Bicarbonate Transport Research Articles

Pro-inflammatory cytokines stimulate CFTR-dependent anion secretion in pancreatic ductal epithelium.

Loss of CFTR-dependent anion and fluid secretion in the ducts of the exocrine pancreas is thought to contribute to the development of pancreatitis, but little is known about the impact of inflammation on ductal CFTR function. Here we used adult stem cell-derived cell cultures (organoids) obtained from porcine pancreas to evaluate the effects of pro-inflammatory cytokines on CFTR function. Organoids were cultured from porcine pancreas and used to prepare ductal epithelial monolayers. Monolayers were characterized by immunocytochemistry. Epithelial bicarbonate and chloride secretion, and the effect of IL-1β, IL-6, IFN-γ, and TNF-α on CFTR function was assessed by electrophysiology. Immunolocalization of ductal markers, including CFTR, keratin 7, and zonula occludens 1, demonstrated that organoid-derived cells formed a highly polarized epithelium. Stimulation by secretin or VIP triggered CFTR-dependent anion secretion across epithelial monolayers, whereas purinergic receptor stimulation by UTP, elicited CFTR-independent anion secretion. Most of the anion secretory response was attributable to bicarbonate transport. The combination of IL-1β, IL-6, IFN-γ, and TNF-α markedly enhanced CFTR expression and anion secretion across ductal epithelial monolayers, whereas these cytokines had little effect when tested separately. Although TNF-α triggered apoptotic signaling, epithelial barrier function was not significantly affected by cytokine exposure. Pro-inflammatory cytokines enhance CFTR-dependent anion secretion across pancreatic ductal epithelium. We propose that up-regulation of CFTR in the early stages of the inflammatory response, may serve to promote the removal of pathogenic stimuli from the ductal tree, and limit tissue injury.

Multiple Regulatory Signals and Components in the Modulation of Bicarbonate Transporters.

Bicarbonate transporters are responsible for the appropriate flux of bicarbonate across the plasma membrane to perform various fundamental cellular functions. The functions of bicarbonate transporters, including pH regulation, cell migration, and inflammation, are highlighted in various cellular systems, encompassing their participation in both physiological and pathological processes. In this review, we focused on recently identified modulatory signaling components that regulate the expression and activity of bicarbonate transporters. Moreover, we addressed recent advances in our understanding of cooperative systems of bicarbonate transporters and channelopathies. This current review aims to provide a new, in-depth understanding of numerous human diseases associated with the dysfunction of bicarbonate transporters.

Inorganic carbon sensing and signalling in cyanobacteria

AbstractCyanobacteria utilize CO2 and HCO3− as inorganic carbon (Ci) sources. In low Ci, like in ambient air, cyanobacteria efficiently collect Ci using a carbon concentrating mechanism (CCM). The CCM includes bicarbonate transporters SbtA, BicA and BCT1; the specialized NDH complexes NDH‐13 and NDH‐14, which convert CO2 to HCO3− in the cytoplasm; and carboxysomes that are protein shell encapsulated ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCo) and carbonic anhydrase containing bodies in which the first reaction of carbon fixation occurs. Ci‐dependent regulation of bicarbonate transporters and specialized NDH complexes, especially the regulation of the SbtA transporter, are well understood. CcmR (also called NdhR), CyAbrB2, CmpR and RbcR act as transcription factors regulating CCM genes. Ci signalling molecules detecting the metabolic status of the cells include 2‐oxoglutarate, which accumulates when the Ci/nitrogen ratio of the cell is high, and 2‐phosphoglycolate, the first intermediate of the photorespiration pathway, whose accumulation indicates low Ci. These signalling molecules act as corepressors and coactivators of the CcmR repressor protein, whereas 2‐phosphoglycolate and ribulose‐1,5‐bisphosphate activate transcription activator CmpR. In addition, bicarbonate or CO2 activates the adenylyl cyclase that produces cAMP, and ATP/ADP/AMP provide information about the energy status of the cell. Less is known about the molecular mechanisms regulating carboxysome dynamics or how production, activity and degradation of photosynthetic complexes are regulated by prevailing Ci conditions or which mechanisms adjust cell division according to Ci. This minireview summarizes the present knowledge about molecular mechanisms regulating cyanobacterial acclimation to prevailing Ci.

Solute Carrier Family 26 Member 4 (SLC26A4), A Potential Therapeutic Target for Asthma.

