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. 2021 Mar 8;22(1):18.
doi: 10.1186/s12860-021-00353-x.

Intracellular pH regulation: characterization and functional investigation of H+ transporters in Stylophora pistillata

Laura Capasso  1   2 , Philippe Ganot  1 , Víctor Planas-Bielsa  1 , Sylvie Tambutté  1 , Didier Zoccola  3
Affiliations

Intracellular pH regulation: characterization and functional investigation of H+ transporters in Stylophora pistillata

Laura Capasso et al. BMC Mol Cell Biol. .

Abstract

Background: Reef-building corals regularly experience changes in intra- and extracellular H+ concentrations ([H+]) due to physiological and environmental processes. Stringent control of [H+] is required to maintain the homeostatic acid-base balance in coral cells and is achieved through the regulation of intracellular pH (pHi). This task is especially challenging for reef-building corals that share an endosymbiotic relationship with photosynthetic dinoflagellates (family Symbiodinaceae), which significantly affect the pHi of coral cells. Despite their importance, the pH regulatory proteins involved in the homeostatic acid-base balance have been scarcely investigated in corals. Here, we report in the coral Stylophora pistillata a full characterization of the genomic structure, domain topology and phylogeny of three major H+ transporter families that are known to play a role in the intracellular pH regulation of animal cells; we investigated their tissue-specific expression patterns and assessed the effect of seawater acidification on their expression levels.

Results: We identified members of the Na+/H+ exchanger (SLC9), vacuolar-type electrogenic H+-ATP hydrolase (V-ATPase) and voltage-gated proton channel (HvCN) families in the genome and transcriptome of S. pistillata. In addition, we identified a novel member of the HvCN gene family in the cnidarian subclass Hexacorallia that has not been previously described in any species. We also identified key residues that contribute to H+ transporter substrate specificity, protein function and regulation. Last, we demonstrated that some of these proteins have different tissue expression patterns, and most are unaffected by exposure to seawater acidification.

Conclusions: In this study, we provide the first characterization of H+ transporters that might contribute to the homeostatic acid-base balance in coral cells. This work will enrich the knowledge of the basic aspects of coral biology and has important implications for our understanding of how corals regulate their intracellular environment.

