The role of the STAS domain in SLC26A9 for chloride ion transporter function.

The anion exchanger solute carrier family 26 (SLC26)A9, consisting of the transmembrane (TM) domain and the cytoplasmic STAS domain, plays an essential role in regulating chloride transport across cell membranes. Recent studies have indicated that C-terminal helices block the entrance of the putative ion transport pathway. However, the precise functions of the STAS domain and C-terminal helix, as well as the underlying molecular mechanisms governing the transport process, remain poorly understood. In this study, we performed molecular dynamics simulations of three distinct models of human SLC26A9, full-length, STAS domain removal (ΔSTAS), and C-terminus removal (ΔC), to investigate their conformational dynamics and ion-binding properties. Stable binding of ions to the binding sites was exclusively observed in the ΔC model in these simulations. Comparing the full-length and ΔC simulations, the ΔC model displayed enhanced motion of the STAS domain. Furthermore, comparing the ΔSTAS and ΔC simulations, the ΔSTAS simulation failed to exhibit stable ion bindings to the sites despite the absence of the C-terminus blocking the ion transmission pathway in both systems. These results suggest that the removal of the C-terminus not only unblocks the access of ions to the permeation pathway but also triggers STAS domain motion, gating the TM domain to promote ions' entry into their binding site. Further analysis revealed that the asymmetric motion of the STAS domain leads to the expansion of the ion permeation pathway within the TM domain, resulting in the stiffening of the flexible TM12 helix near the ion-binding site. This structural change in the TM12 helix stabilizes chloride ion binding, which is essential for SLC26A9's alternate-access mechanism. Overall, our study provides new insights into the molecular mechanisms of SLC26A9 transport and may pave the way for the development of novel treatments for diseases associated with dysregulated ion transport.

The molecular principles underlying diverse functions of the SLC26 family of proteins.

Mammalian SLC26 proteins are membrane-based anion transporters that belong to the large SLC26/SulP family, and many of their variants are associated with hereditary diseases. Recent structural studies revealed a strikingly similar homodimeric molecular architecture for several SLC26 members, implying a shared molecular principle. Now a new question emerges as to how these structurally similar proteins execute diverse physiological functions. In this study, we sought to identify the common versus distinct molecular mechanism among the SLC26 proteins using both naturally occurring and artificial missense changes introduced to SLC26A4, SLC26A5, and SLC26A9. We found: (i) the basic residue at the anion binding site is essential for both anion antiport of SLC26A4 and motor functions of SLC26A5, and its conversion to a nonpolar residue is crucial but not sufficient for the fast uncoupled anion transport in SLC26A9; (ii) the conserved polar residues in the N- and C-terminal cytosolic domains are likely involved in dynamic hydrogen-bonding networks and are essential for anion antiport of SLC26A4 but not for motor (SLC26A5) and uncoupled anion transport (SLC26A9) functions; (iii) the hydrophobic interaction between each protomer's last transmembrane helices, TM14, is not of functional significance in SLC26A9 but crucial for the functions of SLC26A4 and SLC26A5, likely contributing to optimally orient the axis of the relative movements of the core domain with respect to the gate domains within the cell membrane. These findings advance our understanding of the molecular mechanisms underlying the diverse physiological roles of the SLC26 family of proteins.

Substrate binding plasticity revealed by Cryo-EM structures of SLC26A2.

SLC26A2 is a vital solute carrier responsible for transporting essential nutritional ions, including sulfate, within the human body. Pathogenic mutations within SLC26A2 give rise to a spectrum of human diseases, ranging from lethal to mild symptoms. The molecular details regarding the versatile substrate-transporter interactions and the impact of pathogenic mutations on SLC26A2 transporter function remain unclear. Here, using cryo-electron microscopy, we determine three high-resolution structures of SLC26A2 in complexes with different substrates. These structures unveil valuable insights, including the distinct features of the homodimer assembly, the dynamic nature of substrate binding, and the potential ramifications of pathogenic mutations. This structural-functional information regarding SLC26A2 will advance our understanding of cellular sulfate transport mechanisms and provide foundations for future therapeutic development against various human diseases.

