RESUMEN
We have proposed that the cytotoxic action of Alzheimer's amyloid beta protein might be initiated by the interaction with the neuronal cell membrane, and subsequent formation of toxic ion channels. Consequently, A beta P toxicity can be explained on the basis of harmful ion fluxes across A beta P channels. The conformation of A beta P in membranes is not known. However, several models suggests that a transmembrane annular polymeric structure is responsible for the ion channel properties of the membrane-bound A beta P. To identify that portion of the A beta P molecule making up the conducting pore we have hypothesized that the region of the A beta P sequence in the vicinity of the hypothetical pore might interact with complementary regions in the adjacent A beta P subunits. We have further hypothesized that an interaction by a peptide segment would block A beta P conductance. To test this hypothesis we synthesized peptides that encompass the histidine dyad (H-H) previously hypothesized to line the pore. We report here that peptides designed to most closely match the proposed pore are, in fact, the most effective at blocking ion currents through the membrane-incorporated A beta P channel. As previously shown for Zn(2+) blockade, peptide blockade is also asymmetric. The results also provide additional evidence for the asymmetric insertion of the A beta P molecules into lipid membranes, and give support to the concept that rings of histidines line the entry to one side of the A beta P pore.
Asunto(s)
Péptidos beta-Amiloides/química , Histidina/química , Activación del Canal Iónico , Membrana Dobles de Lípidos/química , Liposomas/química , Péptidos/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Conductividad Eléctrica , Potenciales de la Membrana , Membranas Artificiales , Datos de Secuencia Molecular , Permeabilidad , Porosidad , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Heat shock proteins are molecular chaperones that participate in different cellular processes, particularly the folding and translocation of polypeptides across membranes. In this regard, members of the Hsp70 family of heat shock proteins have been observed in close proximity to cellular membranes. In this study, the direct interaction between Hsc70, which is constitutively expressed in cells, and lipid membranes was investigated. Recombinant Hsc70 was incorporated into artificial lipid bilayers, and a transmembrane ion flow was detected, suggesting the incorporation of an ion pathway. This ion flow was very stable and occurred in well defined, multilevel discrete electrical current events, indicating the formation of a multiconductance ion channel. The Hsc70 channel activity is ATP-dependent and is reversibly blocked by ADP. This channel has cationic selectivity. Thus, Hsc70 can directly interact with lipid membranes to create functionally stable ATP-dependent cationic pathways.
Asunto(s)
Adenosina Difosfato/fisiología , Adenosina Trifosfato/fisiología , Proteínas Portadoras/metabolismo , Cationes/metabolismo , Proteínas HSP70 de Choque Térmico , Canales Iónicos/metabolismo , Lípidos de la Membrana/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Portadoras/química , Conductividad Eléctrica , Proteínas del Choque Térmico HSC70 , Membrana Dobles de Lípidos/metabolismo , Técnicas de Placa-Clamp , Unión Proteica , Proteolípidos/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
A growing number of reports suggest that elevated levels of extracellular Alzheimer's beta-amyloid protein alter the homeostasis of free [Ca(2+)](i) in different cell types of the mammalian brain. In line with these results, we have previously shown that AbetaP[1-40] forms cation-selective channels (Ca(2+) included) across artificial planar bilayers formed from acidic phospholipids and across excised membrane patches from immortalized hypothalamic GnRH neurons (GT1-7 cells), suggesting that the nonregulated Ca(2+)-influx through these spontaneously formed "amyloid channels" may provide a mechanism to explain its toxicity (1). We have now found and report here that the application of AbetaP[1-40] to GT1-7 neurons consistently elevates [Ca(2+)](i) levels. We also found that human islet amylin and the prion protein fragment (PrP106-126), peptides that acquire beta-pleated sheet conformation in water solutions and have been reported to form ion channels across planar bilayer membranes, also increase cytosolic free calcium in GT1-7 neurons. Searching for protective agents, we found that soluble cholesterol, known to decrease the fluidity of the cell membrane, inhibits AbetaP[1-40]-evoked [Ca(2+)](i) rise. These results suggest that unregulated Ca(2+) entry across amyloid channels may be a common mechanism causing cell death, not only in diseases of the third age, including Alzheimer's disease and type 2 diabetes mellitus, but also in prion-induced diseases.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Calcio/metabolismo , Hipotálamo/metabolismo , Islotes Pancreáticos/metabolismo , Priones/metabolismo , Enfermedad de Alzheimer/metabolismo , Línea Celular Transformada , Colesterol/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Homeostasis , Humanos , Hipotálamo/citología , Polipéptido Amiloide de los Islotes Pancreáticos , Neuronas/metabolismoRESUMEN
