Biochemical characterization of two novel mutations in the human high-affinity choline transporter 1 identified in a patient with congenital myasthenic syndrome.

Congenital myasthenic syndrome (CMS) is a heterogeneous condition associated with 34 different genes, including SLC5A7, which encodes the high-affinity choline transporter 1 (CHT1). CHT1 is expressed in presynaptic neurons of the neuromuscular junction where it uses the inward sodium gradient to reuptake choline. Biallelic CHT1 mutations often lead to neonatal lethality, and less commonly to non-lethal motor weakness and developmental delays. Here, we report detailed biochemical characterization of two novel mutations in CHT1, p.I294T and p.D349N, which we identified in an 11-year-old patient with a history of neonatal respiratory distress, and subsequent hypotonia and global developmental delay. Heterologous expression of each CHT1 mutant in human embryonic kidney cells showed two different mechanisms of reduced protein function. The p.I294T CHT1 mutant transporter function was detectable, but its abundance and half-life were significantly reduced. In contrast, the p.D349N CHT1 mutant was abundantly expressed at the cell membrane, but transporter function was absent. The residual function of the p.I294T CHT1 mutant may explain the non-lethal form of CMS in this patient, and the divergent mechanisms of reduced CHT1 function that we identified may guide future functional studies of the CHT1 myasthenic syndrome. Based on these in vitro studies that provided a diagnosis, treatment with cholinesterase inhibitor together with physical and occupational therapy significantly improved the patient's strength and quality of life.

Unraveling the molecular landscape of kAE1: a narrative review.

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

Claudin-2 and claudin-12 form independent, complementary pores required to maintain calcium homeostasis.

Calcium (Ca2+) homeostasis is maintained through coordination between intestinal absorption, renal reabsorption, and bone remodeling. Intestinal and renal (re)absorption occurs via transcellular and paracellular pathways. The latter contributes the bulk of (re)absorption under conditions of adequate intake. Epithelial paracellular permeability is conferred by tight-junction proteins called claudins. However, the molecular identity of the paracellular Ca2+ pore remains to be delineated. Claudins (Cldn)-2 and -12 confer Ca2+ permeability, but deletion of either claudin does not result in a negative Ca2+ balance or increased calciotropic hormone levels, suggesting the existence of additional transport pathways or parallel roles for the two claudins. To test this, we generated a Cldn2/12 double knockout mouse (DKO). These animals have reduced intestinal Ca2+ absorption. Colonic Ca2+ permeability is also reduced in DKO mice and significantly lower than single-null animals, while small intestine Ca2+ permeability is unaltered. The DKO mice display significantly greater urinary Ca2+ wasting than Cldn2 null animals. These perturbations lead to hypocalcemia and reduced bone mineral density, which was not observed in single-KO animals. Both claudins were localized to colonic epithelial crypts and renal proximal tubule cells, but they do not physically interact in vitro. Overexpression of either claudin increased Ca2+ permeability in cell models with endogenous expression of the other claudin. We find claudin-2 and claudin-12 form partially redundant, independent Ca2+ permeable pores in renal and colonic epithelia that enable paracellular Ca2+ (re)absorption in these segments, with either one sufficient to maintain Ca2+ balance.

Urinary Tract Infections: Renal Intercalated Cells Protect against Pathogens.

Urinary tract infections affect more than 1 in 2 women during their lifetime. Among these, more than 10% of patients carry antibiotic-resistant bacterial strains, highlighting the urgent need to identify alternative treatments. While innate defense mechanisms are well-characterized in the lower urinary tract, it is becoming evident that the collecting duct (CD), the first renal segment encountered by invading uropathogenic bacteria, also contributes to bacterial clearance. However, the role of this segment is beginning to be understood. This review summarizes the current knowledge on CD intercalated cells in urinary tract bacterial clearance. Understanding the innate protective role of the uroepithelium and of the CD offers new opportunities for alternative therapeutic strategies.

