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J. Biol. Chem., Vol. 281, Issue 31, 22332-22341, August 4, 2006

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Association of Catsper1 or -2 with Ca_v3.3 Leads to Suppression of T-type Calcium Channel Activity^*

From the Neuroscience Research and Advanced Technology, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois 60064

Received for publication, October 17, 2005 , and in revised form, May 1, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Sperm-specific CatSper1 and CatSper2 proteins are critical to sperm-hyperactivated motility and male fertility. Although architecturally resembling voltage-gated ion channels, neither CatSper1 nor CatSper2 alone forms functional ion channels in heterologous expression systems, which may be related to the absence of yet unidentified accessory subunits. Here we isolated CatSper1- and CatSper2-associated protein(s) from human sperm and analyzed their identities by a multidimensional protein identification technology approach. We identified the T-type voltage-gated calcium channel Ca_v3.3 as binding to both CatSper1 and CatSper2. The specificity of their interactions was verified by co-immunoprecipitation in transfected mammalian cells. Electrophysiological studies revealed that the co-expression of CatSper1 or CatSper2 specifically inhibited the amplitude of Ca_v3.3-evoked T-type calcium current without altering other biophysical properties of Ca_v3.3. Immunostaining studies revealed co-localization of CatSper1 and Ca_v3.3 on the principal piece of human sperm tail. Furthermore, fluorescence resonance energy transfer analysis revealed close proximity and physical association of these two proteins on the sperm tail. These studies demonstrate that CatSper1 and CatSper2 can associate with and modulate the function of the Ca_v3.3 channel, which might be important in the regulation of sperm function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Sperm must swim long distances in the female reproductive tract to reach the site of fertilization. In addition, penetration through the gelatinous zona pellucida layer of the oocyte requires the sperm to swim in a hyperactivated state at the time and site of fertilization. This process is characterized by high amplitude and asymmetric beating of the sperm tail. Many studies have indicated that Ca²⁺ serves as a key regulator in the initiation and maintenance of motility, including the hyperactivated motility (for reviews see Refs. 1 and 2).

Extensive efforts have been undertaken to identify and characterize Ca²⁺ entry pathways, particularly Ca²⁺ channels, involved in sperm motility processes. Two such ion channel-like proteins, CatSper1 and CatSper2, were shown to be specifically expressed in the principal piece of the sperm tail (3, 4). Targeted disruption of CatSper1 led to sterile phenotypes in otherwise normal male mice. Further studies revealed that mutant sperm lack hyperactivated motility (5). In vitro fertilization assays revealed that CatSper1 mutant sperm could not fertilize eggs with an intact zona pellucida layer but could fertilize eggs whose outer layers had been enzymatically removed (3). Targeted disruption of CatSper2 also led to male sterile phenotypes, and the null sperm has identical loss-of-function phenotypes as does the CatSper1 null sperm (6, 7). Therefore, CatSper1 and CatSper2 proteins appear to be essential for hyperactivated motility needed late for the sperm to penetrate the zona pellucida.

CatSper1 and CatSper2 represent a unique class of putative ion channel proteins (for a review see Ref. 8). They contain a single domain comprised of six transmembrane-spanning regions, akin to the voltage-gated potassium channels (3). However, their ion selectivity pore sequences between transmembrane regions 5 and 6 are closest to a single domain of the much larger voltage-gated Ca²⁺-selective channels. Residues lining the fourth transmembrane region of CatSper resemble a voltage sensor, as described for voltage-gated ion channels. However, recording ion channel activity following expression of CatSper subunits in heterologous expression systems, including Xenopus oocytes, HEK,² and Chinese hamster ovary K1 cells have not been successful (3, 4). Attempts to measure whole cell currents from the sperm have proven difficult until very recently, where an alkaline-activated Ca²⁺ current was recorded from sperm by patch clamp measurements (9). Interestingly, this current is absent in sperm lacking CatSper1. However, considering the co-dependent expression of CatSper1 and CatSper2 (7), it is still not clear which subunit or subunit complexes mediate this current.

Although several possibilities may be envisaged, the inability to functionally express CatSper subunits may be due to the following: (i) the absence of sperm-specific accessory proteins necessary for a putative ion channel complex in the heterologous systems, as observed with other ion channel complexes, or (ii) that CatSper may function as an accessory subunit to modulate the function of the principal subunit of an undetermined ion channel. The potential for either scenario exists, and it is indeed noteworthy that coiled-coil protein-protein interaction domains are present in the C-terminal regions of each of the CatSper subunits (10). In this study, we adopted a GST pull-down approach to isolate CatSper-associated proteins from human sperm extracts, and we have identified the T-type calcium channel subunit Ca_v3.3 as an interacting protein. Electro-physiological studies revealed that the co-expression of either CatSper1 or CatSper2 specifically inhibited the amplitude of Ca_v3.3-evoked T-type Ca²⁺ current without altering other biophysical properties of Ca_v3.3. Considering that CatSper1 and Ca_v3.3 subunits are co-expressed and associated with each other on the tail of the human sperm, our observations suggest that CatSper-Ca_v3.3 interactions could play an important role in regulating sperm functions such as hyperactivated motility.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cloning—To generate FLAG-tagged human CatSper1 (GenBank^TM accession number AF407333 [GenBank] ) and CatSper2 (GenBank^TM accession number AF411818 [GenBank] ) expression constructs, the coding sequences of CatSper with an in-frame FLAG tag sequence were amplified by PCR and cloned into the NotI site of pIRESneo3 (Clontech). To generate pGEX-4T-1-Cat1-C to express the C terminus of CatSper1 as a GST fusion protein, the coding sequence covering the C-terminal 111 amino acids of human CatSper1 was amplified by PCR and cloned in-frame with GST in pGEX-4T-1 vector (Amersham Biosciences). To prepare pGEX-4T-1-Cat2-N and pGEX-4T-1-Cat2-C constructs for the expression of the N and C termini of CatSper2 as GST fusion proteins, respectively, the coding sequences covering the N-terminal 103 amino acids and C-terminal 190 amino acids of human CatSper2 were amplified by PCR and cloned in-frame with GST in pGEX-4T-1.

