SLC26 Family Of Anion Transporters Research Articles

Folding of prestin's anion-binding site and the mechanism of outer hair cell electromotility.

Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin's voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl- anion at a conserved binding site formed by the amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen-deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl- binding. This event shortens the TM3-TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin's mechanical expansion. We observe helix fraying at prestin's anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin's fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and help define prestin's unique voltage-sensing mechanism and electromotility.

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.

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.

Tumor necrosis factor-α acts reciprocally with solute carrier family 26, member 3, (downregulated-in-adenoma) and reduces its expression, leading to intestinal inflammation.

Solute carrier family 26, member 3 (Slc26a3), also termed downregulated-in-adenoma (DRA) is a member of the Slc26 family of anion transporters and is mutated in congenital chloride diarrhea. Our previous study demonstrated that DRA deficiency is associated with severely reduced colonic HCO3− secretion, a loss of colonic fluid absorption, a lack of a firmly adherent mucus layer and a severely reduced colonic mucosal resistance to dextran sodium sulfate (DSS) damage. However, the direct effect of mediators that trigger intestinal inflammatory factors on DRA has not been fully investigated. Tumor necrosis factor (TNF)-α is a central mediator of intestinal inflammation in inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease. However, to the best of our knowledge, whether TNF-α acts reciprocally with DRA leading to the development of gut inflammation in IBD has not been reported. The present study identified that the expression level of DRA was reduced in active UC patients and DSS-induced colitis mice with high expression levels of TNF-α identified in the peripheral blood serum. In addition, TNF-α may affect the expression level of DRA in human colonic Caco2BBE cells in a dose-dependent manner, including in DRA overexpressed Caco2BBE cells. Furthermore, knockdown of TNF-α in Caco2BBE cells led to a higher expression level of DRA and a markedly reduced secretion of TNF-α in the culture media. In addition, knockdown of DRA in Caco2BBE cells led to a higher secretion of TNF-α in the culture media compared with the control cells, which could be reversed by overexpression of DRA. Overall, these results indicate that TNF-α may act reciprocally with DRA, leading to the development of intestinal inflammation. Based on the pivotal position of TNF-α in IBD, DRA is hypothesized to have therapeutic potential against colitis serving as an important target.

Molecular analysis of human solute carrier SLC26 anion transporter disease-causing mutations using 3-dimensional homology modeling

The availability of the first crystal structure of a bacterial member (SLC26Dg) of the solute carrier SLC26 family of anion transporters has allowed us to create 3-dimensional models of all 10 human members (SLC26A1-A11, A10 being a pseudogene) of these membrane proteins using the Phyre2 bioinformatic tool. The homology modeling predicted that the SLC26 human proteins, like the SLC26Dg template, all consist of 14 transmembrane segments (TM) arranged in a 7+7 inverted topology with the amino-termini of two half-helices (TM3 and 10) facing each other in the centre of the protein to create the anion-binding site, linked to a C-terminal cytosolic sulfate transporter anti-sigma factor antagonist (STAS) domain. A plethora of human diseases are associated with mutations in the genes encoding human SLC26 transporters, including chondrodysplasias with varying severity in SLC26A2 (~50 mutations, 27 point mutations), congenital chloride-losing diarrhea in SLC26A3 (~70 mutations, 31 point mutations) and Pendred Syndrome or deafness autosomal recessive type 4 in SLC26A4 (~500 mutations, 203 point mutations). We have localized all of these point mutations in the 3-dimensional structures of the respective SLC26A2, A3 and A4 proteins and systematically analyzed their effect on protein structure. While most disease-causing mutations may cause folding defects resulting in impaired trafficking of these membrane glycoproteins from the endoplasmic reticulum to the cell surface – as demonstrated in a number of functional expression studies – the modeling also revealed that a number of pathogenic mutations are localized to the anion-binding site, which may directly affect transport function.

Chapter One - 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.

