RESUMEN
GPR40 (FFAR1 or FFA1) is a G protein-coupled receptor, primarily expressed in pancreatic islet ß-cells and intestinal enteroendocrine cells. When activated by fatty acids, GPR40 elicits increased insulin secretion from islet ß-cells only in the presence of elevated glucose levels. Towards this end, studies were undertaken towards discovering a novel GPR40 Agonist whose mode of action is via Positive Allosteric Modulation of the GPR40 receptor (AgoPAM). Efforts were made to identify a suitable GPR40 AgoPAM tool molecule to investigate mechanism of action and de-risk liver toxicity of GPR40 AgoPAMs due to reactive acyl-glucuronide (AG) metabolites.
Asunto(s)
Indanos/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Diseño de Fármacos , HumanosRESUMEN
G protein-coupled receptor 40 (GPR40) partial agonists lower glucose through the potentiation of glucose-stimulated insulin secretion, which is believed to provide significant glucose lowering without the weight gain or hypoglycemic risk associated with exogenous insulin or glucose-independent insulin secretagogues. The class of small-molecule GPR40 modulators, known as AgoPAMs (agonist also capable of acting as positive allosteric modulators), differentiate from partial agonists, binding to a distinct site and functioning as full agonists to stimulate the secretion of both insulin and glucagon-like peptide-1 (GLP-1). Here we show that GPR40 AgoPAMs significantly increase active GLP-1 levels and reduce acute and chronic food intake and body weight in diet-induced obese (DIO) mice. These effects of AgoPAM treatment on food intake are novel and required both GPR40 and GLP-1 receptor signaling pathways, as demonstrated in GPR40 and GLP-1 receptor-null mice. Furthermore, weight loss associated with GPR40 AgoPAMs was accompanied by a significant reduction in gastric motility in these DIO mice. Chronic treatment with a GPR40 AgoPAM, in combination with a dipeptidyl peptidase IV inhibitor, synergistically decreased food intake and body weight in the mouse. The effect of GPR40 AgoPAMs on GLP-1 secretion was recapitulated in lean, healthy rhesus macaque demonstrating that the putative mechanism mediating weight loss translates to higher species. Together, our data indicate effects of AgoPAMs that go beyond glucose lowering previously observed with GPR40 partial agonist treatment with additional potential for weight loss.
Asunto(s)
Regulación del Apetito/genética , Peso Corporal/genética , Ingestión de Alimentos/genética , Péptido 1 Similar al Glucagón/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Pérdida de Peso/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genéticaRESUMEN
The goal of designing safer, more effective drugs has led to tremendous interest in molecular mechanisms through which ligands can precisely manipulate signaling of G-protein-coupled receptors (GPCRs), the largest class of drug targets. Decades of research have led to the widely accepted view that all agonists-ligands that trigger GPCR activation-function by causing rearrangement of the GPCR's transmembrane helices, opening an intracellular pocket for binding of transducer proteins. Here we demonstrate that certain agonists instead trigger activation of free fatty acid receptor 1 by directly rearranging an intracellular loop that interacts with transducers. We validate the predictions of our atomic-level simulations by targeted mutagenesis; specific mutations which disrupt interactions with the intracellular loop convert these agonists into inverse agonists. Further analysis suggests that allosteric ligands could regulate signaling of many other GPCRs via a similar mechanism, offering rich possibilities for precise control of pharmaceutically important targets.