To clone human Ca_v3.3 (GenBank^TM accession number AF393329 [GenBank] ), portions of the coding sequence were amplified by reverse transcription-PCR against poly(A)⁺ RNA from human cerebellum (Clontech). These fragments were ligated together and cloned in pcDNA3.1/V5-His TOPO expression vector (Invitrogen) so as to generate pcDNA3.1-Ca_v3.3-V5/His with V5 and His tags in-frame with the C terminus of Ca_v3.3.

Antibodies—To generate antibody against human CatSper1, the C-terminal 111 amino acids of CatSper1 as a GST fusion protein were purified from bacteria and were used to immunize rabbits. The CatSper1 C-terminal fragment, which was released from the GST fusion protein by thrombin cleavage, was used to affinity-purify antibodies against the CatSper1 part from the crude serum. The strategy as described by Quill et al. (4) was adopted to generate rabbit antibody against a peptide derived from the C-terminal 27 amino acids of mouse CatSper2. The resulting antibody recognized the human CatSper2 as well. The following antibodies were obtained from the sources indicated: human Ca_v3.3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, and Alomone Labs, Israel), Na⁺/K⁺-ATPase antibody (Abcam, Cambridge, UK), PMCA4 antibody (Sigma), human 14-3-3 antibody (Assay Designs, Ann Arbor, MI), V5 tag antibody (Invitrogen), and FLAG tag antibody (Sigma).

GST Pulldown Assay—To prepare GST fusion proteins, pGEX-4T-1, pGEX-4T-1-Cat1-C, pGEX-4T-1-Cat2-N, and pGEX-4T-1-Cat2-C were transformed into BL21 Star(DE3) pLysS (Invitrogen), and the expressions of recombinant proteins were induced by 0.5 mM isopropyl -D-thiogalactopyranoside. GST fusion proteins were purified from bacteria using glutathione-Sepharose 4B beads (Amersham Biosciences) according to the protocol recommended by the manufacturer. The purified proteins were eluted from the beads by a buffer containing glutathione, dialyzed, and re-conjugated on fresh beads to obtain purer fractions.

To prepare human sperm extract, cryo-preserved sperm samples were washed to remove the seminal fluid and lysed in lysis buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 2 mM EDTA, 0.5 mM DTT and protease inhibitor mixture from Sigma) for 1 h. The extract was centrifuged to remove cell debris and combined with an equal volume of dilution buffer to bring down Nonidet P-40 and sodium deoxycholate concentration to 0.5%. The sperm extract was precleared by incubation with glutathione-Sepharose 4B beads for 1 h. To pull down CatSper-associated proteins, the sperm extract was divided into equal parts, and beads conjugated with GST (as a negative control), GST-Cat1-C, or a mixture of GST-Cat2-N and GST-Cat2-C proteins were added. After incubation at 4 °C with gentle rotation overnight, the beads were washed three times with 10 ml of ice-cold IP buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 2 mM EDTA, 0.5 mM DTT and protease inhibitor mixture from Sigma). The GST fusion proteins along with associated proteins on the washed beads were eluted in a buffer containing glutathione (100 mM Tris, pH 8.0, 150 mM NaCl, 20 mM glutathione, 0.2% Triton X-100).

MudPIT Analysis of Protein Identities—Half of the eluted protein mixtures from GST pulldown experiments were resolved by SDS-PAGE and visualized by silver staining. Selective protein bands on the gel were excised, and the gel slices were destained and chopped into 1-mm size cubes. A slightly modified procedure originally developed by Shevchenko et al. (11) was employed for in-gel digestion. The extracted peptides were lyophilized and resuspended in 15-20 µl of 5% formic acid until further analysis. The other portion of the eluted protein mixtures were subjected to in-solution digestion (12) resulting in complex peptide mixtures. Peptide digests resulting from in-gel digests or in-solution digests were then individually loaded on to a three-phase MudPIT (RP-SCX-RP) column. A three-step MudPIT analysis was used for analyzing the in-gel digests, and a six-step MudPIT analysis was used for in-solution digests (13).

Transfection and Cell Culture—HEK cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified 5% CO₂, 95% O₂ incubator at 37 °C. Cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. To generate a cell line stably expressing Ca_v3.3, pcDNA3.1-Ca_v3.3-V5/His was linearized by SalI and transfected into HEK cells. Stable cell line was generated by antibiotic selection (1 mg/ml G418) 48 h post-transfection. Single cell colonies were selected 14 days post-transfection and amplified, and the expression of Ca_v3.3 was assessed.

