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The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO2-concentrating mechanism. BCT1 could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana , but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis. [ABSTRACT FROM AUTHOR]

Kidney anion exchanger 1 (kAE1) is an isoform of the AE1 protein encoded by the SLC4A1 gene. It is a basolateral membrane protein expressed by α-intercalated cells in the connecting tubules and collecting duct of the kidney. Its main function is to exchange bicarbonate and chloride ions between the blood and urine to maintain blood pH at physiological threshold. The kAE1 protein undergoes multiple post-translational modifications such as phosphorylation and ubiquitination and interacts with many different proteins such as claudin-4 and carbonic anhydrase II. Mutations in the gene may lead to the development of distal renal tubular acidosis, characterized by the failure to acidify the urine, which may result in nephrocalcinosis and in more severe cases, renal failure. In this review, we discuss the structure and function of kAE1, its post-translational modifications, and protein–protein interactions. Finally, we discuss insights gained from the study of kAE1 mutations in humans and in mice. [ABSTRACT FROM AUTHOR]

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function. [ABSTRACT FROM AUTHOR]

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function.

Intracellular pH (pHi) regulation is a challenge for the exocrine pancreas, where the luminal secretion of bicarbonate-rich fluid is accompanied by interstitial flows of acid. This acid–base transport requires a plethora of ion transporters, including bicarbonate transporters and the Na+/H+ exchanger isoform 1 (NHE1), which are dysregulated in Pancreatic Ductal Adenocarcinoma (PDAC). PDAC progression is favored by a Collagen-I rich extracellular matrix (ECM) which exacerbates the physiological interstitial acidosis. In organotypic cultures of normal human pancreatic cells (HPDE), parenchymal cancer cells (CPCs) and cancer stem cells (CSCs) growing on matrices reproducing ECM changes during progression, we studied resting pHi, the pHi response to fluxes of NaHCO3 and acidosis and the role of NHE1 in pHi regulation. Our findings show that: (i) on the physiological ECM, HPDE cells have the most alkaline pHi, followed by CSCs and CPCs, while a Collagen I-rich ECM reverses the acid–base balance in cancer cells compared to normal cells; (ii) both resting pHi and pHi recovery from an acid load are reduced by extracellular NaHCO3, especially in HPDE cells on a normal ECM; (iii) cancer cell NHE1 activity is less affected by NaHCO3. We conclude that ECM composition and the fluctuations of pHe cooperate to predispose pHi homeostasis towards the presence of NaHCO3 gradients similar to that expected in the tumor. [ABSTRACT FROM AUTHOR]

LCIA (low CO2-inducible protein A) is a chloroplast envelope protein associated with the CO2-concentrating mechanism of the green alga Chlamydomonas reinhardtii. LCIA is postulated to be a HCO3– channel, but previous studies were unable to show that LCIA was actively transporting bicarbonate in planta. Therefore, LCIA activity was investigated more directly in two heterologous systems: an Escherichia coli mutant (DCAKO) lacking both native carbonic anhydrases and an Arabidopsis mutant (βca5) missing the plastid carbonic anhydrase βCA5. Neither DCAKO nor βca5 can grow in ambient CO2 conditions, as they lack carbonic anhydrase-catalyzed production of the necessary HCO3– concentration for lipid and nucleic acid biosynthesis. Expression of LCIA restored growth in both systems in ambient CO2 conditions, which strongly suggests that LCIA is facilitating HCO3– uptake in each system. To our knowledge, this is the first direct evidence that LCIA moves HCO3– across membranes in bacteria and plants. Furthermore, the βca5 plant bioassay used in this study is the first system for testing HCO3– transport activity in planta , an experimental breakthrough that will be valuable for future studies aimed at improving the photosynthetic efficiency of crop plants using components from algal CO2-concentrating mechanisms. [ABSTRACT FROM AUTHOR]

