The Protein Sequence Length of CatSper1-4 Remains Highly Variable

We analysed the total length of CatSper1-4 units from multiple species and we noted that the length of CatSper1 remains highly variable whereas the total lengths of CatSper2-4 are not that variable (Fig. 1a–d). The overall lengths of CatSper1-4 were compared across the different phyla of vertebrates (Fig. 1a–d). The data suggest that the majority of CatSper1 from mammals are longer and have gained sequences mainly at the N-terminus. This accords well with the fact that the N-terminus of CatSper1 is significantly longer (300–400 amino acids in different species) in mammals and often shows the incorporation of multiple Histidine residues there. For example, within the entire N-terminal region of CatSper1, the Histidine residue constitutes ~ 16–18% in the case of most of the mammals including humans. We noted minor changes in the length of the TM regions and loop regions of CatSper1-4. We also noted length-wise conservation in certain segments.

Fig. 1

Polypeptide lengths of CatSper1-4 subunits remain variable in different phyla. a–d The polypeptide lengths of CatSper1–4 in Mammals, Birds, Reptiles, and Fishes were shown schematically as per their length and presence or absence in different genomes. The N-terminal region shows much variable sequence length for CatSper1. The Lipid-Water-Interface region is 5–10 Å in thickness on both sides of the lipid bilayer. Reptiles have longer sequence lengths in the C-terminal for CatSper4. In each case, the sequence length of different species is plotted according to the phyla on the right side. The number of species analyzed in each group is indicated in the graphs

The TM Regions and Lipid-Water-Interface Segments are Less Conserved in CatSper1-4

Next, we analysed the conservation of TM segments and associated LWI regions of CatSper1. All the six TM regions of CatSper1 are less conserved, though the TM5 and TM6 are better conserved among all the TM regions (Fig. 2a–b). Notably, the total TM regions are more conserved than the full-length CatSper1 (Fig. 2c). In the same comparatives, the total lipid-water-interface residues are less conserved than the TM regions or even full-length CatSper1, suggesting much less selection pressure in the LWI regions (Fig. 2c). Analysis of individual twelve LWI residues also suggests that none of these LWI regions are conserved (Fig. 2d). Yet N-TM2 and C-TM6 are better conserved among all. Notably, in a comparative manner, inner LWI regions are more conserved than the outer LWI regions (Fig. 2d). Further Seq2Logo analysis also indicates that the LWI regions are not conserved. However, position-specific enrichment of aromatic amino acids (such as Tyrosine, Tryptophan, and Phenylalanine residues) is notable (Fig. 2e).

Fig. 2

Neither full-length CatSper1 nor its fragments are conserved in vertebrates. a Shown is the schematic position of 5 amino acid stretches on both sides of each transmembrane that serves as the lipid-water-interface (LWI) residues. The human CatSper1 (UniProt ID-Q8NEC5) sequence was visualized on the protter website and shown as the reference. The N-terminal and C-terminal sides of the TM regions representing LWI residues are depicted in red and green, respectively. Due to the short sequence, the C-terminal of TM1 (C-TM1) and N-terminal of TM2 (N-TM2) are overlapped (marked in yellow) and such overlapping residues are counted for both C-TM1 as well as for N-TM2. This is also the case for C-TM2—N-TM3 and C-TM3—N-TM4 regions. b Conservation analysis of different regions of CatSper1 across vertebrate evolution is shown. None of the TM regions show high levels of conservation. c The LWI regions are less conserved than the TM or full-length CatSper1, suggesting overall more selection pressure on the TM residues than the LWI segments. d All the LWI regions are diverse. LWI (IN) is more conserved than LWI (OUT). e Conservation of individual residues in the LWI region is analysed and aromatic amino acids such as Tryptophan, Tyrosine, and Phenylalanine are found to be highly enriched. The independence of the group was estimated using the Chi-square test. χ2 = 772,498.138, 56,239.551 and 772,370.550 for b, c and d, respectively

Similarly, we analysed the conservation of CatSper2-4. Neither any of the six TM regions nor any of the twelve LWI regions are conserved in the case of CatSper2 (Fig. 3a–d). The overall LWI regions are less conserved than the associated TM regions, suggesting that for CatSper2, the relative selection pressure is low for LWI residues (Fig. 3c). Though none of the individual twelve LWI are conserved, we noted that the inner LWI region is more conserved than the outer LWI (Fig. 3d). Like the CatSper1, the Seq2Logo of CatSper2 highlights the enrichment of aromatic amino acids like Phenylalanine and Tryptophan. (Fig. 3e).