8-Cyclopentyl-1,3-dipropylxanthine (CPX) and 1,3-diallyl-8-cyclohexylxanthine (DAX) are xanthine adenosine antagonists which activate chloride efflux from cells expressing either wild-type or mutant (DeltaF508) cystic fibrosis transmembrane conductance regulator (CFTR). These drugs are active in extremely low concentrations, suggesting their possible therapeutic uses in treating cystic fibrosis. However, knowledge of the mechanism of action of these compounds is lacking. We report here that the same low concentrations of both CPX and DAX which activate chloride currents from cells also generate a profound activation of CFTR channels incorporated into planar lipid bilayers. The process of activation involves a pronounced increase in the total conductive time of the incorporated CFTR channels. The mechanism involves an increase in the frequency and duration of channel opening events. Thus, activation by these drugs of chloride efflux in cells very likely involves direct interaction of the drugs with the CFTR protein. We anticipate that this new information will contribute fundamentally to the rational development of these and related compounds for cystic fibrosis therapy.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Xantinas/farmacología , Línea Celular , Canales de Cloruro/efectos de los fármacos , Fibrosis Quística/tratamiento farmacológico , Conductividad Eléctrica , Electrofisiología , Humanos , Membrana Dobles de Lípidos/metabolismo , Cloruro de Potasio/metabolismo , Antagonistas de Receptores Purinérgicos P1RESUMEN
We have previously shown that the 40-residue peptide termed amyloid beta-protein (A beta P[1-40]) in solution forms cation-selective channels across artificial phospholipid bilayer membranes. To determine whether A beta P[1-40] also forms channels across natural membranes, we used electrically silent excised membrane patches from a cell line derived from hypothalamic gonadotrophin-releasing hormone GnRH neurons. We found that exposing either the internal or the external side of excised membrane patches to A beta P[1-40] leads to the spontaneous formation of cation-selective channels. With Cs+ as the main cation in both the external as well as the internal saline, the amplitude of the A beta P[1-40] channel currents was found to follow the Cs+ gradient and to exhibit spontaneous conductance changes over a wide range (50-500 pS). We also found that free zinc (Zn2+), reported to bind to amyloid beta-protein in solution, can block the flow of Cs+ through the A beta P[1-40] channel. Because the Zn2+ chelator o-phenanthroline can reverse this blockade, we conclude that the underlying mechanism involves a direct interaction between the transition element Zn2+ and sites in the A beta P[1-40] channel pore. These properties of the A beta P[1-40] channel are rather similar to those observed in the artificial bilayer system. We also show here, by immunocytochemical confocal microscopy, that amyloid beta-protein molecules form deposits closely associated with the plasma membrane of a substantial fraction of the GnRH neurons. Taken together, these results suggest that the interactions between amyloid beta-protein and neuronal membranes also occur in vivo, lending further support to the idea that A beta P[1-40] channel formation might be a mechanism of amyloid beta-protein neurotoxicity.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Péptidos beta-Amiloides/fisiología , Membrana Celular/fisiología , Canales Iónicos/fisiología , Neuronas/fisiología , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/fisiología , Zinc/farmacología , Animales , Línea Celular Transformada , Membrana Celular/efectos de los fármacos , Hipotálamo/fisiología , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Fenantrolinas/farmacologíaRESUMEN
Matrix vesicles (MVs), structures that accumulate Ca2+ during the initiation of mineral formation in growing bone, are rich in annexin V. When MVs are fused with planar phospholipid bilayers, a multiconductance Ca2+ channel is formed, with activity essentially identical to that observed when annexin V is delivered to the bilayer with phosphatidylserine liposomes. Ca2+ currents through this channel, from either MV or annexin V liposomes, are blocked by Zn2+, as is Ca2+ uptake by MV incubated in synthetic cartilage lymph. Blockage by Zn2+ was most effective when applied to the side containing the MV or liposomes. ATP and GTP differentially modulated the activity of this channel: ATP increased the amplitude of the current and the number of conductance states; GTP dramatically reduced the number of events and conductance states, leading to well-defined Ca2+ channel activity from either MV or the annexin V liposomes. In the distinctive effects of ATP, GTP, and Zn2+ on the Ca2+ channel activity observed in both the MV and the liposome systems, the common factor was the presence of annexin V. From this we conclude that Ca2+ entry into MV results from the presence of annexin V in these membrane-enclosed structures.