Single particle cryo-EM structure of the outer hair cell motor protein prestin.

The mammalian outer hair cell (OHC) protein prestin (Slc26a5) differs from other Slc26 family members due to its unique piezoelectric-like property that drives OHC electromotility, the putative mechanism for cochlear amplification. Here, we use cryo-electron microscopy to determine prestin's structure at 3.6 Å resolution. Prestin is structurally similar to the anion transporter Slc26a9. It is captured in an inward-open state which may reflect prestin's contracted state. Two well-separated transmembrane (TM) domains and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains form a swapped dimer. The transmembrane domains consist of 14 transmembrane segments organized in two 7+7 inverted repeats, an architecture first observed in the bacterial symporter UraA. Mutation of prestin's chloride binding site removes salicylate competition with anions while retaining the prestin characteristic displacement currents (Nonlinear Capacitance), undermining the extrinsic voltage sensor hypothesis for prestin function.

Identification and Computational Analysis of Rare Variants of Known Hearing Loss Genes Present in Five Deaf Members of a Pakistani Kindred.

Hearing loss (HL) is the most common neurosensory defect in humans that affects the normal communication. Disease is clinically and genetically heterogeneous, rendering challenges for the molecular diagnosis of affected subjects. This study highlights the phenotypic and genetic complexity of inherited HL in a large consanguineous Pakistan kindred. Audiological evaluation of all affected individuals revealed varying degree of mild to profound sensorineural HL. Whole exome (WES) of four family members followed by Sanger sequencing revealed candidate disease-associated variants in five known deafness genes: GJB2 (c.231G>A; p.(Trp77 *)), SLC26A4 (c.1337A>G; p.(Gln446Arg)), CDH23 (c.2789C>T; p.(Pro930Leu)), KCNQ4 (c.1672G>A; p.(Val558Met)) and MPDZ (c.4124T>C; p.(Val1375Ala)). All identified variants replaced evolutionary conserved residues, were either absent or had low frequencies in the control databases. Our in silico and 3-Dimensional (3D) protein topology analyses support the damaging impact of identified variants on the encoded proteins. However, except for the previously established "pathogenic" and "likely pathogenic" categories for the c.231G>A (p.(Trp77 *)) allele of GJB2 and c.1377A>G (p.(Gln446Arg)) of SLC26A4, respectively, all the remaining identified variants were classified as "uncertain significance" based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant pathogenicity guidelines. Our study highlights the complexity of genetic traits in consanguineous families, and the need of combining the functional studies even with the comprehensive profiling of multiple family members to improve the genetic diagnosis in complex inbred families.

Insights into phenotypic differences between humans and mice with p.T721M and other C-terminal variants of the SLC26A4 gene.

Recessive variants of the SLC26A4 gene are an important cause of hereditary hearing impairment. Several transgenic mice with different Slc26a4 variants have been generated. However, none have recapitulated the auditory phenotypes in humans. Of the SLC26A4 variants identified thus far, the p.T721M variant is of interest, as it appears to confer a more severe pathogenicity than most of the other missense variants, but milder pathogenicity than non-sense and frameshift variants. Using a genotype-driven approach, we established a knock-in mouse model homozygous for p.T721M. To verify the pathogenicity of p.T721M, we generated mice with compound heterozygous variants by intercrossing Slc26a4+/T721M mice with Slc26a4919-2A>G/919-2A>G mice, which segregated the c.919-2A > G variant with abolished Slc26a4 function. We then performed serial audiological assessments, vestibular evaluations, and inner ear morphological studies. Surprisingly, both Slc26a4T721M/T721M and Slc26a4919-2A>G/T721M showed normal audiovestibular functions and inner ear morphology, indicating that p.T721M is non-pathogenic in mice and a single p.T721M allele is sufficient to maintain normal inner ear physiology. The evidence together with previous reports on mouse models with Slc26a4 p.C565Y and p.H723R variants, support our speculation that the absence of audiovestibular phenotypes in these mouse models could be attributed to different protein structures at the C-terminus of human and mouse pendrin.