Co-immunoprecipitation—HEK cells in 10-cm dishes were transiently transfected with equal amounts of the expression constructs (totally 10 µg of DNA) using Lipofectamine 2000. When only a single construct was transfected, pcDNA3.1 vector alone was included to maintain the same final amount of DNA. Cells were lysed 48 h post-transfection with IP buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 2 mM EDTA, 0.5 mM DTT, and protease inhibitor mixture from Sigma). The cell lysates were then centrifuged to remove cell debris, and the antibody used for immunoprecipitation was added to the supernatant. After overnight incubation at 4 °C with gentle agitation, immune complexes were precipitated with protein A- or protein G-agarose beads (Invitrogen) followed by washes in 1 ml of IP buffer for three times. After the final wash, the pellet was resuspended in Laemmli sample buffer, and proteins were resolved by SDS-PAGE (4-12% gel) and transferred to polyvinylidene fluoride membrane for immunoblot analysis.

Whole Cell Patch Clamp—HEK cells stably expressing Ca_v3.3 were transfected with either pcDNA3.1 vector, CatSper1, or CatSper2 expressing constructs using Lipofectamine 2000 (Invitrogen). Cells were also co-transfected with a plasmid encoding a GFP reporter (in a 1:5 ratio) to allow identification of positively transfected cells for whole cell patch clamp measurements. 24-48 h post-transfection, whole cell currents were recorded at room temperature using the standard patch clamp technique with an Axopatch 200B amplifier (Axon Instruments, Union City, CA), controlled with a PC computer using pCLAMP6 software (Axon Instruments). Data were filtered at 5 kHz using the built-in filter of the amplifier. Borosilicate pipettes with a typical resistance of 2-3 megohms were filled with a solution containing the following: 110 mM CsCl, 10 mM EGTA, 10 mM HEPES, 2 mM MgATP, 0.6 mM GTP (pH adjusted to 7.2 with CsOH). Extracellular solution contained 5 mM CaCl₂, 155 mM tetraethylammonium chloride, 10 mM HEPES (pH adjusted to 7.4 with tetraethylammonium-OH).

Data Analysis—In electrophysiology measurements, peak currents were determined using Clampfit 8.0 software (Axon Instruments). The conductance-voltage relationship for activation was deduced by the chord conductance method, wherein conductance was obtained by normalizing peak current at each pulse against driving force, plotted as function of voltage, fit with a single Boltzmann function, and normalized against maximal value. Average data are presented as mean ± S.E., and statistical differences between data sets were assessed by Student's t tests, and significance was accepted at the p < 0.05 level.

Biotinylation of Cell Surface Proteins for Expression Analysis—Cells were transfected with a fixed amount of DNA (10 µg) in 10-cm dishes using Lipofectamine 2000. 0.5 µg of a plasmid expressing an hemagglutinin-tagged protein was included in each transfection to allow normalization of transfection efficiencies among various samples. 48 h post-transfection, cells were harvested, washed with PBS, and then incubated in PBS containing 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) for 45 min at 4 °C to label cell surface proteins. The biotinylation reaction was quenched with 50 mM NH₄Cl for 10 min. The cells were washed with PBS and incubated in lysis buffer (0.5% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM DTT, protease inhibitor mixture in PBS). The cell lysates were then centrifuged to remove cell debris, and proteins in the supernatant were quantitated. Equal amounts of protein lysates were aliquoted, one part for the measurement of total protein expression, and the rest were incubated with beads coated with streptavidin (Pierce) for affinity-capture and purification of biotinylated proteins. The proteins were resolved by SDS-PAGE (4-12% gel) and transferred to polyvinylidene fluoride membrane for immunoblot analysis. The intensities of protein bands within a linear range of detection on the scanned blot were quantitated by ImageQuant image analysis software, and the expression levels of protein were normalized to that of the transfection control protein.

Immunofluorescence—Cryo-preserved human sperm samples were thawed, diluted, spotted onto chamber slides, and air-dried. The sperm were fixed and permeabilized with 4% paraformaldehyde for 10 min. After rinsing twice in PBS, the slides were incubated in blocking buffer (2% fetal bovine serum, 2% bovine serum albumin in PBS) for 30 min to reduce nonspecific binding. The slides were then incubated with either rabbit anti-Ca_v3.3 antibody (Alomone Labs, 1:50 dilution) or rabbit anti-CatSper1 antibody (1:50 dilution) diluted in the blocking buffer for 2 h. To assess specific binding, 2 µg of competing peptide for anti-Ca_v3.3 antibody or 25 µg of purified CatSper1 C-terminal fragment for competing anti-CatSper1 antibody were preincubated with the primary antibodies in the blocking buffer for 30 min and then applied to the slides. After incubating with primary antibodies, the slides were washed once in PBS with 0.25% Nonidet P-40 and then twice in PBS. The slides were then incubated with fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) diluted in blocking buffer (1:200 dilution) for 1 h. After washing one time in PBS with 0.25% Nonidet P-40 followed by two times in PBS, the slides were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) for observation under a fluorescent microscope.