Asunto(s)
Anexina A5/fisiología , Canales de Calcio/fisiología , Matriz Extracelular/fisiología , Membrana Dobles de Lípidos , Microsomas/fisiología , Adenosina Trifosfato/farmacología , Animales , Anexina A5/aislamiento & purificación , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Pollos , Colagenasas , Conductividad Eléctrica , Matriz Extracelular/ultraestructura , Guanosina Trifosfato/farmacología , Fusión de Membrana , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Tripsina , Zinc/farmacologíaRESUMEN
The Alzheimer disease 40-residue amyloid beta protein (AbetaP[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS. Zn2+ has been reported to bind to AbetaP[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional consequences of such Zn2+ binding for the AbetaP[1-40] channel. Provided the AbetaP[1-40] channel is expressed in the low conductance (<400 pS) mode, Zn2+ blocks the open channel in a dose- dependent manner. For AbetaP[1-40] channels in the giant conductance mode (>400 pS), Zn2+ doses in the millimolar range were required to exert substantial blockade. The Zn2+ chelator o-phenanthroline reverses the blockade. We also found that Zn2+ modulates AbetaP[1-40] channel gating and conductance only from one side of the channel. These data are consistent with predictions of our recent molecular modeling studies on AbetaP[1-40] channels indicating asymmetric Zn(2+)-AbetaP[1-40] interactions at the entrance to the pore.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Zinc/metabolismo , Péptidos beta-Amiloides/química , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Sustancias Macromoleculares , Fragmentos de Péptidos/química , Fenantrolinas/farmacología , Estructura Terciaria de Proteína , Zinc/farmacologíaRESUMEN
The annexin (Anx) gene family comprises a set of calcium-dependent membrane binding proteins, which have been implicated in a wide variety of cellular processes including membrane fusion and calcium channel activity. We report here that cAMP activates Ca(2+)-dependent aggregation of both phosphatidylserine (PS) liposomes and bovine chromaffin granules driven by [des 1-12]annexin I (lipocortin I, Anx1). The mechanism of cAMP action involves an increase in AnxI-dependent cooperativity on the rate of such a reaction without affecting the corresponding k1/2 values. Cyclic AMP causes the values of the Hill coefficient (nH) for AnxI to change from 3 to 6 in both PS liposomes and chromaffin granules. By contrast, ATP inhibits the rate of aggregation activity without affecting the cooperativity or the extent of aggregation process. We were also able to photolabel Anx1 specifically with an 8-azido analogue of cAMP by a calcium-independent process. Such a process is saturable, yielding a Kd = 0.8 microM by Scatchard analysis. Specific displacement occurs in the presence of cAMP and ATP. Finally, we found that cAMP alters the conductance of calcium channels formed by AnxI in planar lipid bilayers. We interpret these data to indicate that AnxI binds both calcium and cAMP independently, and that both actions have functional consequences. This is the first report of a nucleotide binding function for a member of the annexin gene family.