The water channel aquaporin-1 (AQP1) is the principal water pathway for isotonic water reabsorption in the kidney proximal tubule (PT). We investigated flow-mediated fluid (Jv) and HCO- 3 (JHCO3 ) reabsorption in PTs of the mouse kidney by microperfusion in wild-type (WT) and AQP1 knockout (KO) mice. Experiments were simulated in an adaptation of a mathematical model of the rat PT. An increase in perfusion rate from 5 to 20 nL/min increased Jv and JHCO3 in PTs of WT mice. AQP1 KO mice significantly decreased Jv at low and high flow rates compared with control. In contrast, JHCO3 was not reduced at either low or high flow rates. Cell volume showed no significant difference between WT and AQP1 KO mice. Renal clearance experiments showed significantly higher urine flow in AQP1 KO mice, but there was no significant difference in either Na+ and K+ or HCO-3 excretion. Acid-base parameters of blood pH, PCO2, HCO-3, and urine pH were the same in both WT and KO mice. In model calculations, tubules whose tight junction (TJ) water permeability (Pf) was that assigned to the rat TJ, showed no difference in Jv between WT and KO, whereas TJ Pf set to 25% of the rat predicted Jv concordant with our observations from AQP1 KO. These results affirm the dominance of AQP1 in mediating isotonic water reabsorption by the mouse PT and demonstrate that flowstimulated HCO3 reabsorption is intact and independent of AQP1. With reference to the model, the findings also suggest that TJ water flux in the Pf is less prominent in the mouse than in the rat kidney. NEW & NOTEWORTHY We found an absence of flow-dependent modulation of fluid absorption but no effect on either proximal tubule (Pf) HCO-3 absorption or acid-base parameters in the aquaporin 1 (AQP1) knockout mouse. We affirmed the dominance of the water channel AQP1 in mediating isotonic water reabsorption by the mouse PT and demonstrated that flow-stimulated HCO-3 reabsorption is independent of AQP1. With reference to the model, the findings also suggest that tight junctional water flux in the PT is less prominent in the mouse than rat kidney. [ABSTRACT FROM AUTHOR]

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO2-concentrating mechanism. BCT1 could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Heterologous synthesis of a biophysical CO2-concentrating mechanism (CCM) in plant chloroplasts offers significant potential to improve the photosynthetic efficiency of C3 plants and could translate into substantial increases in crop yield. In organisms utilizing a biophysical CCM, this mechanism efficiently surrounds a high turnover rate Rubisco with elevated CO2 concentrations to maximize carboxylation rates. A critical feature of both native biophysical CCMs and one engineered into a C3 plant chloroplast is functional bicarbonate (HCO3−) transporters and vectorial CO2-to-HCO3− converters. Engineering strategies aim to locate these transporters and conversion systems to the C3 chloroplast, enabling elevation of HCO3− concentrations within the chloroplast stroma. Several CCM components have been identified in proteobacteria, cyanobacteria, and microalgae as likely candidates for this approach, yet their successful functional expression in C3 plant chloroplasts remains elusive. Here, we discuss the challenges in expressing and regulating functional HCO3− transporter, and CO2-to-HCO3− converter candidates in chloroplast membranes as an essential step in engineering a biophysical CCM within plant chloroplasts. We highlight the broad technical and physiological concerns which must be considered in proposed engineering strategies, and present our current status of both knowledge and knowledge-gaps which will affect successful engineering outcomes.

Heterologous synthesis of a biophysical CO2-concentrating mechanism (CCM) in plant chloroplasts offers significant potential to improve the photosynthetic efficiency of C3 plants and could translate into substantial increases in crop yield. In organisms utilizing a biophysical CCM, this mechanism efficiently surrounds a high turnover rate Rubisco with elevated CO2 concentrations to maximize carboxylation rates. A critical feature of both native biophysical CCMs and one engineered into a C3 plant chloroplast is functional bicarbonate (HCO3−) transporters and vectorial CO2-to-HCO3− converters. Engineering strategies aim to locate these transporters and conversion systems to the C3 chloroplast, enabling elevation of HCO3− concentrations within the chloroplast stroma. Several CCM components have been identified in proteobacteria, cyanobacteria, and microalgae as likely candidates for this approach, yet their successful functional expression in C3 plant chloroplasts remains elusive. Here, we discuss the challenges in expressing and regulating functional HCO3− transporter, and CO2-to-HCO3− converter candidates in chloroplast membranes as an essential step in engineering a biophysical CCM within plant chloroplasts. We highlight the broad technical and physiological concerns which must be considered in proposed engineering strategies, and present our current status of both knowledge and knowledge-gaps which will affect successful engineering outcomes. [ABSTRACT FROM AUTHOR]

