RESUMEN
BACKGROUND: Mutations of voltage-gated sodium channel alpha(II) gene, SCN2A, have been described in a wide spectrum of epilepsies. While inherited SCN2A mutations have been identified in multiple mild epilepsy cases, a de novo SCN2A-R102X mutation, which we previously reported in a patient with sporadic intractable childhood localization-related epilepsy, remains unique. To validate the involvement of de novo SCN2A mutations in the etiology of intractable epilepsies, we sought to identify additional instances. METHODS: We performed mutational analyses on SCN2A in 116 patients with severe myoclonic epilepsy in infancy, infantile spasms, and other types of intractable childhood partial and generalized epilepsies and did whole-cell patch-clamp recordings on Na(v)1.2 channels containing identified mutations. RESULTS: We discovered 2 additional de novo SCN2A mutations. One mutation, SCN2A-E1211K, was identified in a patient with sporadic infantile spasms. SCN2A-E1211K produced channels with altered electrophysiologic properties compatible with both augmented (an approximately 18-mV hyperpolarizing shift in the voltage dependence of activation) and reduced (an approximately 22-mV hyperpolarizing shift in the voltage dependence of steady-state inactivation and a slowed recovery from inactivation) channel activities. The other de novo mutation, SCN2A-I1473M, was identified in a patient with sporadic neonatal epileptic encephalopathy. SCN2A-I1473M caused an approximately 14-mV hyperpolarizing shift in the voltage dependence of activation. CONCLUSIONS: The identified de novo mutations SCN2A-E1211K, -I1473M, and -R102X indicate that SCN2A is an etiologic candidate underlying a variety of intractable childhood epilepsies. The phenotypic variations among patients might be due to the different electrophysiologic properties of mutant channels.
Asunto(s)
Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/fisiopatología , Mutación Missense , Proteínas del Tejido Nervioso/genética , Índice de Severidad de la Enfermedad , Canales de Sodio/genética , Secuencia de Aminoácidos , Línea Celular , Secuencia Conservada , Análisis Mutacional de ADN , Resultado Fatal , Femenino , Haplotipos , Humanos , Recién Nacido , Riñón/citología , Masculino , Canal de Sodio Activado por Voltaje NAV1.2 , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/fisiología , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína , Canales de Sodio/química , Canales de Sodio/fisiología , Espasmos Infantiles/genética , Espasmos Infantiles/fisiopatología , Transfección , Adulto JovenRESUMEN
The channel and chaperone activities of Clostridial botulinum neurotoxin (BoNT) A were investigated in Neuro 2a neuroblastoma cells under conditions that closely emulate those prevalent at the endosome. Channel activity occurs in bursts interspersed between periods of little or no activity. The channels are voltage dependent, opening only at negative voltages. Within bursts, the channel resides preferentially in the open state. The channels open to a main conductance of 105 +/- 5 pS or 65 +/- 4 pS in 200 mM CsCl or NaCl, respectively. The BoNT channels display a conspicuous subconductance of 10 +/- 2 pS. The neuroblastoma cell line appears, therefore, to be a suitable system to characterize the BoNT channel and to pursue evaluation of plausible strategies for targeted drug delivery thereby minimizing the requirement for in vivo animal testing.
Asunto(s)
Toxinas Botulínicas Tipo A/metabolismo , Neoplasias Encefálicas/metabolismo , Chaperonas Moleculares/metabolismo , Neuroblastoma/metabolismo , Animales , Línea Celular Tumoral , Electrofisiología , Endosomas/metabolismo , Canales Iónicos/metabolismo , Ratones , Técnicas de Placa-Clamp , Transporte de ProteínasRESUMEN
Vpu, a membrane protein from human immunodeficiency virus-1, folds into two distinct structural domains with different biological activities: a transmembrane (TM) helical domain involved in the budding of new virions from infected cells, and a cytoplasmic domain encompassing two amphipathic helices, which is implicated in CD4 degradation. The molecular mechanism by which Vpu facilitates virion budding is not clear. This activity of Vpu requires an intact TM helical domain. And it is known that oligomerization of the VPU TM domain results in the formation of sequence-specific, cation-selective channels. It has been shown that the channel activity of Vpu is confined to the TM domain, and that the cytoplasmic helices regulate the lifetime of the Vpu channel in the conductive state. Structure-function correlates based on the convergence of information about the channel activity of Vpu reconstituted in lipid bilayers and on its 3-D structure in membranes by a combination of solution and solid-state nuclear magnetic resonance spectroscopy may provide valuable insights to understand the role of Vpu in the pathogenesis of AIDS and for drug design aimed to block channel activity.
