CryoEM structures of anion exchanger 1 capture multiple states of inward- and outward-facing conformations.

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.

Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE.

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.

Identification of multiple substrate binding sites in SLC4 transporters in the outward-facing conformation: Insights into the transport mechanism.

Solute carrier family 4 (SLC4) transporters mediate the transmembrane transport of HCO3-, CO32-, and Cl- necessary for pH regulation, transepithelial H+/base transport, and ion homeostasis. Substrate transport with varying stoichiometry and specificity is achieved through an exchange mechanism and/or through coupling of the uptake of anionic substrates to typically co-transported Na+. Recently solved outward-facing structures of two SLC4 members (human anion exchanger 1 [hAE1] and human electrogenic sodium bicarbonate cotransporter 1 [hNBCe1]) with different transport modes (Cl-/HCO3- exchange versus Na+-CO32- symport) revealed highly conserved three-dimensional organization of their transmembrane domains. However, the exact location of the ion binding sites and their protein-ion coordination motifs are still unclear. In the present work, we combined site identification by ligand competitive saturation mapping and extensive molecular dynamics sampling with functional mutagenesis studies which led to the identification of two substrate binding sites (entry and central) in the outward-facing states of hAE1 and hNBCe1. Mutation of residues in the identified binding sites led to impaired transport in both proteins. We also showed that R730 in hAE1 is crucial for anion binding in both entry and central sites, whereas in hNBCe1, a Na+ acts as an anchor for CO32- binding to the central site. Additionally, protonation of the central acidic residues (E681 in hAE1 and D754 in hNBCe1) alters the ion dynamics in the permeation cavity and may contribute to the transport mode differences in SLC4 proteins. These results provide a basis for understanding the functional differences between hAE1 and hNBCe1 and may facilitate potential drug development for diseases such as proximal and distal renal tubular acidosis.

Energy Shortage in Human and Mouse Models of SLC4A11-Associated Corneal Endothelial Dystrophies.

Purpose: To elucidate the molecular events in solute carrier family 4 member 11 (SLC4A11)-deficient corneal endothelium that lead to the endothelial dysfunction that characterizes the dystrophies associated with SLC4A11 mutations, congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy 4. Methods: Comparative transcriptomic analysis (CTA) was performed in primary human corneal endothelial cells (pHCEnC) and murine corneal endothelial cells (MCEnC) with normal and reduced levels of SLC4A11 (SLC4A11 KD pHCEnC) and Slc4a11 (Slc4a11-/- MCEnC), respectively. Validation of differentially expressed genes was performed using immunofluorescence staining of CHED corneal endothelium, as well as western blot and quantitative PCR analysis of SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC. Functional analyses were performed to investigate potential functional changes associated with the observed transcriptomic alterations. Results: CTA revealed inhibition of cell metabolism and ion transport function as well as mitochondrial dysfunction, leading to reduced adenosine triphosphate (ATP) production, in SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC. Co-localization of SNARE protein STX17 with mitochondria marker COX4 was observed in CHED corneal endothelium, as was activation of AMPK-p53/ULK1 in both SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC, providing additional evidence of mitochondrial dysfunction and mitophagy. Reduced Na+-dependent HCO3- transport activity and altered NH4Cl-induced membrane potential changes were observed in Slc4a11-/- MCEnC. Conclusions: Reduced steady-state ATP levels and subsequent activation of the AMPK-p53 pathway provide a link between the metabolic functional deficit and transcriptome alterations, as well as evidence of insufficient ATP to maintain the Na+/K+-ATPase corneal endothelial pump as the cause of the edema that characterizes SLC4A11-associated corneal endothelial dystrophies.

Ion channel formation by N-terminally truncated Aß (4-42): relevance for the pathogenesis of Alzheimer's disease.

Aß deposition is a pathological hallmark of Alzheimer's disease (AD). Besides the full-length amyloid forming peptides (Aß1-40 and Aß1-42), biochemical analyses of brain deposits have identified a variety of N- and C-terminally truncated Aß variants in sporadic and familial AD patients. However, their relevance for AD pathogenesis remains largely understudied. We demonstrate that Aß4-42 exhibits a high tendency to form ß-sheet structures leading to fast self-aggregation and formation of oligomeric assemblies. Atomic force microscopy and electrophysiological studies reveal that Aß4-42 forms highly stable ion channels in lipid membranes. These channels that are blocked by monoclonal antibodies specifically recognizing the N-terminus of Aß4-42. An Aß variant with a double truncation at phenylalanine-4 and leucine 34, (Aß4-34), exhibits unstable channel formation capability. Taken together the results presented herein highlight the potential benefit of C-terminal proteolytic cleavage and further support an important pathogenic role for N-truncated Aß species in AD pathophysiology.

