Scleractinian or stony corals are organisms in the phylum Cnidaria that deposit calcium carbonate (CaCO₃) in the form of aragonite to build an exoskeleton. Stony corals are the main calcifying organisms responsible for the construction of coral reefs, which are major ecosystems hosting numerous and diverse organisms. Coral reefs also act as natural barriers from strong ocean currents, waves, and tropical storms, providing coastal protection. This protection centers on the ability of scleractinian corals to produce enough CaCO₃. The increase in atmospheric CO₂ concentrations as a result of human activity poses threats to coral-reef-building organisms due to rising sea surface temperatures (Hoegh-Guldberg, 1999) and because CO₂ is taken up by the ocean, dangerously lowering the pH of the sea water (Caldeira and Wickett, 2003).

It is known that precipitation of the aragonitic form of calcium carbonate is facilitated at elevated pH values, at very low concentrations of protons. Calcification by scleractinian corals is a process that has been shown to be modulated by the pH of the solution in which calcium carbonate is precipitated (Allemand et al., 2011). To this end, corals produce a specialized compartment between the ectoderm and the external substrate or skeleton called calicoblastic compartment, which contains a fluid derived from the surrounding sea water. The composition of this calicoblastic fluid or liquor is strictly regulated by the coral to maintain both an elevated pH, often close to one unit higher than the surrounding sea water, and an increased concentration of Ca²⁺ and carbonates. The molecular details of pH regulation in the calicoblastic fluid are not understood completely. Involvement of proton pumps has been postulated and is likely to be part of proton transport in corals. Both P-type and V-type hydrogen pumps are present in coral transcriptomes and are known to play roles in the physiology of coral-algal symbiosis (Tresguerres et al., 2017). V-type H⁺-ATPases have also been shown to be involved in calcification in foraminifera (Toyofuku et al., 2017). If a proton pump is involved in lowering proton concentration in the calicoblastic fluid to maintain high calcification rates, protons will be transported to the cytoplasm of the ectodermal cells that constitute the calicoblastic epithelium, producing a profound acidification of the cytoplasmic pH (pH_i). Although measurements of the pH_i in corals indicate values of 7.13–7.4 (Venn et al., 2009), it is unknown how coral cells regulate pH_i. Thus, an efficient pH-regulatory mechanism is to be expected to be present in corals. We hypothesized that proton channels might be fundamental to this physiological process and also required for calcification in hard corals.

Although a number of studies have delineated the physiological roles of H_v1 voltage-gated proton channels in vertebrate cells (DeCoursey, 2013), less is known about their role in invertebrates. These channels are potential mediators in processes that are critically dependent on proton homeostasis. As an example, they have been shown to be involved in regulating the synthesis of the calcium carbonate skeleton in coccolithophores, calcifying unicellular phytoplankton (Taylor et al., 2011).

The range of voltages over which channel activation occurs is strongly modulated by the transmembrane proton gradient, characterized by ΔpH = pH_o-pH_i, that is, the difference between the external and internal pH. In the majority of known H_v1 channels, the voltage at which half of the channels are activated, the V_0.5 or the apparent threshold for channel opening (V_Thr), shifts by roughly 40 mV per unit of ΔpH. Thus, the pH gradient strongly biases the voltage-independent free energy of channel activation (Cherny et al., 1995). With few exceptions, channel activation occurs at voltages that are more positive than the reversal potential for protons, implying that protons are always flowing outward under steady-state conditions. The fact that most H_v1s mediate outward currents is the reason these channels are mostly involved in reversing intracellular acidification or producing voltage-dependent cytoplasmic alkalization (Lishko and Kirichok, 2010; DeCoursey, 2013).

Here we report the presence of genes encoding H_v1 channels in two species of reef-building corals. We cloned and characterized the biophysical properties of these channels in an expression system using patch-clamp electrophysiology. The demonstration of the presence of voltage-gated proton channels in corals is an initial step to a deeper understanding of coral calcification and its dysregulation under ocean acidification conditions. We show that some of the coral H_v1’s biophysical properties are different from other known proton channels, and this behavior makes them interesting models to try to understand some basic biophysical mechanisms in these channels. To explain this behavior, we developed a novel activation model to describe voltage- and pH-dependent gating that has general applicability to H_v1 channels.

Ion channels have not been characterized in corals. Here, we have initiated their study by searching the transcriptome of the Indo-Pacific coral Acropora millepora (Moya et al., 2012) for sequences coding for putative voltage-sensing residues present in canonical H_v1 channels with the form RxxRxxRIx, which corresponds to the S4 segment of H_v1 channels and is also found in other voltage-sensitive membrane proteins. Blast searches detected four sequences that seem to correspond to a gene encoding the H_v1 voltage-activated proton-selective ion channel (Ramsey et al., 2006; Sasaki et al., 2006). A. millepora is one of the most widely studied species of scleractinian corals and is well represented in the commercial coral trade (Cleves et al., 2018; Ying et al., 2019). We proceeded to clone this gene from a small specimen of A. millepora obtained from a local aquarium (Reef Services, Mexico City). As indicated in the 'Materials and methods' section, total RNA was extracted from tissue and mRNA was retrotranscribed to obtain complementary DNA (cDNA). We managed to obtain a full-length clone and refer to this sequence as AmH_v1 or H_v1-type proton channel of A. millepora.

