Characterization of mechanisms for Ca²⁺ and HCO₃^–/CO₃^2– acquisition for shell formation in embryos of the freshwater common pond snail Lymnaea stagnalis

Embryos of the freshwater common pond snail Lymnaea stagnalis complete direct development, emerging as shell-bearing hatchlings approximately 10 days post-oviposition in a low-ionic strength, minimally buffered environment. In the absence of ambient total calcium (Ca), they tend to develop at a retarded rate relative to controls but grow and hatch at least as well as controls when reared in carbonate-free water (Ebanks et al., 2010). Although they are provided with maternal stores of calcium and carbonate in the perivitelline fluid and within the gelatinous matrix (tunica interna) of the egg mass, they begin to rely on Ca²⁺ from the surrounding medium as maternal stores in the perivitelline fluid and tunica capsulis become depleted, at approximately day 5 post-oviposition (Ebanks et al., 2010). At this point in embryonic development, embryos increase oxygen consumption and total CO₂ retention (Baldwin, 1935). Metamorphosis and shell formation have been observed for Lymnaea palustris (Morrill, 1982), L. stagnalis (Ebanks et al., 2010) and another freshwater pulmonate snail Biomphalaria glabrata (Bielefeld and Becker, 1991; Marxen et al., 2003). The post-metamorphic Ca²⁺ requirements seem to be met entirely by active uptake, but the carbonate requirements can apparently be accommodated through both uptake and endogenous sources or by endogenous sources exclusively (Ebanks et al., 2010).

The idea of Ca²⁺ uptake in freshwater being by active uptake through Ca²⁺ channels has been presented, studied and reviewed from many angles and by many researchers but mostly in freshwater trout (Flik et al., 1995; Marshall et al., 1992; Perry and Flik, 1988; Shahsavarani and Perry, 2006). An electrochemical gradient for Ca²⁺, established by basolateral Ca²⁺-ATPase and Na⁺/K⁺-ATPase activity, and further facilitated and maintained by an apical, outwardly directed H⁺ pump provides the necessary gradient to allow for Ca²⁺ uptake under these conditions (Perry et al., 2003). Hydrogen ions supplied to this pump are mostly from hydration of CO₂. In addition to acid excretion by the H⁺ pump, protons can be extruded in exchange for monovalent, as well as divalent, cations via Na⁺/H⁺ exchangers (NHE) by electroneutral or electrogenic exchange (for a review, see Ahearn et al., 2001). Recent work by Okech et al. (Okech et al., 2008) indicated cation exchange for hydrogen ions can be coupled with vacuolar H⁺-ATPase (V-ATPase) activity on electrogenic Na⁺/H⁺ antiporters. A further point of consideration that is important to this study is that 2Na⁺/1H⁺ electrogenic exchange, which is apparently unique to invertebrates, can accommodate divalent cations, including Ca²⁺, in place of the two Na⁺ (Ahearn et al., 1994).

Our previous observations of unimpeded growth under HCO₃^–-free conditions led to subsequent experiments examining the transport of acid and base equivalents under calcifying, post-metamorphic conditions. We found that the onset of apparent net titratable alkalinity uptake/acid extrusion was concurrent with metamorphosis, the onset of Ca²⁺ uptake and shell formation. Also, Ca²⁺ uptake was significantly greater and apparent net titratable alkalinity uptake/acid extrusion was significantly reduced, but not inhibited, under HCO ^–₃-free conditions (Ebanks et al., 2010). These findings motivated the series of experiments to determine the possible sources of carbonate for shell formation that are described in this paper.

Considering that carbonic anhydrase-mediated CO₂ hydration produces HCO ^–₃, this was a logical starting point. The possibility of endogenous bicarbonate contributing to calcification in the embryos (Baldwin, 1935) and adults (Greenaway, 1971) of this species has been suggested previously in the literature. However, the point of development at which this pathway is activated has not been studied until recently (Ebanks et al., 2010). Also, the possible role of the proton, resulting from CO₂ hydration and CaCO₃ formation, in the acquisition of the essential shell-forming ions by these embryos remains to be evaluated. Therefore, the objectives of this study were to use a pharmacological approach in which flux was measured on the scale of the whole egg mass and the microscale of single eggs to determine the acquisition pathways for shell-forming ions in post-metamorphic embryos of the freshwater common pond snail Lymnaea stagnalis. Additionally, we targeted the possible relationship between protons produced by carbonic anhydrase activity and Ca²⁺ uptake. Each of the proposed pathways was evaluated on whole egg mass and at microscale levels when possible.