Fig. 3

Neither full-length CatSper2 nor its fragments are conserved in vertebrates. a Shown is the schematic position of 5 amino acid stretches on both sides of each transmembrane that serves as the lipid-water-interface (LWI) residues. The human CatSper2 (UniProt ID-Q96P56) sequence was visualized on the protter website and shown as the reference. The N-terminal and C-terminal sides of the TM regions representing LWI residues are depicted in red and green, respectively. Note that due to the short sequence the C-terminal of TM3 (C-TM3) and N-terminal of TM4 (N-TM4) are overlapped (shown in yellow) and are counted for both C-TM3 as well as for N-TM4. This is also the case for C-TM2 and N-TM3. This is also the case for C-TM1-N-TM2 regions. b Conservation analysis of different regions of CatSper2 during vertebrate evolution is shown. None of the TM regions show high levels of conservation. c The LWI regions are less conserved than the TM or full-length sequence of CatSper2, suggesting overall more selection pressure on the TM residues than the LWI segments. d All the LWI regions are diverse. LWI (IN) is more conserved than LWI (OUT). e Conservation of individual residues in the LWI region is analysed and aromatic amino acids, such as Tryptophan, Tyrosine, and Phenylalanine are found to be highly enriched. The independence of the group was estimated using the Chi-square test. χ2 = 563,141.757, 100,343.304, and 1,753,373.965 for b, c and d, respectively

Neither any of the six TM regions nor any of the twelve LWI regions are conserved in the case of CatSper3 (Fig. 4a–d). The overall LWI regions are less conserved than the associated TM regions also (Fig. 4c). However, among all, TM4 is better conserved (Fig. 4b). Also, C-TM4 is better conserved among all the twelve LWI regions (Fig. 4d). Notably, the LWI regions are more variable than the associated TM regions, suggesting that for CatSper3, the relative selection pressure is low for LWI residues. Incidentally, the inner LWI is more conserved than the outer LWI in CatSper3 (Fig. 4d). The Seq2Logo analysis shows the enrichment of Phenylalanine in N-TM6 (Fig. 4e).

Fig. 4

Neither full-length CatSper3 nor its fragments are conserved in vertebrates. a Shown is the schematic position of 5 amino acid stretches on both sides of each transmembrane that serves as the lipid-water-interface (LWI) residues. The human CatSper3 (UniProt ID- Q86XQ3) sequence was visualized on the protter website. b The N-terminal and C-terminal sides of the TM regions representing LWI are depicted in red and green, respectively. Note that due to the short sequence, the C-terminal of transmembrane (C-TM1) and N-terminal of transmembrane 3 (N-TM2) are overlapped (shown in yellow) and are counted for both C-TM1 as well as for N-TM2. This is also the case for (C-TM2) and (N-TM3). c Box plot analysis of different regions of CatSper3 across vertebrate evolution is shown. d The TM regions are more conserved than the LWI or full-length sequence of CatSper3, suggesting overall more selection pressure on the TM residues than the LWI segments. e Conservation of individual residues in the LWI region is analysed and aromatic amino acids, such as Tryptophan, Tyrosine, and Phenylalanine are found to be highly enriched. The independence of the group was estimated using the Chi-square test. χ2 = 448,996.458, 101,097.642 and 909,394.412 for b, c and d, respectively

Neither any of the six TM regions nor any of the twelve LWI regions are conserved in the case of CatSper4, except N-TM6 (Fig. 5a-d). The overall LWI regions are less conserved than the associated TM regions also (Fig. 5c). However, among all, TM5 is better conserved (Fig. 5b). The N-TM6 is better conserved among all the twelve LWI regions (Fig. 5d). Notably, the LWI regions are more variable than the associated TM regions, suggesting that for CatSper4, the relative selection pressure is low for LWI residues. Like the CatSper1, CatSper2, and CatSper3, the inner LWI is more conserved than the outer LWI in CatSper4. Similarly, Seq2Logo analysis was performed for the twelve individual LWI regions of CatSper2-4. The data indicate that the LWI regions are not conserved (Fig. 5e). However, position-specific enrichment of aromatic amino acids, primarily Phenylalanine and Tryptophan residues are notable (Fig. 5e).

