CatSperβ, a Novel Transmembrane Protein in the CatSper Channel Complex*

Four CatSper ion channel subunit genes (CatSpers 1-4) are required for sperm cell hyperactivation and male fertility. The four proteins assemble (presumably as a tetramer) to form a sperm-specific, alkalinization-activated Ca2+-selective channel. We set out to identify proteins associating with CatSper that might help explain its unique role in spermatozoa. Using a transgenic approach, a CatSper1 complex was purified from mouse testis that contained heat shock protein 70-2, a testis-specific chaperone, and CatSperβ, a novel protein with two putative transmembrane-spanning domains. Like the CatSper ion channel subunits, CatSperβ was restricted to testis and localized to the principal piece of the sperm tail. CatSperβ protein is absent in CatSper1-/- sperm, suggesting that it is required for trafficking or formation of a stable channel complex. CatSperβ is the first identified auxiliary protein to the CatSper channel.

During mammalian fertilization, Ca 2ϩ is crucial for sperm capacitation, motility, the acrosome reaction, sperm-egg fusion, and the activation of the fertilization wave in eggs (1). Depolarization, intracellular alkalization, progesterone, cyclic nucleotides, and egg coat proteins trigger Ca 2ϩ influx in mammalian sperm (1)(2)(3)(4). The dominant Ca 2ϩ -selective current in epididymal sperm is mediated via CatSper, although transient receptor potential (TRP), Ca V , and cyclic nucleotide-gated channel proteins have been detected in sperm cells and their precursors (see Refs. 5-7 for review). Of Ͼ20 ion channel genes disrupted in mice, only CatSper family mutations result in male infertility (6).
CatSpers1-4 are expressed in testis and localized primarily to the principal piece of sperm tail (8 -12). CatSpers are most closely related to the six-transmembrane (TM) 2 voltage-gated sodium channel (Na V BP) in bacteria, with the next closest rel-atives being the large mammalian Ca V and Na V channels (13). CatSpers contain positively charged amino acids interspersed within their S4 transmembrane segments, suggesting they are voltage-sensitive channel subunits. CatSper1 is relatively unique and contains a remarkable abundance of histidine residues in its amino terminus, perhaps related to the known pH sensitivity of the CatSper Ca 2ϩ current (14). CatSpers are also present in sea urchin and the ascidian Ciona intestinalis (15).
Targeted disruption of the mouse CatSper1 resulted in complete male infertility in an otherwise normal mouse (9). Although mutant mouse mating behavior, sperm count, and sperm cell morphology were indistinguishable from wild type (WT) mice, mutant sperm motility was abnormal. These sperm had reduced basal velocity and lacked vigorous beating and bending in the tail region. Mutant spermatozoa failed to fertilize intact eggs but could fertilize those in which their outer layers had been enzymatically removed (9). Further studies showed that CatSper null sperm cells could not be hyperactivated under physiological conditions (16,17). Interestingly, depolarization evoked an increase in intracellular Ca 2ϩ ([Ca 2ϩ ] i ) in WT sperm cells, but not in CatSper1 null spermatozoa (16). The phenotype of CatSper2, 3, and 4 Ϫ/Ϫ mice was indistinguishable from CatSper1 Ϫ/Ϫ mice, and their sperm also lacked the hyperactivated motility needed for fertilization (12,18). Whole sperm patch clamp of epididymal sperm showed that CatSper current is absent in CatSper1, 2, 3, and 4 Ϫ/Ϫ mice (12,14). Finally, CatSper genes appear to have similarly important roles in human fertility. Subfertile men with deficient sperm cell motility had significantly reduced expression of CatSper1 (19). CatSper2 has been implicated by linkage analysis in human asthenoteratozoospermia (20).