Summary: Cyanobacteria are the only prokaryotes that perform plant‐like oxygenic photosynthesis. They evolved an inorganic carbon‐concentrating mechanism to adapt to low CO2 conditions.Quantitative phosphoproteomics was applied to analyze regulatory features during the acclimation to low CO2 conditions in the model cyanobacterium Synechocystis sp. PCC 6803.Overall, more than 2500 proteins were quantified, equivalent to c. 70% of the Synechocystis theoretical proteome. Proteins with changing abundances correlated largely with mRNA expression levels. Functional annotation of the noncorrelating proteins revealed an enrichment of key metabolic processes fundamental for maintaining cellular homeostasis. Furthermore, 105 phosphoproteins harboring over 200 site‐specific phosphorylation events were identified. Subunits of the bicarbonate transporter BCT1 and the redox switch protein CP12 were among phosphoproteins with reduced phosphorylation levels at lower CO2, whereas the serine/threonine protein kinase SpkC revealed increased phosphorylation levels. The corresponding ΔspkC mutant was characterized and showed decreased ability to acclimate to low CO2 conditions. Possible phosphorylation targets of SpkC including a BCT1 subunit were identified by phosphoproteomics.Collectively, our study highlights the importance of posttranscriptional regulation of protein abundances as well as posttranslational regulation by protein phosphorylation for the successful acclimation towards low CO2 conditions in Synechocystis and possibly among cyanobacteria. [ABSTRACT FROM AUTHOR]

Background: Amelogenesis, the formation of dental enamel, is well understood at the histomorphological level but the underlying molecular mechanisms are poorly characterized. Ameloblasts secrete enamel matrix proteins and Ca2+, and also regulate extracellular pH as the formation of hydroxyapatite crystals generates large quantities of protons. Genetic or environmental impairment of transport and regulatory processes (e.g. dental fluorosis) leads to the development of enamel defects such as hypomineralization.Aims: Our aims were to optimize the culture conditions for the three-dimensional growth of ameloblast-derived HAT-7 cells and to test the effects of fluoride exposure on HAT-7 spheroid formation.Methods: To generate 3D HAT-7 structures, cells were dispersed and plated within a Matrigel extracellular matrix scaffold and incubated in three different culture media. Spheroid formation was then monitored over a two-week period. Ion transporter and tight-junction protein expression was investigated by RT-qPCR. Intracellular Ca2+ and pH changes were measured by microfluorometry using the fluorescent dyes fura-2 and BCECF.Results: A combination of Hepato-STIM epithelial cell differentiation medium and Matrigel induced the expansion and formation of 3D HAT-7 spheroids. The cells retained their epithelial cell morphology and continued to express both ameloblast-specific and ion transport-specific marker genes. Furthermore, like two-dimensional HAT-7 monolayers, the HAT-7 spheroids were able to regulate their intracellular pH and to show intracellular calcium responses to extracellular stimulation. Finally, we demonstrated that HAT-7 spheroids may serve as a disease model for studying the effects of fluoride exposure during amelogenesis.Conclusion: In conclusion, HAT-7 cells cultivated within a Matrigel extracellular matrix form three-dimensional, multi-cellular, spheroidal structures that retain their functional capacity for pH regulation and intracellular Ca2+ signaling. This new 3D model will allow us to gain a better understanding of the molecular mechanisms involved in amelogenesis, not only in health but also in disorders of enamel formation, such as those resulting from fluoride exposure.

Sodium dependent bicarbonate transporter, SbtA is a high-affinity, inducible bicarbonate transporter in cyanobacterial cells. Our previous work has shown that overexpression of this transporter can significantly increase growth and glycogen accumulation in Synechococcus sp. PCC 7002 cells. In this work, we have tested the effect of two different RBS sequences (RBS1: GGAGGA and RBS2: AGGAGA) and three different promoters (PcpcB, PcpcB560, and PrbcL2) on the growth and glycogen production in SbtA-overexpressing Synechococcus sp. PCC 7002 cells. Our results show that PcpcB or PcpcB560 were more effective than PrbcL2 in increasing the growth and glycogen content. The choice of RBS sequence had relatively minor effect, though RBS2 was more effective than RBS1. The transformant E, with PcpcB560 and RBS2, showed the highest growth. The biomass after 5 days of growth on air or 1% CO2 was increased by about 90% in the strain E compared to PCC 7002 cells. All transformants overexpressing SbtA had higher glycogen content. However, growing the cells with bubbling of 1% CO2 did not increase cellular glycogen content any further. The strain E had about 80% higher glycogen content compared to WT PCC 7002 cells. Therefore, the glycogen productivity of the strain E grown with air-bubbling was about 2.5-fold that of the WT PCC 7002 cells grown similarly. Additionally, some of the transformants had higher chlorophyll content while all the transformants had higher carotenoid content compared to the PCC 7002 cells, suggesting interaction between carbon transport and pigment levels. Thus, this work shows that the choice of photosynthetic promoters and RBSs sequences can impact growth and glycogen accumulation in SbtA-overexpressing cells.