Comparative Molecular Dynamics Investigation of the Electromotile Hearing Protein Prestin.

The mammalian protein prestin is expressed in the lateral membrane wall of the cochlear hair outer cells and is responsible for the electromotile response of the basolateral membrane, following hyperpolarisation or depolarisation of the cells. Its impairment marks the onset of severe diseases, like non-syndromic deafness. Several studies have pointed out possible key roles of residues located in the Transmembrane Domain (TMD) that differentiate mammalian prestins as incomplete transporters from the other proteins belonging to the same solute-carrier (SLC) superfamily, which are classified as complete transporters. Here, we exploit the homology of a prototypical incomplete transporter (rat prestin, rPres) and a complete transporter (zebrafish prestin, zPres) with target structures in the outward open and inward open conformations. The resulting models are then embedded in a model membrane and investigated via a rigorous molecular dynamics simulation protocol. The resulting trajectories are analyzed to obtain quantitative descriptors of the equilibration phase and to assess a structural comparison between proteins in different states, and between different proteins in the same state. Our study clearly identifies a network of key residues at the interface between the gate and the core domains of prestin that might be responsible for the conformational change observed in complete transporters and hindered in incomplete transporters. In addition, we study the pathway of Cl- ions in the presence of an applied electric field towards their putative binding site in the gate domain. Based on our simulations, we propose a tilt and shift mechanism of the helices surrounding the ion binding cavity as the working principle of the reported conformational changes in complete transporters.

Molecular mechanism of prestin electromotive signal amplification.

Hearing involves two fundamental processes: mechano-electrical transduction and signal amplification. Despite decades of studies, the molecular bases for both remain elusive. Here, we show how prestin, the electromotive molecule of outer hair cells (OHCs) that senses both voltage and membrane tension, mediates signal amplification by coupling conformational changes to alterations in membrane surface area. Cryoelectron microscopy (cryo-EM) structures of human prestin bound with chloride or salicylate at a common "anion site" adopt contracted or expanded states, respectively. Prestin is ensconced within a perimeter of well-ordered lipids, through which it induces dramatic deformation in the membrane and couples protein conformational changes to the bulk membrane. Together with computational studies, we illustrate how the anion site is allosterically coupled to changes in the transmembrane domain cross-sectional area and the surrounding membrane. These studies provide insight into OHC electromotility by providing a structure-based mechanism of the membrane motor prestin.

Structure and function of an Arabidopsis thaliana sulfate transporter.

Plant sulfate transporters (SULTR) mediate absorption and distribution of sulfate (SO42-) and are essential for plant growth; however, our understanding of their structures and functions remains inadequate. Here we present the structure of a SULTR from Arabidopsis thaliana, AtSULTR4;1, in complex with SO42- at an overall resolution of 2.8 Å. AtSULTR4;1 forms a homodimer and has a structural fold typical of the SLC26 family of anion transporters. The bound SO42- is coordinated by side-chain hydroxyls and backbone amides, and further stabilized electrostatically by the conserved Arg393 and two helix dipoles. Proton and SO42- are co-transported by AtSULTR4;1 and a proton gradient significantly enhances SO42- transport. Glu347, which is ~7 Å from the bound SO42-, is required for H+-driven transport. The cytosolic STAS domain interacts with transmembrane domains, and deletion of the STAS domain or mutations to the interface compromises dimer formation and reduces SO42- transport, suggesting a regulatory function of the STAS domain.

Calmodulin binds to the STAS domain of SLC26A5 prestin with a calcium-dependent, one-lobe, binding mode.