Egg masses of the common pond snail Lymnaea stagnalis (L.) were collected from our laboratory snail culture at 24-h intervals, with the day of collection considered day 0 post-oviposition, for age determination. Embryos were incubated at room temperature (20–22°C) in aerated, dechlorinated Miami-Dade County tap water (Grosell et al., 2007). Whole, intact egg masses were selected and scored for viability in accordance with criteria outlined in Ebanks et al. (Ebanks et al., 2010) prior to use in experimental procedures. With an exception of the niphedipine, verapamil and bafilomycin experiments using the ion-specific microelectrodes, all experiments were completed on intact egg masses as defined in Ebanks et al. (Ebanks et al., 2010).

Three Ca²⁺ channel blockers were used independently to determine the type and the role of Ca²⁺ channels that may be involved. Nifedipine, which is a voltage-dependent L-type Ca²⁺ channel blocker, was applied as was verapamil, another voltage-dependent L-type Ca²⁺ channel blocker and α1-adrenoceptor antagonist. Lanthanum, an indiscriminate Ca²⁺ channel blocker was also tested. Day 7 egg masses were scored for normal development and placed in Miami-Dade tap water that was either drug-free control (0.1% dimethylsulphoxide; DMSO) or treated for 24 h with either 10 μmol l^–1 nifedipine (Sigma-Aldrich, St Louis, MO, USA), 10 μmol l^–1 verapamil hydrochloride (Sigma-Aldrich), 10 μmol l^–1 La³⁺ or 100 μmol l^–1 La³⁺ as LaCl₃. Initial and final water samples were collected for Ca²⁺ and titratable alkalinity (TA) net flux determinations, which were completed following the protocol for double-endpoint titration described by Ebanks et al. (Ebanks et al., 2010), and are reported in μmol g^–1 h^–1. Considering that carbonate necessary for calcification could be from aquatic sources or endogenous processes (Ebanks et al., 2010), a multi-pronged experimental approach was required. The role of carbonic anhydrase (CA) in the production of endogenous bicarbonate via CO₂ hydration was evaluated using a lipophilic carbonic anhydrase inhibitor, ethoxzolamide (ETOX; Sigma-Aldrich), dissolved in DMSO. Controls (0.1% DMSO) and 100 μmol l^–1 ETOX-exposed egg masses were incubated for 24 h; water samples were taken before and after incubation for Ca²⁺ and TA net flux determinations. In addition to HCO ^–₃, CA-catalyzed hydration of CO₂ also produces equal quantities of H⁺, which are counterproductive to shell formation and must be excreted to make CO₂ hydration a viable source of CO ^2–₃ for shell formation. Thus we evaluated the role of V-ATPase (a H⁺ pump) in the removal of protons and possibly Ca²⁺ uptake, considering that apical Ca²⁺ channels may be driven by an inward-directed electrochemical gradient fueled in part by electrogenic H⁺ excretion via the H⁺ pump. Bafilomycin (Sigma-Aldrich), a proton-pump inhibitor, was dissolved in DMSO for a final concentration of 1 μmol l^–1 bafilomycin and 0.1% DMSO in each flux chamber. Day-7 egg masses were scored for viability and placed in individual flux chambers for a 24-h incubation, after which we determined net Ca²⁺ and titratable alkalinity flux as described below. Also, because of the potential for direct exchange of Ca²⁺ for H⁺ on an apical invertebrate-specific cation/1H⁺ exchanger, we completed a 24-h flux experiment in which day-7 egg masses were treated with 100 μmol l^–1 of the NHE-specific amiloride analogue, ethylisopropylamiloride (EIPA; Sigma-Aldrich) dissolved in a final concentration of 0.1% DMSO.

