RESUMEN
A decrease in renal phosphate reabsorption with mild hypophosphatemia (phosphate leak) is found in some hypercalciuric stone-formers. The NPT2a gene encodes a sodium-phosphate cotransporter, located in the proximal tubule, responsible for reclaiming most of the filtered phosphate load in a rate-limiting manner. To determine whether genetic variation of the NPT2a gene is associated with phosphate leak and hypercalciuria in a cohort of 98 pedigrees with multiple hypercalciuric stone-formers, we sequenced the entire cDNA coding region of 28 probands, whose tubular reabsorption of phosphate normalized for the glomerular filtration rate (TmP/GFR) was 0.7 mmol/l or lower. We performed genotype/phenotype correlations for each genetic variant in the entire cohort and expressed NPT2a variant RNAs in Xenopus laevis oocytes to test for cotransporter functionality. We identified several variants in the coding region including an in-frame 21 bp deletion truncating the N-terminal cytoplasmic tail of the protein (91del7), as well as other single-nucleotide polymorphisms that were non-synonymous (A133V and H568Y) or synonymous. Levels of TmP/GFR and urine calcium excretion were similar in heterozygote carriers of NPT2a variants compared to the wild-type (wt) homozygotes. The transport activity of the H568Y mutants was identical to the wt, whereas the N-terminal-truncated version and the 91del7 and A133V mutants presented minor kinetic changes and a reduction in the expression level. Although genetic variants of NPT2a are not rare, they do not seem to be associated with clinically significant renal phosphate or calcium handling anomalies in a large cohort of hypercalciuric stone-forming pedigrees.
Asunto(s)
Calcio/orina , Hipofosfatemia/genética , Cálculos Renales/genética , Cálculos Renales/orina , Proteínas Cotransportadoras de Sodio-Fosfato/genética , Adulto , Anciano , Animales , Secuencia de Bases , ADN/análisis , ADN/genética , Exones/genética , Femenino , Pruebas Genéticas , Genotipo , Tasa de Filtración Glomerular/fisiología , Humanos , Hipofosfatemia/sangre , Hipofosfatemia/fisiopatología , Cálculos Renales/fisiopatología , Túbulos Renales Proximales/química , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/fisiopatología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Oocitos/química , Oocitos/fisiología , Linaje , Fosfatos/sangre , Polimorfismo Genético , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Cotransportadoras de Sodio-Fosfato/análisis , Proteínas Cotransportadoras de Sodio-Fosfato/fisiología , Xenopus laevisRESUMEN
In rabbit proximal convoluted tubules, an ATP-sensitive K(+) (K(ATP)) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na(+) transport and basolateral K(+) conductance. This K(+) conductance is inhibited by taurine. We sought to isolate this K(+) channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K(+) conductance, largely inhibited by extracellular Ba(2+) and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K(+) current which was Ba(2+)-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K(+) channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K(+) current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K(+) current. The possible involvement of AK in the K(ATP) channel's regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations.
Asunto(s)
Adenilato Quinasa/biosíntesis , Túbulos Renales Proximales/metabolismo , Oocitos/metabolismo , Canales de Potasio/genética , Adenosina Trifosfato/análisis , Adenilato Quinasa/química , Adenilato Quinasa/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/biosíntesis , ADN Complementario/química , Electroquímica , Biblioteca de Genes , Gliburida , Técnicas In Vitro , Manitol , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Canales de Potasio/química , Canales de Potasio/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Conejos , Alineación de Secuencia , Taurina , Transfección , XenopusRESUMEN
K channels are ubiquitous in animal cells, where they are involved in a variety of physiological functions. In epithelial cells of the kidney, K channels are primarily involved in maintaining membrane potential, recycling and secreting K and regulating cell volume. As many renal K channels have now been studied or identified at the molecular level by means of a variety of approaches, including patch-clamp recordings, cDNA cloning and immunohistochemistry, the purpose of this review is to summarize what is presently known about the molecular identity of renal K channels with an emphasis on their regulatory properties.