SLC26A5 transporter prestin is fundamental for the higher hearing sensitivity and frequency selectivity of mammals. Prestin is a voltage-dependent transporter found in the cochlear outer hair cells responsible for their electromotility. Intracellular chloride binding is considered essential for voltage sensitivity and electromotility. Prestin is composed by a transmembrane domain and by a cytosolic domain called STAS. There is evidence of a calcium/calmodulin regulation of prestin mediated by the STAS domain. Using different biophysical techniques, namely SEC, CD, ITC, MST, NMR and SAXS, here we demonstrate and characterize the direct interaction between calmodulin and prestin STAS. We show that the interaction is calcium-dependent and that involves residues at the N-terminal end of the "variable loop". This is an intrinsically disordered insertion typical of the STAS domains of the SLC26 family of transporters whose function is still unclear. We derive a low-resolution model of the STAS/CaM complex, where only one lobe of calmodulin is engaged in the interaction, and build a model for the entire dimeric prestin in complex with CaM, which can use the unoccupied lobe to interact with other regions of prestin or with other regulatory proteins. We show that also a non-mammalian STAS can interact with calmodulin via the variable loop. These data start to shed light on the regulatory role of the STAS variable loop of prestin.

Digenic inheritance of mutations in EPHA2 and SLC26A4 in Pendred syndrome.

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function.

IRE1α kinase-mediated unconventional protein secretion rescues misfolded CFTR and pendrin.

The most prevalent pathogenic mutations in the CFTR (ΔF508) and SLC26A4/pendrin (p.H723R), which cause cystic fibrosis and congenital hearing loss, respectively, evoke protein misfolding and subsequent defects in their cell surface trafficking. Here, we report that activation of the IRE1α kinase pathway can rescue the cell surface expression of ΔF508-CFTR and p.H723R-pendrin through a Golgi-independent unconventional protein secretion (UPS) route. In mammalian cells, inhibition of IRE1α kinase, but not inhibition of IRE1α endonuclease and the downstream effector XBP1, inhibited CFTR UPS. Treatment with the IRE1α kinase activator, (E)-2-(2-chlorostyryl)-3,5,6-trimethyl-pyrazine (CSTMP), rescued cell surface expression and functional activity of ΔF508-CFTR and p.H723R-pendrin. Treatment with a nontoxic dose of CSTMP to ΔF508-CFTR mice restored CFTR surface expression and CFTR-mediated anion transport in the mouse colon. These findings suggest that UPS activation via IRE1α kinase is a strategy to treat diseases caused by defective cell surface trafficking of membrane proteins, including ΔF508-CFTR and p.H723R-pendrin.

Computational analysis of functional single nucleotide polymorphisms associated with SLC26A4 gene.

Single Nucleotide Polymorphisms (SNPs) are the most common candidate mutations in human beings that play a vital role in the genetic basis of certain diseases. Previous studies revealed that Solute Carrier Family 26 Member 4 (SLC26A4) being an essential gene of the multi-faceted transporter family SLC26 facilitates reflexive movement of Iodide into follicular lumen through apical membrane of thyrocyte. SLC26A4 gene encodes Pendred protein, a membrane glycoprotein, highly hydrophobic in nature, present at the apical membrane of thyrocyte functioning as transporter of iodide for thyroid cells. A minor genetic variation in SLC26A4 can cause Pendred syndrome, a syndrome associated with thyroid glands and deafness. In this study, we performed in-silico analysis of 674 missense SNPs of SLC26A4 using different computational platforms. The bunch of tools including SNPNEXUS, SNAP-2, PhD-SNP, SNPs&GO, I-Mutant, ConSurf, and ModPred were used to predict 23 highly confident damaging and disease causing nsSNPs (G209V, G197R, L458P, S427P, Q101P, W472R, N392Y, V359E, R409C, Q235R, R409P, G139V, G497S, H723R, D87G, Y127H, F667C, G334A, G95R, S427C, R291W, Q383H and E384G) that could potentially alter the SLC26A4 gene. Moreover, protein structure prediction, protein-ligand docking and Molecular Dynamics simulation were performed to confirm the impact of two evident alterations (Y127H and G334A) on the protein structure and function.

Systematic quantification of the anion transport function of pendrin (SLC26A4) and its disease-associated variants.

