Chloride/Multiple Anion Exchanger SLC26A Family: Systemic Roles of SLC26A4 in Various Organs.

Solute carrier family 26 member 4 (SLC26A4) is a member of the SLC26A transporter family and is expressed in various tissues, including the airway epithelium, kidney, thyroid, and tumors. It transports various ions, including bicarbonate, chloride, iodine, and oxalate. As a multiple-ion transporter, SLC26A4 is involved in the maintenance of hearing function, renal function, blood pressure, and hormone and pH regulation. In this review, we have summarized the various functions of SLC26A4 in multiple tissues and organs. Moreover, the relationships between SLC26A4 and other channels, such as cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and sodium chloride cotransporter, are highlighted. Although the modulation of SLC26A4 is critical for recovery from malfunctions of various organs, development of specific inducers or agonists of SLC26A4 remains challenging. This review contributes to providing a better understanding of the role of SLC26A4 and development of therapeutic approaches for the SLC26A4-associated hearing loss and SLC26A4-related dysfunction of various organs.

K+-Driven Cl-/HCO3- Exchange Mediated by Slc4a8 and Slc4a10.

Slc4a genes encode various types of transporters, including Na+-HCO3- cotransporters, Cl-/HCO3- exchangers, or Na+-driven Cl-/HCO3- exchangers. Previous research has revealed that Slc4a9 (Ae4) functions as a Cl-/HCO3- exchanger, which can be driven by either Na+ or K+, prompting investigation into whether other Slc4a members facilitate cation-dependent anion transport. In the present study, we show that either Na+ or K+ drive Cl-/HCO3- exchanger activity in cells overexpressing Slc4a8 or Slc4a10. Further characterization of cation-driven Cl-/HCO3- exchange demonstrated that Slc4a8 and Slc4a10 also mediate Cl- and HCO3--dependent K+ transport. Full-atom molecular dynamics simulation on the recently solved structure of Slc4a8 supports the coordination of K+ at the Na+ binding site in S1. Sequence analysis shows that the critical residues coordinating monovalent cations are conserved among mouse Slc4a8 and Slc4a10 proteins. Together, our results suggest that Slc4a8 and Slc4a10 might transport K+ in the same direction as HCO3- ions in a similar fashion to that described for Na+ transport in the rat Slc4a8 structure.

Novel functions of the anion exchanger AE4 (SLC4A9).

The kidney plays a crucial role in acid-base homeostasis. In the distal nephron, α-intercalated cells contribute to urinary acid (H+) secretion and ß-intercalated cells accomplish urinary base (HCO3-) secretion. ß-intercalated cells regulate the acid base status through modulation of the apical Cl-/HCO3- exchanger pendrin (SLC26A4) activity. In this review, we summarize and discuss our current knowledge of the physiological role of the renal transporter AE4 (SLC4A9). The AE4, as cation-dependent Cl-/HCO3- exchanger, is exclusively expressed in the basolateral membrane of ß-intercalated cells and is essential for the sensing of metabolic acid-base disturbances in mice, but not for renal sodium reabsorption and plasma volume control. Potential intracellular signaling pathways are discussed that might link basolateral acid-base sensing through the AE4 to apical pendrin activity.

Biallelic variants in SLC4A10 encoding a sodium-dependent bicarbonate transporter lead to a neurodevelopmental disorder.

PURPOSE: SLC4A10 encodes a plasma membrane-bound transporter, which mediates Na+-dependent HCO3- import, thus mediating net acid extrusion. Slc4a10 knockout mice show collapsed brain ventricles, an increased seizure threshold, mild behavioral abnormalities, impaired vision, and deafness. METHODS: Utilizing exome/genome sequencing in families with undiagnosed neurodevelopmental disorders and international data sharing, 11 patients from 6 independent families with biallelic variants in SLC4A10 were identified. Clinico-radiological and dysmorphology assessments were conducted. A minigene assay, localization studies, intracellular pH recordings, and protein modeling were performed to study the possible functional consequences of the variant alleles. RESULTS: The families harbor 8 segregating ultra-rare biallelic SLC4A10 variants (7 missense and 1 splicing). Phenotypically, patients present with global developmental delay/intellectual disability and central hypotonia, accompanied by variable speech delay, microcephaly, cerebellar ataxia, facial dysmorphism, and infrequently, epilepsy. Neuroimaging features range from some non-specific to distinct neuroradiological findings, including slit ventricles and a peculiar form of bilateral curvilinear nodular heterotopia. In silico analyses showed 6 of 7 missense variants affect evolutionarily conserved residues. Functional analyses supported the pathogenicity of 4 of 7 missense variants. CONCLUSION: We provide evidence that pathogenic biallelic SLC4A10 variants can lead to neurodevelopmental disorders characterized by variable abnormalities of the central nervous system, including altered brain ventricles, thus resembling several features observed in knockout mice.

SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.

SLC4A10 is a plasma-membrane bound transporter that utilizes the Na+ gradient to drive cellular HCO3- uptake, thus mediating acid extrusion. In the mammalian brain, SLC4A10 is expressed in principal neurons and interneurons, as well as in epithelial cells of the choroid plexus, the organ regulating the production of CSF. Using next generation sequencing on samples from five unrelated families encompassing nine affected individuals, we show that biallelic SLC4A10 loss-of-function variants cause a clinically recognizable neurodevelopmental disorder in humans. The cardinal clinical features of the condition include hypotonia in infancy, delayed psychomotor development across all domains and intellectual impairment. Affected individuals commonly display traits associated with autistic spectrum disorder including anxiety, hyperactivity and stereotyped movements. In two cases isolated episodes of seizures were reported in the first few years of life, and a further affected child displayed bitemporal epileptogenic discharges on EEG without overt clinical seizures. While occipitofrontal circumference was reported to be normal at birth, progressive postnatal microcephaly evolved in 7 out of 10 affected individuals. Neuroradiological features included a relative preservation of brain volume compared to occipitofrontal circumference, characteristic narrow sometimes 'slit-like' lateral ventricles and corpus callosum abnormalities. Slc4a10 -/- mice, deficient for SLC4A10, also display small lateral brain ventricles and mild behavioural abnormalities including delayed habituation and alterations in the two-object novel object recognition task. Collapsed brain ventricles in both Slc4a10-/- mice and affected individuals suggest an important role of SLC4A10 in the production of the CSF. However, it is notable that despite diverse roles of the CSF in the developing and adult brain, the cortex of Slc4a10-/- mice appears grossly intact. Co-staining with synaptic markers revealed that in neurons, SLC4A10 localizes to inhibitory, but not excitatory, presynapses. These findings are supported by our functional studies, which show the release of the inhibitory neurotransmitter GABA is compromised in Slc4a10-/- mice, while the release of the excitatory neurotransmitter glutamate is preserved. Manipulation of intracellular pH partially rescues GABA release. Together our studies define a novel neurodevelopmental disorder associated with biallelic pathogenic variants in SLC4A10 and highlight the importance of further analyses of the consequences of SLC4A10 loss-of-function for brain development, synaptic transmission and network properties.

Acid-adapted cancer cells alkalinize their cytoplasm by degrading the acid-loading membrane transporter anion exchanger 2, SLC4A2.

Acidic environments reduce the intracellular pH (pHi) of most cells to levels that are sub-optimal for growth and cellular functions. Yet, cancers maintain an alkaline cytoplasm despite low extracellular pH (pHe). Raised pHi is thought to be beneficial for tumor progression and invasiveness. However, the transport mechanisms underpinning this adaptation have not been studied systematically. Here, we characterize the pHe-pHi relationship in 66 colorectal cancer cell lines and identify the acid-loading anion exchanger 2 (AE2, SLC4A2) as a regulator of resting pHi. Cells adapt to chronic extracellular acidosis by degrading AE2 protein, which raises pHi and reduces acid sensitivity of growth. Acidity inhibits mTOR signaling, which stimulates lysosomal function and AE2 degradation, a process reversed by bafilomycin A1. We identify AE2 degradation as a mechanism for maintaining a conducive pHi in tumors. As an adaptive mechanism, inhibiting lysosomal degradation of AE2 is a potential therapeutic target.