RESUMEN
Niemann-Pick C1-like protein (NPC1L1) mediates the absorption of dietary cholesterol in the proximal region of the intestine, a process that is blocked by cholesterol absorption inhibitors (CAIs), including ezetimibe (EZE). Using a proteomic approach, we demonstrate that NPC1L1 is the protein to which EZE and its analogs bind. Next, we determined the site of interaction of EZE analogs with NPC1L1 by exploiting the different binding affinities of mouse and dog NPC1L1 for the radioligand analog of EZE, [(3)H]AS. Chimeric and mutational studies indicate that high-affinity binding of [(3)H]AS to dog NPC1L1 depends on molecular determinants present in a 61-aa region of a large extracellular domain (loop C), where Phe-532 and Met-543 appear to be key contributors. These data suggest that the [(3)H]AS-binding site resides in the intestinal lumen and are consistent with preclinical data demonstrating in vivo efficacy of a minimally bioavailable CAI. Furthermore, these determinants of [(3)H]AS binding lie immediately adjacent to a hotspot of human NPC1L1 polymorphisms correlated with hypoabsorption of cholesterol. These observations, taken together with the recently described binding of cholesterol to the N terminus (loop A) of the close NPC1L1 homologue, NPC1, may provide a molecular basis for understanding EZE inhibition of NPC1L1-mediated cholesterol absorption. Specifically, EZE binding to an extracellular site distinct from where cholesterol binds prevents conformational changes in NPC1L1 that are necessary for the translocation of cholesterol across the membrane.
Asunto(s)
Anticolesterolemiantes/farmacología , Azetidinas/farmacología , Colesterol en la Dieta/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/genética , Línea Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Perros , Ezetimiba , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/genética , Proteínas de Transporte de Membrana/genética , Ratones , Mutación , Polimorfismo Genético , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Proteómica/métodosRESUMEN
Voltage-gated sodium (Na(V)1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to Na(V)1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding Na(V)1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two inherited painful neuropathies. Although the human genetic data make Na(V)1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires Na(V)1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with Na(V)1.7 channels, inhibiting Na(V)1.7 with an IC(50) value of 0.3 nM, compared with IC(50) values of 30 to 150 nM for other heterologously expressed Na(V)1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little effect on Abeta-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve, which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II ((125)I-ProTx-II) binds with high affinity (K(d) = 0.3 nM) to recombinant hNa(V)1.7 channels. Binding of (125)I-ProTx-II is insensitive to the presence of other well characterized Na(V)1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the (125)I-ProTx-II binding assay, described here, offers a new tool in the search for novel Na(V)1.7-selective blockers.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Nociceptores/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/efectos de los fármacos , Venenos de Araña/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Activación del Canal Iónico , Masculino , Modelos Animales , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , Canales de Sodio/química , Canales de Sodio/genética , Canales de Sodio/fisiologíaRESUMEN
Absorption of dietary cholesterol in the proximal region of the intestine is mediated by Niemann-Pick C1-like protein (NPC1L1) and is sensitive to the cholesterol absorption inhibitor ezetimibe (EZE). Although a correlation exists between EZE binding to NPC1L1 in vitro and efficacy in vivo, the precise nature of interaction(s) between NPC1L1, EZE, and cholesterol remain unclear. Here, we analyze the direct relationship between EZE analog binding to NPC1L1 and its influence on cholesterol influx in a novel in vitro system. Using the EZE analog [(3)H]AS, an assay that quantitatively measures the expression of NPC1L1 on the cell surface has been developed. It is noteworthy that whereas two cell lines (CaCo-2 and HepG2) commonly used for studying NPC1L1-dependent processes express almost undetectable levels of NPC1L1 at the cell surface, polarized Madin-Darby canine kidney (MDCKII) cells endogenously express 4 x 10(5) [(3)H]AS sites/cell under basal conditions. Depleting endogenous cholesterol with the HMG CoA reductase inhibitor lovastatin leads to a 2-fold increase in the surface expression of NPC1L1, supporting the contention that MDCKII cells respond to changes in cholesterol homeostasis by up-regulating a pathway for cholesterol influx. However, a significant increase in surface expression levels of NPC1L1 is necessary to characterize a pharmacologically sensitive, EZE-dependent pathway of cholesterol uptake in these cells. Remarkably, the affinity of EZE analogs for binding to NPC1L1 is almost identical to the IC(50) blocking cholesterol flux through NPC1L1 in MDCKII cells. From a mechanistic standpoint, these observations support the contention that EZE analogs and cholesterol share the same/overlapping binding site(s) or are tightly coupled through allosteric interactions.
