs. 4-6 examine the actions of four venom toxins on the depolarization-evoked responses of sperm.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Resistance of depolarization-evoked responses to the venom toxin calciseptine. A, calibrated Cai responses from representative cells transferred to and perfused with medium that contained 15 mM NaHCO3 (HSB) except during 2 brief (~10 s) control stimuli with medium K8.6. The bath medium then was exchanged with 2 volumes of HSB that contained 5 µM calciseptine (CSP) and 0.1 mg/ml of cytochrome c. After ~10 min, cells again received brief stimuli by perfusion with K8.6 with or without added 300 µM CdCl2 or 100 µM NiCl2 as indicated. Stimuli were preceded and followed by ~10-s perfusion with medium HSB. B, average rates of rise from regression analysis of the linear segments of the Cai responses in A and 21 similar experiments. C, average rates of rise from Cai responses in eight experiments similar to A, except that the bath medium was exchanged with medium that lacked calciseptine, and subsequent stimuli (Con3 and Con4) were with medium K8.6 alone.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effects of other venom toxins on depolarization-evoked responses. Average rates of rise from experiments similar to those of Fig. 4A except that the indicated toxins replaced calciseptine. A, after two control stimuli with K8.6 (Con1 and Con2), the bath medium was exchanged with medium containing 300 nM kurtoxin. After ~10 min of incubation with the toxin, cells received three more stimuli with K8.6 that lacked or contained Ni2+ or Cd2+ as indicated (n = 9). B, mean rates from 27 experiments with 5 µM -conotoxin MVIIC. C, mean rates from 10 experiments with 5 µM -conotoxin GVIA.

View larger version (46K): [in this window] [in a new window]   Fig. 6.   Immunoreactive Ca2+ channel 1B subunits are regionally distributed on sperm and present in sperm extracts. Representative confocal immunofluorescence images are shown in reverse contrast for mouse sperm treated with antibody directed to 1B applied alone (A and B) or after blocking with the peptide antigen (C). Scale bars are 5 µm. D, immunoblot analysis of sperm extracts (lane 2) and authentic affinity-purified rat brain 1B Ca channel protein (lane 3). Lane 1 shows Coomassie-stained molecular mass markers. Upper and lower arrows mark migration of bands at 250 and 85 kDa.

The mamba toxin calciseptine selectively blocks L-type Ca²⁺ currents (33). As with many venom toxins its action is relatively slow in onset and poorly reversible. Fig. 4A shows a modified protocol adopted to accommodate these properties. As in Figs. 1-3, local perfusion applied depolarizing stimuli. After two control episodes of stimulus and recovery, the bath medium (HSB) was exchanged with 2 volumes of medium that lacked or contained the toxin with the carrier peptide cytochrome c. Cells were incubated without flow for ~10 min and then again perfused locally with the depolarizing medium (K8.6), applied in the absence and presence of Ni²⁺ or Cd²⁺. To minimize dilution and removal of toxin, perfusion with control medium (HSB) was restricted to the first and last 10 s of the recovery period. Even on this compressed time scale, it is apparent that exposure to calciseptine did not diminish the response to depolarization. Fig. 4 (B and C) summarizes the analysis of this and like experiments. On average, the rate of Ca_i rise for the second of the pair of control stimuli applied before toxin treatment was slightly greater than for the first. For cells incubated in the absence of toxin (Fig. 4C), this trend continued for a third and fourth control stimulus. Rates also showed an apparent increase after toxin treatment. The greater blockade by Cd²⁺ than by Ni²⁺ found for untreated cells (Fig. 3A) also occurred in calciseptine-treated cells (Fig. 4B). Therefore, it is unlikely that a toxin-induced increase in the entry of Ca²⁺ through a Ni²⁺-sensitive, LVA channel masked an action of calciseptine on L-type channel activity. The resistance of responses to calciseptine is consistent with the ineffectiveness of dihydropyridines noted in Fig. 3B. Both indicate that responses here do not involve L-type channels.

