Transport Metabolon Research Articles

Can hypoxia marker carbonic anhydrase IX serve as a potential new diagnostic marker and therapeutic target of non-small cell lung cancer?

Lung cancer represents the leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC), the most common form of lung cancer, is a molecularly heterogeneous disease with intratumoral heterogeneity and a significant mutational burden associated with clinical outcome. Tumor microenvironment (TME) plays a fundamental role in the initiation and progression of primary de novo lung cancer and significantly influences the response of tumor cells to therapy. Hypoxia, an integral part of the tumor microenvironment and a serious clinical phenomenon, is associated with increased genetic instability and a more aggressive phenotype of NSCLC, which correlates with the risk of metastasis. Low oxygen concentration influences all components of TME including the immune microenvironment. Hypoxia-inducible pathway activated in response to low oxygen supply mediates the expression of genes important for the adaptation of tumor cells to microenvironmental changes. A highly active transmembrane hypoxia-induced metalloenzyme - carbonic anhydrase IX (CAIX), as a part of transport metabolon, contributes to the maintenance of intracellular pH within physiological values and to the acidification of the extracellular space. CAIX supports cell migration and invasion and plays an important role in NSCLC tumor tissue and pleural effusion. Due to its high expression, it also represents a potential diagnostic differential biomarker and therapeutic target in NSCLC. To test new potential targeted therapeutic compounds, suitable models are required that more faithfully simulate tumor tissue, TME components, and spatial architecture.

Proton Transport in Cancer Cells: The Role of Carbonic Anhydrases.

Intra- and extracellular pH regulation is a pivotal function of all cells and tissues. Net outward transport of H+ is a prerequisite for normal physiological function, since a number of intracellular processes, such as metabolism and energy supply, produce acid. In tumor tissues, distorted pH regulation results in extracellular acidification and the formation of a hostile environment in which cancer cells can outcompete healthy local host cells. Cancer cells employ a variety of H+/HCO3−-coupled transporters in combination with intra- and extracellular carbonic anhydrase (CA) isoforms, to alter intra- and extracellular pH to values that promote tumor progression. Many of the transporters could closely associate to CAs, to form a protein complex coined “transport metabolon”. While transport metabolons built with HCO3−-coupled transporters require CA catalytic activity, transport metabolons with monocarboxylate transporters (MCTs) operate independently from CA catalytic function. In this article, we assess some of the processes and functions of CAs for tumor pH regulation and discuss the role of intra- and extracellular pH regulation for cancer pathogenesis and therapeutic intervention.

Interplay of Carbonic Anhydrase IX With Amino Acid and Acid/Base Transporters in the Hypoxic Tumor Microenvironment.

Solid tumors are challenged with a hypoxic and nutrient-deprived microenvironment. Hence, hypoxic tumor cells coordinatively increase the expression of nutrient transporters and pH regulators to adapt and meet their bioenergetic and biosynthetic demands. Carbonic Anhydrase IX (CAIX) is a membrane-bound enzyme that plays a vital role in pH regulation in the tumor microenvironment (TME). Numerous studies have established the importance of CAIX in mediating tumor progression and metastasis. To understand the mechanism of CAIX in mediating tumor progression, we performed an unbiased proteomic screen to identify the potential interactors of CAIX in the TME using the proximity-dependent biotin identification (BioID) technique. In this review, we focus on the interactors from this BioID screen that are crucial for nutrient and metabolite transport in the TME. We discuss the role of transport metabolon comprising CAIX and bicarbonate transporters in regulating intra- and extracellular pH of the tumor. We also discuss the role of amino acid transporters that are high confidence interactors of CAIX, in optimizing favorable metabolic state for tumor progression, and give our perspective on the coordinative interplay of CAIX with the amino acid transporters in the hypoxic TME.

Experimental Approaches to Identify Selective Picomolar Inhibitors for Carbonic Anhydrase IX.