Structural and functional properties of the transporter SLC26A6 reveal mechanism of coupled anion exchange.

Members of the SLC26 family constitute a conserved class of anion transport proteins, which encompasses uncoupled transporters with channel-like properties, coupled exchangers and motor proteins. Among the 10 functional paralogs in humans, several participate in the secretion of bicarbonate in exchange with chloride and thus play an important role in maintaining pH homeostasis. Previously, we have elucidated the structure of murine SLC26A9 and defined its function as an uncoupled chloride transporter (Walter et al., 2019). Here we have determined the structure of the closely related human transporter SLC26A6 and characterized it as a coupled exchanger of chloride with bicarbonate and presumably also oxalate. The structure defines an inward-facing conformation of the protein that generally resembles known structures of SLC26A9. The altered anion selectivity between both paralogs is a consequence of a remodeled ion binding site located in the center of a mobile unit of the membrane-inserted domain, which also accounts for differences in the coupling mechanism.

SLC26A3 (DRA) is stimulated in a synergistic, intracellular Ca2+-dependent manner by cAMP and ATP in intestinal epithelial cells.

In polarized intestinal epithelial cells, downregulated in adenoma (DRA) is an apical Cl-/[Formula: see text] exchanger that is part of neutral NaCl absorption under baseline conditions, but in cyclic adenosine monophosphate (cAMP)-driven diarrheas, it is stimulated and contributes to increased anion secretion. To further understand the regulation of DRA in conditions mimicking some diarrheal diseases, Caco-2/BBE cells were exposed to forskolin (FSK) and adenosine 5'-triphosphate (ATP). FSK and ATP stimulated DRA in a concentration-dependent manner, with ATP acting via P2Y1 receptors. FSK at 1 µM and ATP at 0.25 µM had minimal to no effect on DRA given individually; however, together, they stimulated DRA to levels seen with maximum concentrations of FSK and ATP alone. In Caco-2/BBE cells expressing the Ca2+ indicator GCaMP6s, ATP increased intracellular Ca2+ (Ca2+i) in a concentration-dependent manner, whereas FSK (1 µM), which by itself did not significantly alter Ca2+i, followed by 0.25 µM ATP produced a large increase in Ca2+ that was approximately equal to the elevation caused by 1 µM ATP. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) pretreatment prevented the ATP and FSK/ATP synergistically increased the DRA activity and the increase in Ca2+i caused by FSK/ATP. FSK/ATP synergistic stimulation of DRA was similarly observed in human colonoids. In Caco-2/BBE cells, subthreshold concentrations of FSK (cAMP) and ATP (Ca2+) synergistically increased Ca2+i and stimulated DRA activity with both being blocked by BAPTA-AM pretreatment. Diarrheal diseases, such as bile acid diarrhea, in which both cAMP and Ca2+ are elevated, are likely to be associated with stimulated DRA activity contributing to increased anion secretion, whereas separation of DRA from Na+/H+ exchanger isoform-3 (NHE3) contributes to reduced NaCl absorption.NEW & NOTEWORTHY The BB Cl-/[Formula: see text] exchanger DRA takes part in both neutral NaCl absorption and stimulated anion secretion. Using intestinal cell line, Caco-2/BBE high concentrations of cAMP and Ca2+ individually stimulated DRA activity, whereas low concentrations, which had no/minimal effect, synergistically stimulated DRA activity that required a synergistic increase in intracellular Ca2+. This study increases understanding of diarrheal diseases, such as bile salt diarrhea, in which both cAMP and elevated Ca2+ are involved.

The Air-Liquid Interface Reorganizes Membrane Lipids and Enhances the Recruitment of Slc26a3 to Lipid-Rich Domains in Human Colonoid Monolayers.

