RESUMEN
Pathogenic variants in HCN1 are associated with a range of epilepsy syndromes including a developmental and epileptic encephalopathy. The recurrent de novo HCN1 pathogenic variant (M305L) results in a cation leak, allowing the flux of excitatory ions at potentials where the wild-type channels are closed. The Hcn1M294L mouse recapitulates patient seizure and behavioral phenotypes. As HCN1 channels are highly expressed in rod and cone photoreceptor inner segments, where they shape the light response, mutated channels are likely to impact visual function. Electroretinogram (ERG) recordings from male and female mice Hcn1M294L mice revealed a significant decrease in the photoreceptor sensitivity to light, as well as attenuated bipolar cell (P2) and retinal ganglion cell responses. Hcn1M294L mice also showed attenuated ERG responses to flickering lights. ERG abnormalities are consistent with the response recorded from a single female human subject. There was no impact of the variant on the structure or expression of the Hcn1 protein in the retina. In silico modeling of photoreceptors revealed that the mutated HCN1 channel dramatically reduced light-induced hyperpolarization, resulting in more Ca2+ flux during the response when compared with the wild-type situation. We propose that the light-induced change in glutamate release from photoreceptors during a stimulus will be diminished, significantly blunting the dynamic range of this response. Our data highlight the importance of HCN1 channels to retinal function and suggest that patients with HCN1 pathogenic variants are likely to have a dramatically reduced sensitivity to light and a limited ability to process temporal information.SIGNIFICANCE STATEMENT Pathogenic variants in HCN1 are emerging as an important cause of catastrophic epilepsy. HCN1 channels are ubiquitously expressed throughout the body, including the retina. Electroretinogram recordings from a mouse model of HCN1 genetic epilepsy showed a marked decrease in the photoreceptor sensitivity to light and a reduced ability to respond to high rates of light flicker. No morphologic deficits were noted. Simulation data suggest that the mutated HCN1 channel blunts light-induced hyperpolarization and consequently limits the dynamic range of this response. Our results provide insights into the role HCN1 channels play in retinal function as well as highlighting the need to consider retinal dysfunction in disease caused by HCN1 variants. The characteristic changes in the electroretinogram open the possibility of using this tool as a biomarker for this HCN1 epilepsy variant and to facilitate development of treatments.
Asunto(s)
Epilepsia , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Humanos , Masculino , Femenino , Ratones , Animales , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Retina/metabolismo , Electrorretinografía , Epilepsia/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Canales de Potasio/fisiologíaRESUMEN
Marine food webs are strongly size-structured and size-based analysis of communities is a useful approach to evaluate food webs in a way that can be compared across systems. Fatty acid analysis is commonly used to identify diet sources of species, offering a powerful complement to stable isotopes, but is rarely applied to size-structured communities. In this study, we used fatty acids and stable isotopes to characterize size-based variation in prey resources and trophic pathways over a nine-month temperate coastal ocean time series of seven plankton size classes, from > 0.7-µm particulate organic matter through > 2000-µm zooplankton. Zooplankton size classes were generally distinguishable by their dietary fatty acids, while stable isotopes revealed more seasonal variability. Fatty acids of zooplankton were correlated with those of their prey (particulate organic matter and smaller zooplankton) and identified trophic pathways, including widespread ties to the microbial food web. Diatom fatty acids also contributed to zooplankton but fall blooms were more important than spring. Concurrent isotope-based trophic position estimates and fatty acid markers of carnivory showed that some indicators (18:1ω9/18:1ω7) are not consistent across size classes, while others (DHA:EPA) are relatively reliable. Both analysis methods provided distinct information to build a more robust understanding of resource use. For example, fatty acid markers showed that trophic position was likely underestimated in 250-µm zooplankton, probably due to their consumption of protists with low isotopic fractionation factors. Applying fatty acid analysis to a size-structured framework provides more insight into trophic pathways than isotopes alone.