We were interested in knowing if the same gene is present in a closely related species from the Caribbean Sea. Thus, we used the same primers to clone the H_v1 channel from Acropora palmata, a widespread coral in the same family, and which we call ApH_v1. The amino acid sequence is almost identical to AmH_v1 (Figure 1—figure supplement 1A); the greatest divergence is found between a few amino acid residues in the C-terminal region. This result suggests that despite the large biogeographic difference, these two genes have not diverged significantly. The ApH_v1 sequence also gives rise to fast-activating voltage-gated proton currents (Figure 1—figure supplement 1B).

The most diagnostic feature of the H_v1 protein is the sequence of the fourth transmembrane domain or S4, which contains three charged amino acids in a characteristic triplet repeat. The presence of these repeats in our sequence allowed us to initially identify our clone as an H_v1 channel. However, we decided to compare our sequence to those of several H_v1 orthologs. We selected a list of 130 H_v1 protein sequences that are well curated in the Gene Bank (https://www.ncbi.nlm.nih.gov/), representing several branches of the eukaryotes, from unicellular plants to mammals. As expected, the protein sequence of AmH_v1 has similarity to several other H_v1 genes from varied organisms (Figure 1A). The identity varies from 98%, when compared to other putative coral and anemone sequences, to less than 30%, when compared to plant and nematode sequences. In spite of this variability, the putative transmembrane domains of all these proteins show high conservation, and consensus sequence logos can detect the presence of highly conserved individual amino acid sequences that can be considered characteristic of H_v1 channels. Figure 1B compares these transmembrane domain consensus logos with our AmH_v1 sequence. It can be gleaned that AmH_v1 contains the highly conserved residues that form the voltage-sensing amino acid residues in S4 as well as their acidic pairs present in S2 and S3. The extracellular histidine residues involved in Zn²⁺ coordination are also present. These results suggest that our sequence is that of a bona fide H_v1 voltage-sensing domain (VSD).

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Apart from canonical voltage-gated channels, several other proteins contain VSDs. Examples are the voltage-sensing phosphatases like VSPs (Iwasaki et al., 2008) and TPTE and TPTE2 (Halaszovich et al., 2012) proteins (transmembrane proteins with tensin homology) and genes like TMEM266. These proteins are relevant to us since some TPTEs have been shown to also mediate proton currents and TMEM266 can be modulated by Zn²⁺ (Papp et al., 2019). We compared the sequence of AmH_v1 with several orthologs of TPTEs and TMEM266. Although there is some similarity within transmembrane domains (Figure 1—figure supplement 2), the overall sequence comparison shows that AmH_v1 and these VSD-containing proteins are different.

As mentioned before, we performed a multiple sequence alignment with 130 H_v1 sequences. In Figure 2, we show the detailed sequence alignment of AmH_v1 with five of these sequences, which represent some of the best studied H_v1 genes. It can be seen that there is a high degree of identity, especially in the transmembrane domains. The least degree of conservation appears when comparing this sequence to the dinoflagellate Karlodinium veneficum H_v1 channel (Figure 2A). A search of available transcriptomes from several coral species allowed us to detect the presence of sequences that are found in H_v1 channels. This suggests that H_v1 proton channels might be found in many families of scleractinian corals (Figure 2—figure supplement 1), as has also been recently shown (Capasso et al., 2021).

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Secondary-structure prediction suggests that AmH_v1 is a canonical H_v1 channel formed by a VSD with four transmembrane segments. The protein sequence was used for 3D modeling using the SWISS MODEL server (Waterhouse et al., 2018), which produced models based on the H_v1 chimera structure (Takeshita et al., 2014) and the Kv1.2 potassium channel VSD (Long et al., 2005). This structural model is shown in Figure 2B. The predicted model indicates a shortened N-terminal region, four transmembrane helices, and a long C-terminal helix.

Voltage-gated proton channels from Ciona (Sasaki et al., 2006) and humans (Lee et al., 2008) have been shown to express as dimers in the plasma membrane, and this dimeric form is understood to be the functional unit of these proton channels. The dimer is stabilized by a coiled-coil interaction mediated by an alpha helical C-terminal domain. As shown by the model in Figure 2B, AmH_v1 has a long C-terminal helix, which is predicted to engage in a coiled coil (Paircoil2; McDonnell et al., 2006). We calculated the probability per residue to form a coiled coil for all the C-terminal residues, both for human and AmH_v1 channels, using the program COILS (Lupas et al., 1991). Figure 3A shows that the coiled-coil probability for AmH_v1 C-terminus is at least as high or higher than that for hH_v1, an established dimer, strongly suggesting that coral H_v1s might also form dimers.

Figure 3

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In order to study the oligomeric state of the coral H_v1, we performed FRET experiments with the AmH_v1 channel tagged with fluorescent proteins (FPs) as a FRET pair. Figure 3B shows that there is significant FRET efficiency between FP-tagged subunits, indicating a very close interaction between monomers. The measured apparent FRET efficiency vs the fluorescence intensity ratio can be fitted to a model where the subunits assemble as a dimer. From this fit, we can estimate a distance between fluorophores of ~60 Å, which is compatible with AmH_v1 being a dimer, at least in HEK293 cells.

Functional expression of AmH_v1 voltage-dependence and kinetics

The cDNA of AmH_v1 was cloned in the pcDNA3 expression vector and transfected into HEK293 cells. Under whole-cell conditions, we recorded large voltage-dependent outward currents. Figure 4A shows a family of such currents. The data suggest that these currents were carried mostly by protons, since the reversal potential, measured from a tail current protocol, closely followed the equilibrium potential for protons, as given by the Nernst equation (Figure 4B).