Asunto(s)
Azetidinas/metabolismo , Colesterol/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Azetidinas/química , Células CACO-2 , Línea Celular , Clonación Molecular , Perros , Ezetimiba , Humanos , Proteínas de Transporte de Membrana/metabolismo , Reproducibilidad de los Resultados , Sitoesteroles/metabolismo , Sulfonamidas/química , Transfección , Tritio , beta-Lactamas/metabolismoRESUMEN
Secondary active glucose transport is mediated by at least four members of the solute-linked carrier 5 gene family (sodium/glucose transporter [SGLT] 1-4). Human genetic disorders of SGLTs including glucose-galactose malabsorption and familial renal glucosuria have increased attention on members of this family of transporters as putative drug targets. Using human SGLT1 (hSGLT1) as a paradigm, we developed a functional assay that should be adaptable to ultra-high-throughput operation and to other SGLTs. Human embryonic kidney (HEK) 293 cells stably expressing hSGLT1 (hSGLT1/HEK293 cells) display a Na(+)-dependent, phlorizin-sensitive alpha-methyl-D-[(14)C]glucopyranoside flux with expected kinetic parameters. In electrophysiological studies with hSGLT1/HEK293 cells, substrate-dependent changes in membrane potential were observed, consistent with the electrogenic operation of hSGLT1. With the use of voltage-sensitive dyes, a membrane potential, fluorescence resonance energy transfer-based functional assay on a voltage/ion probe reader platform has been established for SGLT1. This high-capacity functional assay displays similar characteristics in terms of substrate specificity and phlorizin sensitivity to those determined by more traditional approaches and should provide a means to identify novel and selective SGLT inhibitors.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Transportador 1 de Sodio-Glucosa/análisis , Barbitúricos , Línea Celular , Colorantes , Cumarinas , Interpretación Estadística de Datos , Electrofisiología , Etanolaminas , Humanos , Isoxazoles , Cinética , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Florizina/farmacología , Transportador 1 de Sodio-Glucosa/metabolismo , TiobarbitúricosRESUMEN
A series of biaryl chromans exhibiting potent and selective agonism for the GPR40 receptor with positive allosteric modulation of endogenous ligands (AgoPAM) were discovered as potential therapeutics for the treatment of type II diabetes. Optimization of physicochemical properties through modification of the pendant aryl rings resulted in the identification of compound AP5, which possesses an improved metabolic profile while demonstrating sustained glucose lowering.
RESUMEN
GPR40 (FFA1) is a fatty acid receptor whose activation results in potent glucose lowering and insulinotropic effects in vivo. Several reports illustrate that GPR40 agonists exert glucose lowering in diabetic humans. To assess the mechanisms by which GPR40 partial agonists improve glucose homeostasis, we evaluated the effects of MK-2305, a potent and selective partial GPR40 agonist, in diabetic Goto Kakizaki rats. MK-2305 decreased fasting glucose after acute and chronic treatment. MK-2305-mediated changes in glucose were coupled with increases in plasma insulin during hyperglycemia and glucose challenges but not during fasting, when glucose was normalized. To determine the mechanism(s) mediating these changes in glucose metabolism, we measured the absolute contribution of precursors to glucose production in the presence or absence of MK-2305. MK-2305 treatment resulted in decreased endogenous glucose production (EGP) driven primarily through changes in gluconeogenesis from substrates entering at the TCA cycle. The decrease in EGP was not likely due to a direct effect on the liver, as isolated perfused liver studies showed no effect of MK-2305 ex vivo and GPR40 is not expressed in the liver. Taken together, our results suggest MK-2305 treatment increases glucose stimulated insulin secretion (GSIS), resulting in changes to hepatic substrate handling that improve glucose homeostasis in the diabetic state. Importantly, these data extend our understanding of the underlying mechanisms by which GPR40 partial agonists reduce hyperglycemia.