Asthma is a prevalent respiratory condition with multifaceted pathomechanisms, presenting challenges for therapeutic development. The SLC (Solute Carrier) gene family, encompassing diverse membrane transport proteins, plays pivotal roles in various human diseases by facilitating solute movement across biological membranes. These solutes include ions, sugars, amino acids, neurotransmitters, and drugs. Mutations in these ion channels have been associated with numerous disorders, underscoring the significance of SLC gene families in physiological processes. Among these, the SLC26A4 gene encodes pendrin, an anion exchange protein involved in transmembrane transport of chloride, iodide, and bicarbonate. Mutations in SLC26A4 are associated with Pendred syndrome. Elevated SLC26A4 expression has been linked to airway inflammation, hyperreactivity, and mucus production in asthma. Here, we review novel insights from SLC gene family members into the mechanisms of substrate transport and disease associations, with specific emphasis on SLC26A4. We explore triggers inducing SLC26A4 expression and its contributions to the pathogenesis of pulmonary diseases, particularly asthma. We summarize the inhibitors of SLC26A4 that have shown promise in the treatment of different phenotypes of diseases. While SLC26A4 inhibitors present potential treatments for asthma, further research is imperative to delineate their precise role in asthma pathogenesis and develop efficacious therapeutic strategies targeting this protein.

Mechanistic characterization of waterborne selenite uptake in the water flea, Daphnia magna, indicates water chemistry affects toxicity in coal mine-impacted waters.

Concentrations of selenium that exceed regulatory guidelines have been associated with coal mining activities and have been linked to detrimental effects on aquatic ecosystems and the organisms therein. Although the major route of selenium uptake in macroinvertebrates is via the diet, the uptake of waterborne selenite (HSeO3-), the prominent form at circumneutral pH, can be an important contributor to selenium body burden and thus selenium toxicity. In the current study, radiolabelled selenite (Se75) was used to characterize the mechanism of selenite uptake in the water flea, Daphnia magna. The concentration dependence (1-32μM) of selenite uptake was determined in 1-hour uptake assays in artificial waters that independently varied in bicarbonate, chloride, sulphate, phosphate and selenate concentrations. At concentrations representative of those found in highly contaminated waters, selenite uptake was phosphate-dependent and inhibited by foscarnet, a phosphate transport inhibitor. At higher concentrations, selenite uptake was dependent on waterborne bicarbonate concentration and inhibited by the bicarbonate transporter inhibitor DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid). These findings suggest that concentrations of phosphate in coal mining-affected waters could alter selenite uptake in aquatic organisms and could ultimately affect the toxic impacts of selenium in such waters.

Putting bicarbonate on the spot: pharmacological insights for CFTR correction in the airway epithelium.

Introduction: Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) proteins. CFTR controls chloride (Cl-) and bicarbonate (HCO3 -) transport into the Airway Surface Liquid (ASL). We investigated the impact of F508del-CFTR correction on HCO3 - secretion by studying transepithelial HCO3 - fluxes. Methods: HCO3 - secretion was measured by pH-stat technique in primary human respiratory epithelial cells from healthy subjects (WT) and people with CF (pwCF) carrying at least one F508del variant. Its changes after CFTR modulation by the triple combination VX445/661/770 and in the context of TNF-α+IL-17 induced inflammation were correlated to ASL pH and transcriptional levels of CFTR and other HCO3 - transporters of airway epithelia such as SLC26A4 (Pendrin), SLC26A9 and NBCe1. Results: CFTR-mediated HCO3 - secretion was not detected in F508del primary human respiratory epithelial cells. It was rescued up to ∼ 80% of the WT level by VX-445/661/770. In contrast, TNF-α+IL-17 normalized transepithelial HCO3 - transport and increased ASL pH. This was related to an increase in SLC26A4 and CFTR transcript levels. VX-445/661/770 induced an increase in pH only in the context of inflammation. Effects on HCO3 - transport were not different between F508del homozygous and F508del compound heterozygous CF airway epithelia. Conclusion: Our studies show that correction of F508del-CFTR HCO3 - is not sufficient to buffer acidic ASL and inflammation is a key regulator of HCO3 - secretion in CF airways. Prediction of the response to CFTR modulators by theratyping should take into account airway inflammation.

Potential Theranostic Roles of SLC4 Molecules in Human Diseases.