Figure 4

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The voltage-dependence of channel gating was estimated from a fit of the normalized conductance vs voltage (G-V) to Equation 1. The steepness of the curve corresponds to an apparent charge of ~2 e_o, comparable to other H_v1s under similar recording conditions (Figure 4C).

Interestingly, these channels seem to activate rapidly. This is apparent from the current traces, which approach a steady state within a few hundred ms (Figure 4A), as quantified in Figure 4D. Equation 3 estimates two parameters, an activation time constant (τ) and a delay (δ). Both the time constant and the delay are similarly voltage-dependent at positive potentials. The existence of a delay in the time course implies that activation is a multiple-state process. The delay magnitude is smaller than the time constant at all voltages, which can be interpreted to mean that the rate-limiting step for opening comes late in the activation pathway (Schoppa and Sigworth, 1998).

Comparison to human H_v1 channel properties

Human H_v1 is probably the best characterized of the voltage-gated proton channels (Musset et al., 2008); so we compared some of the properties of AmH_v1 with hH_v1. AmH_v1 channels activate faster than their human counterpart. Figure 5 compares the activation kinetics of these two channels under the same conditions. Steady state is apparently reached sooner after a voltage pulse in AmH_v1 (Figure 5A) when compared to hH_v1 (Figure 5C). The slower kinetics of the human ortholog is also evidenced in the more sluggish deactivation tail currents (Figure 5C). The range of voltages over which activation happens is also different between the two channels, with the coral H_v1 channel activating at more negative voltages than the human clone (Figure 5E; notice that the proton gradient is such that ΔpH = 1 and is the same for recordings of both channel types). Even though AmH_v1 activates at more negative voltages, the activation range is still more positive than the proton reversal potential; thus, coral proton currents activated by depolarization, in the steady state and at least as expressed in HEK293 cells, are always outward.

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In order to better estimate both kinetics and activation, we performed experiments with longer pulse durations. This is especially important for the very slow activation of the human channel. The resulting currents are shown in Figure 5B and D. The normalized conductance for these currents that are closer to steady state are shown in Figure 5E by triangles. These G-V curves are shifted to more negative voltages than the G-V from shorter pulses, as expected. The faster kinetics of AmH_v1 is clearly evidenced when the time constant of activation, τ, estimated using fits of the activation time course to Equation 2, is compared for coral and human H_v1 channels. AmH_v1 is more than tenfold faster at 0 mV and over a range of positive voltages (Figure 5F).

Effects of the pH gradient on gating

Both native and cloned voltage-gated proton channels are characteristically modulated by the pH gradient (Cherny et al., 1995; Sasaki et al., 2006; Ramsey et al., 2006). We carried out experiments to investigate the modulation of the coral H_v1 channels by different pH gradients. We first recorded whole-cell currents at various ΔpH values and estimated the voltage-dependence of the conductance. These G-V curves were fitted to Equation 2 to obtain the voltage of half activation, V_0.5, and apparent gating charge, q, which determines the steepness of the fit. As is the case with other H_v1 channels, the V_0.5 shifts to negative voltages when ΔpH is greater than 0 and to positive voltages when ΔpH is less than 0 (Figure 6A). When we plot the V_0.5 as a function of ΔpH, the relationship seems to be mostly linear over the range of ΔpH −1 to 2. This relationship is somewhat steeper than the generally observed −40 mV/ΔpH (Figure 6B). We tried to obtain recordings over an extended range of ΔpH values. To this end, we performed inside-out recordings in which the composition of solutions can be better controlled, tends to be more stable, and the size of currents is smaller. However, recordings were unstable at extreme pH values, and we only managed to reliably extend the data to a ΔpH value of −2. Figure 6C shows the summary of the inside-out recordings. We have plotted both the V_0.5 and the threshold voltage, V_Thr. To obtain this last parameter, we fitted the exponential rise of the G-V curve to a function of the form

$G\left(V\right)={G\text{'}\bullet exp}^{qV/K_{B}T}$

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V_Thr was calculated as the voltage at which the fit reaches 10% of the maximum conductance. The parameter V_Thr should be less sensitive than V_0.5 to the possible change in the proton gradient that can occur with large currents. It is clear from these data that at extreme values, the dependence of V_0.5 or V_Thr on ΔpH deviates from a simple linear relationship and instead appears to saturate at extreme values of ΔpH.

Allosteric model of voltage- and pH-dependent gating

Currently, there is only one quantitative model that has been used to explain ΔpH gating of H_v1 channels (Cherny et al., 1995). However, this model is euristic and does not provide mechanistic insight into the process of proton modulation of the voltage dependence of proton-permeable channels. In order to explain the modulation of the range of activation by the proton gradient, parameterized by the V_0.5, we developed a structurally inspired allosteric model of voltage and proton activation. As many voltage-sensing domains, H_v1 has two water-occupied cavities exposed to the extracellular and intracellular media (Ramsey et al., 2010; Islas and Sigworth, 2001; Ahern and Horn, 2005). Recent evidence suggests that these cavities function as proton-binding sites through networks of electrostatic interactions (De La Rosa et al., 2018). In our model, we propose that these two proton-binding sites, one intracellular and one extracellular, allosterically modulate the movement of the voltage-sensing S4 segment and, thus, channel activation in opposite ways. The extracellular site is postulated as inhibitory, while the intracellular site is excitatory, facilitating voltage sensor movement. As a first approximation, we employed a simplified allosteric formalism based on a Monod-Wyman-Changeux (MWC) style model (Horrigan and Aldrich, 2002; Changeux, 2012). As a simplifying assumption, in this model we assume that the voltage sensor moves in a single voltage-dependent activation step. We assume the external and internal proton-binding sites have simple protonation given by a single pK_a value. These sites operate as two allosteric modules and are coupled to the voltage sensor according to coupling factors C and D, respectively. These binding sites in turn interact with each other through the coupling factor E. The modular representations of the model are illustrated in Figure 7A, while the full model depicting all open and closed states with all permissible transitions and the corresponding equilibrium constants for each transition is shown in Figure 7B. Full details of equations derived from these schemes are given in supplementary data.