Carbonic anhydrases (CAs) regulate pH homeostasis via the reversible hydration of CO2, thereby emerging as essential enzymes for many vital functions. Among 12 catalytically active CA isoforms in humans, CA IX has become a relevant therapeutic target because of its role in cancer progression. Only two CA IX inhibitors have entered clinical trials, mostly due to low affinity and selectivity properties. The current review presents the design, development, and identification of the selective nano- to picomolar CA IX inhibitors VD11-4-2, VR16-09, and VD12-09. Compounds were selected from our database, composed of over 400 benzensulfonamides, synthesized at our laboratory, and tested for their binding to 12 human CAs. Here we discuss the CA CO2 hydratase activity/inhibition assay and several biophysical techniques, such as fluorescent thermal shift assay and isothermal titration calorimetry, highlighting their contribution to the analysis of compound affinity and structure- activity relationships. To obtain sufficient amounts of recombinant CAs for inhibitor screening, several gene cloning and protein purification strategies are presented, including site-directed CA mutants, heterologous CAs from Xenopus oocytes, and native endogenous CAs. The cancer cell-based methods, such as clonogenicity, extracellular acidification, and mass spectrometric gas-analysis are reviewed, confirming nanomolar activities of lead inhibitors in intact cancer cells. Novel CA IX inhibitors are promising derivatives for in vivo explorations. Furthermore, the simultaneous targeting of several proteins involved in proton flux upon tumor acidosis and the disruption of transport metabolons might improve cancer management.

The devil is in the C-tail: a NBCe1-carbonic anhydrase bicarbonate transport metabolon?

The concept of metabolons is well established in biochemistry as structural–metabolic complexes in cells that facilitate substrate channelling through the complex and is best exemplified by the citric acid cycle enzymes. Similarly, an arrangement of enzymes was suggested to promote the transport of acid/base equivalents across the plasma membrane by the Reithmeier group. The concept was supported by direct evidence of physical interaction of an acid/base transporter and carbonic anhydrase (Vince & Reithmeier, 1998) and emboldened by the demonstration of a functional advantage for such an arrangement called a ‘transport metabolon’ (Sterling et al. 2001). The direct protein interaction was first reported for the C-terminal of anion exchanger AE1 (band 3) and the cytosolic ubiquitous carbonic anhydrase II (CAII). Since then, the field has evolved to other acid/base transporters of the SLC4 gene family of HCO3− transporters, the Na+/H+ exchangers (NHE) and monocarboxylate transporters interacting with soluble or membrane-bound CA isozymes in various expression systems and native tissues. Discrepancies have since evolved both regarding the direct physical interaction of acid/base transporters and CA enzymes, and regarding the functional interaction between the individual molecules of such complexes. This ‘Perspectives’ article only addresses the case of the functional interaction of the human renal form of the electrogenic Na+:HCO3− cotransporter NBCe1-A (kNBCe1) and CAII when expressed in Xenopus laevis oocytes, but may prove highly relevant for the metabolon field as such. In one study, the NBCe1 function determined as the current slope conductance (Gm INBC) was not affected by injection of CAII protein or the expression of either NBCe1-CAII or eGFP-NBCe1-CAII fusion proteins (Lu et al. 2006). No effect of the cell-permeant CA inhibitor ethoxyzolamide (EZA) on the Gm INBC was observed. In a separate and opposing study, NBCe1 function assessed as the changes in membrane current (Im), the Na+ flux (d[Na+]i/dt), as well as the membrane conductance (Gm) was augmented by bovine CA protein injection or CAII co-expression (Becker & Deitmer 2007). The induced changes in current and Na+ fluxes were EZA-sensitive in this study. Thus, although the two studies applied similar methodological set-ups at first glance, the results were contrasting. Each paper is compelling to the reader, and both studies were carefully and elegantly performed. Moss and Boron (2020) first carefully replicate the findings by Lu et al. (2006) and then elegantly rule out differences in the amount of injected cRNA, CAII protein and eGFP tag as contributing factors to conflicting results by the two groups. Furthermore, they show that prolonged EZA exposure or the high EZA concentration by Lu et al. were not causing the inconsistency between the previous studies. It also appears that the variations in voltage-clamping protocols cannot explain the contradicting results per se (Moss & Boron 2020). Remarkably, the authors are able to closely replicate the fundamental observations by Becker & Deitmer (2007) that injection of bovine CA in oocytes expressing untagged NBCe1 increases the Na+ flux (d[Na+]i/dt)max as well as the membrane current Im, effects that are sensitive to CA inhibition by EZA. This is apparently only possible 1) in the absence of eGFP tag, 2) by bovine CA injection instead of human CAII, and 3) applying the prolonged -40mV voltage clamp and 10 s step duration of I-V ramps. Surprisingly, Moss and Boron demonstrate that injection of either human CAII or bovine CA alone in oocytes induces an electroneutral process that increases Na+ uptake and acid extrusion upon CO2-induced acid loading. The authors ascribe the effects to the induction of endogenous NHE activity, as this process is inhibited by EIPA. Interestingly, EIPA also prevents the EZA-sensitive change in Im and (d[Na+]i/dt)max induced by injection of bovine CA using the prolonged -40 mV voltage clamp protocol. Thus, Moss and Boron explain the discrepancy between the previous studies (Lu et al. 2006 vs. Becker & Deitmer 2007) by the induction of native NHE activity by exogenous CA in Xenopus oocytes. With the study by Moss and Boron (2020), the specific controversy regarding enhanced NBCe1 activity by CA in oocytes has most likely been fully resolved. The authors accomplished a systematic comparison of experimental approaches resulting in a study with high internal consistency presented in a respectful and transparent manner. The take-home message is that even the most careful studies by world-renowned laboratories can reach conflicting conclusions and that unexpected sources of discrepancies can be identified by hard and meticulous work. The findings by Moss and Boron (2020) should impact the broader transport metabolon field; however, the conclusions can probably not be extrapolated to all other cases of acid/base transporters, CA forms and cell types for which transport metabolons have been suggested. Hopefully, time will tell. None. Sole author. None.