Determinants of coupled transport and uncoupled current by the electrogenic SLC26 transporters

Members of the SLC26 family of anion transporters mediate the transport of diverse molecules and can function as coupled transporters or as channels. A unique feature of two members of the family, Slc26a3 and Slc26a6, is that they can function as both coupled and uncoupled channel-like modes. To identify features that control these modes of transport we performed in silico modeling of Slc26a6, which revealed high fold similarity of the Slc26a6 and ClC transporters, despite their low sequence identity. Examining the predicted Slc26a6 fold identified a highly conserved Glu- (E357) with a predicted spatial orientation similar to that of ClCec E148, which determines coupled or uncoupled transport by ClCec. This raised the question of whether the conserved Glu- in Slc26a6(E357) and Slc26a3(E367) play a role in the two transport modes of these transporters. Reversing the Glu- charge in a3 and a6 to Lys resulted in the inhibition of all modes of transport. However, neutralizing the charge in Slc26a6(E357A) eliminated all forms of coupled transport and augmented the uncoupled channel-like current activity. The a3(E367A) mutation markedly reduced the coupled transport and converted the stoichiometry of the residual exchange from 2Cl-/1HCO3- to 1:1, while completely sparing the channel-like activity. These findings suggest that a similar structural motif may determine multiple functional modes of these transporters.

Expression and Characterization of E. coli YchM, a Bacterial Member of the SLC26 Family of Anion Transporters

The widely-expressed members of the SLC26 family of anion transporters play a crucial role in the transport of bicarbonate, chloride, sulfate and iodide in epithelial cells and mutations cause a number of serious human genetic disorders including cystic fibrosis, dystropic dysplasia, congenital chloride diarrhea and deafness. To elucidate the molecular structure of SLC26 transporters and their mechanism of action, the E. coli homologue, YchM transporter, was chosen for analysis. YchM consists of two functional domains: the N-terminal membrane domain responsible for transport activity, and the C-terminal STAS domain, whose crystal structure resembles SpoIIAA, the anti-sigma factor antagonist from Bacillus. YchM and its membrane domain with a C-terminal His-tag were expressed in various E. coli strains (Tuner, C41, C43) grown at 20°C and purified using Ni-NTA affinity chromatography and gel filtration. Stability tests were carried out with a range of detergents, with n-dodecyl-β-D-maltopyranoside (DDM) at pH 6.0, 100mM NaCl and 2% glycerol being optimal. The CD-spectrum of YchM revealed a high helical content, and the thermal titration profile revealed that YchM protein has 2-step titration curve at 40°C and 55°C. YchM was prone to aggregation while sparce-matrix screening of the membrane domain gave small crystals that diffracted to ∼12Å. Further refinement of the purification and crystallization conditions are underway. Supported by operating and training grants from CIHR.

Determinants of coupled transport and uncoupled current by the electrogenic SLC26 transporters

Members of the SLC26 family of anion transporters mediate the transport of diverse molecules ranging from halides to carboxylic acids and can function as coupled transporters or as channels. A unique feature of the two members of the family, Slc26a3 and Slc26a6, is that they can function as both obligate coupled and mediate an uncoupled current, in a channel-like mode, depending on the transported anion. To identify potential features that control the two modes of transport, we performed in silico modeling of Slc26a6, which suggested that the closest potential fold similarity of the Slc26a6 transmembrane domains is to the CLC transporters, despite their minimal sequence identity. Examining the predicted Slc26a6 fold identified a highly conserved glutamate (Glu−; Slc26a6(E357)) with the predicted spatial orientation similar to that of the CLC-ec1 E148, which determines coupled or uncoupled transport by CLC-ec1. This raised the question of whether the conserved Glu− in Slc26a6(E357) and Slc26a3(E367) have a role in the unique transport modes by these transporters. Reversing the Glu− charge in Slc26a3 and Slc26a6 resulted in the inhibition of all modes of transport. However, most notably, neutralizing the charge in Slc26a6(E357A) eliminated all forms of coupled transport without affecting the uncoupled current. The Slc26a3(E367A) mutation markedly reduced the coupled transport and converted the stoichiometry of the residual exchange from 2Cl−/1HCO3− to 1Cl−/1HCO3−, while completely sparing the current. These findings suggest the possibility that similar structural motif may determine multiple functional modes of these transporters.