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This allosteric model represents the first attempt at producing a quantitative mechanistic understanding of the interaction of the voltage sensor and protons in H_v1 channels.

From the data shown in Figure 6C, it can be seen that the model is capable of reproducing the very steep dependence of V_0.5 on ΔpH and importantly, the saturation of this relationship at extreme values. Some H_v1 channels from other organisms show a linear dependence of gating over a large range of ΔpH values, while others show a reduced dependence and even saturation over some range of ΔpH (Thomas et al., 2018). Our model can explain these different behaviors as different channels having distinct values of pK_as for the internal or external sites, differences in coupling factors, or differences in the voltage-dependent parameters (Figure 6—figure supplements 1 and 2).

Block by Zn²⁺

The best-characterized blocker of proton channels is the divalent ion zinc (Cherny et al., 2020; De La Rosa et al., 2018; Qiu et al., 2016). We performed experiments to determine if AmH_v1 channels are also inhibited by zinc. We found that indeed, extracellular application of zinc in outside-out patches produced inhibition of the channels, reflected in reduced current amplitudes (Figure 8A). Figure 8B shows average current-voltage (I-V) relationships in the absence and presence of 10 μM external zinc. It can be seen that the fraction of current blocked is not the same at every voltage, indicating that this inhibition might be voltage-dependent. The fraction of blocked channels was calculated and is plotted at each voltage along with the I-V curves (Figure 8B). It can be clearly seen that inhibition by Zn²⁺ is voltage-dependent. A simple mechanism for voltage-dependent blockage was proposed by Woodhull, 1973. This model postulates that a charged blocker molecule interacts with a binding site in the target molecule that is located within the electric field. Fitting the data according to this model, and given that zinc is a divalent ion, its apparent binding site is located at a fraction δ = 0.2 of the membrane electric field from the extracellular side (Figure 8B).

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Zinc blockage proceeds very fast. At 1 mM, the channels are blocked almost instantaneously, and the inhibition washes off very fast as well (Figure 8C). Finally, we report the dose-response curve (Figure 8D). The inhibition dose-response curve can be fit by a Hill equation (Equation 4) with a slope factor of nearly 0.5 and an apparent dissociation constant, K_D, of 27 μM.

A few ion transport mechanisms in reef-building corals have been described, but up to now, no ion channels have been characterized from any scleractinian species. Here we have shown that voltage-gated proton-permeable channels formed by the H_v1 protein are present in corals. In particular, we have cloned these channels from two species of the genus Acropora, A. millepora and A. palmata. It is interesting that the protein sequence of these proteins shows a very high degree of conservation, suggesting that, even when the two species are found in different oceans, they haven’t had time to diverge substantially or alternatively; selective pressures on these channels are very similar in both species. The presence of H_v1 sequences in many other species of corals from disparate clades suggests that H_v1 plays an important role in coral physiology.

H_v1 channels are formed by a protein fold that is structurally equivalent to the VSDs of canonical voltage-gated channels (Sasaki et al., 2006; Ramsey et al., 2006). The VSD is formed by a bundle of four antiparallel alpha helices (Takeshita et al., 2014). In some species, it has been shown that H_v1 channels are dimeric (Lee et al., 2008; Mony et al., 2020; Lee et al., 2008). Accordingly, we have also shown here that the AmH_v1 is a dimer. Our FRET results are consistent with the high propensity to form a coiled coil shown by its C-terminal domain.

H_v1 channels are different from canonical voltage-gated channels in that both voltage sensing and permeation are mediated through a single protein domain. Voltage sensing is thought to occur through the interaction of charged amino acid side chains with the electric field, leading to the outward movement of the fourth domain or S4, in a similar fashion to other voltage-sensing domains (Carmona et al., 2018; De La Rosa and Ramsey, 2018). This outward movement of the S4 is coupled to protons moving through the VSD in a manner that is not completely understood (Randolph et al., 2016). Most proton-permeable channels seem to have evolved to extrude protons from the cell, and toward this end, their voltage dependence is tightly modulated by the proton gradient between extracellular and intracellular solutions (Cherny et al., 1995).