Asunto(s)
Anexina A1/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Gránulos Cromafines/metabolismo , AMP Cíclico/metabolismo , Liposomas/metabolismo , Adenosina Trifosfato/metabolismo , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Anexina A1/análogos & derivados , Azidas/metabolismo , Bovinos , AMP Cíclico/análogos & derivados , Conductividad Eléctrica , Conformación Molecular , Datos de Secuencia Molecular , Nucleótidos/metabolismo , Fosfatidilserinas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
1. Alzheimer's disease (AD) is a chronic dementia and neurodegenerative disorder affecting the oldest portions of the population. Brains of AD patients accumulate large amount of the A beta P peptide in amyloid plaques. 2. The A beta P[1-40] peptide is derived by proteolytic processing from a much larger amyloid precursor protein (APP), and has been circumstantially identified as the toxic principle causing cell damage in the disease. 4. The A beta P[1-40] peptide is able to form quite characteristic calcium channels in planar lipid bilayers. These channels have conductances in the nS range, and can dissipate ion gradients quickly. The peptide can also cause equivalent cation conductances in cells. 5. We suggest that amyloid channel blocking agents might be therapeutically useful in Alzheimer's Disease, and have constructed molecular models of the channels to aid in the design of such compounds.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/fisiología , Péptidos beta-Amiloides/toxicidad , Canales Iónicos/fisiología , Neurotoxinas , Estructura Secundaria de Proteína , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/fisiología , Animales , Encéfalo/patología , Encéfalo/fisiopatología , Humanos , Canales Iónicos/química , Modelos Neurológicos , Modelos Estructurales , Datos de Secuencia MolecularRESUMEN
We report here the presence of a Ca(2+)-independent K(+)-channel of large conductance in adrenal chromaffin cell secretory vesicle membranes which is controlled by inhibitory as well as stimulatory heterotrimeric GTP-binding proteins. Using antibodies against specific alpha subunits for immunoblot analysis, we were able to identify the presence of the inhibitory G(i)2 and G(i)3 subtypes, as well as the stimulatory G(o) and Gs subtypes, but not G(i)1 in adrenal chromaffin granules. Furthermore, functional analysis of the K(+)-channel incorporated into planar lipid bilayers showed that GDP beta S and GTP gamma S have opposite effects on channel activity inducing interconversions between a low and a high open-probability state. Consistent with these findings, the same antibodies antagonized the effects of the nonhydrolyzable analogues on the open probability of the K(+)-channel.
Asunto(s)
Médula Suprarrenal/metabolismo , Gránulos Cromafines/metabolismo , Proteínas de Unión al GTP/metabolismo , Canales de Potasio/metabolismo , Médula Suprarrenal/efectos de los fármacos , Animales , Anticuerpos/farmacología , Bovinos , Gránulos Cromafines/efectos de los fármacos , Conductividad Eléctrica , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/aislamiento & purificación , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Potenciales de la Membrana , Bloqueadores de los Canales de Potasio , Canales de Potasio/efectos de los fármacos , Receptores Adrenérgicos/metabolismo , Fluoruro de Sodio/farmacología , Tionucleótidos/farmacologíaAsunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/fisiología , Canales de Calcio/fisiología , Neuronas/citología , Fragmentos de Péptidos/fisiología , Canales de Calcio/química , Muerte Celular , Humanos , Técnicas In Vitro , Activación del Canal Iónico , Técnicas de Placa-Clamp , Conformación Proteica , Trometamina/farmacologíaRESUMEN
Theoretical methods are used to develop models for the ion channel structure of the membrane-bound amyloid beta-protein. This follows recent observations that the beta-protein forms cation-selective channels in lipid bilayers in vitro. Amyloid beta-protein is the main component of the extracellular plaques in the brain that are characteristic of Alzheimer's disease. Based on the amino acid sequence and the unique environment of the membrane, the secondary structure of the 40-residue beta-protein is predicted to form a beta-hairpin followed by a helix-turn-helix motif. The channel structures were-designed as aggregates of peptide subunits in identical conformations. Three types of models were developed that are distinguished by whether the pore is formed by the beta-hairpins, the middle helices, or by the more hydrophobic C-terminal helices. The latter two types can be converted back and forth by a simple conformational change, which would explain the variable conduction states observed for a single channel. It is also demonstrated how lipid headgroups could be incorporated into the pore lining, and thus affect the ion selectivity. The atomic-scale detail of the models make them useful for designing experiments to determine the real structure of the channel, and thus further the understanding of peptide channels in general. In addition, if beta-protein-induced channel activity is found to be the cause of cell death in Alzheimer's disease, then the models may be helpful in designing counteracting drugs.