Asunto(s)
Cadena Alimentaria , Zooplancton , Animales , Estaciones del Año , Isótopos/metabolismo , Ácidos Grasos/metabolismo , FitoplanctonRESUMEN
Org 34167 is a small molecule hyperpolarization-activated cyclic nucleotide-gated (HCN) channel modulator that has been trialed in humans for its potential antidepressant activity. The precise action of Org 34167 is not fully understood. Here we use two-electrode voltage clamp recordings and an allosteric model to explore the interaction of Org 34167 with human HCN1 channels. The impact of Org 34167 on channel function included a hyperpolarizing shift in activation voltage dependence and a slowing of activation kinetics. Furthermore, a reduction in the maximum open probability at extreme hyperpolarization argued for an additional voltage-independent mechanism. Org 34167 had a similar impact on a truncated HCN1 channel lacking the C-terminal nucleotide binding domain, thus ruling out an interaction with this domain. Fitting a gating model, derived from a 10-state allosteric scheme, predicted that Org 34167 strongly reduced the equilibrium constant for the voltage-independent pore domain to favor a closed pore, as well as reducing the voltage sensing domain-pore domain coupling and shifting the zero voltage equilibrium constant of the voltage sensing domain to favor the inactive state. SIGNIFICANCE STATEMENT: The brain penetrant small molecule Org 34167 has been reported to have an antidepressant action by targeting HCN channels; however, its mode of action is unknown. We used heterologously expressed human HCN1 channels to show that Org 34167 inhibits channel activity by modulating kinetic parameters associated with the channel pore domain, voltage sensing domain, and interdomain coupling.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Activación del Canal Iónico , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Activación del Canal Iónico/fisiología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , AMP Cíclico/metabolismo , Antidepresivos/farmacologíaRESUMEN
Pathogenic variants in HCN1 are associated with developmental and epileptic encephalopathies. The recurrent de novo HCN1 M305L pathogenic variant is associated with severe developmental impairment and drug-resistant epilepsy. We engineered the homologue Hcn1 M294L heterozygous knock-in (Hcn1M294L) mouse to explore the disease mechanism underlying an HCN1 developmental and epileptic encephalopathy. The Hcn1M294L mouse recapitulated the phenotypic features of patients with the HCN1 M305L variant, including spontaneous seizures and a learning deficit. Active epileptiform spiking on the electrocorticogram and morphological markers typical of rodent seizure models were observed in the Hcn1M294L mouse. Lamotrigine exacerbated seizures and increased spiking, whereas sodium valproate reduced spiking, mirroring drug responses reported in a patient with this variant. Functional analysis in Xenopus laevis oocytes and layer V somatosensory cortical pyramidal neurons in ex vivo tissue revealed a loss of voltage dependence for the disease variant resulting in a constitutively open channel that allowed for cation 'leak' at depolarized membrane potentials. Consequently, Hcn1M294L layer V somatosensory cortical pyramidal neurons were significantly depolarized at rest. These neurons adapted through a depolarizing shift in action potential threshold. Despite this compensation, layer V somatosensory cortical pyramidal neurons fired action potentials more readily from rest. A similar depolarized resting potential and left-shift in rheobase was observed for CA1 hippocampal pyramidal neurons. The Hcn1M294L mouse provides insight into the pathological mechanisms underlying hyperexcitability in HCN1 developmental and epileptic encephalopathy, as well as being a preclinical model with strong construct and face validity, on which potential treatments can be tested.