Our electrophysiology experiments show that these coral channels give rise to proton currents when expressed in HEK293 cells and that they retain the functional characteristics that have been shown to define the class in other species, such as very high selectivity for protons, activation by voltage, and modulation of this activation by the proton gradient. The new channels reported here activate faster than the human H_v1 channel. It has been known that different orthologs of H_v1 activate with varying kinetics. For example, sea urchin, dinoflagellate, and recently, fungal H_v1 channels activate rapidly, while most mammalian counterparts have slow activation rates (Musset et al., 2008; Smith et al., 2011; Zhao and Tombola, 2021). A comparative study suggests that two amino acids in the S3 transmembrane segment are important determinants of kinetic differences between sea urchin and mouse H_v1 (Sakata et al., 2016). The authors suggest that the time course of activation is slow in channels containing a histidine and a phenylalanine at positions 164 and 166, respectively (mouse sequence numbering). The AmH_v1 has a histidine at an equivalent position, 132, and a methionine at 134. It is possible that this last amino acid in AmH_v1 confers most of the fast kinetics phenotype. A separate work has shown that a lack of the amino-terminal segment in human sperm H_v1 also produced fast-activating channels (Berger et al., 2017). Interestingly, the Acropora channels have a shorter amino-terminal sequence, which could also contribute to their fast kinetics.

One of the most interesting characteristics found in these new proton channels is their modulation by the proton gradient. As opposed to other H_v1 channels, we can observe a trend toward saturation of the V_0.5 for activation as a function of ΔpH at extreme values of this variable. A tendency toward saturation of the V_0.5-ΔpH relationship has been observed in mutants of the hH_v1 channel (Cherny et al., 2015) or at negative values of ΔpH for a snail H_v1 (Thomas et al., 2018), but it seems it can be fully appreciated in AmH_v1. Since our model explains the observation of saturation of voltage gating at extreme values of ΔpH as a consequence of the existence of two saturable sites for proton binding, we attribute this behavior to the large separation of pK_a values for the extracellular and intracellular proton-binding sites. Thus, channels that do not show saturation might have sites with well-separated and differing pKa values. Evolutionary fine tuning of these pKa values will produce channels with pH activation in ranges adapted to their physiological function.

It is important to point out that the external and internal proton-binding sites can be part of the voltage sensor itself. We envision these sites as being formed not by distinct protonatable amino acids but by a network of electrostatic interactions between amino acids in S4 and other transmembrane segments. In this context, the pK_a values of the sites in our model do not reflect pK_as of individual amino acids but of the whole proton-binding site. Recent works by Carmona et al., 2021 and Schladt and Berger, 2020 suggest that voltage sensing is directly responsible for pH gating. These authors suggest that the energy of the pH gradient is part of the free energy needed for voltage-sensor movement. This purely energetic statement is encompassed in our model as the coupling factors between voltage-sensor movement and proton binding, which are in fact energetic factors.

In our model, the strength of allosteric coupling of these sites and the voltage sensor determines if saturation is observed over a short or extended range of ΔpH values and the range of values of V_0.5 that a particular channel can visit. Coupling also determines the value of the slope of the ΔpH vs V_0.5 curve. Values larger or smaller than the typical ~40 mV/pH are obtained as a consequence of strong or weak coupling between binding and voltage-sensor movement, respectively (Figure 6—figure supplement 2). Our model should provide a framework to better understand the gating mechanisms in future work.

It is clear that more complicated models, with a larger number of voltage-dependent and -independent steps (Villalba-Galea, 2014) and coupling to protonation sites, should be the next step to improve data fitting and explore voltage- and proton-dependent kinetics. In particular, these types of models can help explain mutagenesis experiments exploring the nature of the protonation sites. For example, Villalba-Galea, 2014 proposed the existence of a voltage-independent step in Hv1 gating, and this can be easily incorporated in the model proposed here, as is the fact that hH_v1 and AmH_v1 channels are dimers. The present form of the allosteric model has a single subunit. Since gating in dimeric channels seems to be cooperative, allosteric models have the advantage that cooperativity between subunits can be handled naturally by including coupling between two voltage sensors.

H_v1 proton channels seem fundamental in handling fluctuations in intracellular pH and take part in several well-characterized physiological processes that depend on proton concentration changes, such as intracellular pH regulation, sperm flagellum beating, reactive oxygen species production and bacterial killing in immune cells, initiation of bioluminescence in single-celled algae, and so on Castillo et al., 2015.

What is the function of voltage-gated proton channels in corals? The deposition of a CaCO₃ exoskeleton is one of the main defining characteristics of scleractinians; however, the ionic transport mechanisms involved in this process are mostly unknown. In order for aragonite precipitation to occur favorably, the pH of the calicoblastic fluid, right next to the skeleton, is maintained at high levels, between 8.5 and 9 and above the pH of sea water (Le Goff et al., 2017). It has been posited that corals control this pH via vectorial transport of protons to the gastrodermal cavity (Jokiel, 2013). Since proton transport away from the site of calcification would incur a drastically lower intracellular pH in the cells of the aboral region, we propose that, given their ability to rapidly regulate the intracellular pH (De-la-Rosa et al., 2016), H_v1 proton channels contribute by transporting protons from the cells. Thus, these proton channels could be a major component of the mechanisms of intracellular pH regulation in corals. Given that the activation range of H_v1 is controlled by the pH gradient, a large intracellular acidification would facilitate opening of these channels at the resting potential of cells, which is presumably negative.

The finding that coral H_v1 channels retain their sensitivity to Zn²⁺ opens the possibility of using this ion as a pharmacological tool to study the role of proton channels in pH homeostasis. It is interesting that a recent report has shown detrimental effects of zinc supplementation on coral growth (Tijssen et al., 2017), a result that could be explained by zinc inhibition of H_v1.