Despite the essential roles of CatSper proteins in sperm Ca 2ϩ signaling and mammalian fertilization, detailed biophysical and structure-function studies have been hindered by the lack of function in heterologous expression systems essential for these studies. None of the four mammalian CatSper channel subunits expressed in heterologous expression systems, alone (HEK293 cells, Chinese hamster ovary-K1 cells) or in combination (Xenopus oocytes) (9), 3 produced detectable I CatSper . Similarly, two CatSper homologs from sea urchin testis and two from ascidian C. intestinalis did not yield current when expressed in mammalian cells and Xenopus oocytes. 3 In contrast, the functions of the majority of the several hundred channels (K ϩ , Cl Ϫ , Ca 2ϩ , Na ϩ , CNG (cyclic nucleotide-gated), and * This work was supported by grants from the NICHD, National Institutes of Health. 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. transient receptor potential (TRP)) found in somatic cells have been faithfully reproduced in heterologous systems (21,22). One potential reason for the failure of functional heterologous expression is the lack of auxiliary subunits needed for proper channel trafficking, assembly, or gating. In this report, we describe a transgenic strategy to purify proteins associating with CatSper1. We found that a partial CatSper1 complex contained HSP70-2, a testis-specific HSP70 protein, and a novel transmembrane protein, CatSper␤.

EXPERIMENTAL PROCEDURES
Transgenic Animal Generation-Animal protocols followed approved institutional regulations and guidelines. The CatSper1 Ϫ/Ϫ mouse was previously described (9). A BAC clone containing the mouse CatSper1 gene (isolated from an sv129 genomic DNA library) was used to engineer a transgenic construct containing a genomic DNA fragment (ϳ4 kb; from an NheI site) 5Ј to the CatSper1 open reading frame (ORF), and a fragment of ϳ9 kb covering the whole ORF, and 0.5 kb 3Ј to the ORF. A HA-eGFP fusion gene without a stop codon was synthesized by PCR using an eGFP vector (Clontech) as template. This mini-gene was inserted in-frame with the CatSper1 ORF (see Fig. 1 for detail). All fragments generated using PCR were sequenced to ensure that no unintended mutations were introduced. The insert, cloned in the pBluescript II SK vector (Stratagene), was excised, gel-purified, and used for pronuclear injection (Children's Hospital, Boston, MA). Founders carrying the transgene were identified by PCR. Males from two independent lines were subsequently crossed to CatSper1 Ϫ/Ϫ females in order to engineer lines carrying the transgene but lacking WT CatSper1. Mice homozygous for the insertion were generated by crossing hemizygous mice and selected by semi-quantitative genomic PCR and mating tests.
Ca 2ϩ Imaging-Ca 2ϩ imaging was as previously described (9). Briefly, caudal sperm were collected, loaded with Fura-2, and seeded onto coverslips coated with Cell-Tak (BD Biosciences). The ratiometric dye Fura-2 was used to minimize contributions from GFP in the transgenic sperm. Signals collected in the head region were analyzed. Imaging and analysis employed an inverted fluorescence microscope (IX-71; Olympus) with monochromator (DeltaRAM V; PTI), a cooled CCD camera (CoolSNAP HQ; Roper Scientific), data acquisition system, and control software (ImageMaster; PTI). Only cells that were evenly loaded with dye and motile were chosen for analysis (T ϭ 20 -25°C).
In the absence of extracellular divalent ions, CatSper channels became permeable to monovalent cations (14), leading to an apparent reversal potential of ϳ0 mV in Fig. 2A. Recordings were made with an Axopatch 200B amplifier controlled by pCLAMP8.2 software through a Digidata 1322A interface (Axon). Signals were low pass-filtered at 1 kHz and sampled at 5 kHz.