Asunto(s)
Encefalopatías/metabolismo , Modelos Animales de Enfermedad , Epilepsia/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Neuronas/metabolismo , Canales de Potasio/metabolismo , Animales , Encefalopatías/genética , Epilepsia/genética , Femenino , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Masculino , Ratones , Ratones Mutantes , Mutación , Neuronas/patología , Canales de Potasio/genética , Células Piramidales/metabolismo , Xenopus laevisRESUMEN
RATIONALE: Stable isotope analysis (SIA) can provide important insights into food web structure and is a widely used tool in ecological conservation and management. It has recently been augmented by compound-specific stable isotope analysis of amino acids (CSIA-AA), an innovation that can provide greater precision when analyzing trophic level and food web connectivity. The utility of SIA rests on confidence in its constituent parameters such as the trophic enrichment factor (TEF). There is increasing emphasis on the need to experimentally derive species and tissue specific TEFs for studies utilizing SIA. Chinook salmon, Oncorhynchus tshawytscha, is a species with high potential for study using SIA due to the difficulty in observing its ecology during its marine phase and the significance of the conservation consequences of recent population declines. METHODS: Bulk and amino acid-specific TEFs were determined for juvenile and adult Chinook salmon fed specific diets. Three controlled feeding studies were performed: adult salmon were fed a biofeed, juvenile salmon were fed a biofeed, and juvenile salmon were fed krill. Bulk and compound-specific stable isotope data were collected from diet samples and from salmon muscle tissue after a minimum of 8 weeks of controlled feeding. Bulk isotope signatures were measured using EA-IRMS and CSIA-AA signatures using GC/C-IRMS, allowing the TEFs to be calculated. RESULTS: The bulk isotope TEFs were higher than those predicted for similar marine organisms and averaged 3.5 for ∆15 N and 1.3 for ∆13 C. The TEFs derived for nitrogen isotopes of amino acids were in line with expectations for this approach: the mean value for ∆15 NGlu - ∆15 NPhe was 7.06 and, using a multi-AA approach, the value for ∆15 NTrophic - ∆15 NSource was 6.67. For carbon isotopes of amino acids, the derived TEFs of Iso, Leu and Phe were near 0, as was that of Met, supporting their use of as source amino acids in future CSIA studies. CONCLUSIONS: This study presents Chinook salmon-specific TEFs for bulk and amino acid SIA. It supports the application of future research applying SIA to the study of Chinook salmon and validates previous research on species-specific TEFs.
Asunto(s)
Aminoácidos/análisis , Dieta/veterinaria , Cadena Alimentaria , Salmón/metabolismo , Alimentación Animal/análisis , Animales , Isótopos de Carbono/análisis , Conservación de los Recursos Naturales , Espectrometría de Masas/veterinaria , Músculos/química , Isótopos de Nitrógeno/análisisRESUMEN
The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the α3-isoform of Na+,K+-ATPase. Here, using biochemical and electrophysiological approaches, we examined the functional characteristics of E818K, as well as of E818Q and E818A mutants. We found that these amino acid substitutions reduce the apparent Na+ affinity at the cytoplasmic-facing sites of the pump protein and that this effect is more pronounced for the lysine and glutamine substitutions (3-4-fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, â¼30-fold reduction of apparent Na+ affinity for the extracellular-facing sites in the CAPOS mutant, which was related to an accelerated transition between the phosphoenzyme intermediates E1P and E2P. The apparent affinity for K+ activation of the ATPase activity was unaffected by these substitutions, suggesting that primarily the Na+-specific site III is affected. Furthermore, the apparent affinities for ATP and vanadate were WT-like in E818K, indicating a normal E1-E2 equilibrium of the dephosphoenzyme. Proton-leak currents were not increased in E818K. However, the CAPOS mutation caused a weaker voltage dependence of the pumping rate and a stronger inhibition by cytoplasmic K+ than the WT enzyme, which together with the reduced Na+ affinity of the cytoplasmic-facing sites precluded proper pump activation under physiological conditions. The functional deficiencies could be traced to the participation of Glu-818 in an intricate hydrogen-bonding/salt-bridge network, connecting it to key residues involved in Na+ interaction at site III.