The solute carrier family 4 (SLC4) is an important protein responsible for the transport of various ions across the cell membrane and mediating diverse physiological functions, such as the ion transporting function, protein-to-protein interactions, and molecular transduction. The deficiencies in SLC4 molecules may cause multisystem disease involving, particularly, the respiratory system, digestive, urinary, endocrine, hematopoietic, and central nervous systems. Currently, there are no effective strategies to treat these diseases. SLC4 proteins are also found to contribute to tumorigenesis and development, and some of them are regarded as therapeutic targets in quite a few clinical trials. This indicates that SLC4 proteins have potential clinical prospects. In view of their functional characteristics, there is a critical need to review the specific functions of bicarbonate transporters, their related diseases, and the involved pathological mechanisms. We summarize the diseases caused by the mutations in SLC4 family genes and briefly introduce the clinical manifestations of these diseases as well as the current treatment strategies. Additionally, we illustrate their roles in terms of the physiology and pathogenesis that has been currently researched, which might be the future therapeutic and diagnostic targets of diseases and a new direction for drug research and development.

Genomic Analysis of Leptolyngbya boryana CZ1 Reveals Efficient Carbon Fixation Modules.

Cyanobacteria, one of the most widespread photoautotrophic microorganisms on Earth, have evolved an inorganic CO2-concentrating mechanism (CCM) to adapt to a variety of habitats, especially in CO2-limited environments. Leptolyngbya boryana, a filamentous cyanobacterium, is widespread in a variety of environments and is well adapted to low-inorganic-carbon environments. However, little is currently known about the CCM of L. boryana, in particular its efficient carbon fixation module. In this study, we isolated and purified the cyanobacterium CZ1 from the Xin'anjiang River basin and identified it as L. boryana by 16S rRNA sequencing. Genome analysis revealed that L. boryana CZ1 contains β-carboxysome shell proteins and form 1B of Rubisco, which is classify it as belonging to the β-cyanobacteria. Further analysis revealed that L. boryana CZ1 employs a fine CCM involving two CO2 uptake systems NDH-13 and NDH-14, three HCO3- transporters (SbtA, BicA, and BCT1), and two carboxysomal carbonic anhydrases. Notably, we found that NDH-13 and NDH-14 are located close to each other in the L. boryana CZ1 genome and are back-to-back with the ccm operon, which is a novel gene arrangement. In addition, L. boryana CZ1 encodes two high-affinity Na+/HCO3- symporters (SbtA1 and SbtA2), three low-affinity Na+-dependent HCO3- transporters (BicA1, BicA2, and BicA3), and a BCT1; it is rare for a single strain to encode all three bicarbonate transporters in such large numbers. Interestingly, L. boryana CZ1 also uniquely encodes two active carbonic anhydrases, CcaA1 and CcaA2, which are also rare. Taken together, all these results indicated that L. boryana CZ1 is more efficient at CO2 fixation. Moreover, compared with the reported CCM gene arrangement of cyanobacteria, the CCM-related gene distribution pattern of L. boryana CZ1 was completely different, indicating a novel gene organization structure. These results can enrich our understanding of the CCM-related gene arrangement of cyanobacteria, and provide data support for the subsequent improvement and increase in biomass through cyanobacterial photosynthesis.

Substrate binding and inhibition of the anion exchanger 1 transporter.

Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO2 transport between lungs and tissues. Previous studies characterized its role in erythrocyte structure and provided insight into transport regulation. However, key questions remain regarding substrate binding and transport, mechanisms of drug inhibition and modulation by membrane components. Here we present seven cryo-EM structures in apo, bicarbonate-bound and inhibitor-bound states. These, combined with uptake and computational studies, reveal important molecular features of substrate recognition and transport, and illuminate sterol binding sites, to elucidate distinct inhibitory mechanisms of research chemicals and prescription drugs. We further probe the substrate binding site via structure-based ligand screening, identifying an AE1 inhibitor. Together, our findings provide insight into mechanisms of solute carrier transport and inhibition.

