RESUMEN
Several wasp venoms contain philanthotoxins (PhTXs) that act as noncompetitive inhibitors (NCIs) on cation-selective ion channels including the nicotinic acetylcholine receptor (nAChR). In the search for a ligand with high affinity and specificity for the nAChR we tested a series of newly developed PhTX analogues. Modulation of the structural elements of PhTXs can significantly influence their binding affinities. This approach resulted in the development of the photolabile compound MR44. In photoaffinity labelling studies 125I-MR44 was used to map the ligand-binding site at the Torpedo californica nAChR. Upon UV irradiation of the receptor-ligand complex, 125I-MR44 was mainly incorporated into the receptor alpha-subunit. Proteolytic mapping and microsequencing identified the site of 125I-MR44 cross-linking within the sequence alphaHis-186 to alphaLeu-199 that in its C-terminal region partially overlaps with the agonist-binding site. Since bound agonists had only minor influence on 125I-MR44 photocrosslinking, the site where the hydrophobic head group of 125I-MR44 binds must be located outside the zone that is sterically influenced by agonists bound at the nAChR. A possible site of interaction of 125I-MR44 would be the N-terminal region of the labelled sequence, in which aromatic amino-acid residues are accumulated. We suggest that the polyamine moiety of 125I-MR44 interacts with the high affinity non-competitive inhibitor site deep in the ion channel, while the aromatic ring of this compound binds in the vestibule of the nAChR to a hydrophobic region on the alpha-subunit that is located close to the agonist binding site.
Asunto(s)
Canales Iónicos/metabolismo , Antagonistas Nicotínicos/metabolismo , Poliaminas/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Sitios de Unión , Humanos , Etiquetas de FotoafinidadRESUMEN
To map the structure of a ligand-gated ion channel, we used the photolabile polyamine-containing toxin MR44 as photoaffinity label. MR44 binds with high affinity to the nicotinic acetylcholine receptor in its closed channel conformation. The binding stoichiometry was two molecules of MR44 per receptor monomer. Upon UV irradiation of the receptor-ligand complex, (125)I-MR44 was incorporated into the receptor alpha-subunit. From proteolytic mapping studies, we conclude that the site of (125)I-MR44 cross-linking is contained in the sequence alpha His-186 to alpha Leu-199, which is part of the extracellular domain of the receptor. This sequence partially overlaps in its C-terminal region with one of the three loops that form the agonist-binding site. The agonist carbachol and the competitive antagonist alpha-bungarotoxin had only minor influence on the photocross-linking of (125)I-MR44. The site where the hydrophobic head group of (125)I-MR44 binds must therefore be located outside the zone that is sterically influenced by agonist bound at the nicotinic acetylcholine receptor. In binding and photocross-linking experiments, the luminal noncompetitive inhibitors ethidium and triphenylmethylphosphonium were found to compete with (125)I-MR44. We conclude that the polyamine moiety of (125)I-MR44 interacts with the high affinity noncompetitive inhibitor site deep in the channel of the nicotinic acetylcholine receptor, while the aromatic ring of this compound binds in the upper part of the ion channel (i.e. in the vestibule) to a hydrophobic region on the alpha-subunit that is located in close proximity to the agonist binding site. The region of the alpha-subunit labeled by (125)I-MR44 should therefore be accessible from the luminal side of the vestibule.
Asunto(s)
Canales Iónicos/química , Poliaminas/metabolismo , Receptores Nicotínicos/química , Sitios de Unión , Calcio/metabolismo , Células Cultivadas , Hexosaminidasas/farmacología , Activación del Canal Iónico , Canales Iónicos/metabolismo , Subunidades de Proteína , Receptores Nicotínicos/metabolismo , Relación Estructura-ActividadRESUMEN
Astroglia-rich primary cultures and brain slices rapidly metabolize branched-chain amino acids (BCAAs), in particular leucine, as energy substrates. To allocate the capacity to degrade leucine oxidatively in neural cells, we have purified beta-methylcrotonyl-CoA carboxylase (beta-MCC) from rat liver as one of the enzymes unique for the irreversible catabolic pathway of leucine. Polyclonal antibodies raised against beta-MCC specifically cross-reacted with both enzyme subunits in liver and brain homogenates. Immunocytochemical examination of astroglia-rich rat primary cultures demonstrated the presence of beta-MCC in astroglial cells, where the enzyme was found to be located in the mitochondria, the same organelle that the mitochondrial isoform of the BCA(A) aminotransferase (BCAT) is located in. This colocalization of the two enzymes supports the hypothesis that mitochondrial BCAT is the isoenzyme that in brain energy metabolism prepares the carbon skeleton of leucine for irreversible degradation in astrocytes. Analysis of neuron-rich primary cultures revealed also that the majority of neurons contained beta-MCC. The presence of beta-MCC in most neurons demonstrates their ability to degrade the alpha-ketoisocaproate that could be provided by neighboring astrocytes or could be generated locally from leucine by the action of the cytosolic isoform of BCAT that is known to occur in neurons.