Asunto(s)
Adenosina Trifosfato/metabolismo , Ataxia Cerebelosa/metabolismo , Deformidades Congénitas del Pie/metabolismo , Pérdida Auditiva Sensorineural/metabolismo , Potenciales de la Membrana , Mutación Missense , Atrofia Óptica/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfato/genética , Sustitución de Aminoácidos , Animales , Ataxia Cerebelosa/genética , Deformidades Congénitas del Pie/genética , Pérdida Auditiva Sensorineural/genética , Humanos , Atrofia Óptica/genética , Dominios Proteicos , Reflejo Anormal/genética , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/genética , Vanadatos/farmacología , Xenopus laevisRESUMEN
The expression cloning some 25 years ago of the first member of SLC34 solute carrier family, the renal sodium-coupled inorganic phosphate cotransporter (NaPi-IIa) from rat and human tissue, heralded a new era of research into renal phosphate handling by focussing on the carrier proteins that mediate phosphate transport. The cloning of NaPi-IIa was followed by that of the intestinal NaPi-IIb and renal NaPi-IIc isoforms. These three proteins constitute the main secondary-active Na+-driven pathways for apical entry of inorganic phosphate (Pi) across renal and intestinal epithelial, as well as other epithelial-like organs. The key role these proteins play in mammalian Pi homeostasis was revealed in the intervening decades by numerous in vitro and animal studies, including the development of knockout animals for each gene and the detection of naturally occurring mutations that can lead to Pi-handling dysfunction in humans. In addition to characterising their physiological regulation, research has also focused on understanding the underlying transport mechanism and identifying structure-function relationships. Over the past two decades, this research effort has used real-time electrophysiological and fluorometric assays together with novel computational biology strategies to develop a detailed, but still incomplete, understanding of the transport mechanism of SLC34 proteins at the molecular level. This review will focus on how our present understanding of their molecular mechanism has evolved in this period by highlighting the key experimental findings.
Asunto(s)
Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II/metabolismo , Animales , Humanos , Transporte Iónico , Potenciales de la Membrana , Dominios Proteicos , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II/química , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II/genéticaRESUMEN
Idiopathic infantile hypercalcemia (IIH) is characterized by severe hypercalcemia with failure to thrive, vomiting, dehydration, and nephrocalcinosis. Recently, mutations in the vitamin D catabolizing enzyme 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) were described that lead to increased sensitivity to vitamin D due to accumulation of the active metabolite 1,25-(OH)2D3. In a subgroup of patients who presented in early infancy with renal phosphate wasting and symptomatic hypercalcemia, mutations in CYP24A1 were excluded. Four patients from families with parental consanguinity were subjected to homozygosity mapping that identified a second IIH gene locus on chromosome 5q35 with a maximum logarithm of odds (LOD) score of 6.79. The sequence analysis of the most promising candidate gene, SLC34A1 encoding renal sodium-phosphate cotransporter 2A (NaPi-IIa), revealed autosomal-recessive mutations in the four index cases and in 12 patients with sporadic IIH. Functional studies of mutant NaPi-IIa in Xenopus oocytes and opossum kidney (OK) cells demonstrated disturbed trafficking to the plasma membrane and loss of phosphate transport activity. Analysis of calcium and phosphate metabolism in Slc34a1-knockout mice highlighted the effect of phosphate depletion and fibroblast growth factor-23 suppression on the development of the IIH phenotype. The human and mice data together demonstrate that primary renal phosphate wasting caused by defective NaPi-IIa function induces inappropriate production of 1,25-(OH)2D3 with subsequent symptomatic hypercalcemia. Clinical and laboratory findings persist despite cessation of vitamin D prophylaxis but rapidly respond to phosphate supplementation. Therefore, early differentiation between SLC34A1 (NaPi-IIa) and CYP24A1 (24-hydroxylase) defects appears critical for targeted therapy in patients with IIH.
Asunto(s)
Hipercalcemia/genética , Enfermedades del Recién Nacido/genética , Errores Innatos del Metabolismo/genética , Mutación , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Proteínas Cotransportadoras de Sodio-Fosfato/genética , Animales , Genes Recesivos , Humanos , Lactante , Recién Nacido , Ratones , Ratones NoqueadosRESUMEN
Plasma phosphate concentration is maintained within a relatively narrow range by control of renal reabsorption of filtered inorganic phosphate (P(i)). P(i) reabsorption is a transcellular process that occurs along the proximal tubule. P(i) flux at the apical (luminal) brush border membrane represents the rate-limiting step and is mediated by three Na(+)-dependent P(i) cotransporters (members of the SLC34 and SLC20 families). The putative proteins responsible for basolateral P(i) flux have not been identified. The transport mechanism of the two kidney-specific SLC34 proteins (NaPi-IIa and NaPi-IIc) and of the ubiquitously expressed SLC20 protein (PiT-2) has been studied by heterologous expression to reveal important differences in kinetics, stoichiometry, and substrate specificity. Studies on the regulation of the abundance of the respective proteins highlight significant differences in the temporal responses to various hormonal and nonhormonal factors that can influence P(i) homeostasis. The phenotypes of mice deficient in NaPi-IIa and NaPi-IIc indicate that NaPi-IIa is responsible for most P(i) renal reabsorption. In contrast, in the human kidney, NaPi-IIc appears to have a relatively greater role. The physiological relevance of PiT-2 to P(i) reabsorption remains to be elucidated.