Asunto(s)
Péptidos beta-Amiloides/química , Canales Iónicos/química , Modelos Moleculares , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/genética , Fenómenos Biofísicos , Biofisica , Humanos , Canales Iónicos/genética , Membrana Dobles de Lípidos/química , Datos de Secuencia Molecular , Estructura Molecular , Ovillos Neurofibrilares/química , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de ProteínaRESUMEN
The Alzheimer's Disease (AD) amyloid protein (A beta P[1-40]) forms cation selective channels when incorporated into planar lipid bilayers by fusion with liposomes containing the peptide. Since the peptide has been proposed to occur in vivo in both membrane-bound and soluble forms, we also tested the possibility of direct incorporation of the soluble A beta P[1-40] into the membrane. We found the peptide can also form similar channels in acidic phospholipid bilayers formed at the tip of a patch pipet, as well as in the planar lipid bilayer system. As in the case of liposome mediated incorporation, the A beta P[1-40]-channel in the solvent-free membrane patch exhibits multiple cation selectivity (Cs+ > Li+ > Ca2+ > or = K+), and sensitivity to tromethamine. The fact that equivalent A beta P[1-40] amyloid channels can be detected by two different methods thus provides additional validation of our original observation. Further studies with a beta P-channels incorporated into planar lipid bilayers from the liposome complex have also revealed that the channel activity can express spontaneous transitions to a much higher range of conductances between 400 and 4000 pS. Under these conditions, the amyloid channel continues to be cation selective but loses its tromethamine sensitivity. By contrast, amyloid channels were insensitive to nitrendipine at either conductance range. We calculate that if such channels were expressed in cells, the ensuing ion fluxes down their electrochemical potential gradients would disrupt cellular homeostasis. We therefore interpret these data as providing further support for our beta-amyloid Ca(2+)-channel hypothesis for neuronal death in Alzheimer's Disease.
Asunto(s)
Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/fisiología , Encéfalo/fisiopatología , Canales de Calcio/fisiología , Neuronas/fisiología , Animales , Encéfalo/patología , Cationes/metabolismo , Muerte Celular , Membrana Celular/fisiología , Humanos , Membrana Dobles de Lípidos , Potenciales de la Membrana , Modelos Neurológicos , Neuronas/patología , Fosfolípidos/fisiologíaRESUMEN
Ion channels can express multiple conductance levels that are not integer multiples of some unitary conductance, and that interconvert among one another. We report here that for 26 different types of multiple conductance channels, all allowed conductance levels can be calculated accurately using the geometric sequence gn = g(o) (3/2)n, where gn is a conductance level and n is an integer > or = 0. We refer to this relationship as the "3/2 Rule," because the value of any term in the sequence of conductances (gn) can be calculated as 3/2 times the value of the preceding term (gn-1). The experimentally determined average value for "3/2" is 1.491 +/- 0.095 (sample size = 37, average +/- SD). We also verify the choice of a 3/2 ratio on the basis of error analysis over the range of ratio values between 1.1 and 2.0. In an independent analysis using Marquardt's algorithm, we further verified the 3/2 ratio and the assignment of specific conductances to specific terms in the geometric sequence. Thus, irrespective of the open time probability, the allowed conductance levels of these channels can be described accurately to within approximately 6%. We anticipate that the "3/2 Rule" will simplify description of multiple conductance channels in a wide variety of biological systems and provide an organizing principle for channel heterogeneity and differential effects of channel blockers.
