Bicarbonate Transport Research Articles

TMIC-24. BICARBONATE RECEPTOR SLC4A4 SENSITIZES INFILTRATING GLIOBLASTOMA CELLS TO CHANGES IN EXTRACELLULAR PH

Abstract BACKGROUND Glioblastoma progression involves changes in extracellular pH (pHe) related to rapid cell turnover and growth. There exist differences in pH between contrast-enhancing solid tumor and non-enhancing infiltrating tumor. SLC4A4 is a two-way bicarbonate transporter used in cortical astrocytes to buffer intracellular pH. METHODS In patients undergoing surgery for newly diagnosed IDH wild-type glioblastoma, we obtained tissue from solid tumor and infiltrating tumor. Single-nucleus RNA sequencing (n=15), immunohistochemistry (n=20) were carried out to identify SLC4A4 in infiltrating tumor beyond the contrast enhancing edge. SLC4A4 knock-down models were used to evaluate functional biology of this bicarbonate receptor using our tumor – cortical organoid model of infiltrating glioma and gliomaspheres. pH measurements were recorded using pH sensitive microelectrodes to evaluate changes on pH buffering ability of tumor cells during acid titration assays. RESULTS Single-nucleus RNA sequencing (n=15) and spatial RNA sequencing (n=3) showed SLC4A4 to be the highest expressed transmembrane receptor on infiltrating glioblastoma cells but not solid tumor. IHC (n=20) confirmed higher protein expression in infiltrating tumor (p=0.0390). Infiltrating cells were specifically sensitive to decreasing pHe (p=0.0105). In the infiltrating glioma cortical-organoid model, the slc4a4- had increased proliferation (GFP+ cell quantitation; n=10, p=0.0410). There was also increased proliferation in wild-type cells treated with SLC4A4 inhibitor DIDS (n=10, p=0.0016). Immunostaining of these models showed that downmodulation of SLC4A4 was associated with increased Ki67 and decreased caspase staining. CONCLUSIONS SLC4A4 plays a role in pH buffering in glioblastoma. We hypothesize that loss of SLC4A4 in solid tumor allows for desensitization of these cells to an acidic extracellular environment and allows for increased tumor growth and density while infiltrating tumor remains sensitivity to changes in pHe. An understand of pH regulation may lead to therapeutic efficacy through pH modulation of tumor.

The LysR-type transcriptional factor PacR controls heterocyst differentiation and C/N metabolism in the cyanobacterium Anabaena PCC 7120

PacR (All3953) has previously been identified as a global transcriptional regulator of carbon assimilation in cyanobacteria. In the facultative diazotrophic and filamentous cyanobacterium Anabaena PCC 7120 (Anabaena), inactivation of pacR has been shown to affect cell growth under various conditions. Nitrogen fixation in Anabaena occurs in heterocysts, cells differentiated semiregularly along the filaments following deprivation of combined nitrogen such as nitrate or ammonium. Here, we created a markerless deletion mutant of pacR. In addition to its growth defects observed under different light and nitrogen conditions, the mutant could form a high frequency of heterocysts, including heterocyst doublets, even in the presence of nitrate. Inactivation of pacR led to the upregulation of ntcA, a global regulator of nitrogen metabolism and heterocyst formation, as well as downregulation of genes involved in nitrate uptake and assimilation. These changes led to N-limited cells in the presence of nitrate. PacR also regulates most of the genes encoding bicarbonate transport systems. The promoter regions of ntcA, and several other genes involved in nitrogen or carbon uptake and assimilation, as well as patS and hetN involved in heterocyst patterning can be directly recognized by PacR in vitro. These findings, along with previously reported ChIP-seq data, establish PacR as a crucial transcriptional regulator for balancing carbon and nitrogen metabolism in cyanobacteria.

The sodium-bicarbonate cotransporter Slc4a5 mediates feedback at the first synapse of vision.

Feedback at the photoreceptor synapse is the first neuronal circuit computation in vision, which influences downstream activity patterns within the visual system. Yet, the identity of the feedback signal and the mechanism of synaptic transmission are still not well understood. Here, we combined perturbations of cell-type-specific genes of mouse horizontal cells with two-photon imaging of the result of light-induced feedback in cones and showed that the electrogenic bicarbonate transporter Slc4a5, but not the electroneutral bicarbonate transporter Slc4a3, both expressed specifically in horizontal cells, is necessary for horizontal cell-to-cone feedback. Pharmacological blockage of bicarbonate transporters and buffering pH also abolished the feedback but blocking sodium-proton exchangers and GABA receptors did not. Our work suggests an unconventional mechanism of feedback at the first visual synapse: changes in horizontal cell voltage modulate bicarbonate transport to the cell, via Slc4a5, which leads to the modulation of feedback to cones.