Asunto(s)
Homeostasis/fisiología , Túbulos Renales Proximales/fisiología , Proteínas de Transporte de Fosfato/fisiología , Animales , Humanos , Ratones , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/fisiología , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/fisiologíaRESUMEN
Voltage-dependence of Na(+)-coupled phosphate cotransporters of the SLC34 family arises from displacement of charges intrinsic to the protein and the binding/release of one Na(+) ion in response to changes in the transmembrane electric field. Candidate coordination residues for the cation at the Na1 site were previously predicted by structural modeling using the x-ray structure of dicarboxylate transporter VcINDY as template and confirmed by functional studies. Mutations at Na1 resulted in altered steady-state and presteady-state characteristics that should be mirrored in the conformational changes induced by membrane potential changes. To test this hypothesis by functional analysis, double mutants of the flounder SLC34A2 protein were constructed that contain one of the Na1-site perturbing mutations together with a substituted cysteine for fluorophore labeling, as expressed in Xenopus oocytes. The locations of the mutations were mapped onto a homology model of the flounder protein. The effects of the mutagenesis were characterized by steady-state, presteady-state, and fluorometric assays. Changes in fluorescence intensity (ΔF) in response to membrane potential steps were resolved at three previously identified positions. These fluorescence data corroborated the altered presteady-state kinetics upon perturbation of Na1, and furthermore indicated concomitant changes in the microenvironment of the respective fluorophores, as evidenced by changes in the voltage dependence and time course of ΔF. Moreover, iodide quenching experiments indicated that the aqueous nature of the fluorophore microenvironment depended on the membrane potential. These findings provide compelling evidence that membrane potential and cation interactions induce significant large-scale structural rearrangements of the protein.
Asunto(s)
Potenciales de la Membrana/fisiología , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/metabolismo , Sodio/metabolismo , Animales , Cationes Monovalentes/metabolismo , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Lenguado , Fluorometría , Cinética , Microscopía Fluorescente , Modelos Moleculares , Mutación , Oocitos , Técnicas de Placa-Clamp , Conformación Proteica , Homología de Secuencia de Aminoácido , Sodio/química , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/química , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/genética , Relación Estructura-Actividad , Agua/química , Xenopus laevisRESUMEN
L-arginine is a semi-essential amino acid that serves as precursor for the production of urea, nitric oxide (NO), polyamines, and other biologically important metabolites. Hence, a fast and reliable assessment of its intracellular concentration changes is highly desirable. Here, we report on a genetically encoded Förster resonance energy transfer (FRET)-based arginine nanosensor that employs the arginine repressor/activator ahrC gene from Bacillus subtilis. This new nanosensor was expressed in HEK293T cells, and experiments with cell lysate showed that it binds L-arginine with high specificity and with a K d of â¼177 µM. Live imaging experiments showed that the nanosensor was expressed throughout the cytoplasm and displayed a half maximal FRET increase at an extracellular L-arginine concentration of â¼22 µM. By expressing the nanosensor together with SLC7A1, SLC7A2B, or SLC7A3 cationic amino acid transporters (CAT1-3), it was shown that L-arginine was imported at a similar rate via SLC7A1 and SLC7A2B and slower via SLC7A3. In contrast, upon withdrawal of extracellular L-arginine, intracellular levels decreased as fast in SLC7A3-expressing cells compared with SLC7A1, but the efflux was slower via SLC7A2B. SLC7A4 (CAT4) could not be convincingly shown to transport L-arginine. We also demonstrated the impact of membrane potential on L-arginine transport and showed that physiological concentrations of symmetrical and asymmetrical dimethylarginine do not significantly interfere with L-arginine transport through SLC7A1. Our results demonstrate that the FRET nanosensor can be used to assess L-arginine transport through plasma membrane in real time.