Cholesterol-rich membrane domains, also called lipid rafts (LRs), are specialized membrane domains that provide a platform for intracellular signal transduction. Membrane proteins often cluster in LRs that further aggregate into larger platform-like structures that are enriched in ceramides and are called ceramide-rich platforms (CRPs). The role of CRPs in the regulation of intestinal epithelial functions remains unknown. Down-regulated in adenoma (DRA) is an intestinal Cl-/HCO3- antiporter that is enriched in LRs. However, little is known regarding the mechanisms involved in the regulation of DRA activity. The air-liquid interface (ALI) was created by removing apical media for a specified number of days; from 12-14 days post-confluency, Caco-2/BBe cells or a colonoid monolayer were grown as submerged cultures. Confocal imaging was used to examine the dimensions of membrane microdomains that contained DRA. DRA expression and activity were enhanced in Caco-2/BBe cells and human colonoids using an ALI culture method. ALI causes an increase in acid sphingomyelinase (ASMase) activity, an enzyme responsible for enhancing ceramide content in the plasma membrane. ALI cultures expressed a larger number of DRA-containing platforms with dimensions >2 µm compared to cells grown as submerged cultures. ASMase inhibitor, desipramine, disrupted CRPs and reduced the ALI-induced increase in DRA expression in the apical membrane. Exposing normal human colonoid monolayers to ALI increased the ASMase activity and enhanced the differentiation of colonoids along with basal and forskolin-stimulated DRA activities. ALI increases DRA activity and expression by increasing ASMase activity and platform formation in Caco-2/BBe cells and by enhancing the differentiation of colonoids.

SLC4A2, another gene involved in acid-base balancing machinery of osteoclasts, causes osteopetrosis.

SLC4A2 belongs to the Na+-independent solute carrier family 4 (SLC4) of anion exchangers, which regulate electroneutral exchange of Cl- for HCO3- and mediate intra- and extra-cellular pH, chloride concentration and cell volume. Slc4a2 also participates in gastric acid secretion, spermatogenesis and osteoclastogenesis. During osteoclast differentiation, Slc4a2 is exclusively expressed at the contra-lacunar membrane and is up-regulated with osteoclast maturation. Bi-allelic Slc4a2 loss-of-function mutations have been known to cause osteopetrosis in mice and cattle, but not in human. Recently, we have identified bi-allelic pathogenic variants in SLC4A2 in a patient affected by osteopetrosis with severe renal insufficiency, suggesting SLC4A2 deficiency causes a new type of autosomal recessive osteopetrosis (osteopetrosis, Ikegawa type). In this article, we review the advances in exploring the multiple functions of SLC4A2 with emphasis on its roles in osteoclast. Our review would contribute to understanding of the phenotypic spectrum and the pathomechanism of SLC4A2-associated osteopetrosis.

Vitamin D receptor involves in the protection of intestinal epithelial barrier function via up-regulating SLC26A3.

BACKGROUND: Vitamin D receptor (VDR) and SLC26A3 (DRA) have been identified as pivotal protective factors in maintaining gut homeostasis in IBD patients. However, the specific mechanism underlying the increased intestinal susceptibility to inflammation induced by the loss of VDR and whether DRA participates in the role of VDR regulating intestinal epithelial barrier function are undefined. AIM: The current study is undertaken to elucidate the regulatory effects of VDR on DRA and VDR prevents intestinal epithelial barrier dysfunction via up-regulating the expression of DRA. METHODS: WT and VDR-/- mice are used as models for intestinal epithelial response. Paracellular permeability is measured by TEER and FD-4 assays. Immunohistochemistry, immunofluorescence, qPCR and immunoblotting are performed to determine the effects of VDR and DRA on gut epithelial barrier function. RESULTS: VDR-/- mice exhibits significant hyperpermeability of intestine with greatly decreased levels of ZO-1 and Claudin1 proteins. DRA is located on the intestinal epithelial apical membrane and is tightly modulated by VDR in vivo and in vitro via activating ERK1/2 MAPK signaling pathway. Notably, the current study for the first time demonstrates that VDR maintains intestinal epithelial barrier integrity via up-regulating DRA expression and the lack of DRA induced by VDR knockdown leads to a more susceptive condition for intestine to DSS-induced colitis. CONCLUSION: Our study provides evidence and deep comprehension regarding the role of VDR in modulating DRA expression in gut homeostasis and makes novel contributions to better generally understanding the links between VDR, DRA and intestinal epithelial barrier function.

