Identification of human and mouse CatSper3 and CatSper4 genes: Characterisation of a common interaction domain and evidence for expression in testis
© Lobley et al; licensee BioMed Central Ltd. 2003
Received: 01 July 2003
Accepted: 01 August 2003
Published: 01 August 2003
CatSper1 and CatSper2 are two recently identified channel-like proteins, which show sperm specific expression patterns. Through targeted mutagenesis in the mouse, CatSper1 has been shown to be required for fertility, sperm motility and for cAMP induced Ca2+ current in sperm. Both channels resemble a single pore forming repeat from a four repeat voltage dependent Ca2+ /Na+ channel. However, neither CatSper1 or CatSper2 have been shown to function as cation channels when transfected into cells, singly or in conjunction. As the pore forming units of voltage gated cation channels form a tetramer it has been suggested that the known CatSper proteins require additional subunits and/or interaction partners to function.
Using in silico gene identification and prediction techniques, we have identified two further members of the CatSper family, CatSper3 and Catsper4. Each carries a single channel-forming domain with the predicted pore-loop containing the consensus sequence T×D×W. Each of the new CatSper genes has evidence for expression in the testis. Furthermore we identified coiled-coil protein-protein interaction domains in the C-terminal tails of each of the CatSper channels, implying that CatSper channels 1,2,3 and 4 may interact directly or indirectly to form a functional tetramer.
The topological and sequence relationship of CatSper1 and CatSper2 to the four repeat Ca2+ /Na+ channels suggested other members of this family may exist. We have identified a further two novel CatSper genes, conserved in both the human and mouse genomes. Furthermore, all four of the CatSper proteins are predicted to contain a common coiled-coil protein-protein interaction domain in their C-terminal tail. Coupled with expression data this leads to the hypothesis that the CatSper proteins form a functional hetero-tetrameric channel in sperm.
Channel activities, particularly those of calcium channels, have been linked to the process of sperm maturation, motility and to the sperm egg interaction. A number of candidate channels have been associated with these processes, these include the voltage operated Ca2+ channels alpha1A, 1C and 1E plus beta subunits [1, 2], the T-type voltage operated Ca2+ channels alpha 1G and 1H, cyclic nucleotide gated (CNG) channels  and the transient receptor potential (TRP) channel TRP2 , The evidence being primarily based on transcript and immuno-staining studies. However, none of the channels expressed in testes and sperm have been directly associated with sperm mobility. Recently, two novel channel-like proteins, CatSper1 and CatSper2 (Cat ion channel of Sper m), were identified to be specifically expressed in spermatozoa and to be linked to sperm mobility [[5, 6]; reviewed in ].
The human and mouse CatSper channels, CatSper1 and CatSper2, both carry a single six-transmembrane spanning unit analogous to one of the four repeats found in voltage-dependent Ca2+ channels [5, 6]. Analysis of the pore forming region within the repeat suggested that CatSper1 and 2 are Ca2+ selective [5, 6]. Further evidence supporting channel activity has been provided for CatSper1 by gene-targeting experiments in the mouse . Notably in sperm from the mice carrying two null alleles for the CatSper1 gene, cAMP and cGMP induced Ca2+ influx is lost. Moreover, CatSper1 has been shown to be required for normal sperm motility and egg penetration. However, attempts to define channel activity for CatSper1 and CatSper2 – singly or in conjunction – in heterologous expression systems have failed [5, 6]. This suggests that additional factor or factors are required to form a functional channel. The fact that CatSper1 and 2 share features of a single repeat of a four repeat channel suggests that an additional two members might exist.
Here we describe the prediction of two additional CatSper channels in the human and mouse genomes, CatSper3 and CatSper4. Both channels contain a single six-transmembrane repeat domain, which contain the T×D×W pore-lining consensus sequence present in CatSper1 and CatSper2. Based on accompanying EST, cDNA library source information and Taqman data, both genes are expressed in testis. Furthermore, we noted that each CatSper channel is predicted to contain a coiled-coil motif, a protein-protein interaction interface, in its intra-cellular C-terminal tail. Based on a common expression pattern and the fact that each CatSper protein is predicted to contain a coiled-coil domain, we hypothesise that the CatSpers come together to form functional tetrameric channels either by direct interaction of their coiled-coil motif or through interaction with additional factors.