Boosting endoplasmic reticulum folding capacity reduces unfolded protein response activation and intracellular accumulation of human kidney anion exchanger 1 in Saccharomyces cerevisiae.

Human kidney anion exchanger 1 (kAE1) facilitates simultaneous efflux of bicarbonate and absorption of chloride at the basolateral membrane of α-intercalated cells. In these cells, kAE1 contributes to systemic acid-base balance along with the proton pump v-H+ -ATPase and the cytosolic carbonic anhydrase II. Recent electron microscopy analyses in yeast demonstrate that heterologous expression of several kAE1 variants causes a massive accumulation of the anion transporter in intracellular membrane structures. Here, we examined the origin of these kAE1 aggregations in more detail. Using various biochemical techniques and advanced light and electron microscopy, we showed that accumulation of kAE1 mainly occurs in endoplasmic reticulum (ER) membranes which eventually leads to strong unfolded protein response (UPR) activation and severe growth defect in kAE1 expressing yeast cells. Furthermore, our data indicate that UPR activation is dose dependent and uncoupled from the bicarbonate transport activity. By using truncated kAE1 variants, we identified the C-terminal region of kAE1 as crucial factor for the increased ER stress level. Finally, a redistribution of ER-localized kAE1 to the cell periphery was achieved by boosting the ER folding capacity. Our findings not only demonstrate a promising strategy for preventing intracellular kAE1 accumulation and improving kAE1 plasma membrane targeting but also highlight the versatility of yeast as model to investigate kAE1-related research questions including the analysis of structural features, protein degradation and trafficking. Furthermore, our approach might be a promising strategy for future analyses to further optimize the cell surface targeting of other disease-related PM proteins, not only in yeast but also in mammalian cells.

Claudin-12 Knockout Mice Demonstrate Reduced Proximal Tubule Calcium Permeability.

The renal proximal tubule (PT) is responsible for the reabsorption of approximately 65% of filtered calcium, primarily via a paracellular pathway. However, which protein(s) contribute this paracellular calcium pore is not known. The claudin family of tight junction proteins confers permeability properties to an epithelium. Claudin-12 is expressed in the kidney and when overexpressed in cell culture contributes paracellular calcium permeability (PCa). We therefore examined claudin-12 renal localization and its contribution to tubular paracellular calcium permeability. Claudin-12 null mice (KO) were generated by replacing the single coding exon with ß-galactosidase from Escherichia coli. X-gal staining revealed that claudin-12 promoter activity colocalized with aquaporin-1, consistent with the expression in the PT. PTs were microperfused ex vivo and PCa was measured. PCa in PTs from KO mice was significantly reduced compared with WT mice. However, urinary calcium excretion was not different between genotypes, including those on different calcium containing diets. To assess downstream compensation, we examined renal mRNA expression. Claudin-14 expression, a blocker of PCa in the thick ascending limb (TAL), was reduced in the kidney of KO animals. Thus, claudin-12 is expressed in the PT, where it confers paracellular calcium permeability. In the absence of claudin-12, reduced claudin-14 expression in the TAL may compensate for reduced PT calcium reabsorption.

The novel p.Ser263Phe mutation in the human high-affinity choline transporter 1 (CHT1/SLC5A7) causes a lethal form of fetal akinesia syndrome.

A subset of a larger and heterogeneous class of disorders, the congenital myasthenic syndromes (CMS) are caused by pathogenic variants in genes encoding proteins that support the integrity and function of the neuromuscular junction (NMJ). A central component of the NMJ is the sodium-dependent high-affinity choline transporter 1 (CHT1), a solute carrier protein (gene symbol SLC5A7), responsible for the reuptake of choline into nerve termini has recently been implicated as one of several autosomal recessive causes of CMS. We report the identification and functional characterization of a novel pathogenic variant in SLC5A7, c.788C>T (p.Ser263Phe) in an El Salvadorian family with a lethal form of a congenital myasthenic syndrome characterized by fetal akinesia. This study expands the clinical phenotype and insight into a form of fetal akinesia related to CHT1 defects and proposes a genotype-phenotype correlation for the lethal form of SLC5A7-related disorder with potential implications for genetic counseling.