Asunto(s)
Astrocitos/enzimología , Ligasas de Carbono-Carbono/metabolismo , Neuronas/enzimología , Animales , Encéfalo/enzimología , Células Cultivadas , Inmunohistoquímica , Mitocondrias/metabolismo , Ratas , Ratas Wistar , Distribución Tisular , Transaminasas/metabolismoRESUMEN
Several wasp venoms contain philanthotoxins (PhTXs), natural polyamine amides, which act as noncompetitive inhibitors (NCIs) on the nicotinic acetylcholine receptor (nAChR). Effects of varying the structure of PhTXs and poly(methylene tetramine)s on the binding affinity have been investigated. Using the fluorescent NCI ethidium in a displacement assay Kapp values of these compounds have been determined. We found that an increase in size of the PhTX's hydrophobic head group significantly increased the binding affinity, while inserting positive charge almost completely destroyed it. Elongating the PhTX polyamine chain by introducing an additional aminomethylene group decreased the binding affinity, whereas a terminal lysine improved it. In general, poly(methylene tetramine)s showed higher binding affinities than PhTX analogues. The stoichiometry of PhTX binding was determined to be two PhTX molecules per receptor monomer. PhTXs appeared to bind to a single class of nonallosterically interacting binding sites and bound PhTX was found to be completely displaced by well-characterized luminal NCIs. To elucidate the site of PhTX binding, a photolabile, radioactive PhTX derivative was photocross-linked to the nAChR in its closed channel conformation resulting in labeling yields for the two alpha and the beta, gamma and delta subunits of 10.4, 11.1, 4.0 and 7.4%, respectively. Based on these findings we suggest that PhTXs and poly(methylene tetramine)s enter the receptor's ionic channel from the extracellular side. The hydrophobic head groups most likely bind to the high-affinity NCI site, while the positively charged polyamine chains presumably interact with the negatively charged selectivity filter located deep in the channel lumen.
Asunto(s)
Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/metabolismo , Poliaminas/química , Poliaminas/metabolismo , Receptores Nicotínicos/metabolismo , Sitios de Unión , Carbamazepina/análogos & derivados , Carbamazepina/metabolismo , Etidio/metabolismo , Fluorescencia , Glutaral/metabolismo , Concentración 50 Inhibidora , Ligandos , Peso Molecular , Receptores Nicotínicos/química , Electricidad Estática , Relación Estructura-Actividad , Termodinámica , Volumetría , Venenos de Avispas/química , Venenos de Avispas/metabolismoRESUMEN
The universal template approach to drug design foresees that a polyamine can be modified in such a way to recognize any neurotransmitter receptor. Thus, hybrids of polymethylene tetraamines and philanthotoxins, exemplified by methoctramine (1) and PhTX-343 (2), respectively, were synthesized to produce novel inhibitors of muscular nicotinic acetylcholine receptors. Polyamines 3-25 were synthesized and their biological profiles were evaluated at frog rectus abdominis muscle nicotinic receptors and guinea pig left atria (M(2)) and ileum longitudinal muscle (M(3)) muscarinic acetylcholine receptors. All of the compounds, like prototypes 1 and 2, were noncompetitive antagonists of nicotinic receptors while being, like 1, competitive antagonists at muscarinic M(2) and M(3) receptor subtypes. Interestingly, polyamines bearing a low number of methylenes between the nitrogen atoms, as in 3, 6, and 7, displayed a biological profile similar to that of 2: a noncompetitive antagonism at nicotinic receptors in the 7-25 microM range while not showing any antagonism for muscarinic receptors up to 10 microM. Increasing the number of methylenes separating these nitrogen atoms in methoctramine-related tetraamines resulted in a significant improvement in potency at nicotinic receptors. The most potent tetraamine was 19, bearing a 12 methylene spacer between the nitrogen atoms, which was 12-fold and 250-fold more potent than prototypes 1 and 2, respectively. Tetraamines 9-11, bearing a rather rigid spacer between the nitrogen atoms instead of the very flexible polymethylene chain, displayed a profile similar to that of 1 at nicotinic receptors, whereas a significant decrease in potency was observed at muscarinic M(2) receptors. This finding may have relevance in understanding the mode of interaction with these receptors. Similarly, the constrained analogue 12 of methoctramine showed a decrease in potency at nicotinic and muscarinic M(2) receptors, revealing that the tricyclic system, which incorporates the 2-methoxybenzylamine moiety of 1, does not represent a good pharmacophore for activity at these sites. A most intriguing finding was the observation that the photolabile tetraamine 22 was more potent than methoctramine at nicotinic receptors and, what is more important, it inhibited a closed state of the receptor.