Abstract P427: Novel SNVs of NBCe1, D405E and I803M mutants, reduced cell-surface expression and transport activity.

Introduction: The electrogenic Na + /HCO 3 - cotransporter NBCe1 is mainly expressed in proximal renal tubule, and regulate pH homeostasis and plasma volume. NBCe1 homozygous mutation causes many symptoms, including severe hypotension, proximal renal tubular acidosis (RTA), and extrarenal phenotype (migraine, mental retardation, mineral bone disorder and ocular abnormalities). Hypothesis: Undetermined single nucleotide variants (SNVs) in NBCe1 will be crucial role of sodium and bicarbonate transport. Methods: We identified the missense mutations (D405E and I803M) in NBCe1 variant A (kidney type) from the NCBI database. We conducted a comparative analysis of cellular localization and cell protein expression using confocal microscopy and western blotting. Furthermore, we performed functional analysis in D405E and I803M SNVs using two-electrode voltage clamp method. Results: Immunofluorescence analysis with confocal microscopy revealed that the D405E and I803M variants were present predominantly in the cell-surface in HEK293 cells. Western blotting in HEK293 cells confirmed that the D405E and I803M variants were significantly reduced protein expression level (25% in D405E and 57% in I803M) compared with WT, respectively. Moreover, electrophysiological analysis revealed that D405E and I803M variants were significantly reduced transport activity. These consisting data suggest that cell-expression level and transport activity are comparable. Conclusion: These data illustrate the diverse physiological consequences of distinct SNVs and underscore the importance of functional characterization in membrane transport proteins.

Role of Kir5.1 (Kcnj16) Channels in Regulating Renal Ammonia Metabolism during Metabolic Acidosis in Dahl Salt-Sensitive Rats

Maintaining acid-base homeostasis is critical for normal physiological function. The kidneys are essential for regulating acid-base homeostasis through maintaining systemic bicarbonate concentration. Chronic metabolic acidosis is an independent risk factor for chronic kidney diseases. Renal inwardly rectifying potassium channel Kir5.1 plays an essential role in maintaining resting membrane potential. Patients with loss-of-function mutations in KCNJ16 gene, which encodes Kir5.1, reveal tubulopathy with hypokalemia, salt wasting, and hearing loss. Importantly, these mutations also disrupt acid-base balance, particularly causing metabolic acidosis. This study aimed to use Dahl salt-sensitive rats with a knockout of the Kcnj16 gene (SSKcnj16-/-) to investigate how the deletion of Kir5.1 affects the regulation of acid-base balance in salt-sensitive hypertension. Results indicated that SSKcnj16-/- rats displayed metabolic acidosis under a normal salt (NS) diet. Further analysis using RNA-Sequncing and Western blotting showed unchanged expression of proteins responsible for ammonia metabolism in the kidney of SSKcnj16-/- rats despite observed acidosis. However, there was a significant increase in the expression of bicarbonate transporter NBCe1 while a significant decrease in pendrin. In conclusion, the current study demonstrated that the loss of Kir5.1 impairs the sensitivity of ammonia metabolism in the kidney in response to metabolic acidosis, which provides mechanistic insights into developing potential therapeutics for conditions involving hypokalemia and acid-base abnormalities.

Insights into yeast response to chemotherapeutic agent through time series genome-scale metabolic models.

Organism-specific genome-scale metabolic models (GSMMs) can unveil molecular mechanisms within cells and are commonly used in diverse applications, from synthetic biology, biotechnology, and systems biology to metabolic engineering. There are limited studies incorporating time-series transcriptomics in GSMM simulations. Yeast is an easy-to-manipulate model organism for tumor research.Here, a novel approach (TS-GSMM) was proposed to integrate time-series transcriptomics with GSMMs to narrow down the feasible solution space of all possible flux distributions and attain time-series flux samples. The flux samples were clustered using machine learning techniques, and the clusters' functional analysis was performed using reaction set enrichment analysis.A time series transcriptomics response of Yeast cells to a chemotherapeutic reagent-doxorubicin-was mapped onto a Yeast GSMM. Eleven flux clusters were obtained with our approach, and pathway dynamics were displayed. Induction of fluxes related to bicarbonate formation and transport, ergosterol and spermidine transport, and ATP production were captured.Integrating time-series transcriptomics data with GSMMs is a promising approach to reveal pathway dynamics without any kinetic modeling and detects pathways that cannot be identified through transcriptomics-only analysis. The codes are available at https://github.com/karabekmez/TS-GSMM.