Fig. 5

Neither full-length CatSper4 nor its fragments are conserved in vertebrates. a Shown is the schematic position of 5 amino acid stretches on both sides of each transmembrane that serves as the lipid-water-interface (LWI) residues. The human CatSper4 (UniProt ID-Q7RTX7) sequence was visualized on the protter website. b The N-terminal and C-terminal sides of the TM regions representing LWI are depicted in red and green, respectively. Note that due to the short sequence, the C-terminal of transmembrane (C-TM2) and N-terminal of transmembrane 3 (N-TM3) are overlapped and are counted for both C-TM2 as well as for N-TM3. This is also the case for C-TM3 and N-TM4. c Conservation analysis of different regions of CatSper4 during vertebrate evolution is shown. d The TM regions are more conserved than the LWI or full-length sequence of CatSper4, suggesting overall more selection pressure on the TM residues than the LWI segments. e Conservation of individual residues in the LWI region is analysed and aromatic amino acids i.e. Tryptophan, are found to be highly enriched. The independence of the group was estimated using the Chi-square test. χ2 = 465,244.406, 94,188.9201042255.276 for b, c, and d, respectively

Amino Acids Present in the Lipid-Water-Interface Show no Conservation

We calculated the frequencies of amino acids present in the LWI region (Fig S1–4). The frequencies of different amino acids were plotted for total, inner, and outer LWI regions. The apparent lack of conservation was noticeable in the frequency plot too. Also, the absence of CatSper1 in birds and amphibians allowed us to compare the frequencies from fishes, reptiles, and mammals only (Fig S1). Similarly, in amphibians, all other subunits (i.e. CatSper2-4) remain missing (Fig S2-4).

In the case of CatSper1, the frequencies of Phenylalanine and Leucine are higher at the inner LWI as compared to the outer LWI in all the taxon analyzed here. Also, the frequencies of Phenylalanine and Leucine are higher than the natural abundance of these two amino acids, suggesting a true positive selection of these two amino acids, especially at the inner LWI. Notably, the frequency of Leucine is low in the outer LWI region and lower than the natural abundance, suggesting a true negative selection there. The frequencies of Lysin and Proline are lower than the natural abundance of these amino acids in total, inner and outer LWI regions. Notably, the frequency of Cysteine remains nil in outer as well as almost nil in inner LWI regions, suggesting that Cysteine residue is excluded from the LWI regions of CatSper1 (Fig S1).

A similar analysis was performed for CatSper 2–4 (Fig S2-4). In contrast to CatSper1, in CatSper2, the frequency of Phenylalanine (and not Leucine) is higher than the natural abundance, especially, at the inner LWI (Fig S2). Although the frequencies of Phenylalanine and Leucine are higher at the inner LWI than the outer LWI, a trend that matches with CatSper1. The frequencies of Glutamine and Proline are higher at the outer LWI than the inner LWI and also lower than the natural abundance in the inner LWI region. In CatSper2, the frequency pattern of Cysteine matches well with CatSper1, suggesting that Cysteine residue is excluded from the LWI regions of CatSper2 also.

In the case of CatSper3, the frequency of Phenylalanine is similar to their natural abundance but other aromatic amino acids like Tyrosine, and Tryptophan have higher frequencies in inner and outer LWI respectively. In CatSper3 the frequencies of Alanine and Proline are lower than natural abundance in the total and inner LWI. Cysteine was again excluded from the LWI region from CatSper3, just like CatSper1 and 2 (Fig S3).

In the case of CatSper4 the aromatic amino acid Tryptophan has higher frequency in the inner LWI region in all taxa. Other aromatic amino acids like Tyrosine and Phenylalanine are present in higher frequencies in total LWI in all taxon except in fishes. Frequencies of Aspartic acid, and Proline are lower than their natural abundance in the inner LWI. The frequency of Cysteine shows a similar pattern of exclusion which is observed in other subunits (i.e. CatSper1, 2, and 3) (Fig S4).