Transport Metabolons and Acid/Base Balance in Tumor Cells.

Solid tumors are metabolically highly active tissues, which produce large amounts of acid. The acid/base balance in tumor cells is regulated by the concerted interplay between a variety of membrane transporters and carbonic anhydrases (CAs), which cooperate to produce an alkaline intracellular, and an acidic extracellular, environment, in which cancer cells can outcompete their adjacent host cells. Many acid/base transporters form a structural and functional complex with CAs, coined “transport metabolon”. Transport metabolons with bicarbonate transporters require the binding of CA to the transporter and CA enzymatic activity. In cancer cells, these bicarbonate transport metabolons have been attributed a role in pH regulation and cell migration. Another type of transport metabolon is formed between CAs and monocarboxylate transporters, which mediate proton-coupled lactate transport across the cell membrane. In this complex, CAs function as “proton antenna” for the transporter, which mediate the rapid exchange of protons between the transporter and the surroundings. These transport metabolons do not require CA catalytic activity, and support the rapid efflux of lactate and protons from hypoxic cancer cells to allow sustained glycolytic activity and cell proliferation. Due to their prominent role in tumor acid/base regulation and metabolism, transport metabolons might be promising drug targets for new approaches in cancer therapy.

Carbonic Anhydrase XII Expression Is Modulated during Epithelial Mesenchymal Transition and Regulated through Protein Kinase C Signaling.

Members of the carbonic anhydrase family are functionally involved in the regulation of intracellular and extracellular pH in physiological and pathological conditions. Their expression is finely regulated to maintain a strict control on cellular homeostasis, and it is dependent on the activation of extracellular and intracellular signaling pathways. Combining RNA sequencing (RNA-seq), NanoString, and bioinformatics data, we demonstrated that the expression of carbonic anhydrase 12 (CAXII) is significantly different in luminal and triple negative breast cancer (BC) models and patients, and is associated with the activation of an epithelial mesenchymal transition (EMT) program. In BC models, the phorbol ester 12-myristate 13-acetate (PMA)-mediated activation of protein kinase C (PKC) induced a down-regulation of CAXII with a concomitant modulation of other members of the transport metabolon, including CAIX and the sodium bicarbonate cotransporter 3 (NBCn1). This is associated with a remodeling of tumor glycolytic metabolism induced after PKC activation. Overall, this analysis highlights the dynamic nature of transport metabolom and identifies signaling pathways finely regulating this plasticity.