Staphylococcus aureus Stress Response to Bicarbonate Depletion.

Bicarbonate and CO2 are essential substrates for carboxylation reactions in bacterial central metabolism. In Staphylococcus aureus, the bicarbonate transporter, MpsABC (membrane potential-generating system) is the only carbon concentrating system. An mpsABC deletion mutant can hardly grow in ambient air. In this study, we investigated the changes that occur in S. aureus when it suffers from CO2/bicarbonate deficiency. Electron microscopy revealed that ΔmpsABC has a twofold thicker cell wall thickness compared to the parent strain. The mutant was also substantially inert to cell lysis induced by lysostaphin and the non-ionic surfactant Triton X-100. Mass spectrometry analysis of muropeptides revealed the incorporation of alanine into the pentaglycine interpeptide bridge, which explains the mutant's lysostaphin resistance. Flow cytometry analysis of wall teichoic acid (WTA) glycosylation patterns revealed a significantly lower α-glycosylated and higher ß-glycosylated WTA, explaining the mutant's increased resistance towards Triton X-100. Comparative transcriptome analysis showed altered gene expression profiles. Autolysin-encoding genes such as sceD, a lytic transglycosylase encoding gene, were upregulated, like in vancomycin-intermediate S. aureus mutants (VISA). Genes related to cell wall-anchored proteins, secreted proteins, transporters, and toxins were downregulated. Overall, we demonstrate that bicarbonate deficiency is a stress response that causes changes in cell wall composition and global gene expression resulting in increased resilience to cell wall lytic enzymes and detergents.

Calcification increases carbon supply, photosynthesis, and growth in a globally distributed coccolithophore

AbstractCoccolithophores fix organic carbon and produce calcite plates (coccoliths) that ballast organic matter and facilitate carbon export. Photosynthesis consumes carbon dioxide, while calcification produces it, raising questions about whether coccolithophores are a net sink or source of carbon. We characterized the physiology of calcified and noncalcified (“naked”) phenotypes of Emiliania huxleyi (CCMP374) and investigated the relationship between calcification and photosynthesis across a gradient of light (25–2000 μmol photons m−2 s−1) spanning the euphotic zone. Growth and photophysiological parameters increased with light until reaching a mid‐light (150 μmol photons m−2 s−1) maximum for both phenotypes. Calcified cells were characterized by enhanced photophysiology and less photoinhibition. Further, enhanced bicarbonate transport in calcified cells led to higher rates of particulate organic carbon fixation and growth compared to naked cells at mid‐light to high light (150–2000 μmol photons m−2 s−1). Coccolith production was similarly high at mid and high light, but the rate of coccolith shedding was >3‐fold lower at high‐light (1.2 vs. 0.35 coccoliths cell−1 h−1). The cellular mechanims(s) of this differential shedding remain unknown and underly light‐related controls on coccosphere maintenance. Our data suggest coccoliths shade cells at high light and that enhanced bicarbonate transport associated with calcification increases internal carbon supplies available for organic carbon fixation.

Clinical manifestations of cystic fibrosis

Cystic fibrosis is a severe genetic disease that is inherited in an autosomal recessive manner, affecting the short arm of chromosome 7 of the CFTR gene, causing alterations in the transmembrane conductance regulator protein (CFTR), This causes dysfunctional transport of chloride, sodium and bicarbonate leading to the generation of thick and viscous secretions in the lungs, pancreas, liver, intestine and reproductive tract while in the sweat glands there will be increased salt content. The clinical manifestations depend on the degree of mutation present in the gene, and may appear within a few months of birth or may even take decades. Life expectancy in patients with cystic fibrosis in Ecuador is 9.5 years and is mainly related to the quality of treatment and especially with early diagnosis since lung damage begins at birth. A retrospective study was made with various databases and keywords related to Cystic Fibrosis and it was found that the main system affected is the respiratory system, its main clinical manifestations, persistent productive cough, hyperinflation of lung fields and airway obstruction, as the disease progresses and exposure to infections, there will be a progressive deterioration of the lung that will manifest itself in acute exacerbations of cough, tachypnea, dyspnea and increased sputum production. With respect to the digestive system, two thirds of CF patients have pancreatic insufficiency and most have signs of poor or deficient fat absorption, including symptoms and signs such as steatorrhea, growth retardation or weight gain, and in infants edema, hypoproteinemia, electrolyte loss and anemia may occur.