Renal collecting duct physiology and pathophysiology 1.

In the kidney, the collecting duct (CD) is composed of at least four cell types: principal, type-A intercalated cells (IC), type-B IC, and non-A and non-B IC. Although this heterogeneous composition has been recognized since the end of the nineteenth century, the physiological role of the various cell types in the CD continues to be deciphered as of today. Principal and ICs are essential in ion-water balance and acid-base homeostasis, respectively. However, recent research has revealed a striking interplay and overlap between the specific functions of these cell types. This review summarizes the recent findings on CD cells and their role in multiple pathophysiologies.

Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.

Adaptor protein 1 B mu subunit does not contribute to the recycling of kAE1 protein in polarized renal epithelial cells.

Mutations in the gene encoding the kidney anion exchanger 1 (kAE1) can lead to distal renal tubular acidosis (dRTA). dRTA mutations reported within the carboxyl (C)-terminal tail of kAE1 result in apical mis-targeting of the exchanger in polarized renal epithelial cells. As kAE1 physically interacts with the µ subunit of epithelial adaptor protein 1 B (AP-1B), we investigated the role of heterologously expressed µ1B subunit of the AP-1B complex for kAE1 retention to the basolateral membrane in polarized porcine LLC-PK1 renal epithelial cells that are devoid of endogenous AP-1B. We confirmed the interaction and close proximity between kAE1 and µ1B using immunoprecipitation and proximity ligation assay, respectively. Expressing the human µ1B subunit in these cells decreased significantly the amount of cell surface kAE1 at the steady state, but had no significant effect on kAE1 recycling and endocytosis. We show that (i) heterologous expression of µ1B displaces the physical interaction of endogenous GAPDH with kAE1 WT supporting that both AP-1B and GAPDH proteins bind to an overlapping site on kAE1 and (ii) phosphorylation of tyrosine 904 within the potential YDEV interaction motif does not alter the kAE1/AP-1B interaction. We conclude that µ1B subunit is not involved in recycling of kAE1.

Intercalated Cell Depletion and Vacuolar H+-ATPase Mistargeting in an Ae1 R607H Knockin Model.

Distal nephron acid secretion is mediated by highly specialized type A intercalated cells (A-ICs), which contain vacuolar H+-ATPase (V-type ATPase)-rich vesicles that fuse with the apical plasma membrane on demand. Intracellular bicarbonate generated by luminal H+ secretion is removed by the basolateral anion-exchanger AE1. Chronically reduced renal acid excretion in distal renal tubular acidosis (dRTA) may lead to nephrocalcinosis and renal failure. Studies in MDCK monolayers led to the proposal of a dominant-negative trafficking mechanism to explain AE1-associated dominant dRTA. To test this hypothesis in vivo, we generated an Ae1 R607H knockin mouse, which corresponds to the most common dominant dRTA mutation in human AE1, R589H. Compared with wild-type mice, heterozygous and homozygous R607H knockin mice displayed incomplete dRTA characterized by compensatory upregulation of the Na+/HCO3- cotransporter NBCn1. Red blood cell Ae1-mediated anion-exchange activity and surface polypeptide expression did not change. Mutant mice expressed far less Ae1 in A-ICs, but basolateral targeting of the mutant protein was preserved. Notably, mutant mice also exhibited reduced expression of V-type ATPase and compromised targeting of this proton pump to the plasma membrane upon acid challenge. Accumulation of p62- and ubiquitin-positive material in A-ICs of knockin mice suggested a defect in the degradative pathway, which may explain the observed loss of A-ICs. R607H knockin did not affect type B intercalated cells. We propose that reduced basolateral anion-exchange activity in A-ICs inhibits trafficking and regulation of V-type ATPase, compromising luminal H+ secretion and possibly lysosomal acidification.

