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SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) transporters revealed an identical overall fold despite their different transport modes (chloride/bicarbonate exchange versus sodium-carbonate cotransport). However, the exact mechanism determining the different transport modes in the SLC4 family remains unknown. In this work, we report the cryo-EM 3.4 Å structure of the OF conformation of NDCBE (SLC4A8), which shares transport properties with both AE1 and NBCe1 by mediating the electroneutral exchange of sodium-carbonate with chloride. This structure features a fully resolved extracellular loop 3 and well-defined densities corresponding to sodium and carbonate ions in the tentative substrate binding pocket. Further, we combine computational modeling with functional studies to unravel the molecular determinants involved in NDCBE and SLC4 transport. The mechanisms involved in SLC4-mediated ion transport are still under debate. Here, the authors present a cryoEM structure of the Sodium-driven Chloride/Bicarbonate Exchanger NDCBE, which - together with computational modeling and mutagenesis - reveals molecular determinants of ion transport by SLC4.

The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell identity remain, and a comprehensive characterization of the cultured CEnC across serial passages has not been performed. To this end, we compared two established CEnC culture methods by assessing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion.

Na + -coupled acid–base transporters play essential roles in human biology. Their dysfunction has been linked to cancer, heart, and brain disease. High-resolution structures of mammalian Na + -coupled acid–base transporters are not available. The sodium-bicarbonate cotransporter NBCe1 functions in multiple organs and its mutations cause blindness, abnormal growth and blood chemistry, migraines, and impaired cognitive function. Here, we have determined the structure of the membrane domain dimer of human NBCe1 at 3.9 Å resolution by cryo electron microscopy. Our atomic model and functional mutagenesis revealed the ion accessibility pathway and the ion coordination site, the latter containing residues involved in human disease-causing mutations. We identified a small number of residues within the ion coordination site whose modification transformed NBCe1 into an anion exchanger. Our data suggest that symporters and exchangers utilize comparable transport machinery and that subtle differences in their substrate-binding regions have very significant effects on their transport mode. Na + -coupled acid-base membrane transport proteins regulate blood pressure, ion homeostasis and acid-base chemistry. Here the authors present the 3.9 Å resolution cryoEM structure of the sodium-bicarbonate cotransporter NBCe1 and characterize its ion coordination site and ion accessibility pathway with mutagenesis experiments.

Breast cancer cells survive despite being exposed to a toxic acidic extracellular environment, by utilizing the NBCn1 transporter. The molecular basis for this phenomenon is unknown, given the lack of an NBCn1 atomic structural model. We therefore determined the 3.3 Å cryoEM structure of the human NBCn1 outward facing (OF) conformational state with densities corresponding to the transported ions in the ion coordination site. We further generated NBCn1 inward facing (IF) and intermediate (occluded) structures and characterized the transport cycle and the ion dynamics in the IF and OF states. The results showed that NBCn1 utilizes an elevator-type transport mechanism with a small vertical shift of the ion coordination site between OF and IF conformational states and that the transported ions permeate without significant energy barriers. Functional experiments showed that NBCn1 has an extremely high ion turnover rate (TOR) of ~15,000 s −1 . The unusually high NBCn1 TOR value associated with the small protein structural changes during the OF to IF transitions and the favorable ion permeation energetics provides breast cancer cells with a highly efficient base loading mechanism contributing to their survival advantage. NBCn1 plays an important role as a base loader allowing breast cancer cells to survive in an acidic environment. Here, Wang et al report its near atomic structure and transport cycle involving minimal structural changes associated with an exceptionally high turnover rate, enabling efficient cellular base loading and tumor survival

The zinc finger e-box binding homeobox 1 (ZEB1) transcription factor is a master regulator of the epithelial to mesenchymal transition (EMT), and of the reverse mesenchymal to epithelial transition (MET) processes. ZEB1 plays an integral role in mediating cell state transitions during cell lineage specification, wound healing and disease. EMT/MET are characterized by distinct changes in molecular and cellular phenotype that are generally context-independent. Posterior polymorphous corneal dystrophy (PPCD), associated with ZEB1 insufficiency, provides a new biological context in which to understand and evaluate the classic EMT/MET paradigm. PPCD is characterized by a cadherin-switch and transition to an epithelial-like transcriptomic and cellular phenotype, which we study in a cell-based model of PPCD generated using CRISPR-Cas9-mediated ZEB1 knockout in corneal endothelial cells (CEnCs). Transcriptomic and functional studies support the hypothesis that CEnC undergo a MET-like transition in PPCD, termed endothelial to epithelial transition (EnET), and lead to the conclusion that EnET may be considered a corollary to the classic EMT/MET paradigm.