Identification of CatSper3 and 4
PSI-BLAST profiles were constructed from sequence alignments of the ion-transport domain of CatSper protein sequences and calcium channel protein sequences. These profiles were used as input to the PSI-BLAST algorithm  to search large human and mouse genome based protein databases for potential novel members of the CatSper protein family. Two GENSCAN  human gene predictions partially covering the ion transport domain region were identified. The GENEWISE program  was used to improve and extend the original gene predictions into full-length proteins using CatSper2 protein sequence as a template. Orthologous mouse CatSper3 and mouse CatSper4 sequences were identified by mapping syntenic regions of CatSper3 and CatSper4 human loci to the mouse genome. The GENEWISE program was again used to craft the final mouse proteins from their DNA, seeded by human CatSper3 and human CatSper4 predictions. Overlapping EST sequences listed in Table 5 were also used to improve predictions.
Human RNA prepared from non-diseased organs was purchased from either Ambion Europe or Clontech. cDNA was prepared from 500 ng RNA using random hexamers and Multiscribe (Applied Biosystems), following manufacturer's instructions.
Oligonucleotide primers and probes were designed using Primer Express software (Applied Biosystems) with a GC-content of 40–60%, no G-nucleotide at the 5'-end of the probe, and no more than 4 contiguous Gs. Each primer and probe was analysed using BLAST (Basic Local Alignment Search Tool) . Results confirmed that each oligonucleotide recognises the target sequence with a specificity >3 bp when compared to other known cDNA's or genomic sequence represented in the NCBI publicly available databases .
The sequence of the primers and probes directed against human CatSper4 exon 9 are as follows:
Forward primer: 5'-AAGGACATCCGCCAGATGTC-3'
Reverse primer: 5'-GGCACACCTTTTCCATGCTAA-3'
Expected amplicon size is 70 bp, a test PCR reaction was carried out under the following conditions; 40 cycles, 95°C 30 seconds, 58°C 30 seconds, 72°C 30 seconds. Expected amplicon size was confirmed on an agarose gel.
18S rRNA pre-optimised primers and probe were purchased from Applied Biosystems, Foster City, CA.
25 μl PCR reactions were carried out using TaqMan Universal Master Mix (Applied Biosystems) following manufacturer's instructions and as described in Lobenhofer et al .
Each sample reaction contained 100 nM Taqman probe; 300 nM forward primer; 900 nM reverse primer and 15 ng of cDNA template. Within each experiment, a standard curve was carried out of a typical tissue sample, from 50 ng to 0.78 ng of cDNA template. From this standard curve, the amount of actual starting target or 18S cDNA in each test sample was determined. The levels of target cDNA in each sample were normalised to the level of expression of target in a comparative sample. The levels of 18S cDNA in each sample were normalised to the level of expression of 18S in a comparative sample. The data was then represented as fold expression of target sequence normalised to 18S expression relative to the level of expression in the comparative sample, which was set arbitrarily to 1.
Characterisation of the CatSper channel family
CatSper3 and CatSper4 transmembrane regions were predicted using the TMHMM program  and delineated by analogy with other CatSper and calcium-channel family members. Coiled-coils were predicted using the COILS algorithm for each member of the CatSper channel family .
Sequence Alignments and Phylogenetic Tree
Sequence alignments of the CatSper protein family, and calcium channels were created using CLUSTALW multiple sequence alignment program  and hand-crafted using the JALVIEW sequence alignment editor . Pairwise sequence identities presented in tables 6 and 7 were calculated using the pairwise sequence alignment algorithm present in the JALVIEW software.
The phylogenetic tree shown in figure 8 was constructed from sequence alignments of the CatSper protein family with calcium channel sequences CCAA_HUMAN, CCAH_HUMAN, CCAG_HUMAN, CCAS_HUMAN, CCAC_HUMAN over the ion-transport domain region. PHYLIP  PROT-PARS maximum parsimony programme was used to build 1000 bootstrap trees from the sequence alignment. The final tree was obtained using the CONSENSE programme to select the best tree by majority rule.
Identification of CatSper 3 and 4
As part of an on going program to identify novel ion channel encoding genes, ion-channel family sequence profiles have been used to search sets of human gene predictions. Two initial GENSCAN  predictions of 264 aa and 185 aa mapping to human chromosomes 5q31.1 and 1p35.3 respectively contain features related to the previously described CatSper genes – namely a single ion-transport domain and a pore-loop containing the consensus T × D × W. The predictions have been hand polished using a combination of GENSCAN and GENEWISE  analysis, coupled with Expressed Sequence Tag (EST) data and homology between human and mouse chromosomes to obtain full-length gene models.