Trafficking of carbonic anhydrase 12 and bicarbonate transporters by histamine stimulation mediates intracellular acidic scenario in lung cancer cells

Carbonic anhydrase 12 is considered an oncogenic and acidic microenvironmental factor in cancer cells. To verify the role of histamine signalling as an anti-cancer signal, we determined the roles of CA12 and its associated bicarbonate transporters. In this study, histamine stimulation mediated mislocalization of CA12 in lung cancer cells. Histamine receptor activation-mediated CA12 endocytosis and pH were restored by CaMKII inhibition. CA12-associated AE2 expression was enhanced, whereas NBCn1 expression and its activity were reduced by histamine stimulation. Histamine receptor activation-mediated acidification was induced by internalised CA12 and NBCn1 and, at the same time by increased bicarbonate efflux through enhanced AE2 expression. Inhibition of protein trafficking by bafilomycin restored CA12 and AE2 localisation and diminished cellular acidosis. Thus, we verified that histamine stimulation induced an acidic scenario, which revealed trafficking of CA12 and its associated bicarbonate transporters in lung cancer cells and its dysregulated pH modulation may be involved in the histamine signalling-mediated anti-cancer process.

Cystic fibrosis

Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by variants in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR dysfunction results in abnormal chloride and bicarbonate transport in epithelial cells, leading to a multiorgan disease dominated by respiratory and digestive manifestations. The respiratory disease, which is characterized by airway mucus plugging, chronic bacterial infection and progressive development of bronchiectasis, may lead to chronic respiratory failure, which is the main cause of premature death in people with CF. Over the past 50 years, major progress has been obtained by implementing multidisciplinary care, including nutritional support, airway clearance techniques and antibiotics in specialized CF centers. The past 10 years have further seen the progressive development of oral medications, called CFTR modulators, that partially restore ion transport and lead to a major improvement in clinical manifestations and lung function, presumably resulting in longer survival. Although an increasing proportion of people with CF are being treated with CFTR modulators, challenges remain regarding access to CFTR modulators due to their high cost, and their lack of marketing approval and/or effectiveness in people with rare CFTR variants. The anticipated increase in the number of adults with CF and their aging also challenge the current organization of CF care. The purpose of this review article is to describe current status and future perspective of CF disease and care.

Polyphasic characterization of a novel hot-spring cyanobacterium Thermocoleostomius sinensis gen et sp. nov. and genomic insights into its carbon concentration mechanism.

Thermophilic cyanobacteria play a crucial role as primary producers in hot spring ecosystems, yet their microbiological, taxonomic, and ecological characteristics are not extensively studied. This study aimed to characterize a novel strain of thermophilic cyanobacteria, PKUAC-SCTA174 (A174), using a combination of traditional polyphasic methods and modern genomic-based approaches. The study included 16S rRNA-based phylogeny, ITS secondary structure prediction, morphological and habitat analyses, as well as high-quality genome sequencing with corresponding phylogenomic analyses. The results of the 16S rRNA, 16S-23S ITS secondary structure, morphology, and habitat analyses supported the classification of the strain as a member of a novel genus within the family Oculatellaceae, closely related to Albertania and Trichotorquatus. Genomic analysis revealed the presence of a sophisticated carbon-concentrating mechanism (CCM) in the strain, involving two CO2 uptake systems NDH-I3, and NDH-I4, three types of bicarbonate transporters (BCT1, bicA, sbtA,) and two distinct putative carboxysomal carbonic anhydrases (ccaA1 and ccaA2). The expression of CCM genes was investigated with a CO2 shift experiment, indicating varying transcript abundance among different carbon uptake systems. Based on the comprehensive characterization, the strain was delineated as Thermocoleostomius sinensis, based on the botanical code. The study of the complete genome of strain A174 contributes valuable insights into the genetic characteristics of the genus Thermocoleostomius and related organisms and provides a systematic understanding of thermophilic cyanobacteria. The findings presented here offer valuable data that can be utilized for future research in taxogenomics, ecogenomics, and geogenomics.

ECM Composition Differentially Regulates Intracellular and Extracellular pH in Normal and Cancer Pancreatic Duct Epithelial Cells

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.