The structural insight into the functional modulation of human anion exchanger 3

Anion exchanger 3 (AE3) is pivotal in regulating intracellular pH across excitable tissues, yet its structural intricacies and functional dynamics remain underexplored compared to other anion exchangers. This study unveils the structural insights into human AE3, including the cryo-electron microscopy structures for AE3 transmembrane domains (TMD) and a chimera combining AE3 N-terminal domain (NTD) with AE2 TMD (hAE3NTD2TMD). Our analyzes reveal a substrate binding site, an NTD-TMD interlock mechanism, and a preference for an outward-facing conformation. Unlike AE2, which has more robust acid-loading capabilities, AE3’s structure, including a less stable inward-facing conformation due to missing key NTD-TMD interactions, contributes to its moderated pH-modulating activity and increased sensitivity to the inhibitor DIDS. These structural differences underline AE3’s distinct functional roles in specific tissues and underscore the complex interplay between structural dynamics and functional specificity within the anion exchanger family, enhancing our understanding of the physiological and pathological roles of the anion exchanger family.

Analysis of CFTR mRNA and Protein in Peripheral Blood Mononuclear Cells via Quantitative Real-Time PCR and Western Blot.

The Cystic Fibrosis Conductance Transmembrane Regulator gene encodes for the CFTR ion channel, which is responsible for the transport of chloride and bicarbonate across the plasma membrane. Mutations in the gene result in impaired ion transport, subsequently leading to perturbed secretion in all exocrine glands and, therefore, the multi-organ disease cystic fibrosis (CF). In recent years, several studies have reported on CFTR expression in immune cells as demonstrated by immunofluorescence, flow cytometry, and immunoblotting. However, these data are mainly restricted to single-cell populations and show significant variation depending on the methodology used. Here, we investigated CFTR transcription and protein expression using standardized protocols in a comprehensive panel of immune cells. Methods: We applied a high-resolution Western blot protocol using a combination of highly specific monoclonal CFTR antibodies that have been optimized for the detection of CFTR in epithelial cells and healthy primary immune cell subpopulations sorted by flow cytometry and used immortalized cell lines as controls. The specificity of CFTR protein detection was controlled by peptide competition and enzymatic Peptide-N-Glycosidase-F (PNGase) digest. CFTR transcripts were analyzed using quantitative real-time PCR and normalized to the level of epithelial T84 cells as a reference. Results: CFTR mRNA expression could be shown for primary CD4+ T cells, NK cells, as well as differentiated THP-1 and Jurkat T cells. In contrast, we failed to detect CFTR transcripts for CD14+ monocytes and undifferentiated THP-1 cells, as well as for B cells and CD8+ T cells. Prominent immunoreactive bands were detectable by immunoblotting with the combination of four CFTR antibodies targeting different epitopes of the CFTR protein. However, in biosamples of non-epithelial origin, these CFTR-like protein bands could be unmasked as false positives through peptide competition or PNGase digest, meaning that the observed mRNA transcripts were not necessarily translated into CFTR proteins, which could be detected via immunoblotting. Our results confirm that mRNA expression in immune cells is many times lower than in that cells of epithelial origin. The immunoreactive signals in immune cells turned out to be false positives, and may be provoked by the presence of a high-affinity protein with a similar epitope. Non-specific binding (e.g., Fab-interaction with glycosyl branches) might also contribute to false positive signals. Our findings highlight the necessity of accurate controls, such as CFTR-negative cells, as well as peptide competition and glycolytic digest in order to identify genuine CFTR protein by immunoblotting. Our data suggest, furthermore, that CFTR protein expression data from techniques such as histology, for which the absence of a molecular weight or other independent control prevents the unmasking of false positive immunoreactive signals, must be interpreted carefully as well.

Slc26a12-A novel member of the slc26 family, is located in tandem with slc26a2 in coelacanths, amphibians, reptiles, and birds.