Asunto(s)
Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/fisiología , Síndrome de Inmunodeficiencia Adquirida , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antígenos CD4/biosíntesis , Cationes , Citoplasma/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana , Iones , Membrana Dobles de Lípidos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , ViriónRESUMEN
The structures of functional peptides corresponding to the predicted channel-lining M2 segment of the nicotinic acetylcholine (AChR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. The AChR M2 peptide forms a straight transmembrane alpha-helix, with no kinks. M2 inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminus of the peptide, which is assigned to be intracellular. A molecular model of the AChR channel pore, constructed from the solid-state NMR 3-D structure of the AChR M2 helix in the membrane assuming a pentameric organization, results in a funnel-like architecture for the channel with the wide opening on the N-terminal intracellular side. A central narrow pore has a diameter ranging from about 3.0 A at its narrowest, to 8.6 A at its widest. Nonpolar residues are predominantly on the exterior of the bundle, while polar residues line the pore. This arrangement is in fair agreement with evidence collected from permeation, mutagenesis, affinity labeling and cysteine accessibility measurements. A pentameric M2 helical bundle may, therefore, represent the structural blueprint for the inner bundle that lines the channel of the nicotinic AChR.
Asunto(s)
Canales Iónicos/química , Membrana Dobles de Lípidos/análisis , Proteínas de la Membrana/química , Receptores Nicotínicos/química , Animales , Humanos , Espectroscopía de Resonancia Magnética/métodos , Micelas , Modelos Moleculares , Porinas/químicaRESUMEN
Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in beta1 and alpha(I)-subunit genes of voltage-gated Na(+) channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R188W) [corrected] in the gene encoding the alpha-subunit of neuronal voltage-gated Na(+) channel type II (Na(v)1.2) in a patient with FS associated with afebrile seizures. The mutation R188W [corrected] occurring on Arg(187), a highly conserved residue among voltage-gated Na(+) channels, was not found in 224 alleles of unaffected individuals. Whole-cell patch clamp recordings on human embryonic kidney (HEK) cells expressing a rat wild-type (rNa(v)1.2) and the corresponding mutant channels showed that the mutant channel inactivated more slowly than wild-type whereas the Na(+) channel conductance was not affected. Prolonged residence in the open state of the R188W [corrected] mutant channel may augment Na(+) influx and thereby underlie the neuronal hyperexcitability that induces seizure activity. Even though a small pedigree could not show clear cosegregation with the disease phenotype, these findings strongly suggest the involvement of Na(v)1.2 in a human disease and propose the R188W [corrected] mutation as the genetic defect responsible for febrile seizures associated with afebrile seizures.
Asunto(s)
Mutación Missense , Proteínas del Tejido Nervioso/genética , Convulsiones Febriles/genética , Convulsiones/genética , Canales de Sodio/genética , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Niño , ADN Complementario , Electrofisiología , Humanos , Masculino , Datos de Secuencia Molecular , Canal de Sodio Activado por Voltaje NAV1.1 , Canal de Sodio Activado por Voltaje NAV1.2 , Proteínas del Tejido Nervioso/fisiología , Ratas , Canales de Sodio/fisiologíaRESUMEN
Excitotoxicity, resulting from sustained activation of glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype, is considered to play a causative role in the etiology of ischemic stroke and several neurodegenerative diseases. The NMDA receptor is therefore a target for the development of neuroprotective agents. Here, we identify an N-benzylated triamine (denoted as NBTA) as a highly selective and potent NMDA-receptor channel blocker selected by screening a reduced dipeptidomimetic synthetic combinatorial library. NBTA blocks recombinant NMDA receptors expressed in Xenopus laevis oocytes with a mean IC(50) of 80 nM; in contrast, it does not block GluR1, a glutamate receptor of the non-NMDA subtype. The blocking activity of NBTA on NMDA receptors exhibits the characteristics of an open-channel blocker: (i) no competition with agonists, (ii) voltage dependence, and (iii) use dependence. Significantly, NBTA protects rodent hippocampal neurons from NMDA receptor, but not kainate receptor-mediated excitotoxic cell death, in agreement with its selective action on the corresponding recombinant receptors. Mutagenesis data indicate that the N site, a key asparagine on the M2 transmembrane segment of the NR1 subunit, is the main determinant of the blocker action. The results highlight the potential of this compound as a neuroprotectant.