Anion exchanger 1 (AE1, band 3) is a major membrane protein of red blood cells and plays a key role in acid-base homeostasis, urine acidification, red blood cell shape regulation, and removal of carbon dioxide during respiration. Though structures of the transmembrane domain (TMD) of three SLC4 transporters, including AE1, have been resolved previously in their outward-facing (OF) state, no mammalian SLC4 structure has been reported in the inward-facing (IF) conformation. Here we present the cryoEM structures of full-length bovine AE1 with its TMD captured in both IF and OF conformations. Remarkably, both IF-IF homodimers and IF-OF heterodimers were detected. The IF structures feature downward movement in the core domain with significant unexpected elongation of TM11. Molecular modeling and structure guided mutagenesis confirmed the functional significance of residues involved in TM11 elongation. Our data provide direct evidence for an elevator-like mechanism of ion transport by an SLC4 family member. CryoEM structures and computational modeling of bovine anion exchanger 1 in both inward and outward facing conformations reveal an elevator-like mechanism of ion transport by a solute carrier ion transport family 4 member.

Membrane transporter proteins exist in a complex dynamic equilibrium between various oligomeric states that include monomers, dimers, dimer of dimers and higher order oligomers. Given their sub-optical microscopic resolution size, the oligomerization state of membrane transporters is difficult to quantify without requiring tissue disruption and indirect biochemical methods. Here we present the application of a fluorescence measurement technique which combines fluorescence image moment analysis and spatial intensity distribution analysis (SpIDA) to determine the oligomerization state of membrane proteins in situ. As a model system we analyzed the oligomeric state(s) of the electrogenic sodium bicarbonate cotransporter NBCe1-A in cultured cells and in rat kidney. The approaches that we describe offer for the first time the ability to investigate the oligomeric state of membrane transporter proteins in their native state.

Recent data suggested that the osmolal gap attributed to ethanol as determined by the difference between serum osmolality and serum osmolarity is greater than its molar concentration. The increased osmotic activity of ethanol is thought to be due to its binding to water molecules. This study is conducted to determine the true osmotic contribution of ethanol to serum osmolality. Baseline serum osmolality and ethanol concentration were measured on each serum sample. Varying amounts of ethanol were added to aliquots of serum in which the baseline serum ethanol concentration was undetectable. Repeat serum osmolality and serum ethanol concentration were measured after addition of ethanol. The range of serum ethanol concentration was 27.3-429.8 mg/dL. The serum osmolal gap attributed solely to ethanol is calculated based on the difference between serum osmolality before and serum osmolality after addition of ethanol. Our results demonstrated that the contribution of ethanol to serum osmolality can be calculated by dividing the serum ethanol level in mg/dl by 4.6. In addition, the relationship between serum ethanol concentration and osmolal gap due to ethanol was assessed by linear regression analysis. Linear regression analysis relating the osmolal gap due to ethanol and ethanol concentration yielded the following equation: Osmolal Gap (mOsm/kg H O) = 0.23 (Ethanol [mg/dL]) - 1.43. The osmolal concentration of ethanol can be calculated based on its molar concentration. We found no evidence for ethanol binding to water molecules over the range of ethanol concentration in this study.

Background The ion transport stoichiometry (q) of electrogenic transporters is an important determinant of their function. q can be determined by the reversal potential (E rev ) if the transporter under study is the only electrogenic transport mechanism or a specific inhibitor is available. An alternative approach is to calculate delta reversal potential (ΔE rev ) by altering the concentrations of the transported substrates. This approach is based on the hypothesis that the contributions of other channels and transporters on the membrane to E rev are additive. However, E rev is a complicated function of the sum of different conductances rather than being additive. Results We propose a new delta current (ΔI) method based on a simplified model for electrogenic secondary active transport by Heinz ( Electrical Potentials in Biological Membrane Transport , 1981). ΔI is the difference between two currents obtained from altering the external concentration of a transported substrate thereby eliminating other currents without the need for a specific inhibitor. q is determined by the ratio of ΔI at two different membrane voltages (V 1 and V 2 ) where q = 2RT/(F(V 2 –V 1 ))ln(ΔI 2 /ΔI 1 ) + 1. We tested this ΔI methodology in HEK-293 cells expressing the elctrogenic SLC4 sodium bicarbonate cotransporters NBCe2-C and NBCe1-A, the results were consistent with those obtained with the E rev inhibitor method. Furthermore, using computational simulations, we compared the estimates of q with the ΔE rev and ΔI methods. The results showed that the ΔE rev method introduces significant error when other channels or electrogenic transporters are present on the membrane and that the ΔI equation accurately calculates the stoichiometric ratio. Conclusions We developed a ΔI method for estimating transport stoichiometry of electrogenic transporters based on the Heinz model. This model reduces to the conventional reversal potential method when the transporter under study is the only electrogenic transport process in the membrane. When there are other electrogenic transport pathways, ΔI method eliminates their contribution in estimating q. Computational simulations demonstrated that the ΔE rev method introduces significant error when other channels or electrogenic transporters are present and that the ΔI equation accurately calculates the stoichiometric ratio. This new ΔI method can be readily extended to the analysis of other electrogenic transporters in other tissues.