FRET Analysis—Anti-Ca_v3.3, anti-CatSper1, and anti-PMCA4 antibodies were labeled with Alexa Fluor 488 dye or Alexa 555 dye by Zenon IgG labeling kit (Invitrogen) according to the manufacturer's protocol. These labeled antibodies were employed to immunostain sperm samples, as described for indirect immunofluorescence, but without using the secondary antibody. Before mounting, the stained sperm on the slide were fixed again with 4% paraformaldehyde for 10 min. Fluorescent images were acquired using an LSM 5 PASCAL laser scanning confocal imaging system (Carl Zeiss, Thornwood, NY). All images were taken with an oil immersion objective with appropriate filter sets (donor, excitation 488 nm, emission filter BP 505-530 nm; acceptor, excitation 543 nm, emission filter LP 560 nm; FRET, excitation 488 nm, emission filter LP 560 nm). The quantitative FRET analysis was performed using the PASCAL software (Zeiss) according to manufacturer's sensitized emission protocol, which is based on the conventional three-filter method described by Xia and Liu (14). The FRET filter raw image contains the FRET signal, as well as the bleed through of direct donor and acceptor emissions into the FRET channel. To determine bleed through and background corrections, the three track images of sperms stained with donor only and acceptor only were separately obtained. The donor and acceptor images of the samples were then multiplied with the respective correction factor and subtracted from the raw FRET image, and a normalized FRET (NFRET) image was calculated by PASCAL software according to Xia and Liu (14). NFRET values of at least 10 regions on the tails of 3-5 stained sperm were measured and expressed as means ± S.E.

View larger version (42K): [in this window] [in a new window]   FIGURE 1. Identification of CatSper-associated proteins from sperm extracts by GST pulldown. Human sperm extracts were incubated with beads conjugated with GST (negative control), GST-Cat1-C, or a mixture of GST-Cat2-N and GST-Cat2-C proteins, respectively, to isolate CatSper-associated proteins. Shown is the silver staining gel of aliquots of the pulldown products resolved by SDS-PAGE. GST fusion proteins used in pulldown (arrows) appeared as major protein bands on the gel, along with other less abundant protein bands in all three lanes.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

GST fusion protein constructs were prepared and expressed in bacteria. The N terminus of CatSper1 as a GST fusion protein expressed poorly and was largely insoluble, whereas good expression of the C terminus of CatSper1 and the N and C termini of CatSper2 as GST fusion proteins (GST-Cat1-C, GST-Cat2-N, and GST-Cat2-C, respectively) were obtained. These GST fusion proteins were affinity-purified to apparent homogeneity and conjugated to glutathione-Sepharose 4B beads. To pull down CatSper-associated proteins, human sperm extract was incubated with beads conjugated with GST (negative control), GST-Cat1-C, or a mixture of GST-Cat2-N and GST-Cat2-C proteins, respectively. Aliquots of the pull-down products were resolved by SDS-PAGE and visualized by silver staining. GST fusion proteins used in pulldown appeared as major protein bands on the gel. Besides, less abundant protein bands appeared in all three lanes of Fig. 1. Some of these protein bands, with identical molecular weights across all lanes, were apparent nonspecific contaminants during purification. Nonetheless, distinct protein bands were observed specifically in GST-CatSper1 and -2 pulldown lanes but not in GST pull-down controls.

We applied MudPIT, which incorporates on-line two-dimensional capillary chromatography coupled to tandem mass spectrometry to determine the identities of proteins in the pull-down mixtures. As expected, several peptides derived from either GST or CatSper were identified because of the presence of the GST fusion proteins in the pulldown mixtures. To identify proteins specifically associated with CatSper, we subtracted, in silico, proteins identified in GST alone pulldown from those obtained from GST-CatSper1 and -2 pulldowns. This subtractive analysis identified a few proteins present/unique to the GST-Cat1-C or a mixture of GST-Cat2-N and GST-Cat2-C pulldown. Some identified proteins were nonspecific contaminants from semen, including apolipoprotein and semenogelin. One peptide (RTFRLLRVLKLVRFMPALRR) derived from the T-type calcium channel Ca_v3.3 was consistently identified (from two separate experiments) in both in-gel and in-solution digestion of GST-CatSper1 and GST-CatSper2 pulldown complexes but not from pulldown product by GST alone. These findings suggested that Ca_v3.3 might be a likely associated protein for both CatSper1 and CatSper2.

Co-immunoprecipitation of CatSper1 and CatSper2 with Ca_v3.3 in Mammalian Cells—In order to confirm the association of Ca_v3.3 with the CatSper1 and -2 observed in GST pull-down, co-immunoprecipitation experiments were conducted to investigate their interactions in mammalian cells. CatSper1 with a FLAG tag was transiently transfected into HEK cells either alone (negative control) or along with a construct expressing Ca_v3.3 with a V5 tag. Forty eight hours post-transfection, the expression of transfected Ca_v3.3 and CatSper1 could be detected by Western blot analysis of products immunoprecipitated with the corresponding antibodies (Fig. 2A). When Ca_v3.3 antibody was used in the immunoprecipitation, we observed that CatSper1 co-immunoprecipitated with Ca_v3.3 in the case when both were co-transfected but not when CatSper1 alone was transfected (Fig. 2A). To determine the specificity of interactions between Ca_v3.3 and CatSper1, we examined whether other unrelated proteins, such as Na⁺/K⁺-ATPase and 14-3-3, could also co-immunoprecipitate with Ca_v3.3. Although these two proteins were abundantly expressed, they were not present in the products immunoprecipitated by the Ca_v3.3 antibody (Fig. 2A). Similar co-immunoprecipitation studies revealed that CatSper2 also associates with Ca_v3.3 in mammalian cells (Fig. 2B). In reciprocal experiments, when antibodies recognizing the cloned CatSper proteins were used in immunoprecipitation, we observed that Ca_v3.3 could be co-immunoprecipitated along with either CatSper1 or CatSper2 (Fig. 2C).