Fig. 5 summarizes analyses of responses observed in similar experiments with three other venom toxins. The scorpion venom kurtoxin selectively blocks the T-type currents produced by expression of _1G and _1H subunits (34); cone snail -conotoxin MVIIC blocks both N- and P/Q-type currents; and (-conotoxin) GVIA blocks N-type currents selectively (35-38). Incubation with kurtoxin or MVIIC (Fig. 5, A and B) neither diminished the evoked responses of sperm nor altered their relative sensitivities to Ni²⁺ and Cd²⁺. In contrast, incubation with GVIA (Fig. 5C) decreased the rate of evoked responses by ~50%. Moreover, responses evoked after incubation with GVIA were inhibited only slightly more by Cd²⁺ than by Ni²⁺, suggesting that GVIA preferentially blocks the more Ni²⁺-resistant component of sperm responses that was identified in Fig. 3A. The faster recovery of N- than of P/Q-type channels from block by MVIIC (38, 39) may explain why, in the protocols used here, this toxin apparently did not block the N-type channels that are the presumed target of GVIA action on sperm.

Immunological Evidence That Sperm Possess _1B Subunit Proteins-- The presence of a GVIA-sensitive component of response (Fig. 5C), suggests that sperm express Ca²⁺ channel _1B proteins. We tested this hypothesis using antibody CNB-1, directed to a unique region of the rat brain Ca²⁺ channel _1B (Ca_V2.2) subunit (23). Fig. 6 shows representative confocal immunofluorescence images of mouse sperm stained with CNB-1. The chosen, central optical sections show the head and midpiece (Fig. 6A) and the proximal principal piece of the flagellum (Fig. 6B). Controls using antibody blocked by prior exposure to the peptide antigen showed only low, uniform fluorescence (Fig. 6C), indicating a high level of specificity in the detected signal, consistent with past studies using this probe (23, 24).

As found previously for _1C subunits, specific _1B immunoreactivity in the acrosomal region of the sperm head was confined to puncta along the acrosomal crescent. However, unlike _1C and _1E, _1B also appears in the postacrosomal segment (Fig. 6A). In the flagellum, _1B puncta were absent from the midpiece but present at both the dorsal and ventral surfaces of the principal piece (Fig. 6B). Staining was more intense and regular in the proximal than in the distal segment. Thus _1B, like _1A, _1C, and _1E (19), has a distinct pattern of distribution that follows the sharp regional boundaries that compartmentalize major cellular functions in these highly polarized cells. The unique localization of _1B in the postacrosomal segment is intriguing. The egg glycoprotein ZP3 evokes a nifedipine-resistant wave of elevated Ca_i that initiates from this region of the hamster sperm head (29).

Fig. 6D shows that the CNB-1 antibody recognizes a major band with an apparent molecular mass of ~210 kDa in Western blots of sperm extracts (lane 2). A minor component of ~240 kDa also is apparent. Consistent with past findings (23), authentic rat brain _1B Ca²⁺ channel proteins migrated as overlapping bands at ~210 and~240 kDa (lane 3). Thus, sperm possess immunoreactive protein of a size appropriate for _1B.

GVIA Sensitivity of Ca²⁺ Currents in Spermatogenic Cells-- The detection of N-type channels in sperm was surprising because prior studies have not found either HVA Ca²⁺ currents or _1B mRNA in male germ line cells. In preliminary experiments we confirmed that the LVA Ca²⁺ currents found previously in spermatocytes and round spermatids (5-8, 28) also are prominent in elongating spermatids. Fig. 7A is a video image typical of the stage VIII and IX spermatids studied here. Repetitive step depolarizations from 70 mV produced transient inward Ca²⁺ currents, with activation kinetics and voltage dependence similar to those reported in past studies of rodent spermatogenic cells (5, 6, 8). Current amplitude remained stable over many minutes of stepping to 20 mV at 0.1 Hz (data not shown). In contrast, peak current amplitude decreased rapidly after application of 5 µM GVIA (Fig. 7B). The extent of blockade was 38 ± 4% (n = 10) after 5 min of exposure to the toxin. Current amplitude did not recover detectably when GVIA was followed by 5 min of perfusion with bath medium (not shown), indicating a relatively irreversible action. Suprisingly, the GVIA-sensitive and the GVIA-insensitive currents had the same transient kinetics of activation (traces 1 and 2 of the inset) and voltage dependence of current amplitude (not shown). However, Ni²⁺ at 100 µM almost completely blocked the current that remained after application of GVIA (n = 4). In contrast, Ni²⁺ at this concentration blocked only 53 ± 2% (n = 12) of currents recorded prior to treatment with GVIA. We conclude that in elongating spermatids GVIA targets a Ni²⁺-resistant component of response, as it does in mature sperm (Fig. 5C).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   A GVIA-sensitive, Ni2+-resistant component of spermatid Ca2+ currents. Elongating spermatids from acutely dissociated testis preparations were examined in whole cell patch clamp. A, video image of a cell typical of those from which electrical recordings were obtained. B, amplitude of peak currents evoked during 100-ms depolarizations from a holding potential of 70 mV to a test potential of 20 mV, applied at 0.1 Hz. Bars indicate the periods when perfusion with bath medium was replaced by medium supplemented with -conotoxin GVIA, NiCl2, or CdCl2. Arrows mark the sampling time for the current records (from another cell) shown in the inset. Arrow 1, prior to GVIA; arrow 2, after GVIA; arrow 3, after GVIA and Ni2+. V indicates timing of the depolarizing step. Results are representative of 12 cells treated with GVIA alone and 4 cells treated with GVIA and Ni2+.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Early work with cell suspensions provided evidence that membrane depolarization opens surface channels that allow Ca²⁺ to enter sperm (20) and indicated that such channels mediate the exocytotic responses of sperm to glycoproteins of the zona pellucida of the egg (21, 40). Although the presence of voltage-gated Ca²⁺ channels and their importance in sperm preparing for fertilization are not at issue (for recent reviews see Refs. 2 and 3), their identity remains elusive.

