RESUMO
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.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito , Proteínas de Membrana Transportadoras , Bovinos , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/química , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Microscopia Crioeletrônica , Domínios Proteicos , Transporte de ÍonsRESUMO
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.
Assuntos
Simportadores de Sódio-Bicarbonato/ultraestrutura , Células HEK293 , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/isolamento & purificaçãoRESUMO
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.
Assuntos
Proteínas Carreadoras de Solutos/química , Proteínas Carreadoras de Solutos/metabolismo , Sítios de Ligação , Transporte Biológico , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação ProteicaRESUMO
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.
Assuntos
Trifosfato de Adenosina/biossíntese , Endotélio Corneano , Transporte de Íons/fisiologia , Mitocôndrias/metabolismo , Proteínas SLC4A/genética , Quinases Proteína-Quinases Ativadas por AMP , Animais , Células Cultivadas , Distrofias Hereditárias da Córnea/genética , Endotélio Corneano/metabolismo , Endotélio Corneano/patologia , Endotélio Corneano/fisiopatologia , Metabolismo Energético , Perfilação da Expressão Gênica , Humanos , Camundongos , Mutação , Proteínas Quinases/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Técnicas de Cultura de Células/métodos , Endotélio Corneano/citologia , Adolescente , Adulto , Movimento Celular , Proliferação de Células , Senescência Celular , Criança , Pré-Escolar , Biologia Computacional , Transplante de Córnea , Feminino , Humanos , Masculino , Fenótipo , Transdução de Sinais , Transcriptoma , Adulto JovemRESUMO
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.
Assuntos
Amônia/metabolismo , Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Transporte de Íons/fisiologia , Bicarbonatos/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Sódio/metabolismoRESUMO
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.
Assuntos
Endotélio Corneano/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Córnea/metabolismo , Distrofias Hereditárias da Córnea/genética , Distrofias Hereditárias da Córnea/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Humanos , Fatores de Transcrição/metabolismo , Transcriptoma , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Background: Recent data suggested that the osmolal gap attributed to ethanol as determined by the difference between measured serum osmolality and calculated 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. Methods: 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. Results: 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 measured serum osmolality before and measured 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 H2O) = 0.23 (Ethanol [mg/dL]) - 1.43. Conclusion: 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.
RESUMO
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.
Assuntos
Ácidos/metabolismo , Álcalis/metabolismo , Simportadores de Sódio-Bicarbonato/ultraestrutura , Sódio/metabolismo , Transporte Biológico , Microscopia Crioeletrônica , Fenômenos Eletrofisiológicos , Humanos , Troca Iônica , Íons , Modelos Moleculares , Simportadores de Sódio-Bicarbonato/químicaRESUMO
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.
Assuntos
Proteínas de Transporte de Ânions/genética , Antiporters/genética , Variação Genética/genética , Transporte de Íons/genética , Linhagem Celular , Membrana Celular/genética , Distrofias Hereditárias da Córnea/genética , Endotélio Corneano/metabolismo , Células HEK293 , Perda Auditiva Neurossensorial/genética , Humanos , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mutação/genéticaRESUMO
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.
Assuntos
Cisteína/química , Dissulfetos/química , Dobramento de Proteína , Simportadores de Sódio-Bicarbonato/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Cisteína/genética , Cisteína/metabolismo , Dissulfetos/metabolismo , Glicosilação , Células HEK293 , Humanos , Immunoblotting , Transporte de Íons/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Multimerização Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismoRESUMO
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 â¼40-90%. 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.
Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Hidróxidos/metabolismo , Permeabilidade/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Proteínas de Transporte de Ânions/genética , Antiporters/genética , Transporte Biológico/fisiologia , Linhagem Celular , Córnea/efeitos dos fármacos , Córnea/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Mutação/genética , RNA Mensageiro/genéticaRESUMO
Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Recent clinical studies established that >70% of patients with idiopathic (also called primary) MN (IMN) possess circulating autoantibodies targeting the M-type phospholipase A2 receptor-1 (PLA2R) on the surface of glomerular visceral epithelial cells (podocytes). In situ, these autoantibodies trigger the formation of immune complexes, which are hypothesized to cause enhanced glomerular permeability to plasma proteins. Indeed, the level of autoantibody in circulation correlates with the severity of proteinuria in patients. The autoantibody only recognizes the nonreduced form of PLA2R, suggesting that disulfide bonds determine the antigenic epitope conformation. Here, we identified the immunodominant epitope region in PLA2R by probing isolated truncated PLA2R extracellular domains with sera from patients with IMN that contain anti-PLA2R autoantibodies. Patient sera specifically recognized a protein complex consisting of the cysteine-rich (CysR), fibronectin-like type II (FnII), and C-type lectin-like domain 1 (CTLD1) domains of PLA2R only under nonreducing conditions. Moreover, absence of either the CysR or CTLD1 domain prevented autoantibody recognition of the remaining domains. Additional analysis suggested that this three-domain complex contains at least one disulfide bond required for conformational configuration and autoantibody binding. Notably, the three-domain complex completely blocked the reactivity of autoantibodies from patient sera with the full-length PLA2R, and the reactivity of patient sera with the three-domain complex on immunoblots equaled the reactivity with full-length PLA2R. These results indicate that the immunodominant epitope in PLA2R is exclusively located in the CysR-FnII-CTLD1 region.
Assuntos
Autoanticorpos/imunologia , Glomerulonefrite Membranosa/imunologia , Epitopos Imunodominantes/imunologia , Receptores da Fosfolipase A2/imunologia , Autoanticorpos/sangue , Cisteína/imunologia , Fibronectinas/imunologia , Glomerulonefrite Membranosa/sangue , Glomerulonefrite Membranosa/patologia , Células HEK293 , Humanos , Epitopos Imunodominantes/química , Lectinas Tipo C/imunologia , Podócitos/imunologia , Podócitos/patologiaRESUMO
The presence of negatively charged, impermeant proteins in the plasma space alters the distribution of diffusible ions in the plasma and interstitial fluid (ISF) compartments to preserve electroneutrality and is known as Gibbs-Donnan equilibrium. In patients with hypoalbuminemia due to underlying cirrhosis, the decrease in the plasma water albumin concentration ([Alb-]pw) would be expected to result in a decrease in the plasma water sodium concentration ([Na+]pw) due to an alteration in the distribution of Na+ between the plasma and ISF. In addition, cirrhosis-associated hyponatremia may be due to the renal diluting defect resulting from the intravascular volume depletion due to gastrointestinal losses and overdiuresis and/or decreased effective circulatory volume secondary to splanchnic vasodilatation. Therefore, albumin infusion may result in correction of the hyponatremia in cirrhotic patients either by modulating the Gibbs-Donnan effect due to hypoalbuminemia or by restoring intravascular volume in patients with intravascular volume depletion due to gastrointestinal losses and overdiuresis. However, the differential role of albumin infusion in modulating the [Na+]pw in these patients has not previously been analyzed quantitatively. In the present study, we developed an in vitro assay system to examine for the first time the quantitative effect of changes in albumin concentration on the distribution of Na+ between two compartments separated by a membrane that allows the free diffusion of Na+. Our findings demonstrated that changes in [Alb-]pw are linearly related to changes in [Na+]pw as predicted by Gibbs-Donnan equilibrium. However, based on our findings, we predict that the improvement in cirrhosis-associated hyponatremia due to intravascular volume depletion results predominantly from the restoration of intravascular volume rather than alterations in Gibbs-Donnan equilibrium.