View larger version (28K): [in this window] [in a new window]   FIGURE 2. CatSper proteins co-immunoprecipitate with Cav3.3 in transfected HEK cells. A, CatSper1-FLAG construct was transiently transfected in HEK cells either alone (negative control) or along with a construct expressing Cav3.3-V5. Western blot (WB) of products immunoprecipitated (IP) by the anti-Cav3.3 antibody (Santa Cruz Biotechnology) recognized Cav3.3 in transfected cells. Western blot using anti-CatSper1 antibody revealed comparable CatSper1 expression in both transfections by analyzing the proteins immunoprecipitated by the anti-FLAG antibody. CatSper1 was detected in the proteins immunoprecipitated by anti-Cav3.3 antibody only when Cav3.3 and CatSper1 co-expressed in the cells. Western blot analysis using anti-Na+/K+-ATPase antibody or anti-14-3-3 antibody revealed abundant expression of these two proteins in the cell lysates; however, these were not detected in the products immunoprecipitated by Cav3.3. B, similar co-immunoprecipitation experiments demonstrating the association of CatSper2 with Cav3.3 in HEK cells. C, in reciprocal experiments, Cav3.3 were co-immunoprecipitated along with either CatSper1 or CatSper2, utilizing anti-FLAG or anti-CatSper2 antibody, respectively, in immunoprecipitation experiments.

The voltage dependence of channel availability was determined by a two-pulse protocol (Fig. 4A). From a holding potential of -110 mV, a 3-s pulse ranging from -110 to -20 mV was applied to allow channel inactivation, and a second pulse to -30 mV was applied to assess relative channel availability. Peak currents elicited by second pulse were plotted as function of voltage of the first pulse and fit with Boltzmann function to determine the maximal peak currents. The normalized currents against maximal peak currents were plotted and fit with Boltzmann function to derive voltage-dependent inactivation. Neither the V nor slope factors were significantly altered by co-expression with CatSper1 or -2 (Fig. 4B).

We also investigated the effects of co-expression of CatSper1 on the electrophysiological properties of Ca_v3.3 in another expression system, viz. Xenopus oocytes. Current responses were measured following co-injection of Ca_v3.3 and CatSper1 cRNA (90 ng each). Similar to effects observed in HEK cells, co-injection of CatSper1 reduced peak currents at -25 mV by 42 ± 6% (n = 22), whereas other biophysical properties of Ca_v3.3 were largely unaffected (data not shown). To exclude the possibility that the expression of CatSper1 might nonspecifically affect other channels besides Ca_v3.3, we co-expressed CatSper1 with HCN2, which encodes the hyperpolarization-activated cyclic nucleotide-gated channel. Overexpression of CatSper1 does not affect the expression level of HCN2 (data not shown), supporting the idea that the inhibition of Ca_v3.3 currents is because of its specific interaction with CatSper1.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Co-expression of CatSper1 or -2 reduces Cav3.3-evoked current amplitudes. A, voltage protocol and representative current traces from cells expressing Cav3.3 alone, Cav3.3/CatSper1, and Cav3.3/CatSper2. Currents were elicited by voltage steps ranging from -100 to +50 mV in 5-mV increments from a holding potential of -110 mV. B, current-voltage (I-V) relationships were obtained by measuring the peak currents during the 300-ms pulses and plotted as function of voltage (n = 9, 6, and 6 for Cav3.3, Cav3.3/CatSper1, and Cav3.3/CatSper2, respectively). Currents peaked at -25 mV. C, the conductance-voltage relationship for activation was deduced by the chord conductance method, wherein conductance was obtained by normalizing peak current at each pulse against driving force, plotted as function of voltage, fit with a single Boltzmann function, and normalized against maximal value. The mean values were plotted and fitted with Boltzmann function to obtain V and slope factor k.Cav3.3 (n = 9), V =-44.5 ± 0.5 mV, k = 7.8 ± 0.5 mV; Cav3.3/CatSper1 (n = 6), V =-43.2 ± 0.3 mV, k = 8.4 ± 0.4 mV; Cav3.3/CatSper2 (n = 6), V =-41.6 ± 0.9 mV, k = 7.7 ± 0.2 mV.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Co-expression of CatSper1 or -2 does not alter the inactivation properties of Cav3.3. A, representative Cav3.3 currents evoked by two-pulse protocol used to estimate voltage dependence of inactivation. The upper panel shows voltage protocol, and the lower panel shows currents recorded during the interval highlighted by arrows in upper panel. B, relative peak currents for Cav3.3, Cav3.3/CatSper1, and Cav3.3/CatSper2 during the second 300-ms pulse at -30 mV were fitted with Boltzmann function to determine half-maximal (V) and slope factor (k) values for each channel type. Cav3.3 (n = 6), V =-75.9 ± 0.2 mV, k = 5.2 ± 0.3 mV; Cav3.3/CatSper1 (n = 4), V =-75.2 ± 0.2 mV, k = 5.4 ± 0.2 mV; Cav3.3/CatSper2 (n = 4), V = -75.7 ± 0.3 mV, k = 5.4 ± 0.2 mV.