Asunto(s)
Túbulos Renales/fisiología , Canales de Potasio/química , Canales de Potasio/fisiología , Animales , Humanos , Canales de Potasio/clasificaciónRESUMEN
We obtained cDNA chimeras between Na/glucose cotransporter (SGLT1) and the homologous Na(+)/myo-inositol cotransporter (SMIT) by creating random chimeras in plasmids. Of 12 chimeras, two were functional when expressed in Xenopus laevis oocytes but, upon sequencing, only one of them (C1) produced an actual chimeric protein. In C1, the first 69 amino acids of SGLT1 were replaced by the corresponding 50 amino acids of SMIT. C1 transports the same sugars as does SGLT1. C1's affinity for all sugar substrates was systematically increased by a factor of 3.3+/-0.4 but the V(max) was diminished by a factor of 15-40. In contrast, the cotransport affinity for Na(+) was unchanged. The surface expression of C1 was one seventh that of SGLT1, which explains part of the reduced V(max) and implies a significant reduction in turnover rate. N-terminal truncated constructs of SGLT1 cDNA showed that deleting amino acids 2-14 does not affect cotransporter activity, but that the pentapeptide T(14)RPVET(19) is important for normal levels of SGLT1 current. The main result of a kinetic analysis of the systematic increase in apparent affinity for sugars, together with the intact Na apparent affinity, suggests enhanced access to the sugar binding site in C1.
Asunto(s)
Proteínas Portadoras/fisiología , Glucosa/metabolismo , Proteínas de Choque Térmico/fisiología , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana , Proteínas de Transporte de Monosacáridos/fisiología , Sodio/metabolismo , Simportadores , 3-O-Metilglucosa/metabolismo , 3-O-Metilglucosa/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN Complementario , Galactosa/metabolismo , Galactosa/farmacología , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Inositol/metabolismo , Inositol/farmacología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Metilglucósidos/metabolismo , Metilglucósidos/farmacología , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Florizina/metabolismo , Proteínas Recombinantes de Fusión/genética , Transportador 1 de Sodio-Glucosa , Xenopus laevisRESUMEN
1. High-affinity, secondary active transport of glucose in the intestine and kidney is mediated by an integral membrane protein named SGLT1 (sodium glucose cotransporter). Though basic properties of the transporter are now defined, many questions regarding the structure- function relationship of the protein, its biosynthesis and targeting remain unanswered. In order to better address these questions, we produced a functional hSGLT1 protein (from human) containing a reporter tag. 2. Six constructs, made from three tags (myc, haemaglutinin and poly-His) inserted at both the C- and N-terminal positions, were thus tested using the Xenopus oocyte expression system via electrophysiology and immunohistochemistry. Of these, only the hSGLT1 construct with the myc tag inserted at the N-terminal position proved to be of interest, all other constructs showing no or little transport activity. A systematic comparison of transport properties was therefore performed between the myc-tagged and the untagged hSGLT1 proteins. 3. On the basis of both steady-state (affinities for substrate (glucose) and inhibitor (phlorizin) as well as expression levels) and presteady-state parameters (transient currents) we conclude that the two proteins are functionally indistinguishable, at least under these criteria. Immunological detection confirmed the appropriate targeting of the tagged protein to the plasma membrane of the oocyte with the epitope located at the extracellular side. 4. The myc-tagged hSGLT1 was also successfully expressed in polarized MDCK cells. alpha-Methylglucose uptake studies on transfected cells showed an exclusively apical uptake pathway, thus indicating that the expressed protein was correctly targeted to the apical domain of the cell. 5. These comparative studies demonstrate that the myc epitope inserted at the N-terminus of hSGLT1 produces a fully functional protein while other epitopes of similar size inserted at either end of the protein inactivated the final protein.