Secretin alleviates biliary and liver injury during late-stage primary biliary cholangitis via restoration of secretory processes.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. METHODS: At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. RESULTS: Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. CONCLUSION: Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. IMPACT AND IMPLICATIONS: Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

PRDX6: A protein bridging S-palmitoylation and diabetic neuropathy.

Diabetic neuropathy is regarded as one of the most debilitating outcomes of diabetes. It can affect both the peripheral and central nervous systems, leading to pain, decreased motility, cognitive decline, and dementia. S-palmitoylation is a reversible posttranslational lipid modification, and its dysregulation has been implicated in metabolic syndrome, cancers, neurological disorders, and infections. However, the role of S-palmitoylation in diabetic neuropathy remains unclear. Here we demonstrate a potential association between activating protein palmitoylation and diabetic neuropathy. We compared the proteomic data of lumbar dorsal root ganglia (DRG) of diabetes mice and palmitoylome profiling data of the HUVEC cell line. The mapping results identified peroxiredoxin-6 (PRDX6) as a novel target in diabetic neuropathy, whose biological mechanism was associated with S-palmitoylation. Bioinformatic prediction revealed that PRDX6 had two palmitoylation sites, Cys47 and Cys91. Immunofluorescence results indicated PRDX6 translocating between the cytoplasm and cell membrane. Protein function analysis proposed that increased palmitoylation could competitively inhibit the formation of disulfide-bond between Cys47 and Cys91 and change the spatial topology of PRDX6 protein. Cl-HCO3- anion exchanger 3 (AE3) was one of the AE family members, which was proved to express in DRG. AE3 activity evoked Cl- influx in neurons which was generally associated with increased excitability and susceptibility to pain. We demonstrated that the S-palmitoylation status of Cys47 could affect the interaction between PRDX6 and the C-terminal domain of AE3, thereby regulating the activity of AE3 anion exchanger enzyme in the nervous system. The results highlight a central role for PRDX6 palmitoylation in protection against diabetic neuropathy.

The iodide transporter Slc26a7 impacts thyroid function more strongly than Slc26a4 in mice.

SLC26A4 is a known iodide transporter, and is localized at the apical membrane of thyrocytes. Previously, we reported that SLC26A7 is also involved in iodide transport and that Slc26a7 is a novel causative gene for congenital hypothyroidism. However, its detailed role in vivo remains to be elucidated. We generated mice that were deficient in Slc26a7 and Slc26a4 to delineate differences and associations in their roles in iodide transport. Slc26a7-/- mice showed goitrous congenital hypothyroidism and mild growth failure on a normal diet. Slc26a7-/- mice with a low iodine environment showed marked growth failure. In contrast, Slc26a4-/- mice showed no growth failure and hypothyroidism in the same low iodine environment. Double-deficient mice showed more severe growth failure than Slc26a7-/- mice. RNA-seq analysis revealed that the number of differentially expressed genes (DEGs) in Slc26a7-/- mice was significantly higher than that in Slc26a4-/- mice. These indicate that SLC26A7 is more strongly involved in iodide transport and the maintenance of thyroid function than SLC26A4.

Diarrhoeal pathogenesis in Salmonella infection may result from an imbalance in intestinal epithelial differentiation through reduced Notch signalling.