Construction of artificial membrane transport metabolons - an emerging strategy in metabolic engineering.

The term 'membrane transport metabolon' refers to the physical association of membrane transporters with enzymes that metabolize the transported substrates. In naturally evolved systems, physiological relevance of coupling transport with sequential enzymatic reactions resides, for instance, in faster turnover rates, protection of substrates from competing pathways or shielding the cellular environment from toxic compounds. Such underlying principles offer attractive possibilities for metabolic engineering approaches and concepts for constructing artificial transporter-enzyme complexes are recently being developed. In this minireview, the modes of substrate channeling across biological membranes and design principles for artificial transport metabolons are discussed.

Energy Dynamics in the Brain: Contributions of Astrocytes to Metabolism and pH Homeostasis.

Regulation of metabolism is complex and involves enzymes and membrane transporters, which form networks to support energy dynamics. Lactate, as a metabolic intermediate from glucose or glycogen breakdown, appears to play a major role as additional energetic substrate, which is shuttled between glycolytic and oxidative cells, both under hypoxic and normoxic conditions. Transport of lactate across the cell membrane is mediated by monocarboxylate transporters (MCTs) in cotransport with H+, which is a substrate, a signal and a modulator of metabolic processes. MCTs form a “transport metabolon” with carbonic anhydrases (CAs), which not only provide a rapid equilibrium between CO2, HCO3– and H+, but, in addition, enhances lactate transport, as found in Xenopus oocytes, employed as heterologous expression system, as well as in astrocytes and cancer cells. Functional interactions between different CA isoforms and MCTs have been found to be isoform-specific, independent of the enzyme’s catalytic activity, and they require physical interaction between the proteins. CAs mediate between different states of metabolic acidosis, induced by glycolysis and oxidative phosphorylation, and play a relay function in coupling pH regulation and metabolism. In the brain, metabolic processes in astrocytes appear to be linked to bicarbonate transport and to neuronal activity. Here, we focus on physiological processes of energy dynamics in astrocytes as well as on the transfer of energetic substrates to neurons.

CAIX forms a transport metabolon with monocarboxylate transporters in human breast cancer cells

Tumor cells rely on glycolysis to meet their elevated demand for energy. Thereby they produce significant amounts of lactate and protons, which are exported via monocarboxylate transporters (MCTs), supporting the formation of an acidic microenvironment. The present study demonstrates that carbonic anhydrase IX (CAIX), one of the major acid/base regulators in cancer cells, forms a protein complex with MCT1 and MCT4 in tissue samples from human breast cancer patients, but not healthy breast tissue. Formation of this transport metabolon requires binding of CAIX to the Ig1 domain of the MCT1/4 chaperon CD147 and is required for CAIX-mediated facilitation of MCT1/4 activity. Application of an antibody, directed against the CD147-Ig1 domain, displaces CAIX from the transporter and suppresses CAIX-mediated facilitation of proton-coupled lactate transport. In cancer cells, this "metabolon disruption" results in a decrease in lactate transport, reduced glycolysis, and ultimately reduced cell proliferation. Taken together, the study shows that carbonic anhydrases form transport metabolons with acid/base transporters in human tumor tissue and that these interactions can be exploited to interfere with tumor metabolism and proliferation.