A variant in a cis-regulatory element enhances claudin-14 expression and is associated with pediatric-onset hypercalciuria and kidney stones.

The greatest risk factor for kidney stones is hypercalciuria, the etiology of which is largely unknown. A recent genome-wide association study (GWAS) linked hypercalciuria and kidney stones to a claudin-14 (CLDN14) risk haplotype. However, the underlying molecular mechanism was not delineated. Recently, renal CLDN14 expression was found to increase in response to increased plasma calcium, thereby inducing calciuria. We hypothesized therefore that some children with hypercalciuria and kidney stones harbor a CLDN14 variant that inappropriately increases gene expression. To test this hypothesis, we sequenced the CLDN14 risk haplotype in a cohort of children with idiopathic hypercalciuria and kidney stones. An intronic SNP was more frequent in affected children. Dual luciferase and cell-based assays demonstrated increased reporter or CLDN14 expression when this polymorphism was introduced. In silico studies predicted the SNP introduced a novel insulinoma-associated 1 (INSM1) transcription factor binding site. Consistent with this, repeating the dual luciferase assay in the presence of INSM1 further increased reporter expression. Our data suggest that children with the INSM1 binding site within the CLDN14 risk haplotype have a higher likelihood of hypercalciuria and kidney stones. Enhanced CLDN14 expression may play a role in the pathophysiology of their hypercalciuria.

Acidosis and Urinary Calcium Excretion: Insights from Genetic Disorders.

Metabolic acidosis is associated with increased urinary calcium excretion and related sequelae, including nephrocalcinosis and nephrolithiasis. The increased urinary calcium excretion induced by metabolic acidosis predominantly results from increased mobilization of calcium out of bone and inhibition of calcium transport processes within the renal tubule. The mechanisms whereby acid alters the integrity and stability of bone have been examined extensively in the published literature. Here, after briefly reviewing this literature, we consider the effects of acid on calcium transport in the renal tubule and then discuss why not all gene defects that cause renal tubular acidosis are associated with hypercalciuria and nephrocalcinosis.

Far Upstream Element-Binding Protein 1 Binds the 3' Untranslated Region of PKD2 and Suppresses Its Translation.

Autosomal dominant polycystic kidney disease pathogenesis can be recapitulated in animal models by gene mutations in or dosage alterations of polycystic kidney disease 1 (PKD1) or PKD2, demonstrating that too much and too little PKD1/PKD2 are both pathogenic. Gene dosage manipulation has become an appealing approach by which to compensate for loss or gain of gene function, but the mechanisms controlling PKD2 expression remain incompletely characterized. In this study, using cultured mammalian cells and dual-luciferase assays, we found that the 3' untranslated region (3'UTR) of PKD2 mRNA inhibits luciferase protein expression. We then identified nucleotides 691-1044, which we called 3FI, as the 3'UTR fragment necessary for repressing the expression of luciferase or PKD2 in this system. Using a pull-down assay and mass spectrometry we identified far upstream element-binding protein 1 (FUBP1) as a 3FI-binding protein. In vitro overexpression of FUBP1 inhibited the expression of PKD2 protein but not mRNA. In embryonic zebrafish, FUBP1 knockdown (KD) by morpholino injection increased PKD2 expression and alleviated fish tail curling caused by morpholino-mediated KD of PKD2. Conversely, FUBP1 overexpression by mRNA injection significantly increased pronephric cyst occurrence and tail curling in zebrafish embryos. Furthermore, FUBP1 binds directly to eukaryotic translation initiation factor 4E-binding protein 1, indicating a link to the translation initiation complex. These results show that FUBP1 binds 3FI in the PKD2 3'UTR to inhibit PKD2 translation, regulating zebrafish disease phenotypes associated with PKD2 KD.

The carboxyl-terminally truncated kidney anion exchanger 1 R901X dRTA mutant is unstable at the plasma membrane.