Asunto(s)
Proteínas Bacterianas/metabolismo , Técnicas Biosensibles/métodos , Transportador de Aminoácidos Catiónicos 1/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Arginina/metabolismo , Proteínas Bacterianas/química , Células HEK293 , Humanos , Potenciales de la Membrana , Proteínas Represoras/química , Transactivadores/químicaRESUMEN
The epithelial Na+-coupled phosphate cotransporter family Slc34a (NaPi-II) is well conserved in vertebrates and plays an essential role in maintaining whole body levels of inorganic phosphate (Pi). A three-dimensional model of the transport protein has recently been proposed with defined substrate coordination sites. Zebrafish express two NaPi-II isoforms with high sequence identity but a 10-fold different apparent Km for Pi ([Formula: see text]). We took advantage of the two zebrafish isoforms to investigate the contribution of specific amino acids to Pi coordination and transport. Mutations were introduced to gradually transform the low-affinity isoform into a high-affinity transporter. The constructs were expressed in Xenopus laevis oocytes and functionally characterized. Becaue the cotransport of Pi and Na involves multiple steps that could all influence [Formula: see text], we performed a detailed functional analysis to characterize the impact of the mutations on particular steps of the transport cycle. We used varying concentrations of the substrates Pi and its slightly larger analog, arsenate, as well as the cosubstrate, Na+ Moreover, electrogenic kinetics were performed to assess intramolecular movements of the transporter. All of the mutations were found to affect multiple transport steps, which suggested that the altered amino acids induced subtle structural changes rather than coordinating Pi directly. The likely positions of the critical residues were mapped to the model of human Slc34a, and their localization in relation to the proposed substrate binding pockets concurs well with the observed functional data.
Asunto(s)
Aminoácidos/química , Fosfatos/química , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II/química , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II/ultraestructura , Sodio/química , Proteínas de Pez Cebra/química , Animales , Sitios de Unión , Transporte Biológico Activo , Humanos , Modelos Químicos , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , Especificidad de la Especie , Relación Estructura-Actividad , Pez Cebra , Proteínas de Pez Cebra/ultraestructuraRESUMEN
BACKGROUND: Inherited metabolic disorders associated with nephrocalcinosis are rare conditions. The aim of this study was to identify the genetic cause of an Israeli-Arab boy from a consanguineous family with severe nephrocalcinosis and kidney insufficiency. METHODS: Clinical and biochemical data of the proband and family members were obtained from both previous and recent medical charts. Genomic DNA was isolated from peripheral blood cells. The coding sequence and splice sites of candidate genes (CYP24A1, CYP27B1, FGF23, KLOTHO, SLC34A3 and SLC34A1) were sequenced directly. Functional studies were performed in Xenopus laevis oocytes and in transfected opossum kidney (OK) cells. RESULTS: Our patient was identified as having nephrocalcinosis in utero, and at the age of 16.5 years, he had kidney insufficiency but no bone disease. Genetic analysis revealed a novel homozygous missense mutation, Arg215Gln, in SLC34A1, which encodes the renal sodium phosphate cotransporter NaPiIIa. Functional studies of the Arg215Gln mutant revealed reduced transport activity in Xenopus laevis oocytes and increased intracellular cytoplasmic accumulation in OK cells. CONCLUSIONS: Our findings show that dysfunction of the human NaPiIIa causes severe renal calcification that may eventually lead to reduced kidney function, rather than complications of phosphate loss.