The procedures for determination of flux rates were in accordance with those outlined by Ebanks et al. (Ebanks et al., 2010). Briefly, samples of pre- and post-flux media were collected for each treatment and Ca²⁺ net flux was determined by using the difference in [Ca]_total in initial and final flux media following the 24-h flux. Samples were measured by flame atomic absorption spectrophotometry (Varian SpectrAA220, Mulgrave, Victoria, Australia) and flux rates were calculated from the change in [Ca]_total, mass of egg mass (g), and elapsed time (h). Net titratable alkalinity flux was calculated from the change in titratable alkalinity as determined by double endpoint titration (Ebanks et al., 2010), mass of egg mass (g) and elapsed time (h). Final values for both Ca²⁺ and titratable alkalinity net flux parameters are presented as μmol g^–1 h^–1.

Microelectrodes used to measure Ca²⁺ and H⁺ gradients in the unstirred layer next to the surface of egg masses and isolated eggs were pulled from unfilamented 1.5 mm borosilicate capillary glass (A-M Systems, Carlsborg, WA, USA), silanized by exposure to dimethyldichlorosilane vapor at 200°C for 60 min and stored over silica gel until use. Ca²⁺-selective microelectrodes were backfilled with 100 mmol l^–1 CaCl₂ and tip-filled with a 300–500 μm column of Ca²⁺ ionophore I, cocktail A (Fluka, Buchs, Switzerland). H⁺-selective microelectrodes were backfilled with a solution of 100 mmol l^–1 NaCl and 100 mmol l^–1 sodium citrate at pH 6, then tip-filled with a 300–500 μm column of H⁺ ionophore I, cocktail B (Fluka). H⁺ microelectrodes calibrated in solutions buffered with 10 mmol l^–1 Hepes gave Nerstian slopes of 58–60 mV per pH unit.

Transport of calcium ions or protons into or out of egg masses or isolated eggs produces gradients in Ca²⁺ or H⁺ concentration in the unstirred layer adjacent to the surface of the preparation. These gradients can be calculated from the voltages recorded by an ion-selective microelectrode moved between two points within the unstirred layer. Calcium flux can then be calculated from the Ca²⁺ concentration gradients using Fick's law. Measurement of fluxes in this way is the basis of the scanning ion electrode technique (SIET), which allows fluxes to be repeatedly measured in near real-time at sites along the length of an egg mass or an isolated egg (Ebanks et al., 2010).

SIET measurements were made using hardware from Applicable Electronics (Forestdale, MA, USA) and automated scanning electrode technique (ASET) software (version 2.0) from Science Wares Inc. (East Falmouth, MA, USA). Egg masses or isolated eggs were placed in a 35 mm diameter Petri dish filled with 5 ml of artificial Miami-Dade tap water. Reference electrodes were made from 10 cm lengths of 1.5 mm borosilicate glass capillaries that were bent at a 45 deg angle, 1–2 cm from the end, to facilitate placement in the sample dish. Capillaries were filled with boiling 3 mol l^–1 KCl in 3% agar and connected to the ground input of the Applicable Electronics amplifier headstage through a Ag/AgCl half cell.

Calculation of H⁺ fluxes requires knowledge of the buffers that are present (e.g. Donini and O'Donnell, 2005). Ideally, a non-volatile buffer such as Hepes is used. However, because we wished to use the same Miami-Dade tap water as used for the whole egg mass flux experiments, we did not modify the composition of the water and therefore we present H⁺ gradients as microelectrode voltage rather than proton flux in the Results.

Comparisons were completed using either t-test or an ANOVA with contingent significant results evaluated as pairwise comparisons following the Holm–Sidak method after an analysis for whether the datasets were normally distributed. Statements of significance were reserved for those comparisons for which P<0.05.

Absolute flux rates of egg masses of similar age varied from batch to batch even under similar conditions. The reason for this variation is unknown but underscores the importance of performing control and treatment experiments simultaneously and on egg masses from the same batch as was done in the present and previous study (Ebanks et al., 2010). Results from our multi-pronged pharmacological approach indicated that voltage-dependent Ca²⁺ channels, H⁺-pump activity and Na⁺/H⁺ antiport function in conjunction with CA activity allow the post-metamorphic embryonic snail to complete calcification and shell formation in freshwater. The two voltage-dependent Ca²⁺ channel blockers had different effectiveness against net Ca²⁺ uptake with nifedipine being the more effective blocker when examined by net Ca²⁺ flux measurements and SIET techniques (Figs 1 and 2). Verapamil did not significantly block net Ca²⁺ transport at 10 μmol l^–1 but 10 μmol l^–1 nifedipine did in whole egg mass experiments (Fig. 1). Scanning Ca²⁺-selective microelectrode analysis showed that both voltage-dependent Ca²⁺-channel blockers reduced Ca²⁺ transport at 10 μmol l^–1 but that 100 μmol l^–1 verapamil completely blocked net Ca²⁺ uptake (Fig. 2). Neither drug influenced net titratable alkalinity flux (data not shown). Lanthanum blocked net Ca²⁺ uptake at 100 μmol l^–1 but not at 10 μmol l^–1 in whole egg mass experiments (Fig. 3). There was also a significant increase in apparent net titratable alkalinity uptake/acid extrusion in the presence of 100 μmol La³⁺(data not shown). Microelectrode experiments revealed a tendency for reduced Ca²⁺ uptake with 100 μmol l^–1 La³⁺ but just below statistical significance (Fig. 4).