Co-expression of CatSper1 and Ca_v3.3 on the Principal Piece of Human Sperm Tail—To investigate whether CatSper1 and Ca_v3.3 proteins are co-expressed in human sperm, an indirect immunofluorescence technique was used. Unlike the observations by Trevino et al. (16), we could not conclusively determine Ca_v3.3 expression using the same anti-Ca_v3.3 antibody (purchased from Santa Cruz Biotechnology) because immunostaining was only partially blocked by the corresponding antigen. We instead relied on an anti-Ca_v3.3 antibody from a different source (Alomone Labs) in our immunostaining experiments (17). Western blot analysis revealed that this antibody not only recognize cloned Ca_v3.3 expressed in HEK cells but also detected Ca_v3.3 expressed in human sperm and cerebral cortex (Fig. 6A). When this anti-Ca_v3.3 antibody was used in immunostaining experiments, robust staining on the principal piece and a slightly weaker staining on the middle piece of the sperm tail was observed (Fig. 6B, panel 2). The staining was totally blocked by preincubation of the primary antibody with the corresponding antigen peptide (Fig. 6B, panel 4). We next examined the expression of CatSper1 in human sperm. We observed robust fluorescence signals on the principal piece of sperm tail with the antibody raised against the C terminus of human CatSper1 (Fig. 6C, panel 2). This staining was blocked by preincubation of the primary antibody with the recombinant CatSper1 C-terminal fragment used to raise the antibody (Fig. 6C, panel 4), indicating specific staining of CatSper1 expression on the principal piece. This is consistent with the observation of CatSper1 localization on mouse sperm (3). The staining of both Ca_v3.3 and CatSper1 on human sperm tail demonstrates co-localization of these two proteins in this region.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Effects of co-expression of CatSper1 proteins on surface and total expression levels of Cav3.3. HEK cells were transfected with Cav3.3 either with or without CatSper1 as indicated, together with a control plasmid to normalize for differences in transfection efficiencies between samples. A, representative Western blots showing the expression of Cav3.3, CatSper1, Na+/K+-ATPase, and 14-3-3 in the biotinylated surface protein fractions (surface) and the total cell lysates (total). B, normalized relative surface and total expression of Cav3.3 is plotted as means ± S.E. of three separate transfection experiments.

View larger version (82K): [in this window] [in a new window]   FIGURE 6. Immunolocalization of Cav3.3 and CatSper1 on human sperm tail. A, anti-Cav3.3 antibody (Alomone Labs) recognized Cav3.3 expressed in human sperm and cerebral cortex, as well as cloned Cav3.3 expressed in HEK cells by Western blot analysis. B, shown are immunofluorescence images of mature human sperms stained with anti-Cav3.3 antibody preincubated without (panel2) or with (panel4) competing antigenic epitope. The corresponding phase contrast images (panels 1 and 3) are shown on the left. C, shown are immunofluorescence images of mature human sperms stained with anti-CatSper1 antibody preincubated without (2) or with (4) competing antigen. The corresponding phase contrast images (panels 1 and 3) are shown on the left.

We labeled anti-Ca_v3.3 antibody with Alexa Fluor 488 dye as the donor (Ca_v3.3-488) and anti-CatSper1 antibody with Alexa 555 dye as the acceptor (CatSper1-555). These two labeled antibodies were mixed to immunostain human sperm, and FRET analysis was performed using the sensitized emission method (14). A set of representative donors, acceptors, and raw FRET images are shown in Fig. 7. An NFRET image, which shows FRET intensities with high spatial resolution, was calculated from the raw FRET image after correction for the effects of bleed through of direct donor and acceptor emissions into the FRET channel. We observed robust NFRET signals along the sperm tail but not in the head (Fig. 7), indicating a region-specific close molecular association between CatSper1 and Ca_v3.3. Quantitative measurement of 10 regions on the tails of different sperm samples revealed a mean NFRET value of 0.394 ± 0.017. As a positive control, we labeled anti-CatSper1 antibody with either Alexa 488 dye as the donor or Alexa 555 dye as the acceptor and mixed them to immunostain human sperm (Fig. 7). Both labeled antibodies should be in close proximity by recognizing the same protein, and indeed as expected, we detected a robust NFRET signal (mean value of 0.416 ± 0.012), which is comparable with that from the experimental group. To assess the specificity of the FRET analysis, we also immunostained sperm with anti-PMCA4 antibody labeled with Alexa 488 dye and anti-Ca_v3.3 antibody labeled with Alexa 555 dye (Fig. 7). Although PMCA4, a Ca²⁺ -ATPase, is expressed in the sperm tail just like the CatSper1 and Ca_v3.3 (20), only marginal NFRET signals (mean value of 0.075 ± 0.009) were observed in the stained sperm, indicating lack of interaction between Ca_v3.3 and PMCA4. Collectively, these results indicate that Ca_v3.3 specifically associates with CatSper1 on the sperm tail.