#3827 BICARBONATE PROFILING IN HEMODIALYSIS: MATHEMATICAL MODELING OF DATA FROM A CLINICAL TRIAL

Abstract Background and Aims During hemodialysis (HD), bicarbonate and other bases are added to the dialysis fluid to correct chronic acidosis that may develop in kidney-impaired patients, increasing plasma bicarbonate concentration to physiological levels. Previous studies have shown that both exceedingly high and low post-dialysis bicarbonate concentrations are associated with adverse clinical outcomes; however, no clear guidelines for the prescription of bicarbonate concentration in the dialysis fluid are available. Data from a recent controlled clinical trial, carried out at the Department of Nephrology, Dialysis and Internal Medicine, Medical University of Warsaw (Poland), were used to identify transport parameters of a mathematical model of acid-base homeostasis in HD patients, as the first step in the implementation of a tool to optimize bicarbonate profiling during HD. Method The study was carried out in 20 chronic patients, undergoing thrice weekly 4-hr HD. The patients underwent three treatment regimens: A) constant dialysis fluid bicarbonate concentration (HCO3,D) equal to 35 mEq/L; B) HCO3,D decreasing from 35 to 30 mEq/L after 2 hrs; C) HCO3,D increasing from 30 to 35 mEq/L after 2 hrs. The patients were divided in two cohorts: for the first (n = 11) treatment schedule was: treatment A during the first and third week, B during the second week and C during the fourth week. For the second cohort (n = 9) treatment C was carried out during the second week and treatment B during the fourth. Data were collected only during the second and third sessions of the week, and acid-base composition was determined using a blood gas analyzer. The mathematical model describes the transport of bicarbonate and dissolved CO2 from dialysis fluid to the patient and can predict changes in the acid-base equilibrium of arterial and venous plasma and erythrocytes, interstitial fluid, and tissue cells, using mass transport equations. The dialysances for bicarbonate (KHCO3) and dissolved CO2 (KCO2) were estimated via least square minimization. Results No significant difference was found comparing patients in the two cohorts, or comparing sessions of the same week. Pre-dialysis plasma bicarbonate, pH, or CO2 partial pressure (PCO2) were significantly lower for treatment B compared to treatment A. Average plasma bicarbonate concentrations for the different treatments are shown in Figure 1 for data and model simulations. Table 1 reports the average values of the estimated parameters for week. No statistically significant difference was found between sessions, cohorts, or treatments. The model fitted bicarbonate data with an average error of 2.5 ± 1.4%, and PCO2 with an error of 4.5 ± 3.6%. Simulations of mixed-venous blood, interstitial fluid, and tissue cells concentrations showed a similar response to different treatments as in arterial blood. Conclusion The model accurately describes plasma bicarbonate data with high accuracy regardless of the treatment conditions. The parameters estimated showed considerable variability between treatments (less so between the sessions of the same week), but without statistical significance. The model can be used to simulate the effect of dialysate bicarbonate profiling on acid-base equilibrium in HD patients.

Role of the bicarbonate transporter SLC4γ in stony-coral skeleton formation and evolution.

Coral reefs are highly diverse ecosystems of immense ecological, economic, and aesthetic importance built on the calcium-carbonate-based skeletons of stony corals. The formation of these skeletons is threatened by increasing ocean temperatures and acidification, and a deeper understanding of the molecular mechanisms involved may assist efforts to mitigate the effects of such anthropogenic stressors. In this study, we focused on the role of the predicted bicarbonate transporter SLC4γ, which was suggested in previous studies to be a product of gene duplication and to have a role in coral-skeleton formation. Our comparative-genomics study using 30 coral species and 15 outgroups indicates that SLC4γ is present throughout the stony corals, but not in their non-skeleton-forming relatives, and apparently arose by gene duplication at the onset of stony-coral evolution. Our expression studies show that SLC4γ, but not the closely related and apparently ancestral SLC4β, is highly upregulated during coral development coincident with the onset of skeleton deposition. Moreover, we show that juvenile coral polyps carrying CRISPR/Cas9-induced mutations in SLC4γ are defective in skeleton formation, with the severity of the defect in individual animals correlated with their frequencies of SLC4γ mutations. Taken together, the results suggest that the evolution of the stony corals involved the neofunctionalization of the newly arisen SLC4γ for a unique role in the provision of concentrated bicarbonate for calcium-carbonate deposition. The results also demonstrate the feasibility of reverse-genetic studies of ecologically important traits in adult corals.

Semisynthesis, Biological Evaluation and Molecular Docking Studies of Barbatic Acid Derivatives as Novel Diuretic Candidates.

Barbatic acid, a compound isolated from lichen, has demonstrated a variety of biological activities. In this study, a series of esters based on barbatic acid (6a-q') were designed, synthesized, and evaluated for their diuretic and litholytic activity at a concentration of 100 μmol/L in vitro. All target compounds were characterized using 1H NMR, 13C NMR, and HRMS, and the spatial structure of compound 6w was confirmed using X-ray crystallography. The biological results showed that some derivatives, including 6c, 6b', and 6f', exhibited potent diuretic activity, and 6j and 6m displayed promising litholytic activity. Molecular docking studies further suggested that 6b' had an optimal binding affinity to WNK1 kinases related to diuresis, while 6j could bind to the bicarbonate transporter CaSR through a variety of forces. These findings indicate that some barbatic acid derivatives could be further developed into novel diuretic agents.

