Identification of human and mouse CatSper3 and CatSper4 genes: characterisation of a common interaction domain and evidence for expression in testis

Abstract

Background: 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.

Results: 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 TxDxW. 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.

Conclusions: 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.

Figure 1

Genomic organisation of the human and mouse CatSper3 genes. (a) Schematic of human and mouse CatSper3 genes on human chromosome 5q31.1 and mouse chromosome 13 respectively. Horizontal line represent human genome assembly NCBI 31 and mouse genome assembly NCBI 03, filled boxes represent coding regions, un-filled boxes represent non-coding regions (b) Comparison of exon boundaries between human and mouse genes, exons are shaded alternately, MSper3v1 and MSper3v2, represent the predicted splice variants of mouse CatSper3. Predicted transmembrane regions are underlined, the pore forming region is underlined with a dashed line.

Figure 2

Genomic organisation of the human and mouse CatSper4 genes. (a) Gene structure of human and mouse CatSper4 genes on human chromosome 1p35.3 and mouse chromosome 4 band D3 respectively. Horizontal line represent human genome assembly NCBI 31 and mouse genome assembly NCBI 03, filled boxes represent coding regions, un-filled boxes represent non-coding regions. (b) Comparison of exon boundaries between human and mouse genes, exons are shaded alternately. Predicted transmembrane regions are underlined, the pore forming region is underlined with a dashed line.

Figure 3

Normalised expression of Human CatSper4 in 18 normal human tissues. (a) Amplicon size of human CatSper4, amplified from exon9. Lane 1 no template control, Lane 2 15 ng Testis cDNA, Lane 3 40 ng testis cDNA, Lane 4 DNA size marker ascending in 100 bp intervals from 100 bp upwards (Eurogentec). (b) Levels of human CatSper4 mRNA in 18 normal human tissues were determined using Taqman quantitative RT-PCR. Each sample was quantitated in 3 individual experiments, the mean ± SEM for the multiple experiments are shown. Tissue names followed by (2) represents an alternative RNA supplier.

Figure 4

Topology of the CatSper channels. (a) Topology diagram of CatSper1 protein based on Ren et al. (b) Topology diagram of CatSper2,3 and 4 proteins.

Figure 5

Multiple sequence alignment of the CatSper ion channel family ion-transport domain. Transmembrane regions are underlined in black and the S4 voltage sensor transmembrane helix is highlighted in red. The channel pore consensus sequence motif is boxed in blue. Genbank accession codes for the published Catsper genes are as follows: MSper1 AF407332, HSper1 AF407333, MSper2 AF411816, HSper2v1 AF411817, HSper2v2 AF411818, HSper2v3 AF411819.

Figure 6

Alignment of Human CatSper voltage sensor and pore forming regions with selected L- and T-type calcium channels. (a) Voltage sensor region of CatSper human protein sequences aligned with repeats 1–4 of selected human L- and T-type calcium channels; CCAA_Human – calcium channel alpha1A (SWISSPROT O00555), CCAS_Human – calcium channel 1S (SWISSPROT Q13698) and CCAH_Human – calcium channel 1H (SWISSPROT O95180). (b) Pore selectivity region of CatSper ion channel family aligned with selected human L- and T-type calcium channels ion transport repeats.

Figure 7

Results from CatSper channel coiled-coil predictions. (a) CatSper1 human coiled-coil prediction, (b) CatSper2 human coiled-coil prediction, (c) CatSper3 coiled-coil prediction and (d) CatSper4 coiled-coil prediction. X-axis, amino acid residue numbering of query sequence. Y-axis, probability score for a sequence adopting a coiled-coil configuration calculated for a scanning window of 14, 21 or 28 amino acid residues.

Figure 8

Un-rooted phylogenetic tree showing CatSper family members with selected T- and L-type calcium channels. Repeats 1–4 of selected human L- and T-type calcium channels; CCAA_Human – calcium channel alpha1A (SWISSPROT O00555), CCAC_Human – calcium channel alpha1C (SWISSPROT Q13936), CCAG_Human – calcium channel alpha1G (SWISSPROT O43497), CCAH_Human – calcium channel 1H (SWISSPROT O95180) and CCAS_Human – calcium channel alpha1S (SWISSPROT Q13698).

Figure 9

Topology diagram for the L-type and T-type four repeat voltage gated calcium channel families.

Figure 10

Theoretical model for the formation of a CatSper hetero-terameric channel. (a) Diagramatic representation of CatSper subunits 1–4. (b) In this model a tetrameric channel is formed via direct interaction of the coiled-coil domains. (c) In this model the channel is brought together via an auxiliary protein or proteins, interaction being mediated via the coiled-coil domains.