Asunto(s)
Benzopiranos/farmacología , Glucemia/efectos de los fármacos , Diabetes Mellitus Experimental/tratamiento farmacológico , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Receptores Acoplados a Proteínas G/agonistas , Tiazolidinedionas/farmacología , Animales , Benzopiranos/química , Glucemia/metabolismo , Células CHO , Cricetulus , Diabetes Mellitus Experimental/metabolismo , Evaluación Preclínica de Medicamentos , Ayuno/sangre , Células HEK293 , Humanos , Hipoglucemiantes/química , Insulina/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones Noqueados , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Tiazolidinedionas/química , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
GPR40 is a G-protein-coupled receptor expressed primarily in pancreatic islets and intestinal L-cells that has been a target of significant recent therapeutic interest for type II diabetes. Activation of GPR40 by partial agonists elicits insulin secretion only in the presence of elevated blood glucose levels, minimizing the risk of hypoglycemia. GPR40 agoPAMs have shown superior efficacy to partial agonists as assessed in a glucose tolerability test (GTT). Herein, we report the discovery and optimization of a series of potent, selective GPR40 agoPAMs. Compound 24 demonstrated sustained glucose lowering in a chronic study of Goto Kakizaki rats, showing no signs of tachyphylaxis for this mechanism.
RESUMEN
GPR40 agonists are effective antidiabetic agents believed to lower glucose through direct effects on the beta cell to increase glucose stimulated insulin secretion. However, not all GPR40 agonists are the same. Partial agonists lower glucose through direct effects on the pancreas, whereas GPR40 AgoPAMs may incorporate additional therapeutic effects through increases in insulinotrophic incretins secreted by the gut. Here we describe how GPR40 AgoPAMs stimulate both insulin and incretin secretion in vivo over time in diabetic GK rats. We also describe effects of AgoPAMs in vivo to lower glucose and body weight beyond what is seen with partial GPR40 agonists in both the acute and chronic setting. Further comparisons of the glucose lowering profile of AgoPAMs suggest these compounds may possess greater glucose control even in the presence of elevated glucagon secretion, an unexpected feature observed with both acute and chronic treatment with AgoPAMs. Together these studies highlight the complexity of GPR40 pharmacology and the potential additional benefits AgoPAMs may possess above partial agonists for the diabetic patient.
Asunto(s)
Glucosa/metabolismo , Incretinas/metabolismo , Insulina/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Animales , Células CHO , Línea Celular , Cricetulus , Glucagón/metabolismo , Prueba de Tolerancia a la Glucosa , Humanos , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Masculino , Ratones , RatasRESUMEN
Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40-MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.