Exon/intron boundaries of human CATSPER3
Exon Length bp
Intron Length bp
Exon/intron boundaries of mouse CATSPER3
Exon Length bp
Intron Length bp
Notably, exon 1 of human CatSper3 lies within the 3'UTR of the DCOHM gene (dimerisation cofactor of hepatocyte nuclear factor from muscle: Genbank AF499009) such that the two genes are in a head-to-tail orientation. The DCOHM cDNA has been isolated from muscle and kidney libraries, whereas available tissue distribution information for human CatSper3 points to a predominantly testis specific expression. Therefore transcriptional interference is unlikely to occur between the two genes. Using the human DCOHM as a query sequence, an ORF of 90% sequence identity can also be found in the mouse genome 8.5 kb upstream of the mouse CatSper3 start codon. Therefore a similar gene arrangement to the human loci exists in the mouse (data not shown). However, we do have any information relating to the extent of the mouse DCOHM 3'UTR as this has not yet been cloned. Therefore, the mouse DCOHM gene may or may not extend over the mouse CatSper3 coding exons.
Exon/intron boundaries of human CATSPER4
Exon Length bp
Intron Length bp
Exon/intron boundaries of mouse CATSPER4
Exon Length bp
Intron Length bp
Representation of CATSPER 3 and 4 in sequence databases and associated tissue sources.
BI827754 901 bp (Brain-medulla)
AX358304 – WO0194412 (No Tissue distribution)
AI219834 337 bp (Pooled Testis-lung-B cell)
AX047619 – WO0066735 (Predominantly Testis by Northern blot)
AI027609 362 bp (Testis)
AW003058 306 bp (Germ cell tumor)
AW972257 417 bp (Colon cancer)
AW593391 306 bp (Germ cell tumor)
AW590264 306 bp (Germ cell tumor)
AW003002 306 bp (Germ cell tumor)
AW008956 258 bp (Colon)
AW007549 256 bp (Colon)
AA527520 255 bp (Colon)
BX280235 212 bp (Pooled Testis-lung-B-cell)
AK014942 1312 bp (Adult testis)
BY714458 951 bp (Adult testis)
BB679726 390 bp (Adult testis)
BY510167 408 bp (Bone marrow macrophage)
CA465993 758 bp (Testis)
BF147131 335 bp (Testis)
AV043837 252 bp (Testis)
AV280964 263 bp (Adult testis)
BB017226 225 bp (Adult testis)
BB013542 244 bp (Adult testis)
AA421134 518 bp (Testis)
AK077145 1713 bp (Adult testis)
AV280157 622 bp (Adult testis)
BB617038 605 bp (Adult testis)
BY096088 378 bp (Adult testis)
BY088873 365 bp (Pooled adult tissue)
BY454059 444 bp (Pooled adult tissue)
BU961662 795 bp (Testis)
BY459016 398 bp (Adult testis)
AV269930 241 bp (Adult testis)
AV281460 247 bp (Adult testis)
AV263155 176 bp (Adult testis)
Sequence identities shown between human CatSper family members
Sequence identities shown between calcium channel repeats within the same gene and orthologous repeats. CcaH and CcaG sequences correspond with Swissprot identifiers CCAH_HUMAN and CCAG_HUMAN T type calcium channels. CcaC and CcaS represent Swissprot sequences CCAS_HUMAN and CCAC_HUMAN L type calcium channels.
Having found two further members of the CatSper family in human and mouse genomes a search for orthologues in Fugu rubripes and Danio rerio was carried out. Searching with CatSper sequences against raw genomic sequence (TBLASTN)  and ENSEMBL protein predictions (BLASTP) failed to identify any orthologues. Furthermore we failed to identify any channel-like sequence of less than 400 aa containing a pore-forming region of the consensus T×D×W. Given the current coverage of the Fugu genome it is surprising that no CatSper-like sequences were identified.
Tissue distribution of Human CatSper4
Ion specificity is determined by a pore consensus sequence [T/S] × [D/E] × W in voltage gated Calcium channels . Sequence analysis of this region in the CatSpers highlights the presence of a similar conserved motif T×D×W (Figure 5 and 6b) suggesting that the CatSper ion channels may be selective for calcium ions, as previously discussed for CatSper1 . BLASTP homology searches also link the CatSpers most closely with the T-type Calcium channels.