Protein Purification-All purification steps were carried out at 4°C unless otherwise stated. Frozen testes were homogenized in binding buffer (1.5 ml/testis) containing 50 mM NaH 2 PO 4 and 300 mM NaCl, pH 8.0, supplemented with EDTA-free proteinase inhibitor mixture (protease inhibitor cocktail (PIC); Roche Applied Science). The homogenate was spun at 1,000 ϫ g for 10 min at 4°C. The supernatant was centrifuged at 100,000 ϫ g for 50 min to obtain the microsomal fraction. ϳ100 mg of protein was solubilized for 1 h in 40 ml of buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl, 1ϫ detergent and EDTA-free PIC, pH 8.0. Detergent contained 1% Nonidet P-40, 0.05% deoxycholate, and 0.1% SDS (radioimmune precipitation buffer) in one large scale purification, but only 1% Nonidet P-40 in several other such purifications. Following a 15-min spin, the supernatant was added to 1.5 ml of bufferequilibrated cobalt resin and 1.6 ml of imidazole (1 M, pH 7.5), resulting in a final imidazole concentration of 37 mM. The large number of histidine residues in the amino terminus enabled CatSper1 to bind cobalt (9). After mixing 1 h, unbound protein was washed 2ϫ using the same binding buffer supplemented with 40 mM imidazole, followed by wash in pH 6.5 buffer. Bound protein was eluted (22°C) with 3 ml of elution buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl, 300 mM imidazole, 1ϫ detergent, and EDTA-free PIC, pH 7.0. The eluate was concentrated to ϳ0.7 ml (YM-100; Centricon) and the 0.7 ml of concentrate added to 100 l of buffer-equilibrated anti-HA affinity beads (Roche Applied Science) and 3.3 ml of IP buffer containing 150 mM NaCl, 2 mM EDTA, 1ϫ EDTA-free PIC, 0.5ϫ detergent, and 10 mM HEPES, pH 8.0, mixed ϫ 2 h, and washed 3ϫ with IP buffer. Bound protein was eluted with 200 l of HA peptide (5 mg/ml in IP buffer) for 15 min at 37°C. The eluate was concentrated and resuspended in an lithium dodecyl sulfate (LDS) gel loading buffer (Invitrogen). Samples were heated for 15 min at 70°C before being loaded onto a 4 -12% Bis-Tris gradient gel (Invitrogen). After electrophoresis in SDS denaturing buffer, the gel was fixed and stained with Coomassie Blue (R-250) or silver stain for scaled down pilot purifications. Protein bands were excised and stored in 1% acetic acid before protein identification.
Protein Identification-Peptides from in-gel trypsin digestion were separated on a C18 column with a nano liquid chromatography system (Eksigent) and subsequently sequenced online using a nanospray/Qstar-XL mass spectrometer (ABI). Analyst QS software was used for data analysis and Mascot for data base searches (University of Pennsylvania Proteomics Facility).
Cloning of CatSper␤-The 12 peptide sequences (covering 109 amino acids) identified by mass spectrometry were used to search expressed sequence tag and genomic databases. Primers were designed according to the available sequences and used to amplify the whole ORF from mouse testis first strand cDNA by PCR. PCR products were subcloned into a modified pTracer-CMV2 vector (Invitrogen) and multiple clones sequenced. Sequences were blasted against cDNA and genome data bases to ensure that the clones selected for further analysis were free of mutations. The start of the ORF was unambiguously determined by the presence of an inframe stop codon in the 5Ј-untranslated region. The predicted sequences of the human and C. intestinalis CatSper␤ homologs were from NCBI data bases and the C. intestinalis cDNA data base (23).
Multiple Tissue Reverse Transcription PCR-PCR was performed according to standard protocols using commercial multiple panel cDNAs (Clontech) as templates. The sequences of the forward and reverse primers for CatSper␤ amplification were (5Ј to 3Ј) AGGTTCATCGTTTCAAGTTTCCAGTCAC and ACAGTTGTACTTGAGGTGAGTCCAG, respectively. Samples were denatured for 2 min at 94°C, followed by 35-cycle amplification, denature (20 s at 94°C), annealing (20 s at 58°C), and extension (30 s at 72°C). Reactions were incubated at 72°C for 10 min. Aliquots withdrawn from the reactions at 30 amplification cycles were also analyzed. Comparison between products after 30 and 35 cycles indicated no saturation. Mouse G3PDH gene was used as control for cDNA input.