Asunto(s)
Aminas/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Muerte Celular , Células Cultivadas , Femenino , Hipocampo/citología , Neuronas/citología , Receptores de N-Metil-D-Aspartato/genética , Xenopus laevisRESUMEN
Sequence similarity among and electrophysiological studies of known potassium channels, along with the three-dimensional structure of the Streptomyces lividans K(+) channel (KcsA), support the tenet that voltage-gated K(+) channels (Kv channels) consist of two distinct modules: the "voltage sensor" module comprising the N-terminal portion of the channel up to and including the S4 transmembrane segment and the "pore" module encompassing the C-terminal portion from the S5 transmembrane segment onward. To substantiate this modular design, we investigated whether the pore module of Kv channels may be replaced with the pore module of the prokaryotic KcsA channel. Biochemical and immunocytochemical studies showed that chimeric channels were expressed on the cell surface of Xenopus oocytes, demonstrating that they were properly synthesized, glycosylated, folded, assembled, and delivered to the plasma membrane. Unexpectedly, surface-expressed homomeric chimeras did not exhibit detectable voltage-dependent channel activity upon both hyperpolarization and depolarization regardless of the expression system used. Chimeras were, however, strongly dominant-negative when coexpressed with wild-type Kv channels, as evidenced by the complete suppression of wild-type channel activity. Notably, the dominant-negative phenotype correlated well with the formation of stable, glycosylated, nonfunctional, heteromeric channels. Collectively, these findings imply a structural compatibility between the prokaryotic pore module and the eukaryotic voltage sensor domain that leads to the biogenesis of non-responsive channels. Our results lend support to the notion that voltage-dependent channel gating depends on the precise coupling between both protein domains, probably through a localized interaction surface.
Asunto(s)
Proteínas Bacterianas , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/química , Canales de Potasio/fisiología , Streptomyces/fisiología , Animales , Células COS , Membrana Celular/fisiología , Chlorocebus aethiops , Femenino , Canal de Potasio Kv.1.1 , Potenciales de la Membrana/fisiología , Modelos Moleculares , Oocitos/fisiología , Técnicas de Placa-Clamp , Canales de Potasio/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , Transfección , Xenopus laevisRESUMEN
Neural activity is crucial for cell survival and fine patterning of neuronal connectivity during neurodevelopment. To investigate the role in vivo of sodium channels (NaCh) in these processes, we generated knockout mice deficient in brain NaChalpha(II). NaChalpha(II)(-/-) mice were morphologically and organogenically indistinguishable from their NaChalpha(+/-) littermates. Notwithstanding, NaChalpha(II)(-/-) mice died perinatally with severe hypoxia and massive neuronal apoptosis, notably in the brainstem. Sodium channel currents recorded from cultured neurons of NaChalpha(II)(-/-) mice were sharply attenuated. Death appears to arise from severe hypoxia consequent to the brainstem deficiency of NaChalpha(II). NaChalpha(II) expression is, therefore, redundant for embryonic development but essential for postnatal survival.