Carbonic anhydrase II/sodium-proton exchanger 1 metabolon complex in cardiomyopathy of ob−/− type 2 diabetic mice

Heart failure is the leading cause of death among diabetic people. Cellular and molecular entities leading to diabetic cardiomyopathy are, however, poorly understood. Coupling of cardiac carbonic anhydrase II (CAII) and Na+/H+ exchanger 1 (NHE1) to form a transport metabolon was analyzed in obese type 2 diabetic mice (ob−/−) and control heterozygous littermates (ob+/−). Echocardiography showed elevated systolic interventricular septum thickness and systolic posterior wall thickness in ob−/− mice at 9 and 16 weeks. ob−/− mice showed increased left ventricular (LV) weight/tibia length ratio and increased cardiomyocyte cross sectional area as compared to controls, indicating cardiac hypertrophy. Immunoblot analysis showed increased CAII expression in LV samples of ob−/−vs. ob+/− mice, and augmented Ser703 phosphorylation on NHE1 in ob−/− hearts. Reciprocal co-immunoprecipitation analysis showed strong association of CAII and NHE1 in LV samples of ob−/− mice. NHE1-dependent rate of intracellular pH (pHi) normalization after transient acid loading of isolated cardiomyocytes was higher in ob−/− mice vs. ob+/−. NHE transport activity was also augmented in cultured H9C2 rat cardiomyoblasts treated with high glucose/high palmitate, and it was normalized after CA inhibition. We conclude that the NHE1/CAII metabolon complex is exacerbated in diabetic cardiomyopathy of ob−/− mice, which may lead to perturbation of pHi and [Na+] and [Ca2+] handling in these diseased hearts.

Adaptable interaction between aquaporin-1 and band 3 reveals a potential role of water channel in blood CO2 transport.

Human CO2 respiration requires rapid conversion between CO2 and HCO3- Carbonic anhydrase II facilitates this reversible reaction inside red blood cells, and band 3 [anion exchanger 1 (AE1)] provides a passage for HCO3- flux across the cell membrane. These 2 proteins are core components of the CO2 transport metabolon. Intracellular H2O is necessary for CO2/HCO3- conversion. However, abundantly expressed aquaporin 1 (AQP1) in erythrocytes is thought not to be part of band 3 complexes or the CO2 transport metabolon. To solve this conundrum, we used Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging (FLIM-FRET) and identified interaction between aquaporin-1 and band 3 at a distance of 8 nm, within the range of dipole-dipole interaction. Notably, their interaction was adaptable to membrane tonicity changes. This suggests that the function of AQP1 in tonicity response could be coupled or correlated to its function in band 3-mediated CO2/HCO3- exchange. By demonstrating AQP1 as a mobile component of the CO2 transport metabolon, our results uncover a potential role of water channel in blood CO2 transport and respiration.-Hsu, K., Lee, T.-Y., Periasamy, A., Kao, F.-J., Li, L.-T., Lin, C.-Y., Lin, H.-J., Lin, M. Adaptable interaction between aquaporin-1 and band 3 reveals a potential role of water channel in blood CO2 transport.

An artificial transport metabolon facilitates improved substrate utilization in yeast.

Efficient substrate utilization is the first and most important prerequisite for economically viable production of biofuels and chemicals by microbial cell factories. However, production rates and yields are often compromised by low transport rates of substrates across biological membranes and their diversion to competing pathways. This is especially true when common chassis organisms are engineered to utilize nonphysiological feedstocks. Here, we addressed this problem by constructing an artificial complex between an endogenous sugar transporter and a heterologous xylose isomerase in Saccharomyces cerevisiae. Direct feeding of the enzyme through the transporter resulted in acceleration of xylose consumption and substantially diminished production of xylitol as an undesired side product, with a concomitant increase in the production of ethanol. This underlying principle could also likely be implemented in other biotechnological applications.