Phenotypic and functional characterization of corneal endothelial cells during in vitro expansion.

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.

Computed tomography options in the evaluation of hernia repair outcomes using "titanium silk" mesh implants.

BACKGROUND: According to experimental studies, mesh implants may reduce in size up to 50.8% during their integration into soft tissues. This results in impaired mobility of the anterior abdominal wall and hernia recurrences, as well as affects patients' quality of life. Due to unsatisfactory radiographic contrast of polymeric mesh implants, changes in their size can be rarely confirmed using imaging methods. Medical devices made of metal alloys have the best radiographic contrast. OBJECTIVE: The purpose of this study was to evaluate the radiographic contrast of mesh implants made of titanium filaments and to determine the sizes and locations of the implants after the anterior abdominal wall hernia repair. MATERIALS & METHODS: The study included 40 patients with inguinal, umbilical, and postoperative ventral hernias. Surgical hernia repair was performed using a mash implant made of titanium filaments, "titanium silk". Multislice spiral computed tomography (MSCT) was performed on Days 3 and 90 after surgery, followed by an assessment of the size and location of the mesh implant. RESULTS: 90 days after the anterior abdominal wall hernia repair, the locations of the titanium silk mesh implants in the tissues corresponded to their intraoperative setting. There were no statistically significant changes in the sizes of mesh implants on Day 90, compared with Day 3. CONCLUSION: The titanium silk mesh implants have satisfactory radiographic contrast, which allows imaging of their location and size in the tissues using MSCT and comprehensive evaluation of hernia repair outcomes using instrumental methods of examination.

SLC4A11 function: evidence for H+(OH-) and NH3-H+ transport.

Whether SLC4A11 transports ammonia and its potential mode of ammonia transport (NH4+, NH3, or NH3-2H+ transport have been proposed) are controversial. In the absence of ammonia, whether SLC4A11 mediates significant conductive H+(OH-) transport is also controversial. The present study was performed to determine the mechanism of human SLC4A11 ammonia transport and whether the transporter mediates conductive H+(OH-) transport in the absence of ammonia. We quantitated H+ flux by monitoring changes in intracellular pH (pHi) and measured whole cell currents in patch-clamp studies of HEK293 cells expressing the transporter in the absence and presence of NH4Cl. Our results demonstrate that SLC4A11 mediated conductive H+(OH-) transport that was stimulated by raising the extracellular pH (pHe). Ammonia-induced HEK293 whole cell currents were also stimulated by an increase in pHe. In studies using increasing NH4Cl concentrations with equal NH4+ extracellular and intracellular concentrations, the shift in the reversal potential (Erev) due to the addition of ammonia was compatible with NH3-H+ transport competing with H+(OH-) rather than NH3-nH+ (n ≥ 2) transport. The increase in equivalent H+(OH-) flux observed in the presence of a transcellular H+ gradient was also compatible with SLC4A11-mediated NH3-H+ flux. The NH3 versus Erev data fit a theoretical model suggesting that NH3-H+ and H+(OH-) competitively interact with the transporter. Studies of mutant SLC4A11 constructs in the putative SLC4A11 ion coordination site showed that both H+(OH-) transport and ammonia-induced whole cell currents were blocked suggesting that the H+(OH-) and NH3-H+ transport processes share common features involving the SLC4A11 transport mechanism.

ZEB1 insufficiency causes corneal endothelial cell state transition and altered cellular processing.

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.

CryoEM structure of the human SLC4A4 sodium-coupled acid-base transporter NBCe1.

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.

Multifunctional ion transport properties of human SLC4A11: comparison of the SLC4A11-B and SLC4A11-C variants.

Congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (CHED with progressive sensorineural deafness), and potentially a subset of individuals with late-onset Fuchs' endothelial corneal dystrophy are caused by mutations in the SLC4A11 gene that results in corneal endothelial cell abnormalities. Originally classified as a borate transporter, the function of SLC4A11 as a transport protein remains poorly understood. Elucidating the transport function(s) of SLC4A11 is needed to better understand how its loss results in the aforementioned posterior corneal dystrophic disease processes. Quantitative PCR experiments demonstrated that, of the three known human NH2-terminal variants, SLC4A11-C is the major transcript expressed in human corneal endothelium. We studied the expression pattern of the three variants in mammalian HEK-293 cells and demonstrated that the SLC4A11-B and SLC4A11-C variants are plasma membrane proteins, whereas SLC4A11-A is localized intracellularly. SLC4A11-B and SLC4A11-C were shown to be multifunctional ion transporters capable of transporting H+ equivalents in both a Na+-independent and Na+-coupled mode. In both transport modes, SLC4A11-C H+ flux was significantly greater than SLC4A11-B. In the presence of ammonia, SLC4A11-B and SLC4A11-C generated inward currents that were comparable in magnitude. Chimera SLC4A11-C-NH2-terminus-SLC4A11-B experiments demonstrated that the SLC4A11-C NH2-terminus functions as an autoactivating domain, enhancing Na+-independent and Na+-coupled H+ flux without significantly affecting the electrogenic NH3-H(n)+ cotransport mode. All three modes of transport were significantly impaired in the presence of the CHED causing p.R109H (SLC4A11-C numbering) mutation. These complex ion transport properties need to be addressed in the context of corneal endothelial disease processes caused by mutations in SLC4A11.

Ion Channel Formation by Tau Protein: Implications for Alzheimer's Disease and Tauopathies.

Tau is a microtubule associated protein implicated in the pathogenesis of several neurodegenerative diseases. Because of the channel forming properties of other amyloid peptides, we employed planar lipid bilayers and atomic force microscopy to test tau for its ability to form ion permeable channels. Our results demonstrate that tau can form such channels, but only under acidic conditions. The channels formed are remarkably similar to amyloid peptide channels in their appearance, physical and electrical size, permanence, lack of ion selectivity, and multiple channel conductances. These channels differ from amyloid channels in their voltage dependence and resistance to blockade by zinc ion. These channels could explain tau's pathologic role in disease by lowering membrane potential, dysregulating calcium, depolarizing mitochondria, or depleting energy stores. Tau might also combine with amyloid beta peptides to form toxic channels.

Interplay between disulfide bonding and N-glycosylation defines SLC4 Na+-coupled transporter extracellular topography.

The extracellular loop 3 (EL-3) of SLC4 Na(+)-coupled transporters contains 4 highly conserved cysteines and multiple N-glycosylation consensus sites. In the electrogenic Na(+)-HCO3(-) cotransporter NBCe1-A, EL-3 is the largest extracellular loop and is predicted to consist of 82 amino acids. To determine the structural-functional importance of the conserved cysteines and the N-glycosylation sites in NBCe1-A EL-3, we analyzed the potential interplay between EL-3 disulfide bonding and N-glycosylation and their roles in EL-3 topological folding. Our results demonstrate that the 4 highly conserved cysteines form two intramolecular disulfide bonds, Cys(583)-Cys(585) and Cys(617)-Cys(642), respectively, that constrain EL-3 in a folded conformation. The formation of the second disulfide bond is spontaneous and unaffected by the N-glycosylation state of EL-3 or the first disulfide bond, whereas formation of the first disulfide bond relies on the presence of the second disulfide bond and is affected by N-glycosylation. Importantly, EL-3 from each monomer is adjacently located at the NBCe1-A dimeric interface. When the two disulfide bonds are missing, EL-3 adopts an extended conformation highly accessible to protease digestion. This unique adjacent parallel location of two symmetrically folded EL-3 loops from each monomer resembles a domain-like structure that is potentially important for NBCe1-A function in vivo. Moreover, the formation of this unique structure is critically dependent on the finely tuned interplay between disulfide bonding and N-glycosylation in the membrane processed NBCe1-A dimer.

Human SLC4A11-C functions as a DIDS-stimulatable H⁺(OH⁻) permeation pathway: partial correction of R109H mutant transport.