Asunto(s)
Conductividad Eléctrica , Canales Iónicos/fisiología , Modelos Biológicos , Modelos Teóricos , Neuronas/fisiología , Médula Espinal/fisiología , Animales , Anexinas/fisiología , Canales de Calcio/fisiología , Células Cultivadas , Canales de Cloruro/fisiología , Humanos , Activación del Canal Iónico , Modelos Neurológicos , Canales de Potasio/fisiología , Ratas , Receptores Colinérgicos/fisiología , Canales de Sodio/fisiologíaRESUMEN
We have recently shown that the Alzheimer disease 40-residue amyloid beta-protein [A beta P-(1-40)] can form cation-selective channels when incorporated into planar lipid bilayers by fusion of liposomes containing the peptide. Since A beta P-(1-40) comprises portions of the putative extracellular and membrane-spanning domains of the amyloid precursor protein (APP751), we suggested that the channel-forming property could be the underlying cause of amyloid neurotoxicity. The peptide has been proposed to occur in vivo in both membrane-bound and soluble forms, and we now report that soluble A beta P-(1-40) can also form similar channels in solvent-free lipid bilayers formed at the tip of a patch pipet, as well as in the planar lipid bilayer system. As in the case of liposome-mediated incorporation, the amyloid channel activity in the patch pipet exhibits multiple conductance levels between 40 and 400 pS, cation selectivity, and sensitivity to tromethamine (Tris). Further studies with A beta P channels incorporated into planar lipid bilayers from the liposome complex have also revealed that the channel activity can express spontaneous transitions to a much higher range of conductances between 400 and 4000 pS. Under these conditions, the amyloid channel continues to be cation selective. Amyloid channels were insensitive to nitrendipine at either conductance range. We calculate that if such channels were expressed in cells, the ensuing ion fluxes down their electrochemical potential gradients would be homeostatically dissipative. We therefore interpret these data as providing further support for the concept that cell death in Alzheimer disease may be due to amyloid ion-channel activity.
Asunto(s)
Péptidos beta-Amiloides/química , Canales Iónicos/química , Cationes , Conductividad Eléctrica , Técnicas In Vitro , Activación del Canal Iónico , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Potenciales de la Membrana , Membranas Artificiales , Nitrendipino/farmacologíaRESUMEN
Microsomal sarcoplasmic reticulum (SR) fractions from lobster skeletal muscle were found to bind [3H]-ryanodine. [3H]-ryanodine binding was enhanced by AMP, Ca2+ and caffeine, and significantly diminished by ATP, Ba2+ and Sr2+. Furthermore, dantrolene and ruthenium red, two classical inhibitors of Ca2+ release from the SR, blocked [3H]-ryanodine binding. Similarly, tetracaine, known to block the charge movement associated with excitation-contraction coupling in vertebrate muscle, inhibited the binding of the alkaloid. Our lobster SR preparation exhibited a single high-affinity ryanodine binding site (Kd = 6.6 nM, Bmax = 10 pmol/mg protein). Since SDS-PAGE of the SR proteins revealed a major band c. 565 kDa which comigrated with the putative ryanodine receptor from both rat and chicken skeletal muscle, we concluded that lobster skeletal muscle is equipped with the 565 kDa ryanodine receptor. Finally, incorporation of the SR microsomal fraction from lobster into planar bilayer membranes revealed the presence of a ryanodine-sensitive Ca2+ channel activity (160 pS in symmetrical 200 mM CsCl solutions). We concluded that both the crustacean and vertebrate skeletal muscle ryanodine receptor share the relevant properties such as molecular weight and affinity for ryanodine and inositol 1,4,5 triphosphate. However, there are important differences between the two receptors including differential effects of the alkaloid on the Ca2+ release channel and modulation of the receptor by nucleotides.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas Musculares/metabolismo , Nephropidae/metabolismo , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/ultraestructura , Animales , Cafeína/farmacología , Calcio/metabolismo , Calcio/fisiología , Canales de Calcio/efectos de los fármacos , Dantroleno/farmacología , Inositol 1,4,5-Trifosfato/farmacología , Membrana Dobles de Lípidos/metabolismo , Proteínas Musculares/efectos de los fármacos , Fosfolípidos/metabolismo , Rojo de Rutenio/farmacología , Rianodina/metabolismo , Rianodina/farmacología , Canal Liberador de Calcio Receptor de Rianodina , Retículo Sarcoplasmático/efectos de los fármacos , Sensibilidad y Especificidad , Tetracaína/farmacología , TritioRESUMEN