Using 100 μmol l^–1 ETOX to evaluate the role of CA-catalyzed hydration of CO₂ on whole egg mass flux rates, we observed a significant reduction in both net Ca²⁺ uptake and apparent titratable alkalinity uptake/acid extrusion (Fig. 5). This link between CO₂ hydration and Ca²⁺ uptake led to the testing of the possible role of the apical H⁺ pump and Na⁺/H⁺ exchange by evaluating the inhibitory effects of bafilomycin and EIPA at concentrations of 1 μmol l^–1 and 100 μmol l^–1, respectively, on Ca²⁺ and titratable alkalinity transport. Proton-pump inhibition resulted in inhibition of net Ca²⁺ uptake (Fig. 6A) and apparent net titratable alkalinity uptake (equivalent to reduced acid secretion; Fig. 6B). Scanning Ca²⁺-selective microelectrode measurements revealed that bafilomycin had a significant inhibitory effect of up to 80% on Ca²⁺ transport for post-metamorphic egg masses (data not shown).

Furthermore, and in agreement with whole egg mass fluxes, Ca²⁺ uptake was inhibited in isolated eggs containing later-stage day-11 to -13 embryos, treated with 1 μmol l^–1 bafilomycin (Fig. 7A) and H⁺ gradients were effectively reversed (Fig. 7B) as measured with ion-selective microelectrodes in the unstirred boundary layer. Similar to the bafilomycin treatment, both Ca²⁺ (Fig. 8A) and titratable alkalinity (Fig. 8B) net flux were reversed in whole egg mass fluxes where flux medium was treated with 100 μmol l^–1 EIPA.

The system for Ca²⁺ and carbonate acquisition in the freshwater common pond snail Lymnaea stagnalis exhibited sensitivity to several pharmacological agents, which indicate that both Ca²⁺ uptake from the environment as well as endogenous HCO₃^– and/or CO₃^2– production work together directly and indirectly for the post-metamorphic embryo to acquire the necessary ions for calcification in freshwater (Fig. 9). Both the dihydropyridine nifedipine and the phenylalkylamine verapamil are known to block L-type Ca²⁺ channels, with nifedipine displaying relatively greater specificity and efficacy to L-type voltage-dependent Ca²⁺ channels (Hagiwara and Byerly, 1981). These compounds inhibit Ca²⁺ channels in some epithelia such as the distal convoluted tubule of the rabbit kidney (Poncet et al., 1992) but not in others, such as the cortical thick ascending limb (Nitschke et al., 1991). The model that we are proposing here for post-metamorphic embryos compliments findings of Greenaway (Greenaway, 1971) using radioisotopic techniques on adults of the species. In the Greenaway model for adult Lymnaea, blood, fresh tissues and shell are identified as the major compartments between which Ca²⁺ is transported (Greenaway, 1971). In times of shell accretion, Ca²⁺ is taken up from the surrounding medium or from food and into the blood for transport to fresh tissues, particularly the mantle, for deposition as CaCO₃ after combining with endogenously produced HCO ^–₃. Internal production of HCO ^–₃ by the hydration of CO₂, also produces H⁺ ions that are released from the tissues, transported by the blood to excretion sites on the epithelial membrane, and can be exchanged for Ca²⁺. During times of Ca²⁺ depletion, Ca²⁺ can be acquired from the shell, taken into the soft tissues and blood for transport to sites in need to Ca²⁺ supplement. Thus in adult Lymnaea, Ca²⁺ is transported between ambient media, fresh tissues, and shell via the blood (Greenaway, 1971). Considering that these embryos complete direct development, hatching as fully developed snails, it is not surprising to find that the method for acquisition of shell forming ions in embryos apparently is similar to that modeled by Greenaway (Greenaway, 1971).