Combinatorial Cysteine Mutagenesis Reveals a Critical Intramonomer Role for Cysteines in Prestin Voltage Sensing

Prestin is a member of the SLC26 family of anion transporters and is responsible for electromotility in outer hair cells, the basis of cochlear amplification in mammals. It is an anion transporting transmembrane protein, possessing nine cysteine residues, which generates voltage-dependent charge movement. We determine the role these cysteine residues play in the voltage sensing capabilities of prestin. Mutations of any single cysteine residue had little or no effect on charge movement. However, using combinatorial substitution mutants, we identified a cysteine residue pair (C415 and either C192 or C196) whose mutation reduced or eliminated charge movement. Furthermore, we show biochemically that surface expression of mutants with markedly reduced functionality can be near normal; however, we identify two monomers of the protein on the surface of the cell, the larger of which correlates with surface charge movement. Because we showed previously by Förster resonance energy transfer that monomer interactions are required for charge movement, we tested whether disulfide interactions were required for dimerization. Using Western blots to detect oligomerization of the protein in which variable numbers of cysteines up to and including all nine cysteine residues were mutated, we show that disulfide bond formation is not essential for dimer formation. Taken together, we believe these data indicate that intramembranous cysteines are constrained, possibly via disulfide bond formation, to ensure structural features of prestin required for normal voltage sensing and mechanical activity.

Prestin Surface Expression and Activity Are Augmented by Interaction with MAP1S, a Microtubule-associated Protein

Prestin is a member of the SLC26 family of anion transporters that is responsible for outer hair cell (OHC) electromotility. Measures of voltage-evoked charge density (Q(sp)) of prestin indicated that the protein is highly expressed in OHCs, with single cells expressing up to 10 million molecules within the lateral membrane. In contrast, charge density measures in transfected cells indicated that they express, at best, only a fifth as many proteins on their surface. We sought to determine whether associations with other OHC-specific proteins could account for this difference. Using a yeast two-hybrid technique, we found microtubule-associated protein 1S (MAP1S) bound to prestin. The interaction was limited to the STAS domain of prestin and the region connecting the heavy and light chain of MAP1S. Using reciprocal immunoprecipitation and Forster resonance energy transfer, we confirmed these interactions. Furthermore, co-expression of prestin with MAP1S resulted in a 2.7-fold increase in Q(sp) in single cells that was paralleled by a 2.8-fold increase in protein surface expression, indicating that the interactions are physiological. Quantitative PCR data showed gradients in the expression of prestin and MAP1S across the tonotopic axis that may partially contribute to a previously observed 6-fold increase in Q(sp) in high frequency hair cells. These data highlight the importance of protein partner effects on prestin.

AFM Images of Outer Hair Cells' Lateral Plasma Membrane: An Autocorrelation Function Analysis

Our atomic force microscopic study of the cytosolic surface of outer hair cells' lateral plasma revealed images of membrane particles with tip-geometry- corrected diameter of ∼10 nm [1], consistent with 10-nm particles reported by earlier EM studies. These particles were aligned preferentially in one direction and a much weaker alignment consistent with hexagonal packing. The immunoreactivity of these particles to prestin-antibody revealed that these particles involve prestin, a member of the SLC26 family of anion transporters associated with electromotility of outer hair cells. This observation together with reports that prestin forms tetramers consistent with the dimension of these 10-nm particles prompts a question: Are 10-nm particles tetramers of prestin? To address this question, we examined autocorrelation function of AFM images for the detailed structure of these particles. If the slice plain of the peak is adjusted to the dimension that matches the particles, the contours should reveal shapes of the particle. We found the contour at the corresponding height is approximately square, consistent with tetramer symmetry. However, the maximum width of the central peak corresponded to ∼8.2 nm, somewhat smaller than the size of the particles obtained by section analysis. This difference can be attributed to blurring effect of noise. In summary, our observation is consistent with a hypothesis that 10-nm particles are prestin tetramers. [1] Organization of membrane motor in outer hair cells: an atomic force microscopic study, G. Sinha, F. Sabri, E. Dimitriadis, K. Iwasa, Pfugers Archiv European J. of Physiology, 2009.

SLC26A9 is a Cl(-) channel regulated by the WNK kinases.