Asunto(s)
Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Regulación Alostérica , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
A mechanism proposed for lactose/H(+) symport by the lactose permease of Escherichia coli indicates that lactose permease is protonated prior to ligand binding. Moreover, in the ground state, the symported H(+) is shared between His322 (helix X) and Glu269 (helix VIII), while Glu325 (helix X) is charge-paired with Arg302 (helix IX). Substrate binding at the outer surface between helices IV (Glu126) and V (Arg144, Cys148) induces a conformational change that leads to transfer of the H(+) to Glu325 and reorientation of the binding site to the inner surface. After release of substrate, Glu325 is deprotonated on the inside due to re-juxtapositioning with Arg302. The conservative mutation Glu269-->Asp causes a 50-100-fold decrease in substrate binding affinity and markedly reduced active lactose transport, as well as decreased rates of equilibrium exchange and efflux. Gly-scanning mutagenesis of helix VIII was employed systematically with mutant Glu269-->Asp in an attempt to rescue function, and two mutants with increased activity are identified and characterized. Mutant Thr266-->Gly/Met267-->Gly/Glu269-->Asp binds ligand with increased affinity and catalyzes active lactose transport with a marked increase in rate; however, little improvement in efflux or equilibrium exchange is observed. In contrast, mutant Gly262-->Ala/Glu269-->Asp exhibits no improvement in ligand binding but a small increase in the rate of active transport; however, an increase in the steady-state level of accumulation, as well as efflux and equilibrium exchange is observed. Remarkably, when the two sets of mutations are combined, all translocation reactions are rescued to levels approximating those of wild-type permease. The findings support the contention that Glu269 plays a pivotal role in the mechanism of lactose/H(+) symport. Moreover, the results suggest that the two classes of mutants rescue activity by altering the equilibrium between outwardly and inwardly facing conformations of the permease such that impaired protonation and/or H(+) transfer is enhanced from one side of the membrane or the other. When the two sets of mutants are combined, the equilibrium between outwardly and inwardly facing conformations and thus protonation and H(+) transfer are restored.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/enzimología , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos , Simportadores , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Transporte Biológico Activo , Western Blotting , Escherichia coli/genética , Cinética , Lactosa/metabolismo , Proteínas de Transporte de Membrana/genética , Mutagénesis/genética , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Termodinámica , Tiogalactósidos/metabolismoRESUMEN
OBJECTIVES: GPR40 (FFAR1), a clinically proven anti-diabetes target, is a Gq-coupled receptor for long chain fatty acids (LCFA) stimulating insulin secretion directly and mediating a major part of the dietary triglyceride-induced secretion of the incretins GLP-1 and GIP. In phase-II studies the GPR40 agonist TAK-875 decreased blood glucose but surprisingly without stimulating incretins. METHODS AND RESULTS: Here we find that GPR40 can signal through not only Gq and IP3 but also Gs and cAMP when stimulated with certain agonists such as AM-1638 and AM-5262 in contrast to the endogenous LCFA ligands and agonists such as TAK-875 and AM-837, which only signal through Gq. In competition binding against [3H]AM-1638 and [3H]L358 the Gq + Gs and the Gq-only agonists either competed for or showed positive cooperativity by increasing the binding of the two different radio-ligands, in opposite ways. Nevertheless, both the Gq-only and the Gq + Gs agonists all docked surprisingly well into the binding site for TAK-875 in the X-ray structure of GPR40. In murine intestinal primary cell-cultures the endogenous LCFAs and the Gq-only agonists stimulated GLP-1 secretion with rather poor efficacy as compared with the high efficacy Gq + Gs GPR40 agonists and a prototype GPR119 agonist. Similarly, in fasting both male and female mice the Gq + Gs agonists showed significantly higher efficacy than the Gq-only agonists in respect of increasing plasma GLP-1 and plasma GIP in a GPR40-dependent manner. CONCLUSIONS: It is concluded that stimulation of GPR40 by endogenous LCFAs or by Gq-only synthetic agonists result in a rather limited incretin response, whereas Gq + Gs GPR40 agonists stimulate incretin secretion robustly.
RESUMEN
Membrane proteins represent a valuable source of potential drug targets due to their intimate involvement in a wide variety of disease states, including diabetes, cancer and neurological disorders. Defining the proteome of these often rare amphipathic molecules can be accomplished by exploiting the highly accurate and sensitive nature of mass spectrometry (MS). Technical advances have enabled MS to become a valuable tool for detailed mechanistic investigations into membrane proteins of unknown and known structure. The transfer of MS-screening strategies that have already been successfully used to identify interactions between soluble proteins and potential ligands, should allow the identification of drug candidates for membrane proteins in the near future.