Background: Amelogenesis, the formation of dental enamel, is well understood at the histomorphological level but the underlying molecular mechanisms are poorly characterized. Ameloblasts secrete enamel matrix proteins and Ca2+, and also regulate extracellular pH as the formation of hydroxyapatite crystals generates large quantities of protons. Genetic or environmental impairment of transport and regulatory processes (e.g. dental fluorosis) leads to the development of enamel defects such as hypomineralization. Aims: Our aims were to optimize the culture conditions for the three-dimensional growth of ameloblast-derived HAT-7 cells and to test the effects of fluoride exposure on HAT-7 spheroid formation. Methods: To generate 3D HAT-7 structures, cells were dispersed and plated within a Matrigel extracellular matrix scaffold and incubated in three different culture media. Spheroid formation was then monitored over a two-week period. Ion transporter and tight-junction protein expression was investigated by RT-qPCR. Intracellular Ca2+ and pH changes were measured by microfluorometry using the fluorescent dyes fura-2 and BCECF. Results: A combination of Hepato-STIM epithelial cell differentiation medium and Matrigel induced the expansion and formation of 3D HAT-7 spheroids. The cells retained their epithelial cell morphology and continued to express both ameloblast-specific and ion transport-specific marker genes. Furthermore, like two-dimensional HAT-7 monolayers, the HAT-7 spheroids were able to regulate their intracellular pH and to show intracellular calcium responses to extracellular stimulation. Finally, we demonstrated that HAT-7 spheroids may serve as a disease model for studying the effects of fluoride exposure during amelogenesis. Conclusion: In conclusion, HAT-7 cells cultivated within a Matrigel extracellular matrix form three-dimensional, multi-cellular, spheroidal structures that retain their functional capacity for pH regulation and intracellular Ca2+ signaling. This new 3D model will allow us to gain a better understanding of the molecular mechanisms involved in amelogenesis, not only in health but also in disorders of enamel formation, such as those resulting from fluoride exposure. [ABSTRACT FROM AUTHOR]

Propionate, a metabolite from the microbial fermentation of carbohydrates, evokes a release of epithelial acetylcholine in rat caecum resulting in an increase of short-circuit current (Isc) in Ussing chamber experiments. The present experiments were performed in order to characterize the ionic mechanisms underlying this response which has been thought to be due to Cl− secretion. As there are regional differences within the caecal epithelium, the experiments were conducted at oral and aboral rat corpus caeci. In both caecal segments, the propionate-induced Isc (IProp) was inhibited by > 85%, when the experiments were performed either in nominally Cl−- or nominally HCO3−-free buffer. In the case of Cl−, the dependency was restricted to the presence of Cl− in the serosal bath. Bumetanide, a blocker of the Na+-K+-2Cl−-cotransporter, only numerically reduced IProp suggesting that a large part of this current must be carried by an ion other than Cl−. In the aboral caecum, IProp was significantly inhibited by mucosally administered stilbene derivatives (SITS, DIDS, DNDS), which block anion exchangers. Serosal Na+-free buffer reduced IProp significantly in the oral (and numerically also in aboral) corpus caeci. RT-PCR experiments revealed the expression of several forms of Na+-dependent HCO3−-cotransporters in caecum, which might underlie the observed Na+ dependency. These results suggest that propionate sensing in caecum is coupled to HCO3– secretion, which functionally would stabilize luminal pH when the microbial fermentation leads to an increase in the concentration of short-chain fatty acids in the caecal lumen. [ABSTRACT FROM AUTHOR]

Sodium dependent bicarbonate transporter, SbtA is a high-affinity, inducible bicarbonate transporter in cyanobacterial cells. Our previous work has shown that overexpression of this transporter can significantly increase growth and glycogen accumulation in Synechococcus sp. PCC 7002 cells. In this work, we have tested the effect of two different RBS sequences (RBS1: GGAGGA and RBS2: AGGAGA) and three different promoters (PcpcB, PcpcB560, and PrbcL2) on the growth and glycogen production in SbtA-overexpressing Synechococcus sp. PCC 7002 cells. Our results show that PcpcB or PcpcB560 were more effective than PrbcL2 in increasing the growth and glycogen content. The choice of RBS sequence had relatively minor effect, though RBS2 was more effective than RBS1. The transformant E, with PcpcB560 and RBS2, showed the highest growth. The biomass after 5 days of growth on air or 1% CO2 was increased by about 90% in the strain E compared to PCC 7002 cells. All transformants overexpressing SbtA had higher glycogen content. However, growing the cells with bubbling of 1% CO2 did not increase cellular glycogen content any further. The strain E had about 80% higher glycogen content compared to WT PCC 7002 cells. Therefore, the glycogen productivity of the strain E grown with air-bubbling was about 2.5-fold that of the WT PCC 7002 cells grown similarly. Additionally, some of the transformants had higher chlorophyll content while all the transformants had higher carotenoid content compared to the PCC 7002 cells, suggesting interaction between carbon transport and pigment levels. Thus, this work shows that the choice of photosynthetic promoters and RBSs sequences can impact growth and glycogen accumulation in SbtA-overexpressing cells. [ABSTRACT FROM AUTHOR]