In Situ Hybridization-Frozen mouse testis sections (ϳ10-m thick) were used for in situ staining. Single strand, digoxigenin-labeled RNA probes were synthesized from the double strand DNA templates (nucleotides 1940 -3323) generated using PCR with primers attached to a T7 sequence. This fragment had no significant sequence similarity to other genes. A sense probe was used as a negative control. Hybridizations were washed, and signals were visualized using alkaline phosphate-conjugated anti-digoxigenin antibody and 5-bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolium substrate.
Electrophoresis and Western Blotting-Unless otherwise stated, pre-cast 4 -12% Bis-Tris gradient gels without SDS (from Invitrogen) were used for electrophoresis. All the gels were run in electrophoresis buffer containing SDS. A fragment encoding amino acids 648 -984 was cloned into the vector pET-32(a) and used for bacterial expression. The fusion protein was purified under denaturing condition and used for antibody production in rabbits. Because CatSper␤ is only expressed in testis, anti-CatSper␤ sera were preabsorpted against Sepharose beads conjugated with total protein from mouse brain and kidney to minimize non-specificity. Preabsorpted sera were used at 1:2000 for Western blots.
Immunostaining-Sperm were placed on coverslips and fixed with cold methanol at Ϫ20°C for 10 min, followed by acetone for 1 min. Fixed sperm were blocked with 10% normal goat sera in phosphate-buffered saline for 30 min and incubated with anti-CatSper␤ serum (1:50) at 4°C overnight. After being washed three times with 1% Triton X-100 in phosphate-buffered saline, coverslips were incubated with Alex 594-conjugated goat anti-rabbit IgG secondary antibody (4 g/ml) at 22°C for 1 h and washed three times for 5 min each. Washed coverslips were mounted in 90% glycerol with anti-fade reagent and visualized under a Leica laser scanning confocal microscope. WT and CatSper1Ϫ/Ϫ sperm were processed identically.

RESULTS
Transgenic Approach to CatSper1 Complex Purification-Native ion channel plasma membrane proteins are of low abundance, requiring immunoprecipitation methods. Our anti-CatSper1 antibodies worked well for immunoblotting and immunostaining (9), but not for immunoprecipitation. To circumvent this problem we generated mice transgenic for CatSper1-HA⅐GFP on the CatSper1 Ϫ/Ϫ background. The mouse CatSper1 ORF covers 12 exons spanning ϳ9 kb; an HA epitope-tagged green fluorescence protein (HA⅐eGFP) encoding a mini ORF was inserted into the CatSper1 genomic DNA, immediately 5Ј of the translational start site (Fig. 1, A and B). A genomic DNA fragment of ϳ4 kb 5Ј of the first exon was used to drive the expression of the synthetic fusion gene. We established two independent lines with the transgene in the CatSper1 Ϫ/Ϫ background (Fig. 1C). Both these lines were fertile, suggesting that the HA⅐eGFP⅐CatSper1 fusion protein fully rescued WT CatSper1 function. We did not observe any gross abnormality in the transgenic mice for a period of Ͼ4 years. This transgenic rescue experiment further confirmed that the male sterile phenotype in the CatSper1 Ϫ/Ϫ mice was caused by the deficiency in the CatSper1 gene, but not by any potential spurious mutations introduced in the process of mutant generation.
Normal alkalinization-activated, voltage-dependent I CatSper (Fig. 2) was recorded from HA⅐eGFP transgenic mouse epididymal sperm (n ϭ 7), but was absent in all CatSper1 Ϫ/Ϫ sperm (Ref. 14 and not shown). Fusion of the GFP protein to the amino terminus of CatSper1 did not abrogate the pH sensitivity of I CatSper , because intracellular alkalization (induced by bath application of NH 4 Cl) readily potentiated I CatSper current ( Fig. 2A). Membrane-permeant nucleotides increase [Ca 2ϩ ] i by an unknown process requiring CatSper1 expression (9, 24), although not via direct nucleotide activation of the CatSper channel (14). In HA⅐eGFP⅐CatSper1 transgenic sperm, application of cell-permeable cGMP (8-Br-cGMP, 2 mM) elicited an increase in [Ca 2ϩ ] i (Fig. 2B). Finally, based on observations from Ͼ100 litters over 4 years, the transgenic mice were fertile. Taken together, these data suggest that the HA⅐eGFP⅐CatSper1 fusion gene functionally replaced the native CatSper1.