Asunto(s)
Diseño de Fármacos , Espectrometría de Masas/métodos , Proteínas de la Membrana/análisis , Animales , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Proteómica/métodosRESUMEN
Transporters represent attractive targets for drug discovery and are implicated in the pathophysiology of disorders across several therapeutic areas including asthma, cardiovascular disease, diabetes and neuroscience. However, the intrinsic mechanistic properties of transporters present significant challenges to the development of high-throughput screening methodologies. This review provides an update on potential transporter targets and evaluates the impact of available technologies to enable transporter screening, lead optimization and assessment of pharmacokinetics.
Asunto(s)
Descubrimiento de Drogas/métodos , Proteínas de Transporte de Membrana/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , TecnologíaRESUMEN
Inward rectifier potassium channels (Kir) play critical roles in cell physiology. Despite representing the simplest tetrameric potassium channel structures, the pharmacology of this channel family remains largely undeveloped. In this respect, tertiapin (TPN), a 21 amino acid peptide isolated from bee venom, has been reported to inhibit Kir1.1 and Kir3.1/3.4 channels with high affinity by binding to the M1-M2 linker region of these channels. The features of the peptide-channel interaction have been explored electrophysiologically, and these studies have identified ways by which to alter the composition of the peptide without affecting its biological activity. In the present study, the TPN derivative, TPN-Y1/K12/Q13, has been synthesized and radiolabeled to high specific activity with (125)I. TPN-Y1/K12/Q13 and mono-iodo-TPN-Y1/K12/Q13 ([(127)I]TPN-Y1/K12/Q13) inhibit with high affinity rat but not human Kir1.1 channels stably expressed in HEK293 cells. [(125)I]TPN-Y1/K12/Q13 binds in a saturable, time-dependent, and reversible manner to HEK293 cells expressing rat Kir1.1, as well as to membranes derived from these cells, and the pharmacology of the binding reaction is consistent with peptide binding to Kir1.1 channels. Studies using chimeric channels indicate that the differences in TPN sensitivity between rat and human Kir1.1 channels are due to the presence of two nonconserved residues within the M1-M2 linker region. When these results are taken together, they demonstrate that [(125)I]TPN-Y1/K12/Q13 represents the first high specific activity radioligand for studying rat Kir1.1 channels and suggest its utility for identifying other Kir channel modulators.
Asunto(s)
Venenos de Abeja/química , Radioisótopos de Yodo/química , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/metabolismo , Animales , Secuencia de Bases , Venenos de Abeja/aislamiento & purificación , Venenos de Abeja/metabolismo , Venenos de Abeja/farmacología , Fenómenos Fisiológicos Celulares , Células Cultivadas , Electrofisiología/métodos , Humanos , Riñón/citología , Riñón/metabolismo , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Unión Proteica , Ratas , Factores de TiempoRESUMEN
EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu-14) from both EmrE monomers. Carbodiimide modification of EmrE has been studied using functional assays, and the evidence suggests that Glu-14 is the target of the reaction. Here we exploited electrospray ionization mass spectrometry to directly monitor the reaction with each monomer rather than following inactivation of the functional unit. A cyanogen bromide peptide containing Glu-14 allows the extent of modification by the carboxyl-specific modification reagent diisopropylcarbodiimide (DiPC) to be monitored and reveals that peptide 2NPYIYLGGAILAEVIGTTLM(21) is approximately 80% modified in a time-dependent fashion, indicating that each Glu-14 residue in the oligomer is accessible to DiPC. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of Glu-14 with DiPC by up to 80%. Taken together with other biochemical data, the findings support a "time sharing" mechanism in which both Glu-14 residues in a dimer are involved in tetraphenylphosphonium and H(+) binding.