The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer

Mammals incorporate a major proportion of absorbed iron as heme, which is catabolized by the heme oxygenase 1 (HO1)-NADPH-cytochrome P450 reductase (CPR) complex into biliverdin, carbon monoxide, and ferrous iron. Moreover, intestinal iron is incorporated as ferrous iron, which is transported via the iron importer, divalent metal transporter 1 (DMT1). Recently, we demonstrated that the iron chaperone poly(rC)-binding protein 2 (PCBP2) can directly receive ferrous iron from DMT1 or transfer iron to the iron exporter, ferroportin 1. To promote intracellular iron flux, an iron chaperone may be essential for receiving iron generated by heme catabolism, but this hypothesis is untested so far. Herein, we demonstrate that HO1 binds to PCBP2, but not to other PCBP family members, namely PCBP1, PCBP3, or PCBP4. Interestingly, HO1 formed a complex with either CPR or PCBP2, and it was demonstrated that PCBP2 competes with CPR for HO1 binding. Using PCBP2-deletion mutants, we demonstrated that the PCBP2 K homology 3 domain is important for the HO1/PCBP2 interaction. In heme-loaded cells, heme prompted HO1-CPR complex formation and decreased the HO1/PCBP2 interaction. Furthermore, in vitro reconstitution experiments with purified recombinant proteins indicated that HO1 could bind to PCBP2 in the presence of heme, whereas loading of PCBP2 with ferrous iron caused PCBP2 to lose its affinity for HO1. These results indicate that ferrous iron released from heme can be bound by PCBP2 and suggest a model for an integrated heme catabolism and iron transport metabolon.

Analysis of immune molecule expression in mouse adipose tissue and deep fat

Many recent studies indicate that adipose tissue plays a role in innate immunity and inflammation. This lab has been studying the expression of Basigin gene products and the innate immune receptor TLR4 with respect to neural immune functions. Basigin gene products are members of the immunoglobulin superfamily. In the neural retina, Basigin forms a transport metabolon with monocarboxylate transporter 1 (MCT1), which is vital for the function of photoreceptor neurons in mice. Basigin is also expressed on monocytes and epithelial cells throughout the body, but has been less studied in connective tissues like adipose. Recent studies by this laboratory and others suggest that Basigin interacts with the innate immune receptor TLR4, which has the ability to initiate expression of pro‐inflammatory cytokines. The purpose of the present study was to examine the expression of Basigin gene products and TLR4 in mouse adipose and deep visceral fat tissues in an effort to determine whether they participate in immune functions of those tissues. Detergent‐solublized protein lysates and RNA were isolated from mouse adipose and deep visceral fat and used in ELISA and quantitative reverse transcriptase polymerase chain reaction (q‐RT‐PCR), respectively specific for Basigin gene products and TLR4. It was determined that the ubiquitously expressed Basigin is expressed within adipose tissue and deep fat, but the photoreceptor‐specific Basigin‐2 was not. In addition, TLR4 was observed in adipose tissue, but not in the visceral deep fat tissue. The results of this study are consistent with the literature and suggest that Basigin may play a role in obesity‐induced inflammation.Support or Funding InformationUniversity of North Florida Office of Undergraduate Studies and the Office of Academic Affairs

REGRESSION OF CALCIFIC AORTIC VALVE STENOSIS: A NOVEL FUNCTION FOR VALVE INTERSTITIAL CELLS AND CARBONIC ANHYDRASE XII

REGRESSION OF CALCIFIC AORTIC VALVE STENOSIS: A NOVEL FUNCTION FOR VALVE INTERSTITIAL CELLS AND CARBONIC ANHYDRASE XII

Facilitation by intracellular carbonic anhydrase of Na+–HCO3− co‐transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte

Carbonic anhydrase enzymes (CAs) catalyse the reversible hydration of CO2 to H+ and HCO3− ions. This catalysis is proposed to be harnessed by acid/base transporters, to facilitate their transmembrane flux activity, either through direct protein–protein binding (a ‘transport metabolon’) or local functional interaction. Flux facilitation has previously been investigated by heterologous co-expression of relevant proteins in host cell lines/oocytes. Here, we examine the influence of intrinsic CA activity on membrane HCO3− or H+ transport via the native acid-extruding proteins, Na+–HCO3− cotransport (NBC) and Na+/H+ exchange (NHE), expressed in enzymically isolated mammalian ventricular myocytes. Effects of intracellular and extracellular (exofacial) CA (CAi and CAe) are distinguished using membrane-permeant and –impermeant pharmacological CA inhibitors, while measuring transporter activity in the intact cell using pH and Na+ fluorophores. We find that NBC, but not NHE flux is enhanced by catalytic CA activity, with facilitation being confined to CAi activity alone. Results are quantitatively consistent with a model where CAi catalyses local H+ ion delivery to the NBC protein, assisting the subsequent (uncatalysed) protonation and removal of imported HCO3− ions. In well-superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO2/HCO3− buffer is clamped at equilibrium. The CAi insensitivity of NHE flux suggests that, in the native cell, intrinsic mobile buffer-shuttles supply sufficient intracellular H+ ions to this transporter, while intrinsic buffer access to NBC proteins is restricted. Our results demonstrate a selective CA facilitation of acid/base transporters in the ventricular myocyte, implying a specific role for the intracellular enzyme in HCO3− transport, and hence pHi regulation in the heart.