The disease, cystic fibrosis, is caused by the malfunction of the cystic fibrosis transmembrane conductance regulator. Expression of functional CFTR may normally regulate extracellular pH via control of bicarbonate efflux. Reports also suggest that the CFTR may be a Cl-/HCO3- exchanger. If true, this could be very important for treatment of CF given the airway host defense system is quite sensitive to pH, and acidic pH been found to increase mucus viscosity. We compared evidentiary support of four possible models of CFTR's role in the transport of bicarbonate: 1) CFTR as a Cl-channel that permits bicarbonate conductance, 2) CFTR as an anion Cl-/HCO3- exchanger (AE), 3.) CFTR as both a Cl-channel and an AE, and 4.) CFTR as a Cl-channel that allows for transport of bicarbonate and regulates an independent AE. The effect of stimulators and inhibitors of CFTR and AEs were evaluated via iodide efflux and studies of extracellular pH. This data, as well as that published by others, suggest that while CFTR may support and regulate bicarbonate flux it is unlikely it directly performs Cl-/HCO3- anion exchange.

Previous analysis of CFTR-knockout (CFTR−/−) in piglets has provided important insights into the pathology of cystic fibrosis. However, controversies exist as to the true contribution of CFTR to the pH balance in airways and intestine. We therefore compared ion transport properties in newborn wild-type (CFTR+/+) and CFTR-knockout (CFTR−/− piglets). Tracheas of CFTR−/− piglets demonstrated typical cartilage malformations and muscle cell bundles. CFTR−/− airway epithelial cells showed enhanced lipid peroxidation, suggesting inflammation early in life. CFTR was mainly expressed in airway submucosal glands and was absent in lungs of CFTR−/− piglets, while expression of TMEM16A was uncompromised. mRNA levels for TMEM16A, TMEM16F, and αβγENaC were unchanged in CFTR−/− airways, while mRNA for SLC26A9 appeared reduced. CFTR was undetectable in epithelial cells of CFTR−/− airways and intestine. Small intestinal epithelium of CFTR−/− piglets showed mucus accumulation. Secretion of both electrolytes and mucus was activated by stimulation with prostaglandin E2 and ATP in the intestine of CFTR+/+, but not of CFTR−/− animals. pH was measured inside small bronchi using a pH microelectrode and revealed no difference between CFTR+/+ and CFTR−/− piglets. Intracellular pH in porcine airway epithelial cells revealed only a small contribution of CFTR to bicarbonate secretion, which was absent in cells from CFTR−/− piglets. In contrast to earlier reports, our data suggest a minor impact of CFTR on ASL pH. In contrast, enhanced amiloride-sensitive Na+ absorption may contribute to lung pathology in CFTR−/− piglets, along with a compromised CFTR- and TMEM16A-dependent Cl− transport. [ABSTRACT FROM AUTHOR]

The PII‐like protein SbtB has been identified as a regulator of SbtA, which is one of the key bicarbonate transporters in cyanobacteria. While SbtB from Synechocystis sp. PCC 6803 has previously been shown to be a trimer, a new crystal form is reported here which crystallizes in what is thought to be a non‐native tetramer in the crystal, with the C‐terminus in an extended conformation. The crystal structure shows the formation of an intermolecular disulfide bond at Cys94 between SbtB monomers, which may stabilize this conformation in the crystal. This motivates the need for future studies to investigate the potential role that the oxidation and reduction of these cysteines may play in the activation and/or function of SbtB. [ABSTRACT FROM AUTHOR]