Pyruvate orthophosphate dikinase is required for the acclimation to high bicarbonate concentrations in Phaeodactylum tricornutum

The model marine diatom P. tricornutum possesses efficient CO2-concentrating mechanisms (CCMs) involving the bicarbonate transport and the rapid inter-conversion of HCO3− and CO2, and it has a complete set of enzymes essential for biochemical CCM C4 pathway. However, the role of potential C4 pathway in the diatom is still controversial. Based on the analysis of previously published proteome and transcriptome data in P. tricornutum, the expression of pyruvate orthophosphate dikinase (PPDK), a key enzyme generating the primary acceptor for bicarbonate fixation in C4 pathway, was upregulated in long-term high CO2-selected populations. In our study, P. tricornutum PPDK (PtPPDK) transcript abundance was higher with short-term treatment of high NaHCO3 concentrations and overexpression of PtPPDK resulted in a cell density increase of 5–16 % from day 2 to 8. Knockdown (KD) or knockout of PtPPDK inhibited the growth and the maximal photosystem II electron transport rate, and the inhibited growth became more and more evident with the increasing NaHCO3 concentration from 1 to 8 mM in PtPPDK KD lines. Furthermore, elevating NaHCO3 from 1 to 16 mM dramatically decreased the cell density both in wild-type and PtPPDK mutants, suggesting that 1 mM NaHCO3 is enough for the photosynthesis carbon assimilation. PtPPDK was localized in the plastid stroma by eGFP fusion protein analysis at 2 mM NaHCO3, while PtPPDK-eGFP fluorescence was observed both in the stroma and periplasmic space at higher NaHCO3. It is suggested that PPDK is necessary for consuming excessive inorganic carbon (Ci) both from the plastid and extracellular environment to maintain pH homeostasis, indicating a potential function of adapting to high Ci concentrations.

Loss of Kir5.1 disturbs acid-base homeostasis in Dahl salt-sensitive rats

Renal basolateral inwardly-rectifying potassium (K ir ) channels contribute to the maintenance of the resting membrane potential and potassium homeostasis. Heteromeric K ir 4.1/K ir 5.1 channels are more sensitive to intracellular acidification than homomeric K ir 4.1 channels. Our previous study demonstrated that genetic mutation of Kcnj16 (the gene encodes K ir 5.1) causes chronic metabolic acidosis with lower blood pH and bicarbonate levels in Dalh salt-sensitive (SS) rats. Here, we aim to investigate the underlying mechanisms. We hypothesized that loss of K ir 5.1 disturbs acid-base homeostasis in SS rats by impairing ammoniagenesis in the proximal tubule and HCO3 - reabsorption in the distal convoluted tubule (DCT).12-week-old male SS WT and SS Kcnj16-/- rats were separated into two groups (N=5 per group). At the end of the experiment, kidneys were flushed and processed for blood pH and Western blot analysis.Western blot analysis indicated that knock out of K ir 5.1 causes lower expression of sodium-coupled neutral amino acid transporter 3 (SNAT3), which may indicate defective ammoniagenesis in the proximal tubule. On the other hand, increased expression of NBCe1 and NHE3 suggests enhanced HCO3 - reabsorption and H + excretion. Additionally, the expression of vacuolar ATPase (V-ATPase), which is a proton pump responsible for controlling the intracellular and extracellular pH of cells, remained the same. In the following experiments, we will further investigate the ammoniagensis in the proximal tubule as well as key bicarbonate transporters in DCT and collecting duct. Funding sources: National Institute of Health Grants R35 HL135749 (to AS), Department of Veterans Affairs grant I01 BX004024 (to AS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Role of the NaHCO3 Transporter MpsABC in the NaHCO3-β-Lactam-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing concern due to their intrinsic resistance to most standard-of-care β-lactam antibiotics. Recent studies of clinical isolates have documented a novel phenotype, termed NaHCO3 responsiveness, in which a substantial proportion of MRSA strains exhibit enhanced susceptibility to β-lactams such as cefazolin and oxacillin in the presence of NaHCO3. A bicarbonate transporter, MpsAB (membrane potential-generating system), was recently found in S. aureus, where it plays a role in concentrating NaHCO3 for anaplerotic pathways. Here, we investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype. Radiolabeled NaH14CO3 uptake profiling revealed significantly higher accumulation in NaHCO3-responsive vs nonresponsive MRSA strains when grown in ambient air. In contrast, under 5% CO2 conditions, NaHCO3-responsive (but not nonresponsive) strains exhibited repressed uptake. Oxacillin MICs were measured in four prototype strains and their mpsABC deletion mutants in the presence of NaHCO3 supplementation under 5% CO2 conditions. NaHCO3-mediated reductions in oxacillin MICs were observed in the responsive parental strains but not in mpsABC deletion mutants. No significant impact on oxacillin MICs was observed in the nonresponsive strains under the same conditions. Transcriptional and translational studies were carried out using both quantitative reverse transcription-PCR (qRT-PCR) and mpsA-green fluorescent protein (GFP) fusion constructs; these investigations showed that mpsA expression and translation were significantly upregulated during mid-exponential-phase growth in oxacillin-NaHCO3-supplemented medium in responsive versus nonresponsive strains. Taken together, these data show that the NaHCO3 transporter MpsABC is a key contributor to the NaHCO3-β-lactam responsiveness phenotype in MRSA. IMPORTANCE MRSA infections are increasingly difficult to treat, due in part to their resistance to most β-lactam antibiotics. A novel and relatively common phenotype, termed NaHCO3 responsiveness, has been identified in which MRSA strains show increased susceptibility in vitro and in vivo to β-lactams in the presence of NaHCO3. A recently described S. aureus NaHCO3 transporter, MpsAB, is involved in intracellular NaHCO3 concentration for anaplerotic pathways. We investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype in four prototype MRSA strains (two responsive and two nonresponsive). We demonstrated that MpsABC is an important contributor to the NaHCO3-β-lactam responsiveness phenotype. Our study adds to the growing body of well-defined characteristics of this novel phenotype, which could potentially translate to alternative targets for MRSA treatment using β-lactams.