Mutations in the SLC4A1 gene coding for kidney anion exchanger 1 (kAE1) cause distal renal tubular acidosis (dRTA). We investigated the fate of the most common truncated dominant dRTA mutant kAE1 R901X. In renal epithelial cells, we found that kAE1 R901X is less abundant than kAE1 wild-type (WT) at the plasma membrane. Although kAE1 WT and kAE1 R901X have similar half-lives, the decreased abundance of kAE1 R901X at the surface is due to an increased endocytosis rate and a decreased recycling rate of endocytosed proteins. We propose that, in polarized renal epithelial cells, the apically mistargeted kAE1 R901X mutant is endocytosed faster than kAE1 WT and its recycling to the basolateral membrane is delayed. This resets the equilibrium, such that kAE1 R901X resides predominantly in an endomembrane compartment, thereby likely participating in development of dRTA disease.

Carbonic anhydrase II binds to and increases the activity of the epithelial sodium-proton exchanger, NHE3.

Two-thirds of sodium filtered by the renal glomerulus is reabsorbed from the proximal tubule via a sodium/proton exchanger isoform 3 (NHE3)-dependent mechanism. Since sodium and bicarbonate reabsorption are coupled, we postulated that the molecules involved in their reabsorption [NHE3 and carbonic anhydrase II (CAII)] might physically and functionally interact. Consistent with this, CAII and NHE3 were closely associated in a renal proximal tubular cell culture model as revealed by a proximity ligation assay. Direct physical interaction was confirmed in solid-phase binding assays with immobilized CAII and C-terminal NHE3 glutathione-S-transferase fusion constructs. To assess the effect of CAII on NHE3 function, we expressed NHE3 in a proximal tubule cell line and measured NHE3 activity as the rate of intracellular pH recovery, following an acid load. NHE3-expressing cells had a significantly greater rate of intracellular pH recovery than controls. Inhibition of endogenous CAII activity with acetazolamide significantly decreased NHE3 activity, indicating that CAII activates NHE3. To ascertain whether CAII binding per se activates NHE3, we expressed NHE3 with wild-type CAII, a catalytically inactive CAII mutant (CAII-V143Y), or a mutant unable to bind other transporters (CAII-HEX). NHE3 activity increased upon wild-type CAII coexpression, but not in the presence of the CAII V143Y or HEX mutant. Together these studies support an association between CAII and NHE3 that alters the transporter's activity.

Degradation mechanism of a Golgi-retained distal renal tubular acidosis mutant of the kidney anion exchanger 1 in renal cells.

Distal renal tubular acidosis (dRTA) can be caused by mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1). Both recessive and dominant mutations result in mistrafficking of proteins, preventing them from reaching the basolateral membrane of renal epithelial cells, where their function is needed. In this study, we show that two dRTA mutants are prematurely degraded. Therefore, we investigated the degradation pathway of the kidney AE1 G701D mutant that is retained in the Golgi. Little is known about degradation of nonnative membrane proteins from the Golgi compartments in mammalian cells. We show that the kidney AE1 G701D mutant is polyubiquitylated and degraded by the lysosome and the proteosome. This mutant reaches the plasma membrane, where it is endocytosed and degraded by the lysosome via a mechanism dependent on the peripheral quality control machinery. Furthermore, we show that the function of the mutant is rescued at the cell surface upon inhibition of the lysosome and incubation with a chemical chaperone. We conclude that modulating the peripheral quality control machinery may provide a novel therapeutic option for treatment of patients with dRTA due to a Golgi-retained mutant.

Structure, function, and trafficking of SLC4 and SLC26 anion transporters.