The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that helps in successful acclimation to low CO2 conditions. Current models of the CCM postulate that a series of ion transporters bring HCO3 − from outside the cell to the thylakoid lumen, where the carbonic anhydrase 3 (CAH3) dehydrates accumulated HCO3 − to CO2, raising the CO2 concentration for Ribulose bisphosphate carboxylase/oxygenase (Rubisco). Previously, HCO3 − transporters have been identified at both the plasma membrane and the chloroplast envelope, but the transporter thought to be on the thylakoid membrane has not been identified. Three paralogous genes (BST1, BST2, and BST3) belonging to the bestrophin family have been found to be up-regulated in low CO2 conditions, and their expression is controlled by CIA5, a transcription factor that controls many CCM genes. YFP fusions demonstrate that all 3 proteins are located on the thylakoid membrane, and interactome studies indicate that they might associate with chloroplast CCM components. A single mutant defective in BST3 has near-normal growth on low CO2, indicating that the 3 bestrophin-like proteins may have redundant functions. Therefore, an RNA interference (RNAi) approach was adopted to reduce the expression of all 3 genes at once. RNAi mutants with reduced expression of BST1–3 were unable to grow at low CO2 concentrations, exhibited a reduced affinity to inorganic carbon (Ci) compared with the wild-type cells, and showed reduced Ci uptake. We propose that these bestrophin-like proteins are essential components of the CCM that deliver HCO3 − accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen. [ABSTRACT FROM AUTHOR]

The unique composition of the endolymph with a high extracellular K+ concentration is essential for sensory transduction in the inner ear. It is secreted by a specialized epithelium, the stria vascularis, that is connected to the fibrocyte meshwork of the spiral ligament in the lateral wall of the cochlea via gap junctions. In this study, we show that in mice the expression of the bicarbonate transporter Slc4a10/Ncbe/Nbcn2 in spiral ligament fibrocytes starts shortly before hearing onset. Its disruption in a C57BL/6 background results in early onset progressive hearing loss. This hearing loss is characterized by a reduced endocochlear potential from hearing onset onward and progressive degeneration of outer hair cells. Notably, the expression of a related bicarbonate transporter, i.e., Slc4a7/Nbcn1, is also lost in spiral ligament fibrocytes of Slc4a10 knockout mice. The histological analysis of the spiral ligament of Slc4a10 knockout mice does not reveal overt fibrocyte loss as reported for Slc4a7 knockout mice. The ultrastructural analysis, however, shows mitochondrial alterations in fibrocytes of Slc4a10 knockout mice. Our data suggest that Slc4a10 and Slc4a7 are functionally related and essential for inner ear homeostasis. [ABSTRACT FROM AUTHOR]

The article comments on the article "New treatments targeting the basic defects in cystic fibrosis" by I. Fajac and C. E. Wainwright in the 2017 issue of the journal "La Presse médicale." Topics include cystic fibrosis (CF) being the most common life-threatening inherited disorder affecting white people and caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene that generate impaired CFTR anion channels leading to a multi-organ disease.

ATP fuels the removal of metabolic end-products, including H+ ions that profoundly modulate biological activities. Energetic resources in hypoxic tumor regions are constrained by low-yielding glycolysis, and any means of reducing the cost of acid extrusion, without compromising pH homeostasis, would therefore be advantageous for cancer cells. Some cancers express connexin channels that allow solute exchange between cells, and we propose that, via this route, normoxic cells supply hypoxic neighbors with acid-neutralizing HCO3- ions. This hypothesis was tested by imaging cytoplasmic pH in spheroidal tissue growths of connexin43-positive pancreatic cancer Colo357 cells during light-controlled H+ uncaging at the hypoxic core. Cytoplasmic acid retention at the core was halved in the presence of CO2/HCO3-, but this process requires a restorative HCO3-flux. The effect of CO2/HCO3- was ablated by connexin43 inhibition or knockdown. In connexin-decoupled spheroids, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), an inhibitor of HCO3-uptake, had no effect on cytoplasmic [H+] in the H+-uncaging region, indicating that DIDS-sensitive transport is not an adequate pH-regulatory strategy therein. With intact connexin-coupling, acid retention at the core increased upon DIDS treatment, indicating that HCO3- ions are taken up actively by peripheral cells and then transmitted passively to cells at the hypoxic core. Thus, the energetic burden of pH regulation is offloaded from hypoxic cells onto metabolically altruistic normoxic neighbors. [ABSTRACT FROM AUTHOR]