Solute carrier family 26 (Slc26) is a family of anion exchangers with 11 members in mammals (named Slc26a1-a11). Here, we identified a novel member of the slc26 family, slc26a12, located in tandem with slc26a2 in the genomes of several vertebrate lineages. BLAST and synteny analyses of various jawed vertebrate genome databases revealed that slc26a12 is present in coelacanths, amphibians, reptiles, and birds but not in cartilaginous fishes, lungfish, mammals, or ray-finned fishes. In some avian and reptilian lineages such as owls, penguins, egrets, and ducks, and most turtles examined, slc26a12 was lost or pseudogenized. Phylogenetic analysis showed that Slc26a12 formed an independent branch with the other Slc26 members and Slc26a12, Slc26a1 and Slc26a2 formed a single branch, suggesting that these three members formed a subfamily in Slc26. In jawless fish, hagfish have two genes homologous to slc26a2 and slc26a12, whereas lamprey has a single gene homologous to slc26a2. African clawed frogs express slc26a12 in larval gills, skin, and fins. These results show that slc26a12 was present at least before the separation of lobe-finned fish and tetrapods; the name slc26a12 is appropriate because the gene duplication occurred in the distant past.

Protective effect of Albizia chinensis saponin on ethanol-induced gastric mucosal injury in rats focus on mucus and mucosal barriers

This study aims to explore the protective effect of Albizia chinensis saponin on ethanol-induced acute gastric ulcer in rats and elucidate its mechanisms. SD rats were deprived of water for 24 hours before the experiment. The control group and model group were administered water by gavage, and the positive drug group received rabeprazole sodium solution(40 mg·kg~(-1)) by gavage. The experimental groups were given different doses of Albizia chinensis saponin solution(3, 10, and 30 mg·kg~(-1)). After 30 minutes, the control group received 1.5 mL of water by gavage, while the other groups were administered an equal volume of 95% ethanol for modeling. After six hours, the rats were killed by cervical dislocation, and the stomachs were collected. The ulcer area was measured, and the ulcer index was calculated. Hematoxylin-eosin(HE) staining was performed to assess histopathological changes in gastric tissue. Periodic acid-Schiff(PAS) staining was used to evaluate the distribution of gastric mucosal surface mucus. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of phospholipids and aminohexose in the gastric mucosa. Western blot was performed to determine the expression levels of the bicarbonate transporter, matrix metalloproteinase, and tight junction-associated proteins in gastric tissue. Immunohistochemistry(IHC) staining was conducted to quantify the number of positive cells for secreted mucin and tight junction-associated proteins. The results showed that the gastric tissue surface of rats in the control group was smooth without ulceration, and the gastric ulcer index of rats in the model group was 35±11. Albizia chinensis saponin at doses of 3, 10, and 30 mg·kg~(-1) resulted in inhibition rates of gastric ulcer of 46%(P<0.01), 85%(P<0.001), and 100%(P<0.001), respectively. Severe disruption of gastric mucosal structure and absence of the mucus layer were observed in the model group. Compared with the model group, the Albizia chinensis saponin group showed intact gastric mucosal surface mucus layer, significantly increased levels of phospholipids and aminohexose in the mucus, increased number of MUC5AC positive cells, and upregulated expression levels of the bicarbonate transporter SLC26A3 and CFTR. It also showed decreased phosphorylation of JNK and c-Jun, reduced expression levels of MMP-8, elevated expression of TIMP-1, and increased expression levels of Occludin and ZO-1. In conclusion, Albizia chinensis saponin enhances the function of the mucus-bicarbonate barrier by upregulating the content of MUC5AC, phospholipids, and aminohexose and increasing the expression levels of the bicarbonate transporter SLC26A3 and CFTR. Moreover, Albizia chinensis saponin exerts its protective effects on gastric ulcers by inhibiting the JNK signaling pathway to prevent excessive activation of MMP-8, thereby reducing the degradation of Occludin and ZO-1 and enhancing the mucosal barrier function. In summary, Albizia chinensis saponin exerts its anti-gastric ulcer effects by simultaneously enhancing the mucus barrier and the mucosal barrier.

Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts.

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.