Asunto(s)
Encéfalo/metabolismo , Neuronas/patología , Neuronas/fisiología , Canales de Sodio/deficiencia , Canales de Sodio/genética , Animales , Animales Recién Nacidos , Apoptosis , Encéfalo/patología , Tronco Encefálico/patología , Muerte Celular , Células Cultivadas , Muerte Fetal , Hipocampo/fisiología , Ratones , Ratones Noqueados , Neocórtex/patología , Recombinación Genética , Mapeo Restrictivo , Saxitoxina/farmacocinética , Canales de Sodio/fisiologíaRESUMEN
Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release, presumably by cleaving SNAP-25, a protein involved in docking of synaptic vesicles with the presynaptic plasma membrane. Three excitation-secretion uncoupling peptides (ESUPs), which mimic the carboxy-terminal domain of SNAP-25 and span or adjoin the cleavage sites for BoNT/A and BoNT/E, also inhibit transmitter release from permeabilized bovine chromaffin cells. In this study, these peptides were tested for effects on acetylcholine (ACh) release at an identified cholinergic synapse in isolated buccal ganglia of Aplysia californica. The presynaptic neuron was stimulated electrically to elicit action potentials. The postsynaptic neuron was voltage-clamped, and evoked inhibitory postsynaptic currents (IPSCs) were recorded. The ESUPs were pressure-injected into the presynaptic neuron, and their effects on the amplitude of the IPSCs were studied. Acetylcholine release from presynaptic cells, as measured by IPSC amplitudes, was gradually inhibited by the ESUPs. All three peptides caused ca. 40% reduction in IPSC amplitude in 2 h. Random-sequence peptides of the same amino acid composition had no effect. Injection of BoNT/E, in contrast, caused ca. 50% reduction in IPSC amplitude in 30 min and almost complete inhibition in 2 h. These results are the first demonstration that ESUPs block neuronal cholinergic synaptic transmission. They are consistent with the concept that ESUPs compete with the intact SNAP-25 for binding with other fusion proteins, thus inhibiting stimulus-evoked exocytosis of neurotransmitter.
Asunto(s)
Acetilcolina/metabolismo , Toxinas Botulínicas/toxicidad , Proteínas de la Membrana , Proteínas del Tejido Nervioso/farmacología , Fragmentos de Péptidos/farmacología , Sinapsis/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Aplysia , Toxinas Botulínicas Tipo A , Sinapsis/metabolismo , Proteína 25 Asociada a SinaptosomasRESUMEN
Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic alpha-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly (15)N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane alpha-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu(2-51) (residues 2-51), which contains the transmembrane alpha-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu(28-81) (residues 28-81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2-81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 +/- 3 pS and 12 +/- 3 pS in 0.5 M KCl and 29 +/- 3 pS and 12 +/- 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu(2-51), which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu(28-81), which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.
Asunto(s)
VIH-1/química , Proteínas Reguladoras y Accesorias Virales/química , Secuencia de Aminoácidos , Antígenos CD4/metabolismo , VIH-1/genética , Proteínas del Virus de la Inmunodeficiencia Humana , Canales Iónicos/química , Membrana Dobles de Lípidos/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Proteínas Reguladoras y Accesorias Virales/fisiologíaRESUMEN
Sequence similarity among known potassium channels indicates the voltage-gated potassium channels consist of two modules: the N-terminal portion of the channel up to and including transmembrane segment S4, called in this paper the 'sensor' module, and the C-terminal portion from transmembrane segment S5 onwards, called the 'pore' module. We investigated the functional role of these modules by constructing chimeric channels which combine the 'sensor' from one native voltage-gated channel, mKv1.1, with the 'pore' from another, Shaker H4, and vice versa. Functional studies of the wild type and chimeric channels show that these modules can operate outside their native context. Each channel has a unique conductance-voltage relation. Channels incorporating the mKv1.1 sensor module have similar rates of activation while channels having the Shaker pore module show similar rates of deactivation. This observation suggests the mKv1.1 sensor module limits activation and the Shaker pore module determines deactivation. We propose a model that explains the observed equilibrium and kinetic properties of the chimeric constructs in terms of the characteristics of the native modules and a novel type of intrasubunit cooperativity. The properties ascribed to the modules are the same whether the modules function in their native context or have been assembled into a chimera.
Asunto(s)
Canales de Potasio/química , Animales , Técnicas Genéticas , Cinética , Oocitos , Técnicas de Placa-Clamp , Canales de Potasio/genética , Canales de Potasio/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Relación Estructura-Actividad , XenopusRESUMEN
Depletion of Ca2+ stores in Xenopus oocytes activated entry of Ca2+ across the plasma membrane, which was measured as a current I(soc) in subsequently formed cell-attached patches. I(soc) survived excision into inside-out configuration. If cell-attached patches were formed before store depletion, I(soc) was activated outside but not inside the patches. I(soc) was potentiated by microinjection of Clostridium C3 transferase, which inhibits Rho GTPase, whereas I(soc) was inhibited by expression of wild-type or constitutively active Rho. Activation of I(soc) was also inhibited by botulinum neurotoxin A and dominant-negative mutants of SNAP-25 but was unaffected by brefeldin A. These results suggest that oocyte I(soc) is dependent not on aqueous diffusible messengers but on SNAP-25, probably via exocytosis of membrane channels or regulatory molecules.