The SLC4A11 gene mutations cause a variety of genetic corneal diseases, including congenital hereditary endothelial dystrophy 2 (CHED2), Harboyan syndrome, some cases of Fuchs' endothelial dystrophy (FECD), and possibly familial keratoconus. Three NH2-terminal variants of the human SLC4A11 gene, named SLC4A11-A, -B, and -C are known. The SLC4A11-B variant has been the focus of previous studies. Both the expression of the SLC4A11-C variant in the cornea and its functional properties have not been characterized, and therefore its potential pathophysiological role in corneal diseases remains to be explored. In the present study, we demonstrate that SLC4A11-C is the predominant SLC4A11 variant expressed in human corneal endothelial mRNA and that the transporter functions as an electrogenic H(+)(OH(-)) permeation pathway. Disulfonic stilbenes, including 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS), and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonate (SITS), which are known to bind covalently, increased SLC4A11-C-mediated H(+)(OH(-)) flux by 150-200% without having a significant effect in mock-transfected cells. Noncovalently interacting 4,4'-diaminostilbene-2,2'-disulfonate (DADS) was without effect. We tested the efficacy of DIDS on the functionally impaired R109H mutant (SLC4A11-C numbering) that causes CHED2. DIDS (1 mM) increased H(+)(OH(-)) flux through the mutant transporter by ∼40-90%. These studies provide a basis for future testing of more specific chemically modified dilsulfonic stilbenes as potential therapeutic agents to improve the functional impairment of specific SLC4A11 mutant transporters.

Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis.

Mutations in SLC4A4, the gene encoding the electrogenic Na(+)-HCO3(-) cotransporter NBCe1, cause severe proximal renal tubular acidosis (pRTA), growth retardation, decreased IQ, and eye and teeth abnormalities. Among the known NBCe1 mutations, the disease-causing mechanism of the T485S (NBCe1-A numbering) mutation is intriguing because the substituted amino acid, serine, is structurally and chemically similar to threonine. In this study, we performed intracellular pH and whole cell patch-clamp measurements to investigate the base transport and electrogenic properties of NBCe1-A-T485S in mammalian HEK 293 cells. Our results demonstrated that Ser substitution of Thr485 decreased base transport by ~50%, and importantly, converted NBCe1-A from an electrogenic to an electroneutral transporter. Aqueous accessibility analysis using sulfhydryl reactive reagents indicated that Thr485 likely resides in an NBCe1-A ion interaction site. This critical location is also supported by the finding that G486R (a pRTA causing mutation) alters the position of Thr485 in NBCe1-A thereby impairing its transport function. By using NO3(-) as a surrogate ion for CO3(2-), our result indicated that NBCe1-A mediates electrogenic Na(+)-CO3(2-) cotransport when functioning with a 1:2 charge transport stoichiometry. In contrast, electroneutral NBCe1-T485S is unable to transport NO3(-), compatible with the hypothesis that it mediates Na(+)-HCO3(-) cotransport. In patients, NBCe1-A-T485S is predicted to transport Na(+)-HCO3(-) in the reverse direction from blood into proximal tubule cells thereby impairing transepithelial HCO3(-) absorption, possibly representing a new pathogenic mechanism for generating human pRTA.

Topology of NBCe1 protein transmembrane segment 1 and structural effect of proximal renal tubular acidosis (pRTA) S427L mutation.

In the kidney proximal tubule, NBCe1-A plays a critical role in absorbing HCO3(-) from cell to blood. NBCe1-A transmembrane segment 1 (TM1) is involved in forming part of the ion permeation pathway, and a missense mutation S427L in TM1 impairs ion transport, causing proximal renal tubular acidosis. In the present study, we examined the topology of NBCe1-A-TM1 in detail and its structural perturbation induced by S427L. We analyzed the N-terminal cytoplasmic region (Cys-389-Gln-424) of NBCe1-A-TM1 using the substituted cysteine scanning accessibility method combined with extensive chemical stripping, in situ chemical probing, and functional transport assays. NBCe1-A-TM1 was previously modeled on the anion exchanger 1 TM1 (AE1-TM1); however, our data demonstrated that the topology of AE1-TM1 differs significantly from NBCe1-A-TM1. Our findings revealed that NBCe1-A-TM1 is unusually long, consisting of 31 membrane-embedded amino acids (Phe-412 to Thr-442). The linker region (Arg-394-Pro-411) between the N terminus of TM1 and the cytoplasmic domain is minimally exposed to aqueous and is potentially folded in a helical structure that intimately interacts with the NBCe1-A cytoplasmic domain. In contrast, AE1-TM1 contains 25 amino acids connected to an aqueous-exposed cytoplasmic region. Based on our new NBCe1-A-TM1 model, Ser-427 resides in the middle of TM1. Leucine substitution at Ser-427 blocks the normal aqueous access to Thr-442, Ala-435, and Lys-404, implying a significant alteration of NBCe1-TM1 orientation. Our study provides novel structural insights into the pathogenic mechanism of S427L in mediating proximal renal tubular acidosis.