Thanks to the advent of rapid DNA sequencing technology and its prevalence, many disease-associated genetic variants are rapidly identified in many genes from patient samples. However, the subsequent effort to experimentally validate and define their pathological roles is extremely slow. Consequently, the pathogenicity of most disease-associated genetic variants is solely speculated in silico, which is no longer deemed compelling. We developed an experimental approach to efficiently quantify the pathogenic effects of disease-associated genetic variants with a focus on SLC26A4, which is essential for normal inner ear function. Alterations of this gene are associated with both syndromic and nonsyndromic hereditary hearing loss with various degrees of severity. We established HEK293T-based stable cell lines that express pendrin missense variants in a doxycycline-dependent manner, and systematically determined their anion transport activities with high accuracy in a 96-well plate format using a high throughput plate reader. Our doxycycline dosage-dependent transport assay objectively distinguishes missense variants that indeed impair the function of pendrin from those that do not (functional variants). We also found that some of these putative missense variants disrupt normal messenger RNA splicing. Our comprehensive experimental approach helps determine the pathogenicity of each pendrin variant, which should guide future efforts to benefit patients.

Cryo-EM structures and functional characterization of murine Slc26a9 reveal mechanism of uncoupled chloride transport.

The epithelial anion transporter SLC26A9 contributes to airway surface hydration and gastric acid production. Colocalizing with CFTR, SLC26A9 has been proposed as a target for the treatment of cystic fibrosis. To provide molecular details of its transport mechanism, we present cryo-EM structures and a functional characterization of murine Slc26a9. These structures define the general architecture of eukaryotic SLC26 family members and reveal an unusual mode of oligomerization which relies predominantly on the cytosolic STAS domain. Our data illustrates conformational transitions of Slc26a9, supporting a rapid alternate-access mechanism which mediates uncoupled chloride transport with negligible bicarbonate or sulfate permeability. The characterization of structure-guided mutants illuminates the properties of the ion transport path, including a selective anion binding site located in the center of a mobile module within the transmembrane domain. This study thus provides a structural foundation for the understanding of the entire SLC26 family and potentially facilitates their therapeutic exploitation.

Structural basis for functional interactions in dimers of SLC26 transporters.

The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.

Deletion of exons 17 and 18 in prestin's STAS domain results in loss of function.

Cochlear outer hair cells (OHC) express the motor protein, prestin, which is required for sensitivity and frequency selectivity. Because our previous work showed that a calmodulin binding site (CBS) was located in prestin's C-terminal, specifically within the intrinsically disordered region, we sought to delete the IDR to study the functional significance of calcium-dependent, calmodulin binding on OHC function. Although the construct lacking the IDR (∆IDR prestin) demonstrated wildtype-like nonlinear capacitance (NLC) in HEK293T cells, the phenotype in ∆IDR prestin knockins (KI) was similar to that in prestin knockouts: thresholds were elevated, NLC was absent and OHCs were missing from basal regions of the cochlea. Although ∆IDR prestin mRNA was measured, no prestin protein was detected. At the mRNA level, both of prestin's exons 17 and 18 were entirely removed, rather than the smaller region encoding the IDR. Our hybrid exon that contained the targeted deletion (17-18 ∆IDR) failed to splice in vitro and prestin protein lacking exons 17 and 18 aggregated and failed to target the cell membrane. Hence, the absence of prestin protein in ∆IDR KI OHCs may be due to the unexpected splicing of the hybrid 17-18 ∆IDR exon followed by rapid degradation of nonfunctional prestin protein.

Modified U1 snRNA and antisense oligonucleotides rescue splice mutations in SLC26A4 that cause hereditary hearing loss.