Rh proteins and NH4+-activated Na+-ATPase in the Magadi tilapia (Alcolapia grahami), a 100% ureotelic teleost fish

The small cichlid fish Alcolapia grahami lives in Lake Magadi, Kenya, one of the most extreme aquatic environments on Earth (pH ~10, carbonate alkalinity ~300 mequiv l(-1)). The Magadi tilapia is the only 100% ureotelic teleost; it normally excretes no ammonia. This is interpreted as an evolutionary adaptation to overcome the near impossibility of sustaining an NH3 diffusion gradient across the gills against the high external pH. In standard ammoniotelic teleosts, branchial ammonia excretion is facilitated by Rh glycoproteins, and cortisol plays a role in upregulating these carriers, together with other components of a transport metabolon, so as to actively excrete ammonia during high environmental ammonia (HEA) exposure. In Magadi tilapia, we show that at least three Rh proteins (Rhag, Rhbg and Rhcg2) are expressed at the mRNA level in various tissues, and are recognized in the gills by specific antibodies. During HEA exposure, plasma ammonia levels and urea excretion rates increase markedly, and mRNA expression for the branchial urea transporter mtUT is elevated. Plasma cortisol increases and branchial mRNAs for Rhbg, Rhcg2 and Na(+),K(+)-ATPase are all upregulated. Enzymatic activity of the latter is activated preferentially by NH4(+) (versus K(+)), suggesting it can function as an NH4(+)-transporter. Model calculations suggest that active ammonia excretion against the gradient may become possible through a combination of Rh protein and NH4(+)-activated Na(+)-ATPase function.

Transport metabolons with carbonic anhydrases

OPINION article Front. Physiol., 10 October 2013Sec. Membrane Physiology and Membrane Biophysics https://doi.org/10.3389/fphys.2013.00291

Close Association of Carbonic Anhydrase (CA2a and CA15a), Na+/H+ Exchanger (Nhe3b), and Ammonia Transporter Rhcg1 in Zebrafish Ionocytes Responsible for Na+ Uptake

Freshwater (FW) fishes actively absorb salt from their environment to tolerate low salinities. We previously reported that vacuolar-type H+-ATPase/mitochondrion-rich cells (H-MRCs) on the skin epithelium of zebrafish larvae (Danio rerio) are primary sites for Na+ uptake. In this study, in an attempt to clarify the mechanism for the Na+ uptake, we performed a systematic analysis of gene expression patterns of zebrafish carbonic anhydrase (CA) isoforms and found that, of 12 CA isoforms, CA2a and CA15a are highly expressed in H-MRCs at larval stages. The ca2a and ca15a mRNA expression were salinity-dependent; they were upregulated in 0.03 mM Na+ water whereas ca15a but not ca2a was down-regulated in 70 mM Na+ water. Immunohistochemistry demonstrated cytoplasmic distribution of CA2a and apical membrane localization of CA15a. Furthermore, cell surface immunofluorescence staining revealed external surface localization of CA15a. Depletion of either CA2a or CA15a expression by Morpholino antisense oligonucleotides resulted in a significant decrease in Na+ accumulation in H-MRCs. An in situ proximity ligation assay demonstrated a very close association of CA2a, CA15a, Na+/H+ exchanger 3b (Nhe3b), and Rhcg1 ammonia transporter in H-MRC. Our findings suggest that CA2a, CA15a, and Rhcg1 play a key role in Na+uptake under FW conditions by forming a transport metabolon with Nhe3b.