CatSper3 and CatSper4 extend the Calcium channel family
The CatSper ion channel subunits are distant in sequence relationship; sequence identity ranges between 21.6% and 26.5% across the ion transport domain (Table 6). This low sequence identity is in contrast with that observed for the voltage-gated sodium and calcium channel families. Calcium L-type calcium channels generally share ~25% sequence identity over full their length sequence and upwards of 75% sequence identity between their corresponding ion-transport repeat regions (Table 7). These observations are further supported by the phylogenetic tree (Figure 8) which shows that each repeat is more closely related to its analogous repeat in a paralagoue than to the other repeats in the same gene unit, i.e. repeat I in alpha 1S is more closely related to repeat I in alpha 1T than to repeat II in alpha 1S. Figure 9 shows the repeat topology of a voltage-gated cation channel. In addition, repeats 1 and 3, and repeats 2 and 4 share common ancestry with all four repeats stemming from a single common ancestor (Figure 8). In contrast, the CatSper family members do not associate with any one particular repeat, this therefore raises questions over the detailed evolutionary history of the CatSper family.
Here, we applied bioinformatic tools in a focused approach to identify and characterise novel ion-channel genes in both human and mouse genomes. We identified two genes, CatSper3 and CatSper4, which extend the CatSper ion channel-like family to four members in human and mouse. As previously described for CatSper1 and 2 [5, 6], CatSper3 and 4 contain a single ion transport domain comprised of 6 transmembrane spanning regions, where the fourth transmembrane region resembles a voltage sensor and a pore forming region lies between transmembrane regions 5 and 6. The pore contains the consensus sequence T×D×W indicative of a probable calcium selective channel. Available expression data suggest that CatSper3 and 4 are present in testis and may also be found in other tissues. To date, CatSper1 and 2 have not shown channel activity when expressed in heterologous systems alone or when co-expressed. One explanation is that additional factors are required for full function. The identification of two more CatSper like channels both of which show expression in testis and both of which resemble single pore forming repeats from a multi-repeat channel, may well provide the missing factors required for a functional CatSper channel to be formed.
The above model for CatSper subunits function and interactions could be tested in a variety of experiments. Function may be tested through targeted mutagenesis experiments of the new CatSper subunits in mice as described by Ren et al . Expression of all four subunits in an heterologous expression system could be attempted with the aim of reconstituting a functional channel. To identifying interactions via the coiled-coil domain, the intracellular domain of the CatSper subunits could be used as the "bait" in the yest two-hybrid system. This system was successful in identifying the GABABR2 receptor as the co-receptor GABABR1  via a coiled-coil domain. Certainly the identification of two further CatSper subunits provides further possibilities in which to test this family of protein's function in sperm mobility and fertility.
An interesting question posed by identification of four CatSper genes is how did the CatSper family evolve. Sequence comparison between family members show each CatSper paralogoue to be equally distant from each other, i.e. only around 25% sequence ID. Low sequence identity would argue for an early duplication event or that the CatSper subunits have resulted from convergent evolution of ion channel genes at different chromosomes towards a common function. However, we cannot detect any CatSper like channels in species lower than mouse. This observation would argue for a more recent evolutionary event or rapid evolution. It is notable that sequence identity between repeats within a multi-repeat channel share similar identities to those shared between the CatSper channels ie around 25%. We explored the possibility that a particular CatSper channel would represent one of the four repeating units found in channels such as the L-type calcium channel. However, we cannot form a direct one-to-one relationship between a particular channel repeat and a CatSper unit to support this theory.
CatSper ion-channels present themselves as attractive potential targets for non-hormonal contraceptives. Benoff et al  have already illustrated the reversible contraceptive effect of Nifedipine, a widely used calcium-channel blocker in the treatment of high blood pressure and migraine. These effects are mediated via voltage-gated calcium-channels, primarily the L-type voltage-gated channels. The relationship of the CatSper subunits to the voltage-gated calcium channels, their established role in sperm motility and their testis restricted expression pattern, therefore makes them a highly validated target for the identification of novel contraceptives.
Based on our identification of two novel CatSper channels and interaction domains we have presented a theoretical model that suggests the CatSper proteins form subunits of a hetero-tetrameric Ca2+ channel in sperm. We also further suggest that experimental determination of this hypothesis and pharmacological studies may lead to the identification of non-hormonal contraceptives.
Note added in proof
Human CatSper 3 and CatSper 4 predicted sequences have been submitted to EMBL Nucleotides databases under the accession numbers BN000272 and BN000273 respectively.
The authors would like to thank John Overington, Richard Fagan, Janet Allen and colleagues at Inpharmatica for helpful comments and discussions.
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