SLC26A9 is a member of the SLC26 family of anion transporters, which is expressed at high levels in airway and gastric surface epithelial cells. The transport properties and regulation of SLC26A9, and thus its physiological function, are not known. Here we report that SLC26A9 is a highly selective Cl(-) channel with minimal OH(-)/HCO(3)(-) permeability that is regulated by the WNK kinases. Expression in Xenopus oocytes and simultaneous measurement of membrane potential or current, intracellular pH (pH(i)) and intracellular Cl(-) (Cl(-)(i)) revealed that expression of SLC26A9 resulted in a large Cl(-) current. SLC26A9 displays a selectivity sequence of I(-) > Br(-) > NO(3)(-) > Cl(-) > Glu(-), but it conducts Br(-) > Cl(-) > I(-) > NO(3)(-) > Glu(-), with NO(3)(-) and I(-) inhibiting the Cl(-) conductance. Similarly, expression of SLC26A9 in HEK cells resulted in a large Cl(-) current. Although detectable, OH(-) and HCO(3)(-) fluxes in oocytes expressing SLC26A9 were very small. Moreover, HCO(3)(-) had no discernable effect on the Cl(-) current, the reversal potential in the presence or absence of Cl(-)(o) and, importantly, HCO(3)(-) had no effect on Cl(-) fluxes. These findings indicate that SLC26A9 is a Cl(-) channel with minimal OH(-)/HCO(3)(-) permeability. Co-expression of SLC26A9 with the WNK kinases WNK1, WNK3 or WNK4 inhibited SLC26A9 activity, and the inhibition was independent of WNK kinase activity. Immunolocalization in oocytes and cell surface biotinylation in HEK cells indicated that the WNK-mediated inhibition of SLC26A9 activity is caused by reduced SLC26A9 surface expression. Expression of SLC26A9 in the airway and the response of the WNKs to homeostatic stress raise the possibility that SLC26A9 serves to mediate the response of the airway to stress.

Effects of Chlorpromazine and Trinitrophenol on the Membrane Motor of Outer Hair Cells

The motile activity of outer hair cells’ cell body is associated with large nonlinear capacitance due to a membrane motor that couples electric displacement with changes in the membrane area, analogous to piezoelectricity. This motor is based on prestin, a member of the SLC26 family of anion transporters and utilizes the electric energy available at the plasma membrane associated with the sensory function of these cells. To understand detailed mechanism of this motile activity, we examined the effect of amphipathic ions, cationic chlorpromazine and anionic trinitrophenol, which are thought to change the curvature of the membrane in opposite directions. We found that both chemicals reduced cell length at the holding potential of −75 mV and induced positive shifts in the cells’ voltage dependence. The shift observed was ∼10 mV for 500 μM trinitrophenol and 20 mV for 100 μM cationic chlorpromazine. Length reduction at the holding potential and voltage shifts of the motile activity were well correlated. The voltage shifts of nonlinear capacitance were not diminished by eliminating the cells’ turgor pressure or by digesting the cortical cytoskeleton. These observations suggest that the membrane motor undergoes conformational transitions that involve changes not only in membrane area but also in bending stiffness.

The Role of the STAS Domain in the Function and Biogenesis of a Sulfate Transporter as Probed by Random Mutagenesis

Sulfate transporters in plants represent a family of proteins containing transmembrane domains that constitute the catalytic part of the protein and a short linking region that joins this catalytic moiety with a C-terminal STAS domain. The STAS domain resembles an anti-sigma factor antagonist of Bacillus subtilis, which is one distinguishing feature of the SLC26 transporter family; this family includes transporters for sulfate and other anions such as iodide and carbonate. Recent work has demonstrated that this domain is critical for the activity of Arabidopsis thaliana sulfate transporters, and specific lesions in this domain, or the exchange of STAS domains between different sulfate transporters, can severely impair transport activity. In this work we generated a Saccharomyces cerevisiae expression library of the A. thaliana Sultr1;2 gene with random mutations in the linking region-STAS domain and identified STAS domain lesions that altered Sultr1;2 biogenesis and/or function. A number of mutations in the beta-sheet that forms the core of the STAS domain prevented intracellular accumulation of Sultr1;2. In contrast, the linking region and one surface of the STAS domain containing N termini of the first and second alpha-helices have a number of amino acids critical for the function of the protein; mutations in these regions still allow protein accumulation in the plasma membrane, but the protein is no longer capable of efficiently transporting sulfate into cells. These results suggest that the STAS domain is critical for both the activity and biosynthesis/stability of the transporter, and that STAS sub-domains correlate with these specific functions.