Asunto(s)
Histidina , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Monosacáridos/genética , Animales , Transporte Biológico , Línea Celular , Membrana Celular/metabolismo , Perros , Epítopos , Técnica del Anticuerpo Fluorescente , Expresión Génica , Hemaglutininas/genética , Hemaglutininas/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Metilglucósidos/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Oocitos , Técnicas de Placa-Clamp , Péptidos/genética , Péptidos/metabolismo , Florizina/farmacología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transportador 1 de Sodio-Glucosa , Transfección , Xenopus laevisRESUMEN
The HIV-1-specific vpu gene encodes an integral membrane phosphoprotein which affects three aspects of the HIV-1 infectious cycle: it enhances virion release from infected cells; it causes degradation of the CD4 protein in the endoplasmic reticulum; and it delays syncytia formation in HIV-1-infected CD4+ T-cells. Although little is known about how Vpu mediates these effects, it has been proposed to function as a nonspecific cation channel. In this report, voltage clamp measurements of Xenopus oocytes show that Vpu expression is not associated with increased transmembrane currents. Instead, Vpu expression diminishes membrane conductance. Injection of 4.6 ng of Vpu mRNA into these cells reduces endogenous potassium conductance by 50%. Only Vpu mutants which retain the ability to degrade CD4 can diminish K+ conductance. Inhibition by Vpu is not unique to K+ channels as it is also observed on several coexpressed membrane proteins but not on a coexpressed cytoplasmic protein. These results indicate that the CD4 degradative capability of Vpu and the Vpu-mediated modulation of membrane protein expression are mechanistically coupled and that Vpu may contribute to HIV pathogenesis by altering plasma membrane protein expression at the cell surface.
Asunto(s)
Membrana Celular/fisiología , Proteínas Reguladoras y Accesorias Virales/metabolismo , Animales , Conductividad Eléctrica , Expresión Génica , VIH-1 , Proteínas del Virus de la Inmunodeficiencia Humana , Mutagénesis Sitio-Dirigida , Oocitos , Canales de Potasio/fisiología , ARN Mensajero , Proteínas Recombinantes , Proteínas Reguladoras y Accesorias Virales/genética , Xenopus laevisRESUMEN
The Na+-glucose cotransporter (SGLT1) expressed in Xenopus laevis oocytes was shown to generate a phlorizin-sensitive sodium leak in the absence of sugars. Using the current model for SGLT1, where the sodium leak was presumed to occur after two sodium ions are bound to the free carrier before glucose binding, a characteristic concentration constant (Kc) was introduced to describe the relative importance of the sodium leak versus Na+-glucose cotransport currents. Kc represents the glucose concentration at which the Na+-glucose cotransport current is equal to the sodium leak. As both the sodium leak and the Na+-glucose cotransport current are predicted to occur after the binding of two sodium ions, the model predicted that Kc should be sodium-independent. However, by using a two-microelectrode voltage-clamp technique, the observed Kc was shown to depend strongly on the external sodium concentration ([Na+]o): it was four times higher at 5 mM [Na+]o than at 20 mM [Na+]o. In addition, the magnitude of the sodium leak varied as a function of [Na+]o in a Michaelian fashion, and the sodium affinity constant for the sodium leak was 2-4 times lower than that for cotransport in the presence of low external glucose concentrations (50 or 100 microM), whereas the current model predicted a sigmoidal sodium dependence of the sodium leak and identical sodium affinities for the sodium leak and the Na+-glucose cotransport. These observations indicate that the sodium leak occurs after one sodium ion is associated with the carrier and agree with predictions from a model with the binding order sodium-glucose-sodium. This conclusion was also supported by experiments performed where protons replaced Na+ as a "driving cation."