The difficulty of applying patch-clamp methods and the tools of molecular biology to mature sperm has stimulated work upon the germ line cells from which sperm are derived. In spermatogenic cells, evidence for heterogeneity in channel mRNA contrasts with that for apparent homogeneity in the functional properties of the channels detected in electrical recordings. Thus, probes for channel mRNA detect message for the _1A, _1C, and _1E (4, 18, 41, 42) subunits that specify channels activated at high and intermediate voltages and for the _1G and _1H (18) that specify LVA channels (Table I). However, the current-voltage relationships and rapid inactivation observed for Ca²⁺ currents of voltage-clamped spermatogenic cells are typical of T-type, LVA channels (5-8). No HVA component of current has been detected. In the simplest interpretation, the transient Ca²⁺ currents of spermatocytes are T-type channels specified by expression of _1G or _1H subunits. In this explanation, mRNA for HVA channel subunits either is not translated, HVA subunit proteins do not assemble into functional channels, or a low relative abundance of functional channels precludes recording of HVA currents.

In the studies here, with spermatogenic cells examined at a later developmental stage (the elongating spermatid) we also find only LVA, transient Ca²⁺ currents.² However, we observe that -conotoxin GVIA separates those currents into pharmacological components that differ in their sensitivity to Ni²⁺. The relatively high sensitivity of the GVIA-resistant component to Ni²⁺ (Fig. 7) may indicate a more prominent contribution from _1H, whose expression specifies channels much more sensitive to this cation (43-45). The identity of the GVIA-sensitive component is uncertain. If the LVA currents of spermatogenic cells were specified exclusively by _1G and _1H, then one or both must be the target of GVIA action. A similar argument must be invoked to explain the susceptibility of these currents to dihydropyridines observed by others (6-8). Although no precedent for such a doubly unusual sensitivity profile exists, the pharmacology of expressed T-type channels is still sketchy.

Rather than possessing LVA channels with normal biophysical but unique pharmacological properties, the spermatogenic cells might have channels with a more usual pharmacology but unusual electrophysiological properties. A curious observation may provide a precedent for this hypothesis. Reportedly, expression of _1B without cotransfected  subunits can produce solely fast transient currents that are activated at low voltage and thus resemble T-type channels (46). Although mRNA for _1B in spermatogenic cells has not been reported, detection of the _1B immunoreactivity in sperm (Fig. 6) strongly suggests that it will be found. Conceivably, an absence of auxiliary subunits in spermatogenic cells could allow _1B and _1C to specify channels with the observed voltage dependence, kinetic properties, and sensitivities to the GVIA toxin and to dihydropyridines. The possibilities outlined in this and the preceding two paragraphs need to be tested directly.