View larger version (63K): [in this window] [in a new window]   FIGURE 7. FRET analysis of the association of CatSper1 with Cav3.3 on human sperm tail. Human sperm were immunostained either with Alexa 488-labeled anti-CatSper1 antibody (CatSper1-488) and Alexa 555-labeled anti-CatSper1 antibody (CatSper1-555) as a positive control, or with Alexa 488-labeled anti-Cav3.3 antibody (Cav3.3-488) and Alexa 555-labeled anti-CatSper1 antibody (CatSper1-555), or with Alexa 488-labeled anti-PMCA4 antibody (PMCA4-488) and Alexa 555-labeled anti-Cav3.3 antibody (Cav3.3-555) as a negative control. FRET analysis was performed, and donor (Alexa 488), acceptor (Alexa 555), and raw FRET images were taken using corresponding excitation and filter settings. A set of representative images along with the calculated NFRET image is shown. Color scales for NFRET intensity are displayed. Red and blue indicate high and low intensity, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To our knowledge, Ca_v3.3 is the first protein identified that associates with CatSper1 and -2. The presence of coiled-coil domains on the C terminus of CatSper1 and CatSper2 suggests that the C-terminal segments of CatSper proteins likely mediate Ca_v3.3-CatSper interactions. However, the potential promiscuity of coiled-coil interactions with other proteins should be acknowledged, as it is not known whether other proteins with coiled-coil segments associate with Ca_v3.3 as well. The association of both CatSper1 and CatSper2 with Ca_v3.3 initially raised the possibility that these three proteins might form a triple complex. However, when we co-expressed all three proteins in mammalian cells, we failed to detect CatSper2 in the product immunoprecipitated by CatSper1 antibody or vice versa.³ This implies that CatSper1 and CatSper2 may bind competitively to Ca_v3.3, perhaps through a region common in Ca_v3.3.

The physical association of Ca_v3.3 with CatSper1 and -2 suggested functional interactions between these proteins. However, we attempted, but failed, to elicit any currents other than the T-type calcium current from cells expressing both Ca_v3.3 and CatSper1 or -2, suggesting that co-expression of Ca_v3.3 still cannot facilitate the functional expression of CatSper in heterologous systems. It is likely, however, that Ca_v3.3 may not be the sole accessory protein that interacts with CatSper, and we could have missed the identification of other binding partners for CatSper1 or CatSper2 in our GST pulldown experiments. Other proteins in the sperm membrane might associate with CatSper proteins so tightly as a protein complex that precludes its capture by GST-CatSper in the pulldown procedure. We also noticed multiple distinct protein bands specifically in GST-CatSper pulldowns; however, we failed to resolve their identities. This might be due to dynamic range limitation of the MudPIT analysis, considering that the peptides derived from the GST fusion proteins are overwhelmingly abundant and may affect the sampling efficiency of the peptides from other relatively low abundant proteins.

Although co-expression of Ca_v3.3 with CatSper did not reconstitute a novel or distinct ion channel complex, we observed net reduction in Ca_v3.3-evoked T-type Ca²⁺ currents upon co-expression with either CatSper1 or CatSper2 in heterologous expression systems. The whole cell current amplitudes of Ca_v3.3 showed significant reductions of 47 and 40% when co-expressed with CatSper1 and CatSper2, respectively. Interestingly, other biophysical properties of Ca_v3.3 were largely unaltered except for a modest (1-3 mV) shift in activation V of Ca_v3.3 toward the depolarizing direction. The effects of CatSper1 on Ca_v3.3 were replicated using the Xenopus oocyte expression system and are specific because overexpression of CatSper1 does not affect the expression of another channel, viz. HCN2. These studies demonstrate that the association of CatSper proteins with Ca_v3.3 predominantly affects the current amplitude of Ca_v3.3 without altering other channel properties.

Recombinant T-type Ca_v3 channel subunits generally do not require accessory proteins for functional expression in a variety of heterologous expression systems, which is in contrast to L-type calcium channels that are profoundly modulated by and 2 subunits and function as multimeric complexes. It has been reported, however, that _1b and ₂-₁ subunits, typically associated with L-type calcium channels, can also modulate T-type calcium channels, including Ca_v3.3 (21). Unlike CatSper, these auxiliary subunits enhance the current amplitudes of Ca_v3 channels by increasing their cell surface expression. However, physical interactions between T-type Ca²⁺ channel subunits and _1b or ₂-₁ have not been established. In another report (22), it was shown that co-expression of calcium channel ₆ subunit, but not the ₄ or ₇ subunits, with Ca_v3.1 in HEK cells significantly decreases current density without changing the kinetic properties and the protein expression of Ca_v3.1. To date, CatSper1 and CatSper2 remain as the first identified proteins shown to physically associate with and functionally modulate Ca_v3.3 channels. It would be interesting to determine whether CatSper proteins also modulate other T-type Ca²⁺ channel types such as Ca_v3.1 and Ca_v3.2 that are also expressed in sperm.

How does the association of CatSper1 modulate Ca_v3.3 current? It is known that the macroscopic current is proportional to the product of the single channel conductance, the number of channels, the open probability, and the effective driving force. Because we did not see significant changes in the kinetic properties of Ca_v3.3, our data do not support the possibility that the decrease in current density is because of changes in Ca_v3.3 biophysical properties, although this cannot be entirely eliminated without detailed biophysical analysis at a single channel level. In a number of cases, it has been reported that auxiliary proteins may modulate the current density by altering the amount of channel protein on the cell surface (21, 23). To assess whether the reduced amplitude could be attributed to reductions in the number of Ca_v3.3 channels, surface membrane proteins of viable, intact cells were labeled with biotin and quantified. We observed a tendency that co-expression of CatSper1 reduced the surface expression of Ca_v3.3 by about 20%, suggesting that reduction in expression of Ca_v3.3 might contribute to the decreased current density.