Asunto(s)
Glucosa/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Oocitos/metabolismo , Sodio/metabolismo , Animales , Electrofisiología , Expresión Génica , Concentración de Iones de Hidrógeno , Transporte Iónico , Cinética , Microelectrodos , Microinyecciones , Modelos Biológicos , Técnicas de Placa-Clamp , Florizina/farmacología , Unión Proteica , Transportador 1 de Sodio-Glucosa , Xenopus laevisRESUMEN
The human Na(+)-glucose cotransporter (hSGLT1) has been shown to generate, in the absence of sugar, presteady-state currents in response to a change in potential, which could be fitted with single exponentials once the voltage had reached a new constant value. By the cut-open oocyte technique (voltage rising-speed approximately 1 mV/microsecond), phlorizin-sensitive transient currents could be detected with a higher time resolution during continuous intracellular perfusion. In the absence of sugar and internal Na+, and with 90 mM external Na+ concentration ([Na+]o), phlorizin-sensitive currents exhibited two relaxation time-constants: tau 1 increased from 2 to 10 ms when Vm decreased from +60 mV to -80 mV and remained at 10 ms for more negative Vm; tau 2 ranged from 0.4 to 0.8 ms in a weakly voltage-dependent manner. According to a previously proposed model, these two time constants could be accounted for by 1) Na+ crossing a fraction of the membrane electrical field to reach its binding site on the carrier and 2) conformational change of the free carrier. To test this hypothesis, the time constants were measured as [Na+]o was progressively reduced to 0 mM. At 30 and 10 mM external Na+, tau 1 reached the same plateau value of 10 ms but at more negative potentials (-120 and -160 mV, respectively). Contrary to the prediction of the model, two time constants continued to be detected in the bilateral absence of Na+ (at pH 8.0). Under these conditions, tau 1 continuously increased through the whole voltage range and did not reach the 10 ms level even when Vm had attained -200 mV while tau 2 remained in the range of 0.4-0.8 ms. These results indicate that 1) conformational change of the free carrier across the membrane must occur in more than one step and 2) Na+ binding/debinding is not responsible for either of the two observed exponential components of transient currents. By use of the simplest kinetic model accounting for the portion of the hSGLT1 transport cycle involving extracellular Na+ binding/debinding and the dual-step conformational change of the free carrier, tau 1 and tau 2 were fitted throughout the voltage range, and a few sets of parameters were found to reproduce the data satisfactorily. This study shows that 1) tau 1 and tau 2 correspond to two steps in the conformational change of the free carrier, 2) Na+ binding/debinding modulates the slow time constant (tau 1) and 3) a voltage-independent slow conformational change of the free carrier accounts for the observed plateau value of 10 ms.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Sodio/fisiología , Animales , Conductividad Eléctrica , Glucosa/fisiología , Humanos , Potenciales de la Membrana , Oocitos/fisiología , Florizina/farmacología , Proteínas Recombinantes , Transportador 1 de Sodio-Glucosa , Xenopus laevisRESUMEN
The question of sugar transport heterogeneity in the human intestinal Caco-2 cell line was addressed using alpha-methyl-D-glucose (AMG) and 2-deoxy-D-glucose (DG) as substrate analogues for D-glucose, the transport inhibitors phlorizin (PZ) and phloretin (PT), and NaCl or choline chloride uptake media. The data are compatible with the existence of three distinct pathways that can be isolated kinetically according to specific characteristics: 1) an "AMG-strict" system, strictly Na+ dependent and specific for AMG [Michaelis-Menten constant value (K(m)) = 2.0 +/- 0.3 mM] but sensitive to both PZ and PT, with PZ being more potent than PT, 2) a "DG-strict" system, strictly Na+ independent and specific for both DG (K(m) = 5.2 +/- 0.5 mM) and PT; and 3) a "DG/AMG-mixed" system, strictly Na+ dependent, with loose specificities for the glucose analogues DG (K(m) = 0.81 +/- 0.07 mM) and AMG (K(m) = 8.1 +/- 0.8 mM), and the inhibitors PZ and PT, but with PT being more potent than PZ. Since SGLT-1 obtained by polymerase chain reaction from either Caco-2 cells or normal human jejunum demonstrated identical transport properties when expressed in Xenopus laevis oocytes, we conclude that the "AMG-strict" system represents the expression of human SGLT-1 activity in this cell line. Moreover, Western blot analysis revealed that SGLT-1 is located exclusively in the apical membrane. In contrast, neither the nature nor the membrane location of both the DG-strict and DG/AMG-mixed pathways could be resolved unambiguously. Still it has been demonstrated that expression of the latter system is constitutive to all Caco-2 cells and that its Na+ dependence is not the consequence of H(+)-dependent transport activity. Aside from the presence of the DG/AMG-mixed system, a salient feature of Caco-2 cells is that the GLUT-3 protein is located exclusively in the brush-border membrane. Due to these limitations, it is concluded that the Caco-2 cell line cannot be considered as equivalent to either fetal colonic cells or normal enterocytes.