Characterization of a novel thermophilic cyanobacterium within Trichocoleusaceae, Trichothermofontia sichuanensis gen. et sp. nov., and its CO2-concentrating mechanism.

Thermophiles from extreme thermal environments have shown tremendous potential regarding ecological and biotechnological applications. Nevertheless, thermophilic cyanobacteria remain largely untapped and are rarely characterized. Herein, a polyphasic approach was used to characterize a thermophilic strain, PKUAC-SCTB231 (hereafter B231), isolated from a hot spring (pH 6.62, 55.5°C) in Zhonggu village, China. The analyses of 16S rRNA phylogeny, secondary structures of 16S-23S ITS and morphology strongly supported strain B231 as a novel genus within Trichocoleusaceae. Phylogenomic inference and three genome-based indices further verified the genus delineation. Based on the botanical code, the isolate is herein delineated as Trichothermofontia sichuanensis gen. et sp. nov., a genus closely related to a validly described genus Trichocoleus. In addition, our results suggest that Pinocchia currently classified to belong to the family Leptolyngbyaceae may require revision and assignment to the family Trichocoleusaceae. Furthermore, the complete genome of Trichothermofontia B231 facilitated the elucidation of the genetic basis regarding genes related to its carbon-concentrating mechanism (CCM). The strain belongs to β-cyanobacteria according to its β-carboxysome shell protein and 1B form of Ribulose bisphosphate Carboxylase-Oxygenase (RubisCO). Compared to other thermophilic strains, strain B231contains a relatively low diversity of bicarbonate transporters (only BicA for HCO3- transport) but a higher abundance of different types of carbonic anhydrase (CA), β-CA (ccaA) and γ-CA (ccmM). The BCT1 transporter consistently possessed by freshwater cyanobacteria was absent in strain B231. Similar situation was occasionally observed in freshwater thermal Thermoleptolyngbya and Thermosynechococcus strains. Moreover, strain B231 shows a similar composition of carboxysome shell proteins (ccmK1-4, ccmL, -M, -N, -O, and -P) to mesophilic cyanobacteria, the diversity of which was higher than many thermophilic strains lacking at least one of the four ccmK genes. The genomic distribution of CCM-related genes suggests that the expression of some components is regulated as an operon and others in an independently controlled satellite locus. The current study also offers fundamental information for future taxogenomics, ecogenomics and geogenomic studies on distribution and significance of thermophilic cyanobacteria in the global ecosystem.