When applying pharmacological agents that were developed and tested for use on mammals, it is necessary to consider that there may be a reduced degree of specificity of the drug. Additionally, using these chemicals with ion-sensitive microelectrodes can interfere with the sensitivity and function of the electrode. However, our preliminary tests for drug interaction and/or poisoning of the electrode, and the general agreement between results obtained by the two techniques used in the present study, give confidence in the accuracy of our findings. The voltage-dependent Ca²⁺ channel blockers and bafilomycin gave consistent results between whole egg mass net flux measurements and assessment of flux rates by microelectrodes validating the reported observation. With the exception of EIPA, none of the drugs interfered with microelectrode function at the concentrations used in our experiments. Unfortunately, EIPA interfered with both Ca²⁺- and H⁺-selective microelectrodes. EIPA concentrations of 100 μmol l^–1 reduced the slope of the Ca²⁺-selective microelectrodes and caused the H⁺ microelectrode voltage to drift. These effects are consistent with a poisoning of the ionophore cocktail by EIPA at 100 μmol l^–1. We could not use SIET, therefore, to verify the whole egg mass flux observations that suggested the presence of a cation/H⁺ exchanger. Nevertheless, the excellent agreement between observations when made with both techniques is reassuring. L-type voltage-dependent Ca²⁺ channels and an EIPA-sensitive cation exchanger appear to be involved in Ca²⁺ uptake in post-metamorphic embryos (Fig. 9).

In the absence of external HCO ^–₃, Lymnaea stagnalis embryos grow, form shell, develop normally, and hatch in the same time intervals as controls (Ebanks et al., 2010). These observations point to an internal source of HCO ^–₃/CO ^2–₃ for shell formation via the hydration of endogenous CO₂. For this hydration reaction to allow for CO ^2–₃ accumulation within the organism and for shell formation, hydrogen ions resulting from the hydration reaction must be eliminated (Fig. 9). We have demonstrated that the enzyme carbonic anhydrase (CA), which facilitates the hydration of endogenous CO₂ is involved in the acquisition of ions for calcification. Also, though developing snails show an apparent net uptake of Ca²⁺ and HCO ^–₃ equivalents, the movement of acid–base equivalents is in reality a combination of acid secretion and base uptake, because it persists, although at reduced rates, in absence of ambient HCO ^–₃ (Ebanks et al., 2010). Our SIET measurements of H⁺ gradients further confirm the role of H⁺ excretion.

The effects of CA inhibition indicated a direct role for CA in carbonate production in post-metamorphic embryos but also points to an indirect role in Ca²⁺ acquisition that is revealed when considered in conjunction with the bafilomycin A₁ results. Bafilomycin inhibits V-ATPase activity, which has been found to be ubiquitous among eukaryotes (Harvey, 1992; Finbow and Harrison, 1997; Harvey et al., 1998). This includes invertebrates and more directly related to our model species, calcifying organisms such as blue crabs (Cameron and Wood, 1985). This enzyme has also been characterized in tissues of freshwater bivalves Anodonta cygnea (Machado et al., 1990; da Costa et al., 1999; Oliveira et al., 2004), Unio complanatus (Hudson, 1993) and Nucella lamellosa (Clelland and Saleuddin, 2000). The connection between H⁺-pump activity and cation uptake has been demonstrated in epithelia of a variety of species (Harvey et al., 1998), including Ca²⁺ uptake by post-moult blue crabs Callinectes sapidus (Cameron and Wood, 1985; Neufeld and Cameron, 1992). There have also been reviews on the presence of this pathway in crustaceans (Neufeld and Cameron, 1993). Furthermore, H⁺-pump-driven active cation uptake has been documented in adults of this species (Ebanks and Grosell, 2008) as well as in several other freshwater species including carp (Fenwick et al., 1999), tilapia (Fenwick et al., 1999) and trout (Bury and Wood, 1999; Grosell and Wood, 2002; Lin and Randall, 1991). Thus inhibition of Ca²⁺ uptake in the presence of the V-ATPase inhibitor, bafilomycin, in developing snail embryos is persistent across later time points (up to 80% reduction in post-metamorphic Ca²⁺ uptake) and appears to be consistent with investigations in other species. Thus the H⁺ pump plays a significant role in Ca²⁺ flux in post-metamorphic stages. By electrogenic excretion of H⁺ ions, the electrical potential across the apical membrane, and thereby the electrochemical gradient for Ca²⁺ uptake is maintained. In addition, the observations of reduced acid excretion and Ca²⁺ uptake in the presence of the specific Na⁺/H⁺ exchange inhibitor, EIPA, points to an even more direct coupling of H⁺ extrusion to Ca²⁺ uptake. However, Hudson found no effect of 1 mmol l^–1 amiloride or Na⁺-depleted solutions on acid secretion in Ussing-type chamber experiments on mantle epithelia of U. complanatus (Hudson, 1993). EIPA was not included in the study, but there was no detectable coupling between a proxy for acid secretion, cAMP-dependent short-circuit current (I_SC) (Machado et al., 1990), and unidirectional Na⁺, K⁺, Ca²⁺ or Cl^– fluxes in the same species (Hudson, 1993). Nevertheless, our data suggest the presence and involvement of a Ca²⁺/H⁺ exchanger in L. stagnalis. It is interesting to note that electrogenic Ca²⁺/H⁺ exchange would be facilitated by parallel electrogenic H⁺ extrusion by the H⁺ pump.