Asunto(s)
Células CACO-2/metabolismo , Carbohidratos/farmacocinética , Proteínas del Tejido Nervioso , Transporte Biológico , Senescencia Celular , Desoxiglucosa/farmacocinética , Transportador de Glucosa de Tipo 3 , Humanos , Glicoproteínas de Membrana/farmacocinética , Metilglucósidos/farmacocinética , Proteínas de Transporte de Monosacáridos/farmacocinética , Protones , Sodio/fisiología , Transportador 1 de Sodio-GlucosaRESUMEN
The rBAT protein, when expressed in Xenopus oocytes, was previously shown to reproduce the selectivity of the Na(+)-independent neutral and basic amino acid transport system called bo,+. More recently, the capacity of rBAT to generate a transmembrane current was demonstrated when addition of neutral amino acids stimulated the efflux of cations (presumably basic amino acids) in rBAT-injected oocytes. In the present paper, aminoisobutyric acid (AIB), a neutral amino acid analogue, was shown to induce outward currents (efflux of basic amino acids) through rBAT similar to those caused by alanine in terms of affinity, maximal currents and I-V curves. Despite generating similar currents, the AIB transport rate was more than 30 times lower than that of alanine, thus challenging the assumption that rBAT functions as a classical exchanger. Experiments using a cut-open oocyte voltage clamp demonstrated that AIB was capable of stimulating rBAT-mediated currents from either side of the membrane. AIB, like alanine, was able to stimulate the efflux of radiolabeled alanine and arginine while no rBAT-mediated efflux was measurable in the absence of external rBAT substrates. These results demonstrate that (i) the presence of amino acids is required on both sides of the membrane for rBAT to mediate amino acid flux and thus rBAT must be some type of exchanger but (ii) rBAT-mediated amino acid influx is not stoichiometrically related to the efflux. A model of a "double gated pore" is proposed to account for these properties of rBAT, which contravene standard models of exchangers and other transporters.
Asunto(s)
Alanina/metabolismo , Sistemas de Transporte de Aminoácidos Básicos , Ácidos Aminoisobutíricos/metabolismo , Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana/metabolismo , Técnicas de Placa-Clamp , Aminoácidos/metabolismo , Animales , Proteínas Portadoras/genética , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Glicoproteínas de Membrana/genética , Oocitos/fisiología , Xenopus laevisRESUMEN
Phlorizin-sensitive currents mediated by a Na-glucose cotransporter were measured using intact or internally perfused Xenopus laevis oocytes expressing human SGLT1 cDNA. Using a two-microelectrode voltage clamp technique, measured reversal potentials (Vr) at high external alpha-methylglucose (alpha MG) concentrations were linearly related to In[alpha MG]o, and the observed slope of 26.1 +/- 0.8 mV/decade indicated a coupling ratio of 2.25 +/- 0.07 Na ions per alpha MG molecule. As [alpha MG]o decreased below 0.1 mM, Vr was no longer a linear function of In[alpha MG]o, in accordance with the suggested capacity of SGLT1 to carry Na in the absence of sugar (the "Na leak"). A generalized kinetic model for SGLT1 transport introduces a new parameter, Kc, which corresponds to the [alpha MG]o at which the Na leak is equal in magnitude to the coupled Na-alpha MG flux. Using this kinetic model, the curve of Vr as a function of In[alpha MG]o could be fitted over the entire range of [alpha MG]o if Kc is adjusted to 40 +/- 12 microM. Experiments using internally perfused oocytes revealed a number of previously unknown facets of SGLT1 transport. In the bilateral absence of alpha MG, the phlorizin-sensitive Na leak demonstrated a strong inward rectification. The affinity of alpha MG for its internal site was low; the Km was estimated to be between 25 and 50 mM, an order of magnitude higher than that found for the extracellular site. Furthermore, Vr determinations at varying alpha MG concentrations indicate a transport stoichiometry of 2 Na ions per alpha MG molecule: the slope of Vr versus In[alpha MG]o averaged 30.0 +/- 0.7 mV/decade (corresponding to a stoichiometry of 1.96 +/- 0.04 Na ions per alpha MG molecule) whenever [alpha MG]o was higher than 0.1 mM. These direct observations firmly establish that Na ions can utilize the SGLT1 protein to cross the membrane either alone or in a coupled manner with a stoichiometry of 2 Na ions per sugar, molecule.