Asunto(s)
Diaminas/química , Atrios Cardíacos/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Antagonistas Nicotínicos/farmacología , Poliaminas/farmacología , Animales , Anuros , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Estimulación Eléctrica , Cobayas , Atrios Cardíacos/metabolismo , Espectroscopía de Resonancia Magnética , Músculo Esquelético/metabolismo , Antagonistas Nicotínicos/síntesis química , Antagonistas Nicotínicos/química , Etiquetas de Fotoafinidad , Poliaminas/síntesis química , Poliaminas/química , Receptores Muscarínicos/clasificación , Receptores Muscarínicos/efectos de los fármacosRESUMEN
The first step in the catabolism of branched-chain amino acids (BCAA), reversible transamination, is catalyzed by one of the two isoforms of branched-chain amino acid aminotransferase (BCAT). The mitochondrial isoenzyme (BCATm) is widely distributed among tissues, whereas the cytosolic isoenzyme (BCATc) is restricted to only a few organs. Remarkably, BCATc is the prominent isoenzyme found in brain. The physiological significance of the subcellular compartmentation of BCAT is still not understood. To contribute to the elucidation of the cellular distribution of the two isoenzymes in brain, we used cultured rat glial cells in an immunocytochemical study to determine the pattern of BCAT isoenzyme expression by glial cells. Antiserum against BCATm generated a punctate staining pattern of astroglial cells, confirming the mitochondrial location of this isoenzyme. In contrast, the cytosol of galactocerebroside-expressing oligodendroglial cells and O2A progenitor cells displayed intense staining only for BCATc. In addition, subpopulations of astroglial cells exhibited BCATc immunoreactivity. The presence of BCATm in astrocytes is consistent with the known ability of these cells to oxidize BCAA. Furthermore, our results on BCATc provide support for the hypothesis that BCATs are also involved in nitrogen transfer from astrocytes to neurons.
Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Encéfalo/enzimología , Isoenzimas/metabolismo , Neuroglía/enzimología , Transaminasas/metabolismo , Animales , Western Blotting , Encéfalo/citología , Células Cultivadas , Galactosilceramidas/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Neuroglía/citología , Ratas , Ratas WistarRESUMEN
To elucidate the significance of branched-chain amino acids (BCAAs) for brain energy metabolism, the capacity to use BCAAs for oxidative metabolism was investigated in astroglia-rich primary cultures derived from newborn rat brain. The cells selectively removed BCAAs from the culture medium, the disappearance following first-order kinetics. The BCAAs disappeared rapidly in spite of the presence of sufficient glucose as substrate for the generation of energy. Taking into consideration that the ketogenic amino acid leucine could be degraded only to acetyl-CoA and acetoacetate, and with the knowledge that astroglial cells have the capacity to secrete ketone bodies, this amino acid was chosen for further metabolic studies. After incubation of the cells with leucine, acetoacetate, D-beta-hydroxybutyrate, and alpha-ketoisocaproate were found to have accumulated in the culture medium. Identification of the radioactive metabolites generated from [4,5-3H]leucine established that the source of the substances released was indeed leucine. These results indicate that, at least in culture, astroglial cells degrade leucine via the known metabolite alpha-ketoisocaproate, to acetoacetate, which can be further reduced to D-beta-hydroxybutyrate. It is hypothesized that upon release from brain astrocytes, the ketone bodies could serve as fuel molecules for neighboring cells such as neurons and oligodendrocytes. In view of these and other results, astrocytes may be considered the brain's fuel processing plants.