The structure and function of the red cell anion exchanger 1 (AE1, Band 3, SLC4A1), the truncated kidney anion exchanger 1 (kAE1), and the other members of the SLC4 family of bicarbonate transporters are reviewed. Mutations in the AE1 gene cause human diseases like Southeast Asian ovalocytosis and hereditary spherocytosis in the red cell and distal renal tubular acidosis in the kidney. These mutations affect the folding, trafficking, and functional expression of these membrane glycoproteins. In the SLC26 family of anion transporters, mutations also cause trafficking defects and human disease. Membrane glycoproteins are cotranslationally N-glycosylated in the endoplasmic reticulum (ER) and when properly folded, traffic via the secretory pathway to their final destination such as the plasma membrane. Misfolded glycoproteins are retained in ER and are targeted for degradation by the proteasome following retrotranslocation and ubiquitinylation. ER chaperones, like membrane-bound calnexin, interact transiently with glycoproteins and are part of the quality control system that monitors the folding of glycoproteins during their biosynthesis. Recent results have indicated that it is possible to "correct" trafficking defects caused by some mutations in the SLC4 and 26 families through the use of small molecules that interfere with the interaction of glycoproteins with the components of the quality control system. This review summarizes the current knowledge on structure and function of anion transporters from the SLC4 and SLC26 families, and the effect of mutations on their trafficking and functional expression.

Proximal tubular NHEs: sodium, protons and calcium?

Na⁺/H⁺ exchange activity in the apical membrane of the proximal tubule is fundamental to the reabsorption of Na⁺ and water from the filtrate. The role of this exchange process in bicarbonate reclamation and, consequently, the maintenance of acid-base homeostasis has been appreciated for at least half a century and remains a pillar of renal tubular physiology. More recently, apical Na⁺/H⁺ exchange, mediated by Na⁺/H⁺ exchanger isoform 3 (NHE3), has been implicated in proximal tubular reabsorption of Ca²âº and Ca²âº homeostasis in general. Overexpression of NHE3 increased paracellular Ca²âº flux in a proximal tubular cell model. Consistent with this observation, mice with genetic deletion of Nhe3 have a noticable renal Ca²âº leak. These mice also display decreased intestinal Ca²âº uptake and osteopenia. This review highlights the traditional roles of proximal tubular Na⁺/H⁺ exchange and summarizes recent novel findings implicating the predominant isoform, NHE3, in Ca²âº homeostasis.

The Na⁺/H⁺ exchanger isoform 3 is required for active paracellular and transcellular Ca²âº transport across murine cecum.

Intestinal calcium (Ca²âº) absorption occurs via paracellular and transcellular pathways. Although the transcellular route has been extensively studied, mechanisms mediating paracellular absorption are largely unexplored. Unlike passive diffusion, secondarily active paracellular Ca²âº uptake occurs against an electrochemical gradient with water flux providing the driving force. Water movement is dictated by concentration differences that are largely determined by Na⁺ fluxes. Consequently, we hypothesized that Na⁺ absorption mediates Ca²âº flux. NHE3 is central to intestinal Na⁺ absorption. NHE3 knockout mice (NHE3-/-) display impaired intestinal Na⁺, water, and Ca²âº absorption. However, the mechanism mediating this latter abnormality is not clear. To investigate this, we used Ussing chambers to measure net Ca²âº absorption across different segments of wild-type mouse intestine. The cecum was the only segment with net Ca²âº absorption. Quantitative RT-PCR measurements revealed cecal expression of all genes implicated in intestinal Ca²âº absorption, including NHE3. We therefore employed this segment for further studies. Inhibition of NHE3 with 100 µM 5-(N-ethyl-N-isopropyl) amiloride decreased luminal-to-serosal and increased serosal-to-luminal Ca²âº flux. NHE3-/- mice had a >60% decrease in luminal-to-serosal Ca²âº flux. Ussing chambers experiments under altered voltage clamps (-25, 0, +25 mV) showed decreased transcellular and secondarily active paracellular Ca²âº absorption in NHE3-/- mice relative to wild-type animals. Consistent with this, cecal Trpv6 expression was diminished in NHE3-/- mice. Together these results implicate NHE3 in intestinal Ca(2+) absorption and support the theory that this is, at least partially, due to the role of NHE3 in Na⁺ and water absorption.