One of most important factors for messenger RNA (mRNA) transcription is the spliceosomal component U1 small nuclear RNA (snRNA), which recognizes 5' splicing donor sites at specific regions in pre-mRNA. Mutations in these sites disrupt U1 snRNA binding and cause abnormal splicing. In this study, we investigated mutations at splice sites in SLC26A4 (HGNC 8818), one of the major causative genes of hearing loss, which may result in the synthesis of abnormal pendrin, the channel protein encoded by the gene. Seventeen SLC26A4 variants with mutations in the U1 snRNA binding sites were assessed by minigene splicing assays, and 11 were found to result in abnormal splicing. Interestingly, eight of the 11 pathogenic mutations were intronic, suggesting the importance of conserved sequences at the intronic splice site. The application of modified U1 snRNA effectively rescued the abnormal splicing for most of these mutations. Although three were cryptic mutations, they were rescued by cotransfection of modified U1 snRNA and modified antisense oligonucleotides. Our results demonstrate the important role of snRNA in SLC26A4 mutations, suggesting the therapeutic potential of modified U1 snRNA and antisense oligonucleotides for neutralizing the pathogenic effect of the splice-site mutations that may result in hearing loss.

Slc26 Family of Anion Transporters in the Gastrointestinal Tract: Expression, Function, Regulation, and Role in Disease.

SLC26 family members are multifunctional transporters of small anions, including Cl- , HCO3 - , sulfate, oxalate, and formate. Most SLC26 isoforms act as secondary (coupled) anion transporters, while others mediate uncoupled electrogenic transport resembling Cl- channels. Of the 11 described SLC26 isoforms, the SLC26A1,2,3,6,7,9,11 are expressed in the gastrointestinal tract, where they participate in salt and water transport, surface pH-microclimate regulation, affect the microbiome composition, the absorption, and secretion of oxalate and sulfate, and other functions that require further study. Several intestinal or extra-intestinal diseases are related to SLC26A mutations. Patients with congenital chloride diarrhea (CLD) suffer from Cl- -rich acidic diarrhea and systemic alkalosis due to SLC26A3 mutations. Patients with osteochondrodysplastic syndromes experience skeletal defects due to SLC26A2 mutations, resulting in defective sulfate absorption in enterocytes and sulfate uptake in chondrocytes. Because of functional interactions between SLC26 and other proteins, such as the Cl- channel CFTR, some of the intestinal cystic fibrosis manifestations may be attributed to impaired SLC26 isoform localization and function. The altered expression of SLC26 members due to inflammation or operative procedures have important consequences on intestinal transport and barrier function in common diseases as inflammatory bowel disease or bariatric surgery. The present review gives an overview on the current state of knowledge of the intestinally expressed SLC26A isoforms (SLC26A1,2,3,6,7,9,11) from the history of their functional identification, cloning and expression, the insights into their function, interaction partners and regulation gained in heterologous expression systems and Slc26a-deficient mice, to information about their transcriptional regulation and roles in gastrointestinal disease manifestations. © 2019 American Physiological Society. Compr Physiol 9:839-872, 2019.

Role of N-glycosylation in the expression of human SLC26A2 and A3 anion transport membrane glycoproteins 1.

The human solute carrier 26 (SLC26) gene family of anion transporters consists of 10 members (SLC26A1-A11, A10 being a pseudogene) that encode membrane glycoproteins with 14 transmembrane segments and a C-terminal cytoplasmic sulfate transporter anti-sigma antagonist domain. Thus far, mutations in eight members of the SLC26 family (A1-A6, A8, and A9) have been linked to diseases in humans. Our goal is to characterize the role of N-glycosylation and the effect of mutations in SLC26A2 and A3 proteins on their functional expression in transfected HEK-293 cells. We found that certain mutants were retained in the endoplamic reticulum via an interaction with the lectin chaperone calnexin. Some could escape protein quality control and traffic to the cell surface upon removal of the N-glycosylation sites. Furthermore, we found that loss of N-glycosylation reduced expression of SLC26A2 at the cell surface. Loss of N-glycosylation had no effect on the stability of SLC26A3, yet resulted in a profound decrease in transport activity. Thus, N-glycosylation plays three roles in the functional expression of SLC26 proteins: (1) to retain misfolded proteins in the endoplamic reticulum, (2) to stabilize the protein at the cell surface, and (3) to maintain the transport protein in a functional state.