Commercial antibodies that efficiently precipitated the CatSper1 fusion protein from mouse testis were selected for use in fusion protein purification (Fig. 3A). In addition, the numerous amino-terminal histidine residues served as native metal binding sites. Indeed, CatSper1 bound well to a cobalt column under relatively stringent conditions (pH 6.5 wash with 1% Triton X-100, 40 mM imidazole). With these two "handles" on CatSper1, a simple strategy could be designed to purify the channel protein complex (Fig. 3B). The HA⅐eGFP-tagged CatSper1 protein solubilized from the transgenic testes was enriched on a cobalt column, followed by purification on an anti-HA antibody column. The protein eluted with the HA peptide was separated by SDS-PAGE. Proteins from CatSper1 Ϫ/Ϫ (for small scale pilot purification) or WT (untagged CatSper1; for large scale purification) testes were used as negative controls. The purification procedure was highly efficient (silver stain detection with Ͻ5 mg of membrane protein starting material from 1-3 mice).
Identification of the CatSper1 Protein Complex-Three specific bands were identified (Fig. 3C). The bands from a purification using ϳ100 mg of protein (60 testes) were stained with Coomassie Blue, excised, trypsin-digested, and identified using mass spectrometry. Peptides from one band were identified as CatSper1 and GFP proteins. The second band was identified as testis-specific HSP70 (HSP70-2) (25). From the 12 peptides in the third band, a novel protein (CatSper␤) was identified. These three proteins (GFP-CatSper1, HSP70-2, and CatSper␤) were identified in two independent purifications. Using an HSP70-2-specific antibody for Western blot (25), we confirmed that HSP70-2 could be precipitated with anti-HA antibody from the transgenic mouse testis, presumably by the interaction between HSP70-2 and HA⅐eGFP⅐CatSper1, but not with anti-FLAG M2 antibody (negative control; not shown).
CatSper␤, a Novel Transmembrane Protein in the CatSper1 Complex-CatSper␤ was identified from data base searches and reverse transcription PCR. The full-length mouse CatSper␤ (GenBank TM accession number EF199807) encodes an 1109-amino acid protein (126 kDa; calculated pI of 8.6; Fig. 4A). CatSper␤ contains two clear transmembrane domains (6 -22 of the NH 2 terminus, and 1060 -1076 at the end of the COOH domain; Fig. 4B). A large extracellular domain (ϳ1000 amino acids) precedes the carboxyl terminal transmembrane segment. The predicted mouse intracellular carboxyl terminus is only 27 amino acids in length. Eight N-glycosylation sites are predicted in the polypeptide. The overall 2-TM topology is reminiscent of that of the large conductance K ϩ channel (BK) ␤ subunits and the P2X receptor (ATP-gated channel) (26,27), but CatSper␤ (ϳ126 kDa) is significantly larger (BK ␤ subunits Ͻ30 kDa) (28). Data base searches indicate that CatSper␤ is not predicted to be similar to any other protein of established function, although a putative extracellular fragment (Val-166-Gly-294) is weakly similar (42% similarity, 24% identity) to the extracellular loop of a P2X receptor (P2X3b, GenBank TM accession number NP_945337). Human CatSper␤ (GenBank TM accession number AK126034) is located on chromosome 14. Mouse (C57BL/6) has two copies of CatSper␤, separated by ϳ37 kb on chromosome 12. For reasons not fully understood, fertilization-specific proteins seem to be less conserved between species (29). Like CatSper1, CatSper␤ is relatively poorly conserved between human and mouse (amino acid identity ϳ56%; Fig. 4A). A CatSper␤ homo- FIGURE 3. Purification of the CatSper1 protein complex. A, transgenic testis membrane proteins were solubilized and precipitated with anti-GFP or anti-HA antibodies. The eluate and flow-through were probed with anti-CatSper1 antibody. B, strategy used to purify the CatSper1-containing protein complex. C, the purified CatSper1 protein complex was separated by SDS-PAGE and stained with silver. Proteins from CatSper1 Ϫ/Ϫ testes were used as controls. Three specific bands are indicated by arrows. log in the marine chordate C. intestinalis is predicted to encode a 977-amino acid protein. The sequence identity between the mouse and C. intestinalis CatSper␤ homologs is 21%, with highest homology in the region close to the carboxyl terminus (not shown).