Keywords: Gene expression; H+ transport; Ocean acidification; Reef-building corals; pH regulation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
A maximum likelihood phylogenetic (Phyml, LG + I + G) tree of human and cnidarian SLC9s (protein sequences were previously aligned by Clustal Omega). Phylogenetic analysis of S. pistillata SLC9 sequences with functionally characterized SLC9s in H. sapiens grouped cnidarian SLC9s within three different subfamilies: subfamily A, including plasma membrane and organelle homologs, which are represented in blue and red, respectively; subfamily B, which is represented in orange; and subfamily C, which is represented in yellow. Cnidarian species include Stylophora pistillata (Spi), Acropora digitifera (Adi), Nematostella vectensis (Nve), Aiptasia pallida (Apa), Corallium rubrum (Cru), Dendronephthya gigantea (Dgi), Amplexidiscus fenestrafer (Afe), and Discosoma sp. (Dsp). Chordata species include Homo sapiens (Hsa)
Fig. 2
Fig. 2
a Exon/intron organization of V0 V-ATPase subunit a in the genome of S. pistillata. Exons are represented as boxes, whereas introns are depicted as lines. b Sequence comparison of S. pistillata and H. sapiens V0 V-ATPase subunit a. Identical and similar amino acids (aa) are shaded in black and grey, respectively. The boxes represent the predicted transmembrane segments in H. sapiens V0 V-ATPase subunit a. The circles and crosses represent phosphorylation and N-glycosylation sites, respectively, in spiHvCN1.1 and spiHvCN1.2. The asterisk indicates a conserved R relevant to H+ transport
Fig. 3
Fig. 3
Maximum likelihood (Phyml, LG + I + G) phylogenetic tree of V0 V-ATPase subunit-a (protein sequences were previously aligned by Clustal Omega). Cnidarian species include Stylophora pistillata (Spi), Acropora digitifera (Adi), Nematostella vectensis (Nve), Aiptasia pallida (Apa), Corallium rubrum (Cru), Dendronephthya gigantea (Dgi), Amplexidiscus fenestrafer (Afe), and Discosoma sp. (Dsp). Mollusca species include Crassostrea gigas (Cgi). Echinodermata species include Strongylocentrotus purpuratus (Spu) and Acanthaster planci (Apl). Chordata species include Homo sapiens (Hsa) and Ciona intestinalis (Cin). Placozoa species include Trichoplax adhaerens (Tad). Porifera calcarea species include Sycon ciliatum (Sci). Porifera Homoscleromorpha species include Oscarella carmela (Oca)
Fig. 4
Fig. 4
a Exon/intron organization of spiHvCNs in the genome of S. pistillata. Exons are represented as boxes, whereas introns are depicted as lines. b Sequence comparison of S. pistillata and H. sapiens HvCN proteins. Identical and similar amino acids (aa) are shaded in black and grey, respectively, whereas aa that are missing from the other sequence are denoted by dashes. The boxes represent the predicted transmembrane segments in human HvCN1 (S1-S4). The circles and crosses represent phosphorylation and N-glycosylation sites, respectively, in spiHvCN1.1 and spiHvCN1.2
Fig. 5
Fig. 5
Maximum likelihood (Phyml, LG + I + G) phylogenetic tree of voltage-gated proton channels (protein sequences were previously aligned by Clustal Omega). HvCN1.1 and HvCN1.2 are separated into two semicircles. Cnidarian species include Stylophora pistillata (Spi), Acropora digitifera (Adi), Nematostella vectensis (Nve), Aiptasia pallida (Apa), Corallium rubrum (Cru), Dendronephthya gigantea (Dgi), Amplexidiscus fenestrafer (Afe), and Discosoma sp. (Dsp). Mollusca species include Crassostrea gigas (Cgi). Echinodermata species include Strongylocentrotus purpuratus (Spu) and Acanthaster planci (Apl). Chordata species include Homo sapiens (Hsa) and Ciona intestinalis (Cin). Placozoa species include Trichoplax adhaerens (Tad). Porifera calcarea species include Sycon ciliatum (Sci). Porifera Homoscleromorpha species include Oscarella carmela (Oca)
Fig. 6
Fig. 6
Relative mRNA quantification (Rq) of SLC9s, V0 V-ATPase subunit-a and HvCNs measured in total (total colony) and oral (oral fraction) fractions. Box and whisker plots show the first, second (median) and third quartiles (horizontal lines of the boxes) and the respective whiskers (vertical lines spanning the lowest and highest data points of all data, including outliers). The replicate numbers (n = 3) represent separate coral samples. The asterisks and points indicate significant differences (• 0.11 ≤ p-value≤0.10 and ** p-value< 0.05)
Fig. 7
Fig. 7
Relative mRNA quantification (Rq) of SLC9s, V0 V-ATPase subunit-a and HvCNs at 1 week of pCO2 exposure plotted against pH 8.1 and 7.2 (n = 5)
Fig. 8
Fig. 8
Relative mRNA quantification (Rq) of SLC9s, V0 V-ATPase subunit-a and HvCNs at 1 year of pCO2 exposure plotted against pH 8.1 and 7.2 (n = 5). The asterisks indicate significant difference (** p < 0.05)
Fig. 9
Fig. 9
Model of acid-base transporters involved in intracellular pH regulation expressed on the apical (AM) and basolateral (BLM) membranes of coral cells throughout the tissue layers. The roles of other ion channels and transporters involved in other cellular processes are not considered here. Transporters that are more highly expressed in the oral fraction (oral-specific) are coloured in blue, those that are more highly expressed in the total colony (aboral-specific) are coloured in orange, and those that are expressed at the same levels in both fractions (ubiquitous) are coloured in green. Other enzymes (CA = carbonic anhydrase) and transporters (PMCA = Ca+ 2 ATPase) involved in the H+ flux balance are represented in bold letters
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