The physiological role of H_v1 channels in corals might be essential in the response of these organisms to ocean acidification. Although further research is needed to understand the cellular and subcellular localization of these channels, we theorize that as the pH of sea water acidifies, gating of H_v1 should require stronger depolarization, thus hindering its capacity to transport protons from the cell. This will contribute to a diminished calcification rate and less aragonite saturation of the CaCO₃ skeleton. It would be interesting and important to study the effects of acidification on H_v1 physiology and pH regulation in corals in vivo. Essentially nothing is known about the electrophysiological properties of coral cells. This report represents the first time that an ion channel has been cloned and characterized in any coral and should open a new avenue of research, such as uncovering the cellular and possible subcellular localization of these channels and carefully measuring their physiological role in vivo.

All data generated or analyzed during this study are included in the manuscript and supporting files. We have provided an Excel file with source data used for figures.

1. 1. Rangel-Yescas G
   2. Cervantes C
   3. Cervantes-Rocha MA
   4. Suárez-Delgado E
   5. Banaszak AT
   6. Maldonado E
   7. Ramsey IS
   8. Rosenbaum T
   9. Islas LD

   (2021) NCBI GenBank

   ID MZ029046. Acropora millepora Hv1 proton-channel nucleotide sequence. MZ029047 - Acropora palmata Hv1 proton-channel nucleotide sequence.

   https://www.ncbi.nlm.nih.gov/nuccore/MZ029046
2. 1. Rangel-Yescas G
   2. Cervantes C
   3. Cervantes-Rocha MA
   4. Suárez-Delgado E
   5. Banaszak AT
   6. Maldonado E
   7. Ramsey IS
   8. Rosenbaum T
   9. Islas LD

   (2021) NCBI GenBank

   ID MZ029047. Acropora palmata Hv1 proton-channel nucleotide sequence.

   https://www.ncbi.nlm.nih.gov/nuccore/MZ029047

1. 1. Australian National University

   (2019) NCBI Protein

   ID XP_029202331.1. Title voltage-gated hydrogen channel 1-like [Acropora millepora].

   https://www.ncbi.nlm.nih.gov/protein/1666377134/
2. 1. King Abdullah University of Science and Technology

   (2017) NCBI Protein

   ID XP_022795192.1. voltage-gated hydrogen channel 1-like [Stylophora pistillata].

   https://www.ncbi.nlm.nih.gov/protein/XP_022795192.1
3. 1. University of Miami

   (2018) NCBI Protein

   ID XP_027057117.1. voltage-gated hydrogen channel 1-like [Pocillopora damicornis].

   https://www.ncbi.nlm.nih.gov/protein/XP_027057117.1
4. 1. QUT

   (2019) NCBI Protein

   ID XP_031564162.1. voltage-gated hydrogen channel 1-like [Actinia tenebrosa].

   https://www.ncbi.nlm.nih.gov/protein/XP_031564162.1
5. 1. Joint Genome Institute (JGI)

   (2017) NCBI Protein

   ID XP_001626501.1. voltage-gated hydrogen channel 1 [Nematostella vectensis].

   https://www.ncbi.nlm.nih.gov/protein/XP_001626501.1
6. 1. JCVI

   (2009) NCBI Protein

   ID XP_012554112.1. PREDICTED: voltage-gated hydrogen channel 1-like [Hydra vulgaris].

   https://www.ncbi.nlm.nih.gov/protein/828190613/
7. 1. Anon

   (2012) NCBI Protein

   ID NP_001119779.1. voltage-gated proton channel [Strongylocentrotus purpuratus].

   https://www.ncbi.nlm.nih.gov/protein/187282419/
8. 1. Anon

   (2006) NCBI Protein

   ID NP_001071937.1. voltage-gated hydrogen channel 1 [Ciona intestinalis].

   https://www.ncbi.nlm.nih.gov/protein/NP_001071937.1
9. 1. Vertebrate Genomes Project

   (2020) NCBI Protein

   ID XP_032803138.1. voltage-gated hydrogen channel 1 [Petromyzon marinus].

   https://www.ncbi.nlm.nih.gov/protein/XP_032803138.1
10. 1. Wellcome Sanger Institute

    (2021) NCBI Protein

    ID XP_040202566.1. voltage-gated hydrogen channel 1 [Rana temporaria].

    https://www.ncbi.nlm.nih.gov/protein/XP_040202566.1
11. 1. Naturalis Biodiversity Center

    (2013) NCBI Protein

    ID ETE71598.1. Voltage-gated hydrogen channel 1, partial [Ophiophagus hannah].

    https://www.ncbi.nlm.nih.gov/protein/565320699/
12. 1. Baylor College of Medicine

    (2005) NCBI Protein

    ID XP_030847861.1. voltage-gated hydrogen channel 1-like isoform X1 [Strongylocentrotus purpuratus].

    https://www.ncbi.nlm.nih.gov/protein/XP_030847861.1
13. 1. China Agricultural University

    (2010) NCBI Protein

    ID EOA95241.1. Voltage-gated hydrogen channel 1, partial [Anas platyrhynchos].

    https://www.ncbi.nlm.nih.gov/protein/EOA95241.1
14. 1. Anon

    (2002) NCBI Protein

    ID NP_001088875.1. voltage-gated hydrogen channel 1 [Xenopus laevis].

    https://www.ncbi.nlm.nih.gov/protein/NP_001088875.1
15. 1. KAUST

    (2015) NCBI Protein

    ID KXJ27230.1. Voltage-gated hydrogen channel 1 [Exaiptasia diaphana].

    https://www.ncbi.nlm.nih.gov/protein/KXJ27230.1
16. 1. The University of Queensland