Asunto(s)
Glucosa/metabolismo , Glicoproteínas de Membrana/fisiología , Modelos Biológicos , Proteínas de Transporte de Monosacáridos/fisiología , Sodio/metabolismo , Animales , ADN Complementario , Femenino , Humanos , Cinética , Matemática , Glicoproteínas de Membrana/biosíntesis , Potenciales de la Membrana , Microelectrodos , Proteínas de Transporte de Monosacáridos/biosíntesis , Oocitos/fisiología , Técnicas de Placa-Clamp , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Transportador 1 de Sodio-Glucosa , Termodinámica , Xenopus laevisRESUMEN
Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na(+)-K(+)-ATPase activity from the dihydroouabain-sensitive current (IDHO) in the presence of increasing concentrations of tetraethylammonium (TEA+; 0, 5, 10, 20, 40 mM), a well-known blocker of K+ channels. The effects of TEA+ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (VM) and a saturable inhibitory component affecting an outward current easily detectable at positive VM. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA+ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA+ on K+ channels. Interestingly, this latter component disappears when the Na(+)-K(+)-ATPase is inhibited by 10 microM DHO. Conversely, TEA+ inhibits a component of IDHO with a KD of 25 +/- 4 mM at +50 mV. As the TEA(+)-sensitive current present in IDHO reversed at -75 mV, we hypothesized that it could come from an inhibition of K+ channels whose activity varies in parallel with the Na(+)-K(+)-ATPase activity. Supporting this hypothesis, the inward portion of this TEA(+)-sensitive current can be completely abolished by the addition of 1 mM Ba2+ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA(+)-sensitive K+ currents and indicates that, in the absence of effective K+ channel inhibitors, IDHO does not exclusively represent the Na(+)-K(+)-ATPase-generated current.
Asunto(s)
Oocitos/fisiología , Bloqueadores de los Canales de Potasio , Canales de Potasio/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Compuestos de Tetraetilamonio/farmacología , Xenopus laevis/fisiología , Animales , Membrana Celular/química , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Electrofisiología , Femenino , Potenciales de la Membrana/fisiología , Oocitos/química , Oocitos/ultraestructura , Ouabaína/análogos & derivados , Ouabaína/farmacología , Técnicas de Placa-Clamp , Canales de Potasio/análisis , TetraetilamonioRESUMEN
A cDNA clone was isolated from rabbit renal cortex using DNA-mediated expression cloning, which caused alanine-dependent outward currents when expressed in Xenopus oocytes. The cDNA encodes rBAT, a Na-independent amino acid transporter previously cloned elsewhere. Exposure of cDNA-injected oocytes to neutral amino acids led to voltage-dependent outward currents, but inward currents were seen upon exposure to basic amino acids. Assuming one charge/alanine, the outward current represented 38% of the rate of uptake of radiolabelled alanine, and was significantly reduced by prolonged preincubation of oocytes in 5 mM alanine. The currents were shown to be due to countertransport of basic amino acids for external amino acids using the cut-open oocyte system. This transport represents a major mode of action of this protein, and may help in defining a physiological role for rBAT in the apical membrane of renal and intestinal cells.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos , Aminoácidos/metabolismo , Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana/metabolismo , Oocitos/fisiología , Alanina/metabolismo , Alanina/farmacología , Animales , Arginina/metabolismo , Arginina/farmacología , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/biosíntesis , Clonación Molecular , ADN Complementario , Femenino , Corteza Renal/metabolismo , Glicoproteínas de Membrana/biosíntesis , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido Nucleico , Xenopus laevisRESUMEN
We have injected mRNA from rabbit renal cortex into Xenopus oocytes and demonstrated the expression of renal carriers for Na(+)-independent transport of L-phenylalanine and L-lysine and Na(+)-dependent transport of L-alanine and succinate. Maximal expression of renal amino acid transporters occurred 6-8 days following mRNA injection. The proteins responsible for transport of these four substrates were translated from mRNAs which are between 1.5 and 3.0 kb. This information serves as a starting point for expression cloning of these transport proteins.