The low voltage-activated T-type Ca²⁺ channels produce low threshold spikes that have been shown to trigger burst firing in various cell types. They play important physiological roles in diverse tissues, especially in central and peripheral nervous systems and in the heart (for a review see Ref. 24). In mammalian germ cells and sperm, the expression of all three members of the T-type Ca²⁺ channels have been reported (16, 25), and electrophysiological studies have documented the existence of T-type Ca²⁺ current in mouse spermatogenic cells (26). The most prominent role attributed for T-type Ca²⁺ channels is in the acrosome reaction. Blockade of T-type Ca²⁺ channels during gamete interaction inhibited zona pellucida-dependent Ca²⁺ elevation and acrosome reaction (26). However, although T-type Ca²⁺ channels are present in the sperm head (16), the expression of neither CatSper1 nor CatSper2 was localized in the acrosome region (3, 4). Therefore, it is unlikely that Ca_v3.3-CatSper interactions could play a role in sperm acrosome reaction. Instead, the co-expression and association of Ca_v3.3 and CatSper1 on human sperm tail suggests a role for their interactions in sperm motility.

Ca²⁺ is a key regulator in the initiation and maintenance of hyperactivated motility of the sperm (27). During this process, Ca²⁺ concentration in the cytoplasm rises, which regulates the movement of axoneme (28). The release of Ca²⁺ from membrane-bound internal Ca²⁺ stores, such as the redundant nuclear envelope, is critical for hyperactivated motility (29). Moreover, the increase of cytoplasmic Ca²⁺ can also result from the influx of extracellular Ca²⁺ through the plasma membrane; however, the type of Ca²⁺ channels mediating this process remains elusive. Knock-out studies revealed that mice lacking either CatSper1 or CatSper2 have defects in sperm-hyperactivated motility that is accompanied by a reduction in Ca²⁺ concentration in sperm tail (3, 6), indicating a crucial role for these CatSper proteins in Ca²⁺ influx during hyperactivated motility. The detection of Ca_v3.3 expression on sperm flagellum and, more importantly, its association with CatSper imply that Ca_v3.3 may also play a role in mediating calcium influx required for hyperactivated motility. However, studies with Ca_v3.3 knockout or with selective T-type Ca²⁺ channel blockers are currently unavailable to directly assess the role of Ca_v3.3 in sperm function. It has been reported that weak T-type Ca²⁺ channel inhibitors, mibefradil and gossypol, did not significantly affect sperm basal motility at low concentrations but did cause motility alterations at higher concentrations where high voltage-activated Ca²⁺ channels may also be blocked (16). Accordingly, the assessment of the Ca_v3.3 role in sperm function awaits the identification of potent and selective Ca_v3.3 blockers or knock-out studies.

Should Ca_v3.3 indeed contribute to Ca²⁺ influx during sperm-hyperactivated motility, CatSper1 could modulate Ca_v3.3 function by physical interactions in a region-specific manner. Interestingly, our studies demonstrate a decrease, rather than increase, in the amplitude of Ca_v3.3-mediated Ca²⁺ currents upon co-expression of CatSper1 or CatSper2 in heterologous systems. Although it remains to be proven whether this holds true in the native sperm environment, this observation suggests that CatSper1 and -2 may "fine-tune" Ca_v3.3-mediated current to maintain an optimal level of Ca²⁺ in the principal piece during hyperactivated motility. Indeed, recent studies reveal the presence of both major Ca²⁺ influx and efflux mechanisms in the principal piece. PMCA4, which is a plasma membrane Ca²⁺-ATPase that acts as an extrusion pump to mediate the efflux of excess Ca²⁺ from the cytosol, is highly expressed in the principal piece just like CatSper1 and Ca_v3.3. Targeted ablation of PMCA4 in mice resulted in defects in sperm-hyperactivated motility and male fertility (20, 30). Abnormal mitochondria was observed in PMCA4^-/- sperm, which was attributed to Ca²⁺ overload. Therefore, perhaps a controlled regulation of Ca²⁺ levels, rather than mere increase, could be important for regulating hyperactivated motility of the sperm, and it may be speculated that Ca_v3.3 and CatSper proteins partner together to regulate Ca²⁺ influx needed for hyperactivated motility.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Neuroscience Research, Dept. R4PM, Bldg. AP9-1125, Abbott Laboratories, 100 Abbott Park Rd., Abbott Park, IL 60064-6118. Tel.: 847-937-8271; Fax: 847-937-9195; E-mail: di.zhang{at}abbott.com.

² The abbreviations used are: HEK, human embryonic kidney; MudPIT, multi-dimensional protein identification technology; GST, glutathione S-transferase; GFP, green fluorescent protein; PBS, phosphate-buffered saline; FRET, Fluorescence Resonance Energy Transfer; NFRET, normalized FRET; DTT, dithiothreitol; pF, picofarad.

³ D. Zhang, J. Chen, A. Saraf, S. Cassar, P. Han, J. C. Rogers, J. D. Brioni, J. P. Sullivan, and M. Gopalakrishnan, unpublished observations.

	ACKNOWLEDGMENTS

 
We thank Drs. David Clapham (Harvard Medical School, Boston), Magdalene Moran, and Drew Chong (Hydra Biosciences, Cambridge, MA) for CatSper1 and -2 constructs and helpful discussions. We also thank Drs. Jie Tang and Tariq Ghayur (Abbott Bioresearch Center, Worcester, MA) for valuable assistance during the initial phases of CatSper1 antibody generation and Gerard Gagne, Kerry Swift, and Edmund Matayoshi for advice on FRET analysis.

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