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

Lobley A, Pierron V, Reynolds L, Allen L, Michalovich D - Reprod. Biol. Endocrinol. (2003)

Bottom Line: However, neither CatSper1 or CatSper2 have been shown to function as cation channels when transfected into cells, singly or in conjunction.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Target Discovery, Inpharmatica Ltd, 60 Charlotte Street, London W1T 2NU, UK. a.lobley@inpharmatica.co.uk

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.

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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.
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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.

Mentions: The previously described CatSper sequences, CatSper1 and CatSper2 are expressed in testis and more specifically spermatocytes. Data from ESTs and patent literature suggest that CatSper3 also shares a common expression profile. As there was limited evidence supporting the human CatSper4 transcript we carried out a Taqman quantitative PCR analysis to address expression of the Human Catsper4 gene. A primer probe set was designed within exon 9 of human CatSper 4 sequence and tissue expression profiling was carried out in 18 normal human tissues as described in Materials and Methods. Figure 3a shows the correct amplicon size for primers directed against human CatSper4 exon9 and figure 3b shows the normalised level of expression of CatSper 4 in the 18 tissues. These data confirm the prediction of testis specific expression. Low expression levels were detected in placenta and lung, whereas no significant expression was detected in any other tissue.


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

Lobley A, Pierron V, Reynolds L, Allen L, Michalovich D - Reprod. Biol. Endocrinol. (2003)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC184451&req=5

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.
Mentions: The previously described CatSper sequences, CatSper1 and CatSper2 are expressed in testis and more specifically spermatocytes. Data from ESTs and patent literature suggest that CatSper3 also shares a common expression profile. As there was limited evidence supporting the human CatSper4 transcript we carried out a Taqman quantitative PCR analysis to address expression of the Human Catsper4 gene. A primer probe set was designed within exon 9 of human CatSper 4 sequence and tissue expression profiling was carried out in 18 normal human tissues as described in Materials and Methods. Figure 3a shows the correct amplicon size for primers directed against human CatSper4 exon9 and figure 3b shows the normalised level of expression of CatSper 4 in the 18 tissues. These data confirm the prediction of testis specific expression. Low expression levels were detected in placenta and lung, whereas no significant expression was detected in any other tissue.

Bottom Line: However, neither CatSper1 or CatSper2 have been shown to function as cation channels when transfected into cells, singly or in conjunction.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Target Discovery, Inpharmatica Ltd, 60 Charlotte Street, London W1T 2NU, UK. a.lobley@inpharmatica.co.uk

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.

Show MeSH
Related in: MedlinePlus