Asunto(s)
Aminoácidos/metabolismo , Proteínas Portadoras/genética , Corteza Renal/fisiología , Oocitos/fisiología , Sistemas de Transporte de Aminoácidos , Animales , Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/metabolismo , Femenino , Cinética , Microinyecciones , Peso Molecular , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Conejos , Sodio/farmacología , Xenopus laevisRESUMEN
Sodium-dependent glucose transport occurs in the intestine and kidney of most animal species. The cDNA encoding the Na+/glucose cotransporter from rabbit jejunum was used to examine the distribution of homologous mRNA in other rabbit tissues and in the intestines of other species. Northern blots of mRNA extracted from various tissues were probed with radiolabeled cDNA of the cloned rabbit transporter. The probe hybridized with mRNA of approximately 2.2 kb from rabbit jejunum, renal cortex, and renal medulla, indicating that related mRNA of the same size is found in these tissues. With the use of the same cDNA probe, a 1.6-kb partial-length clone encoding 484 amino acids was isolated from a rabbit renal cortex cDNA library. There was greater than 99% identity between the cDNA sequences, and 100% identity between the amino acid sequences, of the renal clone and the rabbit intestinal Na+/glucose cotransporter. The 2.2-kb transcript was seen in mRNA from duodenum, jejunum, and ileum, with a distribution that matched the Na+/glucose transport capacity in these tissues. A faint signal at 2.2 kb was also seen in colon mRNA. There was no detectable hybridization to blots of stomach and heart mRNA. The rabbit probe also hybridized to intestinal mRNA from a number of species from trout to humans. We conclude that a Na+/glucose cotransporter of rabbit renal cortex is very similar to that of the intestine and that the intestinal transporter has been conserved during evolution.
Asunto(s)
Mucosa Gástrica/metabolismo , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Glucosa/metabolismo , Humanos , Corteza Renal/metabolismo , Médula Renal/metabolismo , Datos de Secuencia Molecular , Necturus , ARN Mensajero/genética , Conejos , Homología de Secuencia de Ácido Nucleico , Sodio/metabolismo , Especificidad de la Especie , TruchaRESUMEN
The cloned rabbit intestinal Na+/glucose cotransporter was expressed in Xenopus oocytes, and transmembrane currents associated with this transporter were monitored using a two-electrode voltage clamp. Addition of D-glucose to a Na(+)-containing solution bathing these oocytes generated a current which was blocked by phlorizin. Water-injected control oocytes did not exhibit any currents under these conditions. The magnitude and shape of the currents were dependent on the extracellular glucose and Na+ concentrations and the membrane potential. At Vhold = -50 mV, the Km values for glucose and Na+ were 14 +/- 2 (N = 4) microM and 17 +/- 1 (N = 3) mM, respectively. These Km values and imax exhibited voltage dependence: increasing the membrane potential from -30 to -150 mV increased KGlcm and imax threefold and decreased KNam eightfold. The reversal potential (VR) of the phlorizin-sensitive, glucose-dependent current varied with log Nao+ (slope 46 +/- 6 [N = 9] mV). In the absence of sugar, a Na(+)-dependent, phlorizin-sensitive (Ki = 3 +/- 0.5 microM) current was detected only in RNA-injected oocytes. The amplitude of this current at -50 mV was 6 +/- 1% (N = 13) of the maximum current measured in the presence of D-glucose. The VR of this sugar-independent current varied with log Nao+ (slope 63 +/- 1 [N = 4] mV), indicating that the cotransporter may carry Na+ in the absence of sugar. We conclude that the Na+/glucose cotransporter is electrogenic and that investigations of currents associated with its operation can yield valuable insights into the mechanisms of solute translocation.