Aqueous humor perturbations in chronic smokers: a proteomic study

The detrimental effects of smoking are multisystemic and its effects on the eye health are significant. Smoking is a strong risk factor for age-related nuclear cataract, age-related macular degeneration, glaucoma, delayed corneal epithelial healing and increased risk of cystoid macular edema in patients with intermediate uveitis among others. We aimed to characterize the aqueous humor (AH) proteome in chronic smokers to gain insight into its perturbations and to identify potential biomarkers for smoking-associated ocular pathologies. Compared to the control group, chronic smokers displayed 67 (37 upregulated, 30 downregulated) differentially expressed proteins (DEPs). Analysis of DEPs from the biological point of view revealed that they were proteins involved in complement activation, lymphocyte mediated immunity, innate immune response, cellular oxidant detoxification, bicarbonate transport and platelet degranulation. From the molecular function point of view, DEPs were involved in oxygen binding, oxygen carrier activity, hemoglobin binding, peptidase/endopeptidase/cysteine-type endopeptidase inhibitory activity. Several of the upregulated proteins were acute phase reactant proteins such as clusterin, alpha-2-HS-glycoprotein, fibrinogen, alpha-1-antitrypsin, C4b-binding protein and serum amyloid A-2. Further research should confirm if these proteins might serve as biomarkers or therapeutic target for smoking-associated ocular diseases.

Molecular dynamics simulations of lipid-protein interactions in SLC4 proteins

The SLC4 family of secondary bicarbonate transporters is responsible for the transport of HCO3−, CO32−, Cl−, Na+, K+, NH3, and H+, which are necessary for regulation of pH and ion homeostasis. They are widely expressed in numerous tissues throughout the body and function in different cell types with different membrane properties. Potential lipid roles in SLC4 function have been reported in experimental studies, focusing mostly on two members of the family: AE1 (Cl−/HCO3− exchanger) and NBCe1 (Na+-CO32−cotransporter). Previous computational studies of the outward-facing state of AE1 with model lipid membranes revealed enhanced protein-lipid interactions between cholesterol (CHOL) and phosphatidylinositol bisphosphate (PIP2). However, the protein-lipid interactions in other members of the family and other conformation states are still poorly understood and this precludes the detailed studies of a potential regulatory role for lipids in the SLC4 family. In this work, we performed coarse-grained and atomistic molecular dynamics simulations on three members of the SLC4 family with different transport modes: AE1, NBCe1, and NDCBE (an Na+-CO32−/Cl− exchanger), in model HEK293 membranes consisting of CHOL, PIP2, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. The recently resolved inward-facing state of AE1 was also included in the simulations. Lipid-protein contact analysis of the simulated trajectories was performed with the ProLint server, which provides a multitude of visualization tools for illustration of areas of enhanced lipid-protein contact and identification of putative lipid binding sites within the protein matrix. We observed enrichment of CHOL and PIP2 around all proteins with subtle differences in their distribution depending on the protein type and conformation state. Putative binding sites were identified for CHOL, PIP2, phosphatidylcholine, and sphingomyelin in the three studied proteins, and their potential roles in the SLC4 transport function, conformational transition, and protein dimerization are discussed.

Erythroid anion transport, nitric oxide, and blood pressure.

Glycophorin A and glycophorin B are structural membrane glycoproteins bound in the band 3 multiprotein complexes on human red blood cells (RBCs). Band 3 is an erythroid-specific anion exchanger (AE1). AE1-mediated HCO3 - transport provides the substrate for the enzyme-catalyzed conversion HCO3 - (aq) ⇌ CO2(g), which takes place inside the RBCs. Bicarbonate transport via AE1 supports intravascular acid-base homeostasis and respiratory excretion of CO2. In the past decade, we conducted several comparative physiology studies on Taiwanese people having the glycophorin variant GPMur RBC type (which accompanies greater AE1 expression). We found that increased anion transport across the erythrocyte membrane not only enhances gas exchange and lung functions but also elevates blood pressure (BP) and reduces nitric oxide (NO)-dependent vasodilation and exhaled NO fraction (FeNO) in healthy individuals with GP.Mur. Notably, in people carrying the GPMur blood type, the BP and NO-dependent, flow-mediated vasodilation (FMD) are both more strongly correlated with individual hemoglobin (Hb) levels. As blood NO and nitrite (NO2 -) are predominantly scavenged by intraerythrocytic Hb, and NO2 - primarily enters RBCs via AE1, could a more monoanion-permeable RBC membrane (i.e., GPMur/increased AE1) enhance NO2 -/NO3 - permeability and Hb scavenging of NO2 - and NO to affect blood pressure? In this perspective, a working model is proposed for the potential role of AE1 in intravascular NO availability, blood pressure, and clinical relevance.

The role of Na+-coupled bicarbonate transporters (NCBT) in health and disease.