Studies of Ca²⁺ channel expression and function in mature sperm have required different methods. However, they yield a similarly conflicting picture, with clear evidence for many expressed channels but uncertainty in the assignment of their roles in specific evoked responses. So far, our studies with immunological methods indicate that rodent sperm possess the HVA _1A, _1C, _1E (19), and, now, _1B (Fig. 6) Ca²⁺ channel proteins (Table I). In contrast, the sensitivities of Ca²⁺-mediated exocytosis in sperm (7, 9, 47) to several inorganic and organic channel blockers resemble the sensitivities found for the apparently homogenous LVA Ca²⁺ currents of spermatogenic cells (6, 8). The prevailing interpretation has been that the functional predominance of LVA channels observed in spermatids also applies to sperm. Unfortunately, technical limitations have prevented direct examination of this hypothesis.

With the use of conotoxin GVIA, we now provide evidence that sperm, like spermatids, possess multiple types of functional voltage-gated Ca²⁺ channels. In the simplest interpretation, GVIA inhibits Ca_i responses of sperm to depolarizing stimuli by blocking the _1B channel protein that is present in these cells (Fig. 6) and that forms the GVIA-sensitive N-type channels in neurons. The relative resistance to Ni²⁺ of the GVIA-sensitive component of responses in both sperm (Fig. 5C) and spermatids is consistent with the proposition that both possess functional N-type channels. The identity of the GVIA-resistant component(s) of sperm Ca_i responses is less certain. R-type calcium channels of somatic cells resist dihydropyridines but are blocked by moderate concentrations of Ni²⁺ or mibefradil (10, 31, 43). The GVIA-resistant component of sperm Ca_i response has this sensitivity to Ni²⁺ (Fig. 5C) and is the likely target of mibefradil action (Fig. 3B). In the simplest explanation, the _1E subunits present in sperm (19) specify functional R-type channels. At present, a more direct test of this proposal is hindered by the lack of pharmacological tools selective for R-type channels.

The presence of message for _1G or _1H and the prevalence of LVA currents in spermatogenic cells makes the lack of sensitivity of sperm responses to kurtoxin (Fig. 5A) somewhat surprising. This apparent noninvolvement of the T-type channels in K⁺-evoked Ca²⁺ entry might be explained by the proposal (9) that a relatively depolarized membrane potential of resting sperm places these channels in an inactivated state from which they cannot be activated directly by further depolarization. The methods used here with sperm do not measure membrane potential or its changes as we alter the composition of the external medium. Insensitivity of sperm responses to dihydropyridines (Fig. 3) also was unexpected because the slow inactivation of L-type channels would be well matched to the sustained entry of Ca²⁺ observed with strongly depolarizing stimuli of several seconds duration. Moreover, considerable past work had found dihydropyridines effective in blocking sperm responses, including those to depolarizing stimuli (20, 21, 47). For now, this apparent discrepancy remains unexplained.

In summary, we have increased the number of Ca²⁺ channel proteins found in sperm and shown that at least two are functionally active in our assay. In addition, we have shown that spermatogenic cells also possess more than one functional Ca²⁺ channel type whose presence has been masked by an apparent homogeneity in biophysical properties. These results broaden the repertoire of voltage-gated Ca²⁺ channels in sperm examined prior to capacitation as they begin to prepare for fertilization. Nevertheless, significant questions remain open. The possibility should be kept in mind that the availability and functional properties of channels change as sperm progress toward fertilization.

	ACKNOWLEDGEMENTS

We thank Drs. Kenton J. Swartz and Eric Ertel for the generous gifts of kurtoxin and mibefradil. We also thank Drs. William Moody, John Roche, and Todd Scheuer for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by the NICHD, National Institutes of Health through cooperative agreement U54-HD12629 as part of the Specialized Cooperative Centers Program in Reproduction Research, the W. M. Keck Foundation, the University of Washington Royalty Research Fund, and Fellowship We 2344/1-1 from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: Dept. of Anatomy and Cell Biology, Philipps University Marburg, D35037 Marburg, Germany.

To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Box 357290, University of Washington, Seattle, WA 98195-7290. Tel.: 206-543-6661; Fax: 206-685-0619; E-mail: donner@u.washington.edu.

Published, JBC Papers in Press, May 1, 2000, DOI 10.1074/jbc.M002068200

² T. Xu, D. Babcock, and B. Hille, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: LVA, low voltage-activated; HVA, high voltage-activated; Ca_i, intracellular free [Ca²⁺]; TAPS, N-tris[hydroxymethyl]methyl-4-aminobutanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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