Structural and functional characterization of the C-terminal transmembrane region of NBCe1-A.

NBCe1-A and AE1 both belong to the SLC4 HCO(3)(-) transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala(800)-Lys(967). Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met(858) accessible to both biotin maleimide and TAMRA and Thr(926)-Ala(929) only to TAMRA labeling. The intracellular surface contains a highly exposed (Met(813)-Gly(828)) region and a cryptic (Met(887)-Arg(904)) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp(960). Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro(868)-Leu(967) (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1.

Topological location and structural importance of the NBCe1-A residues mutated in proximal renal tubular acidosis.

NBCe1-A electrogenically cotransports Na(+) and HCO(3)(-) across the basolateral membrane of renal proximal tubule cells. Eight missense mutations and 3 nonsense mutations in NBCe1-A cause severe proximal renal tubular acidosis (pRTA). In this study, the topologic properties and structural importance of the 8 endogenous residues mutated in pRTA and the in situ topology of NBCe1-A were examined by the substituted cysteine accessibility method. Of the 55 analyzed individually introduced cysteines, 8 were labeled with both membrane permeant (biotin maleimide (BM)) and impermeant (2-((5(6)-tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonate (MTS-TAMRA)) sulfhydryl reagents, 4 with only BM, and 3 with only MTS-TAMRA. The location of the labeled and unlabeled introduced cysteines clearly indicates that the transmembrane region of NBCe1-A contains 14 transmembrane segments (TMs). In this in situ based NBCe1-A topology, residues mutated in pRTA (pRTA residues) are assigned as: Ser(427), TM1; Thr(485) and Gly(486), TM3; Arg(510) and Leu(522), TM4; Ala(799), TM10; and Arg(881), TM12. Substitution of pRTA residues with cysteines impaired the membrane trafficking of R510C and R881C, the remaining membrane-processed constructs had various impaired transport function. Surprisingly, none of the membrane-processed constructs was accessible to labeling with BM and MTS-TAMRA, nor were they functionally sensitive to the inhibition by (2-aminoethyl)methanethiosulfonate. Functional analysis of Thr(485) with different amino acid substitutions indicated it resides in a unique region important for NBCe1-A function. Our findings demonstrate that the pRTA residues in NBCe1-A are buried in the protein complex/lipid bilayer where they perform important structural roles.

NBCe1-A Transmembrane Segment 1 Lines the Ion Translocation Pathway.

The electrogenic Na(+)/HCO(3)(-) cotransporter (NBCe1-A) transports sodium and bicarbonate across the basolateral membrane of the renal proximal tubule. In this study the structural requirement of transmembrane segment 1 (TM1) residues in mediating NBCe1-A transport was investigated. Twenty-five introduced cysteine mutants at positions Gln-424 to Gly-448 were tested for their sensitivity to the methanethiosulfonate reagents (2-sulfonatoethyl) methanethiosulfonate (MTSES), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and (2-aminoethyl) methanethiosulfonate (MTSEA). Two mutants, T442C and A435C, showed 100 and 70% sensitivity, respectively, to inhibition by all the three methanethiosulfonate (MTS) reagents, I441C had >50% sensitivity to MTSET and MTSEA, and A428C had 50% sensitivity to MTSEA inhibition. A helical wheel plot showed that mutants T442C, A435C, and A428C are clustered on one face of TM1 within a 100 degrees arc. Topology analysis of TM1 with biotin maleimide and 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) revealed Thr-442 marks the C-terminal end of TM1 and that extracellular FGGLLG stretch is in a small aqueous-accessible cavity. Functional studies indicated that Thr-442 resides in a narrow region of the ion translocation pore with strong delta(-) helical dipole influence. Analysis of the corresponding residue of NBCe1-A-Thr-442 in AE1 (Thr-422) shows it is functionally insensitive to MTSES and unlabeled with MTS-TAMRA, indicating that AE1-TM1 is oriented differently from NBCe1-A. In summary, we have identified residues Thr-442, Ala-435, and Ala-428 in TM1 lining the ion translocation pore of NBCe1-A. Our findings are suggestive of a delta(-) helical dipole at the C-terminal end of TM1 involving Thr-442 that plays a critical role in the function of the cotransporter.