Cellular and organism survival depends upon the regulation of pH, which is regulated by highly specialized cell membrane transporters, the solute carriers (SLC) (For a comprehensive list of the solute carrier family members, see: https://www.bioparadigms.org/slc/ ). The SLC4 family of bicarbonate (HCO3-) transporters consists of ten members, sorted by their coupling to either sodium (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE), chloride (AE1, AE2, AE3), or borate (BTR1). The ionic coupling of SLC4A9 (AE4) remains controversial. These SLC4 bicarbonate transporters may be controlled by cellular ionic gradients, cellular membrane voltage, and signaling molecules to maintain critical cellular and systemic pH (acid-base) balance. There are profound consequences when blood pH deviates even a small amount outside the normal range (7.35-7.45). Chiefly, Na+-coupled bicarbonate transporters (NCBT) control intracellular pH in nearly every living cell, maintaining the biological pH required for life. Additionally, NCBTs have important roles to regulate cell volume and maintain salt balance as well as absorption and secretion of acid-base equivalents. Due to their varied tissue expression, NCBTs have roles in pathophysiology, which become apparent in physiologic responses when their expression is reduced or genetically deleted. Variations in physiological pH are seen in a wide variety of conditions, from canonically acid-base related conditions to pathologies not necessarily associated with acid-base dysfunction such as cancer, glaucoma, or various neurological diseases. The membranous location of the SLC4 transporters as well as recent advances in discovering their structural biology makes them accessible and attractive as a druggable target in a disease context. The role of sodium-coupled bicarbonate transporters in such a large array of conditions illustrates the potential of treating a wide range of disease states by modifying function of these transporters, whether that be through inhibition or enhancement.

CFTR function is impaired in a subset of patients with pancreatitis carrying rare CFTR variants

BackgroundMany affected by pancreatitis harbor rare variants of the cystic fibrosis (CF) gene, CFTR, which encodes an epithelial chloride/bicarbonate channel. We investigated CFTR function and the effect of CFTR modulator drugs in pancreatitis patients carrying CFTR variants. MethodsNext-generation sequencing was performed to identify CFTR variants. Sweat tests and nasal potential difference (NPD) assays were performed to assess CFTR function in vivo. Intestinal current measurement (ICM) was performed on rectal biopsies. Patient-derived intestinal epithelial monolayers were used to evaluate chloride and bicarbonate transport and the effects of a CFTR modulator combination: elexacaftor, tezacaftor and ivacaftor (ETI). ResultsOf 32 pancreatitis patients carrying CFTR variants, three had CF-causing mutations on both alleles and yielded CF-typical sweat test, NPD and ICM results. Fourteen subjects showed a more modest elevation in sweat chloride levels, including three that were provisionally diagnosed with CF. ICM indicated impaired CFTR function in nine out of 17 non-CF subjects tested. This group of nine included five carrying a wild type CFTR allele. In epithelial monolayers, a reduction in CFTR-dependent chloride transport was found in six out of 14 subjects tested, whereas bicarbonate secretion was reduced in only one individual. In epithelial monolayers of four of these six subjects, ETI improved CFTR function. ConclusionsCFTR function is impaired in a subset of pancreatitis patients carrying CFTR variants. Mutations outside the CFTR locus may contribute to the anion transport defect. Bioassays on patient-derived intestinal tissue and organoids can be used to detect such defects and to assess the effect of CFTR modulators.

Increased CO2 fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast

CO2 fixation plays a key role to make biobased production cost competitive. Here, we use 3-hydroxypropionic acid (3-HP) to showcase how CO2 fixation enables approaching theoretical-yield production. Using genome-scale metabolic models to calculate the production envelope, we demonstrate that the provision of bicarbonate, formed from CO2, restricts previous attempts for high yield production of 3-HP. We thus develop multiple strategies for bicarbonate uptake, including the identification of Sul1 as a potential bicarbonate transporter, domain swapping of malonyl-CoA reductase, identification of Esbp6 as a potential 3-HP exporter, and deletion of Uga1 to prevent 3-HP degradation. The combined rational engineering increases 3-HP production from 0.14 g/L to 11.25 g/L in shake flask using 20 g/L glucose, approaching the maximum theoretical yield with concurrent biomass formation. The engineered yeast forms the basis for commercialization of bio-acrylic acid, while our CO2 fixation strategies pave the way for CO2 being used as the sole carbon source.