    (2020) NCBI Protein

    ID XP_035421542.1. voltage-gated hydrogen channel 1 [Cygnus atratus].

    https://www.ncbi.nlm.nih.gov/protein/XP_035421542.1
17. 1. Anon

    (2005) NCBI Protein

    ID NP_001025834.1. voltage-gated hydrogen channel 1 [Gallus gallus].

    https://www.ncbi.nlm.nih.gov/protein/NP_001025834.1
18. 1. Vertebrate Genomes Project

    (2019) NCBI Protein

    ID XP_028914661.1. voltage-gated hydrogen channel 1 [Ornithorhynchus anatinus].

    https://www.ncbi.nlm.nih.gov/protein/XP_028914661.1
19. 1. University of Washington

    (2018) NCBI Protein

    ID XP_025257726.1. voltage-gated hydrogen channel 1 [Theropithecus gelada].

    https://www.ncbi.nlm.nih.gov/protein/XP_025257726.1
20. 1. Anon

    (2002) NCBI Protein

    ID AAH07277.1. HVCN1 protein [Homo sapiens].

    https://www.ncbi.nlm.nih.gov/protein/AAH07277.1
21. 1. The Roslin Institute

    (2020) NCBI Protein

    ID XP_038292573.1. voltage-gated hydrogen channel 1 isoform X1 [Canis lupus familiaris].

    https://www.ncbi.nlm.nih.gov/protein/XP_038292573.1
22. 1. Anon

    (2021) NCBI Protein

    ID NP_001035954.1. voltage-gated hydrogen channel 1 [Mus musculus].

    https://www.ncbi.nlm.nih.gov/protein/NP_001035954.1
23. 1. Beijing Genomics Institute

    (2003) NCBI Protein

    ID XP_005424087.1. voltage-gated hydrogen channel 1 [Mus musculus].

    https://www.ncbi.nlm.nih.gov/protein/XP_005424087.1
24. 1. College of Medicine and Forensics
    2. Xi'an Jiaotong University

    (2014) NCBI Protein

    ID XP_009633183.1. voltage-gated hydrogen channel 1 [Egretta garzetta].

    https://www.ncbi.nlm.nih.gov/protein/XP_009633183.1
25. 1. Vertebrate Genomes Project

    (2020) NCBI Protein

    ID XP_032899181.1. voltage-gated hydrogen channel 1 [Amblyraja radiata].

    https://www.ncbi.nlm.nih.gov/protein/XP_032899181.1
26. 1. US Department of Agriculture
    2. Agriculture Research Service

    (2018) NCBI Protein

    ID RGP61076.1. voltage-gated hydrogen channel 1 [Fusarium longipes].

    https://www.ncbi.nlm.nih.gov/protein/RGP61076.1
27. 1. US Department of Agriculture
    2. Agriculture Research Service

    (2018) NCBI Protein

    ID RFN53390.1. voltage-gated hydrogen channel 1 [Fusarium flagelliforme].

    https://www.ncbi.nlm.nih.gov/protein/RFN53390.1
28. 1. US Department of Agriculture
    2. Agriculture Research Service

    (2020) NCBI Protein

    ID KAF5660113.1. voltage-gated hydrogen channel 1 [Fusarium heterosporum].

    https://www.ncbi.nlm.nih.gov/protein/KAF5660113.1
29. 1. The Institute of Vegetables and Flowers CAAS

    (2019) NCBI Protein

    ID TQW00298.1. voltage-gated hydrogen channel 1 [Cordyceps javanica].

    https://www.ncbi.nlm.nih.gov/protein/TQW00298.1
30. 1. Broad Institute

    (2006) NCBI Protein

    ID XP_005100666.1. voltage-gated hydrogen channel 1 [Aplysia californica].

    https://www.ncbi.nlm.nih.gov/protein/XP_005100666.1
31. 1. The Genomic Institute

    (2017) NCBI Protein

    ID THD25470.1. Voltage-gated hydrogen channel 1 [Fasciola hepatica].

    https://www.ncbi.nlm.nih.gov/protein/THD25470.1
32. 1. Global Invertebrate Genomics Alliance (GIGA)

    (2020) NCBI Protein

    ID KAF6036357.1. HVCN1 [Bugula neritina].

    https://www.ncbi.nlm.nih.gov/protein/KAF6036357.1
33. 1. NINGBO UNIVERSITY

    (2020) NCBI Protein

    ID CAC5426376.1. HVCN1 [Mytilus coruscus].

    https://www.ncbi.nlm.nih.gov/protein/CAC5426376.1
34. 1. McDonnell Genome Institute

    (2020) NCBI Protein

    ID KAF5400532.1. Voltage-gated hydrogen channel 1 [Paragonimus heterotremus].

    https://www.ncbi.nlm.nih.gov/protein/KAF5400532.1
35. 1. Fudan University

    (2019) NCBI Protein

    ID TNN21174.1. Voltage-gated hydrogen channel 1 [Schistosoma japonicum].

    https://www.ncbi.nlm.nih.gov/protein/TNN21174.1
36. 1. BGI-Shenzhen

    (2012) NCBI Protein

    ID XP_012568882.1. voltage-gated hydrogen channel 1 [Cicer arietinum].

    https://www.ncbi.nlm.nih.gov/protein/XP_012568882.1
37. 1. The Cucumber Genome Initiative