Restricted Expression of CatSper␤ mRNA in Testis-Among the eight adult tissues/organs and embryos of four developmental stages examined, only testis had detectable expression of CatSper␤ mRNA (Fig. 5A). In situ mRNA antisense revealed robust, specific staining of CatSper␤ in the seminiferous tubules; no significant signal was detected in the interstitial cells (Fig. 5B). Within the tubules, CatSper␤ appears to be expressed in spermatocytes and spermatids, but not in spermatogonia (Fig. 5B), similar to CatSpers 1-4.
Disruption of CatSper␤ Protein in CatSper1 Ϫ/Ϫ Sperm-We developed a polyclonal antibody against amino acids 648 -984 of CatSper␤ protein. Taking advantage of the restricted expression of CatSper␤, nonspecific activity of the antibody was reduced by preabsorption of the antibody sera by total protein from mouse brain and kidney. The purified antibody specifically recognized a protein band from CatSper␤-transfected HEK293 cells, but not from mock-transfected cells (Fig. 6A). The anti-CatSper␤ antibody also recognized a band of ϳ130 kDa from cobalt-purified testis proteins, presumably by association between CatSper␤ and the histidine-rich CatSper1 (not shown). Anti-GFP antibody (but not anti-FLAG M2) immunoprecipitated CatSper␤ from HA⅐eGFP⅐CatSper transgenic testicular protein, further confirming the association of the two proteins (Fig. 6B).
In mouse sperm, the CatSper␤ antibody recognized a single major band migrating at the same position as recombinant CatSper␤ protein from HEK293 cells (Fig. 6C). Interestingly, CatSper␤ was undetectable in the CatSper1 mutant sperm, but was present in CatSper1 mutant testes. In HA⅐eGFP⅐CatSper1 transgenic mice, CatSper␤ protein was again detected in the mutant sperm (Fig. 6C). Like CatSper1, CatSper␤ protein was localized to the sperm principal piece and was largely absent in the CatSper1 Ϫ/Ϫ sperm (Fig. 6D). These data suggest that CatSper␤ protein is colocalized with, and dependent upon,  1-12); H 2 O served as negative control (lane "-"). Lanes 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, testis; 9, 7-day embryo; 10, 11-day embryo; 11, 15-day embryo; 12, 17-day embryo. B, representative fields of in situ hybridization in mouse testis using antisense probes corresponding to a unique portion of the CatSper␤ sequence. Sense probe served as a background control (lower right panel). Scale bar, 50 m. A, anti-CatSper␤ antibody specifically recognized protein from CatSper␤ cDNA-transfected HEK293 cells, but not from mock-transfected cells. B, interaction between CatSper␤ and HA⅐eGFP⅐CatSper1. Total testis membrane proteins (ϳ100 g) prepared from the HA⅐eGFP⅐CatSper1 transgenic mice were immunoprecipitated with anti-GFP or anti-FLAG M2 antibody (negative control) and blotted with anti-CatSper␤ antibody. The lower bands are presumed immunoglobulins (Ig). C, disruption of CatSper␤ protein in sperm, but not in testis, in the CatSper1 mutant mice. Total sperm (7.5 ϫ 10 5 cells) (upper) or testis (50 g) (lower) proteins prepared from WT (ϩ/ϩ), CatSper1 Ϫ/Ϫ , and the HA⅐eGFP⅐CatSper1 transgenic (Tg; in CatSper1 Ϫ/Ϫ background) mice were blotted with anti-CatSper␤ antibody. Protein expressed from CatSper␤-transfected HEK293 cells was loaded on the same gel as the molecular weight reference (lane ctrl). Prolonged exposure did not reveal a CatSper␤ band in the CatSper1 Ϫ/Ϫ lane (not shown). The migrations of CatSper␤ in panels B and C were aberrant due to lower SDS content of the commercial gel compared with our own in panel A. D, immunofluorescence detection with anti-CatSper␤ antibody in WT (ϩ/ϩ) and CatSper1 (Ϫ/Ϫ) sperm. Immunofluorescence (in green) localizes specifically to the principal piece. Scale bar, 10 m. CatSper1 protein. Because CatSper2, 3, and 4 antibodies are not as specific for their targets by immunohistochemistry, CatSper␤ dependence on these proteins will be examined in more detail using CatSper 2, 3, and 4 Ϫ/Ϫ mice.