Asunto(s)
Intestinos/fisiología , Proteínas de Transporte de Monosacáridos/fisiología , Oocitos/fisiología , Animales , Conductividad Eléctrica , Femenino , Técnicas In Vitro , Canales Iónicos/efectos de los fármacos , Canales Iónicos/fisiología , Cinética , Matemática , Potenciales de la Membrana/efectos de los fármacos , Metilglucósidos/farmacología , Microinyecciones , Modelos Teóricos , Proteínas de Transporte de Monosacáridos/genética , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , Conejos , Análisis de Regresión , Sodio/farmacología , Xenopus laevisRESUMEN
The Na+/glucose cotransporter from rabbit intestinal brush border membranes has been cloned, sequenced, and expressed in Xenopus oocytes. Injection of cloned RNA into oocytes increased Na+/sugar cotransport by three orders of magnitude. In this study, we have compared and contrasted the transport properties of this cloned protein expressed in Xenopus oocytes with the native transporter present in rabbit intestinal brush borders. Initial rates of 14C-alpha-methyl-D-glucopyranoside uptake into brush border membrane vesicles and Xenopus oocytes were measured as a function of the external sodium, sugar, and phlorizin concentrations. Sugar uptake into oocytes and brush borders was Na+-dependent (Hill coefficient 1.5 and 1.7), phlorizin inhibitable (Ki 6 and 9 microM), and saturable (alpha-methyl-D-glucopyranoside Km 110 and 570 microM). The sugar specificity was examined by competition experiments, and in both cases the selectivity was D-glucose greater than alpha-methyl-D-glucopyranoside greater than D-galactose greater than 3-O-methyl-D-glucoside. In view of the close similarity between the properties of the cloned protein expressed in oocytes and the native brush border transporter, we conclude that we have cloned the classical Na+/glucose cotransporter.
Asunto(s)
Intestino Delgado/análisis , Proteínas de Transporte de Monosacáridos/análisis , Animales , Clonación Molecular , Glucosa/metabolismo , Glucosa/farmacocinética , Intestino Delgado/metabolismo , Intestino Delgado/fisiología , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Florizina/farmacología , Conejos , Sodio/metabolismo , Sodio/farmacocinética , Especificidad por SustratoRESUMEN
Injection of Xenopus laevis oocytes with rabbit heart poly(A)+RNA results in expression of Na+ inside (Nai+)-dependent Ca2+ uptake activity. The activity was measured by first loading the oocytes with Na+ using nystatin and then incubating the oocytes in K+ or Na+ medium containing 45Ca. The expressed Na+ gradient-dependent Ca2+ uptake was five to eight times that observed with water-injected oocytes or with poly(A)+RNA-injected oocytes for which the Na+ load step had been omitted. Induced activity was related to the amount of RNA injected and was insensitive to nifedipine. Fractionation of the poly(A)+RNA on a sucrose gradient determined that the active message had a size range between 3 and 5 kb. The properties of the Na+ gradient-dependent Ca2+ uptake indicated that Na+-Ca2+ exchange activity had been expressed in X. laevis oocytes. The result may be useful for cloning and identifying the molecular component responsible for Na+-Ca2+ exchange.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/genética , Miocardio/metabolismo , Oocitos/metabolismo , Sarcolema/metabolismo , Animales , Proteínas Portadoras/metabolismo , Femenino , Técnicas In Vitro , Cinética , Poli A/genética , Poli A/aislamiento & purificación , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/genética , Conejos , Intercambiador de Sodio-Calcio , Xenopus laevisRESUMEN
Organic substrates (sugars, amino acids, carboxylic acids and neutrotransmitters) are actively transported into eukaryotic cells by Na+ co-transport. Some of the transport proteins have been identified--for example, intestinal brush border Na+/glucose and Na+/proline transporters and the brain Na+/CI-/GABA transporter--and progress has been made in locating their active sites and probing their conformational states. The archetypical Na+-driven transporter is the intestinal brush border Na+/glucose co-transporter (see ref. 8), and a defect in the co-transporter is the origin of the congenital glucose-galactose malabsorption syndrome. Here we describe cloning of this co-transporter by a method new to membrane proteins. We have sequenced the cloned DNA and have found no homology between the Na+/glucose co-transporter and either the mammalian facilitated glucose carrier or the bacterial sugar transport proteins. This suggests that the mammalian Na+-driven transporter has no evolutionary relationship to the other sugar transporters.