Amyloid beta protein (A beta P) is the 40-42 residue polypeptide implicated in the pathogenesis of Alzheimer's disease (AD). We have reconstituted this peptide into phosphatidylserine liposomes and then fused the liposomes with a planar lipid bilayer. When incorporated into this bilayer, the A beta P forms cation selective channels capable of transporting calcium and some monovalent cations including cesium, lithium, potassium, and sodium. The channels behave in an ohmic fashion and single channels can be shown to exhibit multiple subconductance states. Hitherto, A beta P has been presumed to be neurotoxic, although direct demonstration of toxicity has proved elusive. On the basis of the present data we suggest that the ion channel activity of the polypeptide may be the basis of its neurotoxic effects.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/fisiología , Amiloide/toxicidad , Canales de Calcio/fisiología , Compuestos de Alumbre/farmacología , Enfermedad de Alzheimer/etiología , Péptidos beta-Amiloides/efectos de los fármacos , Canales de Calcio/efectos de los fármacos , Humanos , Membrana Dobles de Lípidos/metabolismo , Fosfatidilserinas/metabolismo , Trometamina/farmacologíaRESUMEN
Amyloid beta protein (A beta P) is the 40- to 42-residue polypeptide implicated in the pathogenesis of Alzheimer disease. We have incorporated this peptide into phosphatidylserine liposomes and then fused the liposomes with a planar bilayer. When incorporated into bilayers the A beta P forms channels, which generate linear current-voltage relationships in symmetrical solutions. A permeability ratio, PK/PCl, of 11 for the open A beta P channel was estimated from the reversal potential of the channel current in asymmetrical KCl solutions. The permeability sequence for different cations, estimated from the reversal potential of the A beta P-channel current for each system of asymmetrical solutions, is Pcs > PLi > PCa > or = PK > PNa. A beta P-channel current (either CS+ or Ca2+ as charge carriers) is blocked reversibly by tromethamine (millimolar range) and irreversibly by Al3+ (micromolar range). The inhibition of the A beta P-channel current by these two substances depends on transmembrane potential, suggesting that the mechanism of blockade involves direct interaction between tromethamine (or Al3+) and sites within the A beta P channel. Hitherto, A beta P has been presumed to be neurotoxic. On the basis of the present data we suggest that the channel activity of the polypeptide may be responsible for some or all of its neurotoxic effects. We further propose that a useful strategy for drug discovery for treatment of Alzheimer disease may include screening compounds for their ability to block or otherwise modify A beta P channels.
Asunto(s)
Aluminio/farmacología , Péptidos beta-Amiloides/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Trometamina/farmacología , Enfermedad de Alzheimer/etiología , Péptidos beta-Amiloides/efectos de los fármacos , Canales de Calcio/efectos de los fármacos , Cationes/metabolismo , Conductividad Eléctrica , Humanos , Membrana Dobles de Lípidos , Potenciales de la Membrana/fisiología , Permeabilidad , FosfatidilserinasRESUMEN
Intact adrenal chromaffin granules and purified granule membrane ghosts were allowed to fuse with acidic phospholipid planar bilayer membranes in the presence of Ca2+ (1 mM). From both preparations, we were able to detect a large conductance potassium channel (ca. 160 pS in symmetrical 400 mM K+), which was highly selective for K+ over Na+ (PK/PNa = 11) as estimated from the reversal potential of the channel current. Channel activity was unaffected by charybdotoxin, a blocker of the [Ca2+]-activated K+ channel of large conductance. Furthermore, this channel proved quite different from the previously described channels from other types of secretory vesicle preparations, not only in its selectivity and conductance, but also in its insensitivity to both calcium and potential across the bilayer. We conclude that the chromaffin granule membrane contains a K(+)-selective channel with large conductance. We suggest that the role of this channel may include ion movement during granule assembly or recycling, and do not rule out events leading to exocytosis.