ATP fuels the removal of metabolic end‐products, including H+ ions that profoundly modulate biological activities. Energetic resources in hypoxic tumor regions are constrained by low‐yielding glycolysis, and any means of reducing the cost of acid extrusion, without compromising pH homeostasis, would therefore be advantageous for cancer cells. Some cancers express connexin channels that allow solute exchange between cells, and we propose that, via this route, normoxic cells supply hypoxic neighbors with acid‐neutralizing HCO3_ ions. This hypothesis was tested by imaging cytoplasmic pH in spheroidal tissue growths of connexin43‐positive pancreatic cancer Colo357 cells during light‐controlled H+ uncaging at the hypoxic core. Cytoplasmic acid retention at the core was halved in the presence of CO2/HCO3−, but this process requires a restorative HCO3− flux. The effect of CO2/HCO3− was ablated by connexin43 inhibition or knockdown. In connexin‐decoupled spheroids, 4,4'‐diisothiocyano‐2,2'‐stilbenedisulfonic acid (DIDS), an inhibitor of HCO3− uptake, had no effect on cytoplasmic [H+]inthe H+‐uncaging region, indicating that DIDS‐sensitive transport is not an adequate pH‐regulatory strategy therein. With intact connexin‐coupling, acid retention at the core increased upon DIDS treatment, indicating that HCO3− ions are taken up actively by peripheral cells and then transmitted passively to cells at the hypoxic core. Thus, the energetic burden of pH regulation is offloaded from hypoxic cells onto metabolically altruistic normoxic neighbors.—Dovmark, T. H., Hulikova, A., Niederer, S. A., Vaughan‐Jones, R. D., Swietach, P. Normoxic cells remotely regulate the acid‐base balance of cells at the hypoxic core of connexin‐coupled tumor growths. FASEB J. 32,83‐96 (2018). www.fasebj.org [ABSTRACT FROM AUTHOR]

Amelogenesis features two major developmental stages—secretory and maturation. During maturation stage, hydroxyapatite deposition and matrix turnover require delicate pH regulatory mechanisms mediated by multiple ion transporters. Several members of the Slc26 gene family (Slc26a1, Slc26a3, Slc26a4, Slc26a6, and Slc26a7), which exhibit bicarbonate transport activities, have been suggested by previous studies to be involved in maturation-stage amelogenesis, especially the key process of pH regulation. However, details regarding the functional role of these genes in enamel formation are yet to be clarified, as none of the separate mutant animal lines demonstrates any discernible enamel defects. Continuing with our previous investigation of Slc26a1−/− and Slc26a7−/− animal models, we generated a double-mutant animal line with the absence of both Slc26a1 and Slc26a7. We showed in the present study that the double-mutant enamel density was significantly lower in the regions that represent late maturation-, maturation- and secretory-stage enamel development in wild-type mandibular incisors. However, the “maturation” and “secretory” enamel microstructures in double-mutant animals resembled those observed in wild-type secretory and/or pre-secretory stages. Elemental composition analysis revealed a lack of mineral deposition and an accumulation of carbon and chloride in double-mutant enamel. Deletion of Slc26a1 and Slc26a7 did not affect the stage-specific morphology of the enamel organ. Finally, compensatory expression of pH regulator genes and ion transporters was detected in maturation-stage enamel organs of double-mutant animals when compared to wild-type. Combined with the findings from our previous study, these data indicate the involvement of SLC26A1and SLC26A7 as key ion transporters in the pH regulatory network during enamel maturation.

Keywords: acid-based balance; bicarbonate transport; NBCe1; sodium transport; stoichiometry EN acid-based balance bicarbonate transport NBCe1 sodium transport stoichiometry 3005 3006 2 07/04/22 20220701 NES 220701 In 1983 Boron and Boulpaep first described an electrogenic Na SP + sp and HCO SB 3 sb SP - sp cotransport activity in the kidney proximal tubule of the salamander I Ambystoma tigrinum i (Boron & Boulpaep, 1983). This "molecular sequence switch" seemed to conveniently segregate with transport direction and apparent ion coupling; that is Na SP + sp / I n i HCO SB 3 sb SP - sp I efflux i by the kidney as the role of NBCe1-A and the Na SP + sp / I n i HCO SB 3 sb SP - sp I influx i by other tissues as the role of NBCe1-B. Biology is rarely so simple. Acid-based balance, bicarbonate transport, NBCe1, sodium transport, stoichiometry. [Extracted from the article]

Keywords: acid-based balance; bicarbonate transport; NBCe1; sodium transport; stoichiometry EN acid-based balance bicarbonate transport NBCe1 sodium transport stoichiometry 3005 3006 2 07/04/22 20220701 NES 220701 In 1983 Boron and Boulpaep first described an electrogenic Na SP + sp and HCO SB 3 sb SP - sp cotransport activity in the kidney proximal tubule of the salamander I Ambystoma tigrinum i (Boron & Boulpaep, 1983). This "molecular sequence switch" seemed to conveniently segregate with transport direction and apparent ion coupling; that is Na SP + sp / I n i HCO SB 3 sb SP - sp I efflux i by the kidney as the role of NBCe1-A and the Na SP + sp / I n i HCO SB 3 sb SP - sp I influx i by other tissues as the role of NBCe1-B. Biology is rarely so simple. Acid-based balance, bicarbonate transport, NBCe1, sodium transport, stoichiometry. [Extracted from the article]