Asunto(s)
Antiportadores/química , Espectrometría de Masas/métodos , Proteínas de la Membrana/química , Antiportadores/ultraestructura , Sitios de Unión , Transporte Biológico , Carbodiimidas/farmacología , Bromuro de Cianógeno/química , Dimerización , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Ácido Glutámico/química , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/ultraestructura , Modelos Biológicos , Modelos Químicos , Compuestos Onio/química , Compuestos Organofosforados/química , Péptidos/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Protones , Espectrometría de Masa por Ionización de Electrospray , Factores de TiempoRESUMEN
Luminescence resonance energy transfer with a lanthanide like Tb(3+) as donor is a useful technique for estimating intra- and intermolecular distances in macromolecules. However, the technique usually requires the use of a bulky chelator with a flexible linker attached to a Cys residue to bind Tb(3+) and, for intramolecular studies, an acceptor fluorophor attached to another Cys residue in the same protein. Here, an engineered EF- hand motif is incorporated into the central cytoplasmic loop of the lactose permease of Escherichia coli generating a high-affinity site for Tb(3+) (K(Tb)(3+) approximately 4.5 microM) or Gd(3+) (K(Gd)(3+) approximately 2.3 microM). By exciting a Trp residue in the coordination sequence, Tb(3+) bound to the EF-hand motif is sensitized specifically, and the efficiency of energy transfer to strategically placed Cys residues labeled with fluorophors is measured. In this study, we use the technique to measure distance from the EF-hand in the central cytoplasmic loop of lactose permease to positions 179 or 169 at the center or periplasmic end of helix VI, respectively. The average calculated distances of approximately 23 A (position 179) and approximately 33 A (position 169) observed with three different fluorophors as acceptors agree well with the geometry of a slightly tilted alpha-helix. The approach should be of general use for studying static and dynamic aspects of polytopic membrane protein structure and function.
Asunto(s)
Motivos EF Hand , Transferencia de Energía , Proteínas de Escherichia coli , Mediciones Luminiscentes , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos , Ingeniería de Proteínas , Simportadores , Terbio/metabolismo , Sitios de Unión , Citoplasma/metabolismo , Motivos EF Hand/genética , Escherichia coli/enzimología , Escherichia coli/genética , Fluorescencia , Proteínas de Transporte de Membrana/genética , Mutación/genética , Estructura Secundaria de Proteína , Proteolípidos/química , Proteolípidos/genética , Proteolípidos/metabolismo , Análisis Espectral , Relación Estructura-ActividadRESUMEN
Enzyme IIA(Glc), encoded by the crr gene of the phosphoenolpyruvate:sugar phosphotransferase system, plays an important role in regulating intermediary metabolism in Escherichia coli ("catabolite repression"). One function involves inhibition of inducible transport systems ("inducer exclusion"), and with lactose permease, a galactoside is required for unphosphorylated IIA(Glc) binding to cytoplasmic loops IV/V and VI/VII [Sondej, M., Sun, J. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3525-3530]. With inside-out membrane vesicles containing the permease, [(125)I]IIA(Glc) binding promoted by melibiose exhibits an affinity (K(D)(IIA)) of approximately 1 microM and a stoichiometry of one mole of IIA(Glc) per six moles of lactose permease. Both the quantity of [(125)I]IIA(Glc) bound and the sugar concentration required for half-maximal IIA(Glc) binding (K(0.5)(IIA)(sug)) was measured for eight permease substrates. Differences in maximal IIA(Glc) binding are observed, and the K(0.5)(IIA)(sug) does not correlate with the affinity of LacY for sugar. Furthermore, K(0.5)(IIA)(sug) does not correlate with sugar affinities for various permease mutants. IIA(Glc) does not bind to a mutant (Cys154 --> Gly), which is locked in an outwardly facing conformation, binds with increased stoichiometry to mutant Lys131 --> Cys, and binds only weakly to two other mutants which appear to be predominantly in either an outwardly or an inwardly facing conformation. When the latter two mutations are combined, sugar-dependent IIA(Glc) binding returns to near wild-type levels. The findings suggest that binding of various substrates to lactose permease results in a collection of unique conformations, each of which presents a specific surface toward the inner face of the membrane that can interact to varying degrees with IIA(Glc).