DISCUSSION
We used a tagged CatSper1 transgenic mouse to enable unambiguous purification of CatSper1-associating proteins from testes. Previous work has suggested that CatSpers1-4 form a heterotetrameric complex surrounding the Ca 2ϩ -selective pore (12). In this report, we have shown that a novel auxiliary subunit, CatSper␤, accompanies CatSper proteins. We suggest that CatSper␤ is a subunit of the CatSper complex based on its in vitro co-purification, in vivo colocalization, disappearance in CatSper1 Ϫ/Ϫ sperm, and reappearance in sperm from mice rescued with tagged CatSper1. Interestingly both CatSpers and CatSper␤ are lacking in Caenorhabditis elegans and Drosophila but both appear in the ascidian C. intestinalis, suggesting that the origin of this molecular complex structure can be traced back to before the divergence among deuterosomes.
The CatSper channel complex is reminiscent of the maxi-K Ca 2ϩ -activated K ϩ channel, which consists of a pore-forming ␣ subunit (7-TM) and a 2-TM ␤ subunit. Many other channel complexes have transmembrane auxiliary subunits. In Ca V channels, the single TM ␣2/␦ subunit not only increases channel protein surface expression but also affects current kinetics (30). The 1-TM ␤ subunit of Na V channels has a short intracellular tail but large extracellular domains that also mediate cell-cell interaction (31,32). The CatSper␤ protein is predicted to have a large extracellular fragment (ϳ1000 amino acids) with several putative N-glycosylation sites, and we speculate that these extracellular domains could be "sensors" for sperm interactions with other cell types or surfaces.
Not surprisingly, heat shock proteins have been implicated in channel assembly, including the HERG K ϩ channel (33), the ClC-2 Cl Ϫ channel (34), and the CFTR Cl Ϫ channel (35). Here, we find that the testis-specific HSP70-2 purifies with CatSper1, suggesting that sperm-specific membrane proteins such as channels require unique chaperones for their correct folding/assembly or trafficking. Unfortunately, preliminary experiments in which HSP70-2, CatSper␤, and all four CatSper subunits were expressed in Xenopus oocytes did not yield functional currents. Similarly, we did not observe changes in the subcellular localization of CatSper1 protein when co-expressed with CatSper␤ in cultured HEK293 cells. Functional expression of sperm-specific proteins commonly fails in heterologous systems, as it does in proteins specific to other ciliated structures (e.g. olfactory receptors) (36). We can only speculate as to why this might be so; functional heterologous CatSper channel formation may require 1) additional unknown protein subunits, 2) an intact intraflagellar transport system (37), 3) the unique lipid composition of sperm plasma membrane (38), 4) sperm-specific cytosolic composition (39), or 5) all the above. Whatever the reasons may be for this complexity, the CatSper protein complex appears to consist of some of the most unique and diverse elements in the field of ion channels.