The frequency of hydrophobic and hydrophilic amino acids presents in the lipid-water-interface show specific patterns in certain cases

Recently we reported that certain ion channels such as TRPV1 and TRPV4 maintain a specific ratio of total positively charged to total negatively charged residues in their LWI regions (Das et al. 2023; Saha et al. 2017, 2022). Accordingly, we performed a similar analysis for CatSper1-4. We found that in the case of CatSper1, the total positively charged amino acids had a lower frequency in all the taxon than the natural abundance (Fig. 6a). Total negatively charged residues remain variable. Thus, the ratio of positive to negative charged residues remains variable, in inner, outer, and total LWI regions. Similarly, we analyzed the total hydrophobic and total hydrophilic residues of CatSper1. We noted that these frequencies are not conserved. However, at the inner LWI region, the abundance of total hydrophobic residues is more than the natural frequencies and total hydrophilic residues are lower than the natural frequencies (Fig. 6b).

Fig. 6

In CatSper1-4, the ratio of positive to negative as well as hydrophobic to hydrophilic residues is not conserved during vertebrate evolution. Values from each species belonging to different vertebrate phyla (fishes: F, amphibians A, reptiles: R, birds: B, and mammals: M) are shown in violet, blue, yellow, green, and red dots, respectively. The total abundance of these amino acids in nature is indicated as a blue dotted line. a, c, e, and g The total frequency of positively charged amino acids and negatively charged residues as well as their relative ratio are shown for all CatSper subunits. The frequency for the inner LWI (rightmost) as well as for outer LWI (middle) as well for total (left side) are shown. There is no conservation or pattern observed in the ratio of positive to negative amino acids in the LWI regions. b, d, f, and h The total frequency of hydrophobic amino acids and hydrophilic residues as well as their relative ratio are shown for all CatSper subunits. The frequencies of the inner LWI (rightmost) as well as for outer LWI (middle) as well for total (left side) are shown. The conserved ratio of hydrophobic to hydrophilic amino acids in the LWI region indicates a selectivity pressure on the overall hydrophobicity in the LWI region

A similar analysis was performed for CatSper2-4 (Fig. 6c–h). Unlike CatSper1, in CatSper2, the frequency of all positively charged amino acids is higher than their natural abundance, and the frequency of all negatively charged amino acids is higher than their natural abundance in the inner LWI. Also, the ratio of all positive to all negative charged residues remains higher in the inner LWI (Fig. 6c). The frequency of all hydrophobic amino acids is higher than the natural abundance in the inner LWI in all taxa (except reptiles) in CatSper2 and this trend is similar to CatSper1 (Fig. 6d). In the case of CatSper3, the frequency of all positively and all negatively charged residues remain variable in total, inner and outer LWI regions. The ratio of positively and negatively charged amino acids also remains variable in all the LWI regions (Fig. 6e). In CatSper3, the frequency of all hydrophobic amino acids is higher than natural abundance in the total and inner LWI regions and this trend matches well with CatSper1 and CatSper2 (Fig. 6f). In the case of CatSper4, the frequency of all positively and all negatively charged amino acids remains variable and their ratio also remains variable in the total, inner, and outer LWI regions (Fig. 6g). In the case of CatSper4, the frequency of all hydrophobic residues remains higher in the inner and total LWI and this trend matches well with other subunits (i.e. CatSper1, 2, 3). In CatSper4, frequency of the all these hydrophilic residues remains low in the inner and total LWI regions. Also, the ratio of hydrophobic to hydrophilic residues is higher at the inner and in total LWI regions and this trend matches well with other sub-units (i.e. CatSper1, 2, 3) (Fig. 6h).

Full-Length CatSper1-4 Subunits are Less Conserved

As P4 serves as a key molecule/ligand involved in reproduction, we analyzed the conservation of full-length CatSper1, CatSper2, CatSper3, and CatSper4 across the vertebrate species for comparison. We also compared the CatSper proteins with TRPV4, another ion channel that we have reported to be activated by P4 (Dubey et al. 2023). For this purpose, the pairwise distance was calculated using Mega 11.0 and plotted as a boxplot. There is a large diversity in all CatSper across the species. The maximum diversity is observed in the Fish taxon in the case of all the CatSper1-4 units (Fig. 8). The same sub-units remain highly variable in reptilians (but lower than fishes). In mammals, these sub-units are relatively more conserved. In contrast, TRPV4 remains more conserved than any of the CatSper subunits in all the taxon including fishes.