Asunto(s)
Nefrocalcinosis/genética , Insuficiencia Renal/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Adolescente , Animales , Simulación por Computador , ADN/genética , Factor-23 de Crecimiento de Fibroblastos , Humanos , Riñón/metabolismo , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Masculino , Mutación , Mutación Missense , Oocitos/metabolismo , Zarigüeyas , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Transfección , Xenopus laevisRESUMEN
Transporters of the SLC34 family (NaPi-IIa,b,c) catalyze uptake of inorganic phosphate (Pi) in renal and intestinal epithelia. The transport cycle requires three Na(+) ions and one divalent Pi to bind before a conformational change enables translocation, intracellular release of the substrates, and reorientation of the empty carrier. The electrogenic interaction of the first Na(+) ion with NaPi-IIa/b at a postulated Na1 site is accompanied by charge displacement, and Na1 occupancy subsequently facilitates binding of a second Na(+) ion at Na2. The voltage dependence of cotransport and presteady-state charge displacements (in the absence of a complete transport cycle) are directly related to the molecular architecture of the Na1 site. The fact that Li(+) ions substitute for Na(+) at Na1, but not at the other sites (Na2 and Na3), provides an additional tool for investigating Na1 site-specific events. We recently proposed a three-dimensional model of human SLC34a1 (NaPi-IIa) including the binding sites Na2, Na3, and Pi based on the crystal structure of the dicarboxylate transporter VcINDY. Here, we propose nine residues in transmembrane helices (TM2, TM3, and TM5) that potentially contribute to Na1. To verify their roles experimentally, we made single alanine substitutions in the human NaPi-IIa isoform and investigated the kinetic properties of the mutants by voltage clamp and (32)P uptake. Substitutions at five positions in TM2 and one in TM5 resulted in relatively small changes in the substrate apparent affinities, yet at several of these positions, we observed significant hyperpolarizing shifts in the voltage dependence. Importantly, the ability of Li(+) ions to substitute for Na(+) ions was increased compared with the wild-type. Based on these findings, we adjusted the regions containing Na1 and Na3, resulting in a refined NaPi-IIa model in which five positions (T200, Q206, D209, N227, and S447) contribute directly to cation coordination at Na1.
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Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Humanos , Datos de Secuencia Molecular , Unión Proteica , Sodio/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , XenopusAsunto(s)
Epilepsia Generalizada , Epilepsia , Discapacidad Intelectual , Humanos , Receptores de GABA-A , ConvulsionesRESUMEN
To gain insight into the steady-state and dynamic characteristics of structural rearrangements of an electrogenic secondary-active cotransporter during its transport cycle, two measures of conformational change (pre-steady-state current relaxations and intensity of fluorescence emitted from reporter fluorophores) were investigated as a function of membrane potential and external substrate. Cysteines were substituted at three believed-new sites in the type IIb Na(+)-coupled inorganic phosphate cotransporter (SLC34A2 flounder isoform) that were predicted to be involved in conformational changes. Labeling at one site resulted in substantial suppression of transport activity, whereas for the other sites, function remained comparable to the wild-type. For these mutants, the properties of the pre-steady-state charge relaxations were similar for each, whereas fluorescence intensity changes differed significantly. Fluorescence changes could be accounted for by simulations using a five-state model with a unique set of apparent fluorescence intensities assigned to each state according to the site of labeling. Fluorescence reported from one site was associated with inward and outward conformations, whereas for the other sites, including four previously indentified sites, emissions were associated principally with one or the other orientation of the transporter. The same membrane potential change induced complementary changes in fluorescence at some sites, which suggested that the microenvironments of the respective fluorophores experience concomitant changes in polarity. In response to step changes in voltage, the pre-steady-state current relaxation and the time course of change in fluorescence intensity were described by single exponentials. For one mutant the time constants matched well with and without external Na(+), providing direct evidence that this label reports conformational changes accompanying intrinsic charge movement and cation interactions.