    (2009) NCBI Protein

    ID XP_011656484.2. voltage-gated hydrogen channel 1 [Cucumis sativus].

    https://www.ncbi.nlm.nih.gov/protein/XP_011656484.2
38. 1. African Centre of excellence in Phytomedicine Resaerch

    (2018) NCBI Protein

    ID RDX63547.1. Voltage-gated hydrogen channel 1, partial [Mucuna pruriens].

    https://www.ncbi.nlm.nih.gov/protein/RDX63547.1
39. 1. Xi'an Jiaotong University

    (2018) NCBI Protein

    ID XP_026460796.1. voltage-gated hydrogen channel 1 [Papaver somniferum].

    https://www.ncbi.nlm.nih.gov/protein/XP_026460796.1
40. 1. International peanut genome project

    (2018) NCBI Protein

    ID QHO09623.1. Voltage-gated hydrogen channel [Arachis hypogaea].

    https://www.ncbi.nlm.nih.gov/protein/QHO09623.1
41. 1. Chinese Academy of Agricultural Sciences

    (2019) NCBI Protein

    ID XP_038886538.1. voltage-gated hydrogen channel 1 [Benincasa hispida].

    https://www.ncbi.nlm.nih.gov/protein/XP_038886538.1
42. 1. University of Veterinary Medicine Hannover

    (2018) NCBI Protein

    ID RDD43770.1. Voltage-gated hydrogen channel 1 [Trichoplax sp. H2].

    https://www.ncbi.nlm.nih.gov/protein/RDD43770.1
43. 1. Fisheries and Oceans Canada

    (2020) NCBI Protein

    ID XP_035634051.1. voltage-gated hydrogen channel 1-like [Oncorhynchus keta].

    https://www.ncbi.nlm.nih.gov/protein/XP_035634051.1
44. 1. Deakin University

    (2017) NCBI Protein

    ID XP_023152539.1. voltage-gated hydrogen channel 1 [Amphiprion ocellaris].

    https://www.ncbi.nlm.nih.gov/protein/XP_023152539.1
45. 1. Wellcome Sanger Institute

    (2019) NCBI Protein

    ID XP_028830549.1. voltage-gated hydrogen channel 1 [Denticeps clupeoides].

    https://www.ncbi.nlm.nih.gov/protein/XP_028830549.1
46. 1. Yellow Sea Fisheries Research Institute
    2. Chinese Academy of Fishery Sciences

    (2019) NCBI Protein

    ID XP_033488479.1. voltage-gated hydrogen channel 1 [Epinephelus lanceolatus].

    https://www.ncbi.nlm.nih.gov/protein/XP_033488479.1
47. 1. Wellcome Sanger Institute

    (2019) NCBI Protein

    ID XP_029918050.1. voltage-gated hydrogen channel 1 [Myripristis murdjan].

    https://www.ncbi.nlm.nih.gov/protein/XP_029918050.1
48. 1. King Abdullah University of Science and technology

    (2017) NCBI Protein

    ID XP_022070642.1. voltage-gated hydrogen channel 1 [Acanthochromis polyacanthus].

    https://www.ncbi.nlm.nih.gov/protein/XP_022070642.1
49. 1. BGI-SZ

    (2018) NCBI Protein

    ID XP_027031192.1. voltage-gated hydrogen channel 1-like [Tachysurus fulvidraco].

    https://www.ncbi.nlm.nih.gov/protein/XP_027031192.1
50. 1. Mammalian Gene Collection Program Team

    (2002) NCBI Protein

    ID AAH32672.1. HVCN1 protein [Homo sapiens].

    https://www.ncbi.nlm.nih.gov/protein/AAH32672.1
51. 1. Anon

    (2011) NCBI Protein

    ID AEQ59286.1. voltage-gated proton channel kHv1 [Karlodinium veneficum].

    https://www.ncbi.nlm.nih.gov/protein/AEQ59286.1
52. 1. Okinawa Institute of Science and Technology Graduate University (OIST)

    (2015) NCBI Protein

    ID XP_013413952.1. voltage-gated hydrogen channel 1 [Lingula anatina].

    https://www.ncbi.nlm.nih.gov/protein/XP_013413952.1

1. 1. Ahern CA
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Author details

1. Gisela Rangel-Yescas

   Departmento de Fisiología, Facultad of Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico

   Contribution

   Resources, Investigation, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

2. Cecilia Cervantes

   Departmento de Fisiología, Facultad of Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico

   Contribution

   Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

3. Miguel A Cervantes-Rocha

   Departmento de Fisiología, Facultad of Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico

   Contribution

   Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7401-7567

4. Esteban Suárez-Delgado

   Departmento de Fisiología, Facultad of Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico

   Contribution

   Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0147-3451

5. Anastazia T Banaszak

   Unidad Académica de Sistemas Arrecifales, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Puerto Morelos, Mexico

   Contribution

   Resources, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6667-3983

6. Ernesto Maldonado

   EvoDevo Research Group, Unidad Académica de Sistemas Arrecifales, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Puerto Morelos, Mexico

   Contribution

   Resources, Funding acquisition, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9627-967X

7. Ian Scott Ramsey

   Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, United States

   Contribution

   Conceptualization, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6432-4253

8. Tamara Rosenbaum

   Departmento of Neurociencia Cognitiva, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico

   Contribution

   Resources, Software, Formal analysis, Validation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

9. Leon D Islas

   Departmento de Fisiología, Facultad of Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   leon.islas@gmail.com

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-7461-5214

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