Taken together, our results suggest that through the use of the ionic products of CO₂ hydration, H⁺ and HCO₃^–, the high Ca²⁺ and carbonate demands for shell formation in freshwater are met and maintained with notable efficiency. The higher the rate of CO₂ hydration, the higher the rate of HCO ^–₃ formation, and the higher the amount of H⁺ excretion by the proton pumps and/or cation exchange, which in turn provides for Ca²⁺ uptake. Inhibition of CA activity leads to inhibition of transport of both shell-forming ions. Admittedly, inhibition of the ability to hydrate respiratory CO₂ can have broader impacts on the physiological level. However, when the results of CA inhibition are taken in conjunction with the response to H⁺ pump and NHE inhibition as well as the ability of the embryos to develop in HCO ^–₃-free conditions (Ebanks et al., 2010), there is substantial evidence that endogenously produced carbonate can be an exclusive carbonate source for these post-metamorphic embryos. A further point to consider is that in addition to protons produced from hydration of metabolic CO₂, H⁺ ions are liberated by the calcification process (HCO₃^–+Ca²⁺→CaCO₃+H⁺) adding to the need for acid secretion by these animals. By coupling Ca²⁺ uptake and HCO₃^– production with the generation of inside alkaline conditions via H⁺ extrusion, the conditions for calcification within the egg and egg mass are enhanced. Furthermore, the depletion of Ca²⁺ within the perivitelline fluid and gelatinous matrix (tunica interna) between the eggs to levels below water concentrations during later embryonic stages (Ebanks et al., 2010) creates an inward Ca²⁺ gradient and thus can enhance the influx of Ca²⁺ from the surrounding water while decreasing the energy required for ion uptake.

Acquisition of the necessary Ca²⁺ and carbonate for calcification by embryos of the freshwater common pond snail Lymnaea stagnalis are intricately intertwined. These embryos take up Ca²⁺ from ambient media after maternal stores in the perivitelline fluid have been depleted; but they also directly utilize endogenously produced bicarbonate from the CA-catalyzed hydration of CO₂. Protons generated from CO₂ hydration are excreted, which fuels continued Ca²⁺ uptake and allows for accumulation of bicarbonate and maintenance of alkaline internal conditions that are favorable for calcification. With endogenous production of HCO ^–₃ for calcification perpetuating continued Ca²⁺ uptake, these embryos are capable of efficient calcification under low ionic strength conditions.

With global climate change, studies are necessary to determine how ambient pH and bicarbonate buffering affect the ability of these freshwater snails to calcify. Furthermore, evaluating the extent to which acid secretion rather than base uptake is the basis for calcification in other freshwater as well as marine calcifiers offers an exciting area for comparative studies.

 
• CA

  carbonic anhydrase

 
• EIPA

  ethylisopropylamiloride (C₁₁H₁₈ClN₇O)

 
• ETOX

  ethoxzolamide (C₉H₁₀N₂O₃S₂)

 
• NHE

  Na⁺/H⁺ exchanger