Asunto(s)
Sodio/metabolismo , Simportadores/química , Simportadores/metabolismo , Animales , Cisteína/química , Cisteína/genética , Fenómenos Electrofisiológicos , Lenguado , Fluorescencia , Humanos , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Conformación Proteica , Rodaminas/metabolismo , Coloración y Etiquetado , Compuestos de Sulfhidrilo/metabolismo , Factores de Tiempo , Xenopus laevisRESUMEN
Phosphate plays essential biological roles and its plasma level in humans requires tight control to avoid bone loss (insufficiency) or vascular calcification (excess). Intestinal absorption and renal reabsorption of phosphate are mediated by members of the SLC34 family of sodium-coupled transporters (NaPi-IIa,b,c) whose membrane expression is regulated by various hormones, circulating proteins, and phosphate itself. Consequently, NaPi-II proteins are also potentially important pharmaceutical targets for controlling phosphate levels. Their crucial role in Pi homeostasis is underscored by pathologies resulting from naturally occurring SLC34 mutations and SLC34 knockout animals. SLC34 isoforms have been extensively studied with respect to transport mechanism and structure-function relationships; however, the three-dimensional structure is unknown. All SLC34 transporters share a duplicated motif comprising a glutamine followed by a stretch of threonine or serine residues, suggesting the presence of structural repeats as found in other transporter families. Nevertheless, standard bioinformatic approaches fail to clearly identify a suitable template for molecular modeling. Here, we used hydrophobicity profiles and hidden Markov models to define a structural repeat common to all SLC34 isoforms. Similar approaches identify a relationship with the core regions in a crystal structure of Vibrio cholerae Na(+)-dicarboxylate transporter VcINDY, from which we generated a homology model of human NaPi-IIa. The aforementioned SLC34 motifs in each repeat localize to the center of the model, and were predicted to form Na(+) and Pi coordination sites. Functional relevance of key amino acids was confirmed by biochemical and electrophysiological analysis of expressed, mutated transporters. Moreover, the validity of the predicted architecture is corroborated by extensive published structure-function studies. The model provides key information for elucidating the transport mechanism and predicts candidate substrate binding sites.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II/metabolismoRESUMEN
The SLC34 family of sodium-driven phosphate cotransporters comprises three members: NaPi-IIa (SLC34A1), NaPi-IIb (SLC34A2), and NaPi-IIc (SLC34A3). These transporters mediate the translocation of divalent inorganic phosphate (HPO4 (2-)) together with two (NaPi-IIc) or three sodium ions (NaPi-IIa and NaPi-IIb), respectively. Consequently, phosphate transport by NaPi-IIa and NaPi-IIb is electrogenic. NaPi-IIa and NaPi-IIc are predominantly expressed in the brush border membrane of the proximal tubule, whereas NaPi-IIb is found in many more organs including the small intestine, lung, liver, and testis. The abundance and activity of these transporters are mostly regulated by changes in their expression at the cell surface and are determined by interactions with proteins involved in scaffolding, trafficking, or intracellular signaling. All three transporters are highly regulated by factors including dietary phosphate status, hormones like parathyroid hormone, 1,25-OH2 vitamin D3 or FGF23, electrolyte, and acid-base status. The physiological relevance of the three members of the SLC34 family is underlined by rare Mendelian disorders causing phosphaturia, hypophosphatemia, or ectopic organ calcifications.
Asunto(s)
Fosfatos/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Animales , Factor-23 de Crecimiento de Fibroblastos , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/metabolismo , Humanos , Absorción Intestinal , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiología , Túbulos Renales/metabolismo , Túbulos Renales/fisiología , Fosfatos/deficiencia , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/química , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genéticaRESUMEN
We used voltage clamp fluorometry to probe the movement of the S4 helix in the voltage-sensing domain of the sea urchin HCN channel (spHCN) expressed in Xenopus oocytes. We obtained markedly different fluorescence responses with either ALEXA-488 or MTS-TAMRA covalently linked to N-terminal Cys332 of the S4 helix. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly, consistent with it reporting the initial inward movement of S4, as previously described. In contrast, MTS-TAMRA fluorescence increased more slowly and its early phase correlated with that of channel opening. Additionally, a slow fluorescence component that tracked the development of the mode shift, or channel hysteresis, could be resolved with both labels. We quantitated this component as an increased deactivation tail current delay with concomitantly longer activation periods and found it to depend strongly on the presence of K+ ions in the pore. Using collisional quenching experiments and structural predictions, we established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. We propose that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal distinct biophysical properties. Our findings underscore the need to apply caution when interpreting voltage clamp fluorometry data and demonstrate the potential utility of different labels to interrogate distinct biophysical properties of voltage-gated membrane proteins.