Rising CO2 concentrations may have large effects on aquatic microorganisms. In this study, we investigated how elevated pCO2 affects the harmful freshwater cyanobacterium Microcystis aeruginosa. This species is capable of producing dense blooms and hepatotoxins called microcystins. Strain PCC 7806 was cultured in chemostats that were shifted from low to high pCO2 conditions. This resulted in a transition from a C-limited to a light-limited steady state, with a ~2.7 fold increase of the cyanobacterial biomass and ~2.5 fold more microcystin per cell. Cells increased their chlorophyll a and phycocyanin content, and raised their PSI/PSII ratio at high pCO2. Surprisingly, cells had a lower dry weight and contained less carbohydrates, which might be an adaptation to improve the buoyancy of Microcystis when light becomes more limiting at high pCO2. Only 234 of the 4,691 genes responded to elevated pCO2. For instance, expression of the carboxysome, RuBisCO, photosystem and C metabolism genes did not change significantly, and only a few N assimilation genes were expressed differently. The lack of large-scale changes in the transcriptome could suit a buoyant species that lives in eutrophic lakes with strong CO2 fluctuations very well. However, we found major responses in inorganic carbon uptake. At low pCO2, cells were mainly dependent on bicarbonate uptake, whereas at high pCO2 gene expression of the bicarbonate uptake systems was down-regulated and cells shifted to CO2 and low-affinity bicarbonate uptake. These results show that the need for high-affinity bicarbonate uptake systems ceases at elevated CO2. Moreover, the combination of an increased cyanobacterial abundance, improved buoyancy, and higher toxin content per cell indicates that rising atmospheric CO2 levels may increase the problems associated with the harmful cyanobacterium Microcystis in eutrophic lakes.

The two major interfaces separating brain and blood have different primary roles. The choroid plexuses secrete cerebrospinal fluid into the ventricles, accounting for most net fluid entry to the brain. Aquaporin, AQP1, allows water transfer across the apical surface of the choroid epithelium; another protein, perhaps GLUT1, is important on the basolateral surface. Fluid secretion is driven by apical Na+-pumps. K+ secretion occurs via net paracellular influx through relatively leaky tight junctions partially offset by transcellular efflux. The blood-brain barrier lining brain microvasculature, allows passage of O2, CO2, and glucose as required for brain cell metabolism. Because of high resistance tight junctions between microvascular endothelial cells transport of most polar solutes is greatly restricted. Because solute permeability is low, hydrostatic pressure differences cannot account for net fluid movement; however, water permeability is sufficient for fluid secretion with water following net solute transport. The endothelial cells have ion transporters that, if appropriately arranged, could support fluid secretion. Evidence favours a rate smaller than, but not much smaller than, that of the choroid plexuses. At the blood-brain barrier Na+ tracer influx into the brain substantially exceeds any possible net flux. The tracer flux may occur primarily by a paracellular route. The blood-brain barrier is the most important interface for maintaining interstitial fluid (ISF) K+ concentration within tight limits. This is most likely because Na+-pumps vary the rate at which K+ is transported out of ISF in response to small changes in K+ concentration. There is also evidence for functional regulation of K+ transporters with chronic changes in plasma concentration. The blood-brain barrier is also important in regulating HCO3- and pH in ISF: the principles of this regulation are reviewed. Whether the rate of blood-brain barrier HCO3- transport is slow or fast is discussed critically: a slow transport rate comparable to those of other ions is favoured. In metabolic acidosis and alkalosis variations in HCO3- concentration and pH are much smaller in ISF than in plasma whereas in respiratory acidosis variations in pHISF and pHplasma are similar. The key similarities and differences of the two interfaces are summarized. [ABSTRACT FROM AUTHOR]

The crystal structure of the dimeric membrane domain of human Band 3 1 , the red cell chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1), provides a structural context for over four decades of studies into this historic and important membrane glycoprotein. In this review, we highlight the key structural features responsible for anion binding and translocation and have integrated the following topological markers within the Band 3 structure: blood group antigens, N-glycosylation site, protease cleavage sites, inhibitor and chemical labeling sites, and the results of scanning cysteine and N-glycosylation mutagenesis. Locations of mutations linked to human disease, including those responsible for Southeast Asian ovalocytosis, hereditary stomatocytosis, hereditary spherocytosis, and distal renal tubular acidosis, provide molecular insights into their effect on Band 3 folding. Finally, molecular dynamics simulations of phosphatidylcholine self-assembled around Band 3 provide a view of this membrane protein within a lipid bilayer. [ABSTRACT FROM AUTHOR]