Infections with non-typhoidal Salmonella spp. represent the most burdensome foodborne illnesses worldwide, yet despite their prevalence, the mechanism through which Salmonella elicits diarrhoea is not entirely known. Intestinal ion transporters play important roles in fluid and electrolyte homeostasis in the intestine. We have previously shown that infection with Salmonella caused decreased colonic expression of the chloride/bicarbonate exchanger SLC26A3 (down-regulated in adenoma; DRA) in a mouse model. In this study, we focused on the mechanism of DRA downregulation during Salmonella infection, by using murine epithelial enteroid-derived monolayers (EDMs). The decrease in DRA expression caused by infection was recapitulated in EDMs and accompanied by increased expression of Atonal Homolog 1 (ATOH1), the goblet cell marker Muc2 and the enteroendocrine cell marker ChgA. This suggested biased epithelial differentiation towards the secretory, rather than absorptive phenotype. In addition, the downstream Notch effector, Notch intracellular domain (NICD) and Hes1 were decreased following Salmonella infection. The relevance of Notch signalling was further investigated using a γ-secretase inhibitor, which recapitulated the downregulation in Hes1 and DRA as well as upregulation in ATOH1 and Muc2 seen following infection. Our findings suggest that Salmonella infection may result in a shift from absorptive to secretory cell types through Notch inhibition, which explains why there is a decreased capacity for absorption and ultimately the accumulation of diarrhoeal fluid. Our work also shows the value of EDMs as a model to investigate mechanisms that might be targeted for therapy of diarrhoea caused by Salmonella infection. KEY POINTS: Salmonella is a leading foodborne pathogen known to cause high-chloride-content diarrhoea. Salmonella infection of murine enteroid-derived monolayers decreased DRA expression. Salmonella infection resulted in upregulation of the secretory epithelial marker ATOH1, the goblet cell marker Muc2 and the enteroendocrine cell marker ChgA. Downregulation of DRA may result from infection-induced Notch inhibition, as reflected by decreased expression of Notch intracellular domain and Hes1, as well as from decreased HNF1α signalling. The imbalance in intestinal epithelial differentiation favouring secretory over absorptive cell types is a possible mechanism by which Salmonella elicits diarrhoea and may be relevant therapeutically.

SLC4A2 Deficiency Causes a New Type of Osteopetrosis.

Osteopetrosis is a group of rare inherited skeletal disorders characterized by a marked increase in bone density due to deficient bone resorption. Pathogenic variants in several genes involved in osteoclast differentiation and/or function have been reported to cause osteopetrosis. Solute carrier family 4 member 2 (SLC4A2, encoding anion exchanger 2) plays an important role in osteoclast differentiation and function by exchange of Cl- with HCO3- . Biallelic Slc4a2 loss-of-function mutations in mice and cattle lead to osteopetrosis with osteoclast deficiency; however, pathogenic SLC4A2 variants in humans have not been reported. In this study, we describe a patient with autosomal recessive osteopetrosis due to biallelic pathogenic variants in SLC4A2. We identified novel compound heterozygous variants in SLC4A2 (NM_003040.4: c.556G>A [p.A186T] and c.1658T>C [p.V553A]) by exome sequencing. The measurement of intracellular Cl- showed that the variants decrease the anion exchange activity of SLC4A2. The impact of the variants on osteoclast differentiation was assessed by a gene knockout-rescue system using a mouse macrophage cell line, RAW 264.7. The Slc4a2-knockout cells show impaired osteoclastogenesis, which was rescued by the wild-type SLC4A2, but not by the mutant SLC4A2s. Immunofluorescence and pit assay revealed that the mutant SLC4A2s leads to abnormal podosome belt formation with impaired bone absorption. This is the first report on an individual affected by SLC4A2-associated osteopetrosis (osteopetrosis, Ikegawa type). With functional studies, we prove that the variants lead to SLC4A2 dysfunction, which altogether supports the importance of SLC4A2 in human osteoclast differentiation. © 2021 American Society for Bone and Mineral Research (ASBMR).

Alterations in SLC4A2, SLC26A7 and SLC26A9 Drive Acid-Base Imbalance in Gastric Neuroendocrine Tumors and Uncover a Novel Mechanism for a Co-Occurring Polyautoimmune Scenario.