Assuntos
Albuminas/administração & dosagem , Líquido Extracelular/metabolismo , Hipoalbuminemia/terapia , Hiponatremia/terapia , Cirrose Hepática/complicações , Substitutos do Plasma/administração & dosagem , Sódio/metabolismo , Albuminas/metabolismo , Difusão , Humanos , Hipoalbuminemia/sangue , Hipoalbuminemia/etiologia , Hiponatremia/sangue , Hiponatremia/etiologia , Infusões Parenterais , Modelos Lineares , Cirrose Hepática/sangue , Modelos Biológicos , Substitutos do Plasma/metabolismo , Volume Plasmático , Albumina Sérica/metabolismo , Sódio/sangueRESUMO
BACKGROUND: The ion transport stoichiometry (q) of electrogenic transporters is an important determinant of their function. q can be determined by the reversal potential (Erev) 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 (ΔErev) 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 Erev are additive. However, Erev 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 (V1 and V2) where q = 2RT/(F(V2 -V1))ln(ΔI2/ΔI1) + 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 Erev inhibitor method. Furthermore, using computational simulations, we compared the estimates of q with the ΔErev and ΔI methods. The results showed that the ΔErev 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 ΔErev 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.
RESUMO
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.
Assuntos
Acidose Tubular Renal/metabolismo , Túbulos Renais Proximais/metabolismo , Mutação de Sentido Incorreto , Simportadores de Sódio-Bicarbonato/metabolismo , Acidose Tubular Renal/genética , Bicarbonatos/metabolismo , Carbonatos/metabolismo , Metanossulfonato de Etila/análogos & derivados , Metanossulfonato de Etila/farmacologia , Etilmaleimida/farmacologia , Predisposição Genética para Doença , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Túbulos Renais Proximais/efeitos dos fármacos , Potenciais da Membrana , Mesilatos/farmacologia , Mutagênese Sítio-Dirigida , Nitratos/metabolismo , Técnicas de Patch-Clamp , Fenótipo , Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/efeitos dos fármacos , Simportadores de Sódio-Bicarbonato/genética , TransfecçãoRESUMO
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.
Assuntos
Acidose Tubular Renal/genética , Mutação , Simportadores de Sódio-Bicarbonato/química , Acidose Tubular Renal/metabolismo , Aminoácidos/química , Bicarbonatos/química , Transporte Biológico , Biotina/química , Membrana Celular/metabolismo , Citoplasma/metabolismo , Células HEK293 , Humanos , Bicamadas Lipídicas/química , Maleimidas/química , Mutagênese Sítio-Dirigida , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismoRESUMO
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.
Assuntos
Proteínas de Membrana Transportadoras/química , Imagem Molecular/métodos , Multimerização Proteica , Espectrometria de Fluorescência/métodos , Animais , Anticorpos/imunologia , Células CHO , Cricetinae , Cricetulus , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Rim/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Microscopia Confocal , Fótons , Estrutura Quaternária de Proteína , Ratos , Simportadores de Sódio-Bicarbonato/química , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/imunologia , Simportadores de Sódio-Bicarbonato/metabolismoRESUMO
Integrins are a family of heterodimeric adhesion receptors that transmit signals bi-directionally across the plasma membranes. The transmembrane domain (TM) of integrin plays a critical role in mediating transition of the receptor from the default inactive to the active state on the cell surfaces. In this study, we successfully applied the substituted cysteine scanning accessibility method to determine the intracellular border of the integrin α(IIb)ß(3) TM in the inactive and active states in living cells. We examined the aqueous accessibility of 75 substituted cysteines comprising the C terminus of both α(IIb) and ß(3) TMs, the intracellular membrane-proximal regions, and the whole cytoplasmic tails, to the labeling of a membrane-permeable, cysteine-specific chemical biotin maleimide (BM). The active state of integrin α(IIb)ß(3) heterodimer was generated by co-expression of activating partners with the cysteine-substituted constructs. Our data revealed that, in the inactive state, the intracellular lipid/aqueous border of α(IIb) TM was at Lys(994) and ß(3) TM was at Phe(727) respectively; in the active state, the border of α(IIb) TM shifted to Pro(998), whereas the border of ß(3) TM remained unchanged, suggesting that complex conformational changes occurred in the TMs upon α(IIb)ß(3) inside-out activation. On the basis of the results, we propose a new inside-out activation mechanism for integrin α(IIb)ß(3) and by inference, all of the integrins in their native cellular environment.