Fig. 7

CatSper is not the only ion channel responsible for P4-mediated sperm motility in bull sperm. a Bull sperm collected form frozen semen sample were incubated with or without following drugs (NNC, 10 µM; RN1734, 10 µM; P4, 100 nM; GSK, 100 nM) for 1 min and motility was measured in CASA. The percentage of motile sperm were plotted with more than 100 sperm in each condition. In some condition the cells are preincubated for 1 min with drug as mentioned in figure. Test of significance were calculated using Unpaired t-test and P values were calculated by two tailed test with * = p < 0.05, *** = p ≤ 0.0001, **** = p < 0.0001. b Fold-change was calculated based on sperm motility in P4-incubated sample (considered as 1). Inhibition of TRPV4 by RN1734 is more effective than inhibition of CatSper by NNC to reduce the P4-mediated motility

Fig. 8

CatSper units are less conserved than TRPV4, another P4-responsive Ca2+-ion channel. a Shown are the conservation patterns of CatSper1, CatSper2, CatSper3, CatSper4, and TRPV4 for mammals, birds, reptiles, amphibians, and fishes. Higher values indicate less conservation and lesser values indicate higher conservation and are indicated by different background colours. The “X” sign indicates the loss of CatSper unit/s from specific phylogenetic group/s. b In birds, only CatSper1 is absent. Similarly, in amphibians, all the CatSper units are absent, suggesting the presence of other proteins that can act as P4-responsive Ca2+-ion channels there. TRPV4 is present in all these vertebrate groups and multiple copies of the TRPV4 gene are present in amphibians

Taken together, the data suggest loss-of-gene of certain CatSper subunits in specific taxa, less conservation of full-length CatSper sub-units, and more variability in the LWI regions as compared to the corresponding TM regions. All these suggest that CatSper sub-units are less likely to be the sole P4-responsive molecules present in the sperm cells responsible for reproduction.

P4 can Increase the Bull Sperm Motility in CatSper-Inhibited Conditions

Sperm motility is an important parameter for the normal physiology of the sperm and it is proposed the CatSper ion channel plays a prominent role in its regulation. It was reported previously that the P4 increases the sperm motility in a Catsper-depended manner (Lishko et al.. 2011). To compare the effect of pharamacological inhibition of CatSper, we performed CASA analysis with bull sample. Here our data indicate that in CatSper-inhibited condition (i.e. NNC-incubated, 10 µM), the motility in bull sperm decreases as compared to P4 (100 nM) incubated conditions (Fig. 7). However, the motility increases further (although non-significantly) when the sperm cells are stimulated by P4 in the CatSper-pre-inhibited condition. This suggests that the inhibition of the CatSper by NNC is insufficient to block the P4-medaited motility regulation, even the inhibitor is used at high concentration (10 µM). This may suggest the involvement of other Ca2+ ion channels which could also be activated by P4. Recently we have reported that TRPV4 channel can be activated by P4 and TRPV4 is also present in mature sperm from all the vertebrates (Dubey et al. 2023; Kumar et al. 2016). To check for the role of the TRPV4 ion channel in motility, sperm motility by TRPV4 modulation was also analysed. In the presence of a TRPV4 activator (GSK101, 100 nM), the motility increases significantly and in the presence of a TRPV4 inhibitor (RN, 10 µM), the motility decreases. Notably, in the presence of TRPV4 inhibitor, further application of P4 does not increase the motility further (in fact decreases). Further analysis suggests that pharmacological pre-incubation of CatSper inhibition causes ~ 56% decrease in the motility (Fig. 7b). Comparatively, pharmacological pre-incubation of TRPV4 inhibition causes ~ 90% decrease in the motility. As a proof-of-concept, this data suggest that TRPV4 inhibition is more effective than CatSper inhibition, at least in bull sample.