Autoimmune polyendocrine syndrome (APS) is assumed to involve an immune system malfunction and entails several autoimmune diseases co-occurring in different tissues of the same patient; however, they are orphans of its accurate diagnosis, as its genetic basis and pathogenic mechanism are not understood. Our previous studies uncovered alterations in the ATPase H+/K+ Transporting Subunit Alpha (ATP4A) proton pump that triggered an internal cell acid-base imbalance, offering an autoimmune scenario for atrophic gastritis and gastric neuroendocrine tumors with secondary autoimmune pathologies. Here, we propose the genetic exploration of APS involving gastric disease to understand the underlying pathogenic mechanism of the polyautoimmune scenario. The whole exome sequencing (WES) study of five autoimmune thyrogastric families uncovered different pathogenic variants in SLC4A2, SLC26A7 and SLC26A9, which cotransport together with ATP4A. Exploratory in vitro studies suggested that the uncovered genes were involved in a pathogenic mechanism based on the alteration of the acid-base balance. Thus, we built a custom gene panel with 12 genes based on the suggested mechanism to evaluate a new series of 69 APS patients. In total, 64 filtered putatively damaging variants in the 12 genes of the panel were found in 54.17% of the studied patients and none of the healthy controls. Our studies reveal a constellation of solute carriers that co-express in the tissues affected with different autoimmune diseases, proposing a unique genetic origin for co-occurring pathologies. These results settle a new-fangled genetics-based mechanism for polyautoimmunity that explains not only gastric disease, but also thyrogastric pathology and disease co-occurrence in APS that are different from clinical incidental findings. This opens a new window leading to the prediction and diagnosis of co-occurring autoimmune diseases and clinical management of patients.

Mouse organoid culture is a suitable model to study esophageal ion transport mechanisms.

Altered esophageal ion transport mechanisms play a key role in inflammatory and cancerous diseases of the esophagus, but epithelial ion processes have been less studied in the esophagus because of the lack of a suitable experimental model. In this study, we generated three-dimensional (3D) esophageal organoids (EOs) from two different mouse strains and characterized the ion transport processes of the EOs. EOs form a cell-filled structure with a diameter of 250-300 µm and were generated from epithelial stem cells as shown by FACS analysis. Using conventional PCR and immunostaining, the presence of Slc26a6 Cl-/HCO3- anion exchanger (AE), Na+/H+ exchanger (NHE), Na+/HCO3- cotransporter (NBC), cystic fibrosis transmembrane conductance regulator (CFTR), and anoctamin 1 Cl- channels was detected in EOs. Microfluorimetric techniques revealed high NHE, AE, and NBC activities, whereas that of CFTR was relatively low. In addition, inhibition of CFTR led to functional interactions between the major acid-base transporters and CFTR. We conclude that EOs provide a relevant and suitable model system for studying the ion transport mechanisms of esophageal epithelial cells, and they can be also used as preclinical tools to assess the effectiveness of novel therapeutic compounds in esophageal diseases associated with altered ion transport processes.

Activation of the Ae4 (Slc4a9) cation-driven Cl-/HCO3- exchanger by the cAMP-dependent protein kinase in salivary gland acinar cells.

Ae4 transporters are critical for Cl- uptake across the basolateral membrane of acinar cells in the submandibular gland (SMG). Although required for fluid secretion, little is known about the physiological regulation of Ae4. To investigate whether Ae4 is regulated by the cAMP-dependent signaling pathway, we measured Cl-/HCO3- exchanger activity in SMG acinar cells from Ae2-/- mice, which only express Ae4, and found that the Ae4-mediated activity was increased in response to ß-adrenergic receptor stimulation. Moreover, pretreatment with H89, an inhibitor of the cAMP-activated kinase (PKA), prevented the stimulation of Ae4 exchangers. We then expressed Ae4 in CHO-K1 cells and found that the Ae4-mediated activity was increased when Ae4 is coexpressed with the catalytic subunit of PKA (PKAc), which is constitutively active. Ae4 sequence analysis showed two potential PKA phosphorylation serine residues located at the intracellular NH2-terminal domain according to a homology model of Ae4. NH2-terminal domain Ser residues were mutated to alanine (S173A and S273A, respectively), where the Cl-/HCO3- exchanger activity displayed by the mutant S173A was not activated by PKA. Conversely, S273A mutant kept the PKA dependency. Together, we conclude that Ae4 is stimulated by PKA in SMG acinar cells by a mechanism that probably depends on the phosphorylation of S173.NEW & NOTEWORTHY We found that Ae4 exchanger activity in secretory salivary gland acinar cells is increased upon ß-adrenergic receptor stimulation. The activation of Ae4 was prevented by H89, a nonselective PKA inhibitor. Protein sequence analysis revealed two residues (S173 and S273) that are potential targets of cAMP-dependent protein kinase (PKA). Experiments in CHO-K1 cells expressing S173A and S273A mutants showed that S173A, but not S273A, is not activated by PKA.