Asunto(s)
Toxinas Botulínicas , Señalización del Calcio , Proteínas de la Membrana , Proteínas del Tejido Nervioso/fisiología , ADP Ribosa Transferasas/farmacología , Animales , Toxinas Botulínicas Tipo A/farmacología , Calcio/metabolismo , Canales de Calcio/metabolismo , Difusión , Exocitosis , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/fisiología , Transporte Iónico , Modelos Biológicos , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Oocitos/citología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Proteína 25 Asociada a Sinaptosomas , Xenopus laevis , Proteínas de Unión al GTP rhoRESUMEN
The assignment of amide resonances in the two-dimensional PISEMA (Polarization Inversion with Spin Exchange at the Magic Angle) spectrum of uniformly 15N labeled M2 peptide corresponding to the channel-lining segment of the acetylcholine receptor in oriented phospholipid bilayers is described. The majority of the resonances were assigned through comparisons with spectra from selectively 15N labeled recombinant peptides and specifically 15N labeled synthetic peptides. Some resonances were assigned to specific amino acid residues by means of homonuclear 15N spin-exchange spectroscopy. A modification to the conventional spin-exchange pulse sequence that significantly shortens the length of the experiments by combining the intervals for 15N spin-exchange and 1H magnetization recovery is described.
Asunto(s)
Membrana Dobles de Lípidos , Receptores Muscarínicos/química , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular/instrumentación , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/química , Conformación Proteica , Receptor Muscarínico M2RESUMEN
SNARE proteins appear to be involved in homotypic and heterotypic membrane fusion events [Sollner et al. (1993) Nature 362, 318-324]. The crystal structure of the synaptic SNARE complex exhibits a parallel four-helical bundle fold with two helices contributed by SNAP-25, a target SNARE (t-SNARE), and the other two by a different t-SNARE, syntaxin, and a donor vesicle SNARE (v-SNARE), synaptobrevin. The carboxy-terminal boundary of the complex, predicted to occur at the closest proximity between the apposed membranes, displays a high density of positively charged residues. This feature combined with the enrichment of negatively charged phospholipids in the cytosolic exposed leaflet of the membrane bilayer suggest that electrostatic attraction between oppositely charged interfaces may be sufficient to induce dynamic and discrete micellar discontinuities of the apposed membranes with the transient breakdown at the junction and subsequent reformation. Thus, the positively charged end of the SNARE complex in concert with Ca2+ may be sufficient to generate a transient 'fusion pore'.
Asunto(s)
Fusión de Membrana/fisiología , Proteínas de Transporte Vesicular , Calcio/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Fosfolípidos/metabolismo , Proteínas SNARE , Electricidad EstáticaRESUMEN
The structures of functional peptides corresponding to the predicted channel-lining M2 segments of the nicotinic acetylcholine receptor (AChR) and of a glutamate receptor of the NMDA subtype (NMDAR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. Both M2 segments form straight transmembrane alpha-helices with no kinks. The AChR M2 peptide inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminal side of the membrane, which is assigned to be intracellular. A model built from these solid-state NMR data, and assuming a symmetric pentameric arrangement of M2 helices, results in a funnel-like architecture for the channel, with the wide opening on the N-terminal intracellular side.
Asunto(s)
Canales Iónicos/química , Fragmentos de Péptidos/química , Receptores de N-Metil-D-Aspartato/química , Receptores Nicotínicos/química , Secuencia de Aminoácidos , Escherichia coli/genética , Activación del Canal Iónico , Marcaje Isotópico , Membrana Dobles de Lípidos , Lípidos/química , Espectroscopía de Resonancia Magnética/métodos , Micelas , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolucionesRESUMEN
Voltage-sensitive cation-selective ion channels of the voltage-gated ion channel (VGC) superfamily were examined by a combination of sequence alignment and phylogenetic tree construction procedures. Segments of the alpha-subunits of K+-selective channels homologous to the structurally elucidated KcsA channel of Streptomyces lividans were multiply aligned, and this alignment provided the database for computer-assisted structural analyses and phylogenetic tree construction. Similar analyses were conducted with the four homologous repeats of the alpha-subunits from representative Ca2+- and Na+-selective channels, as well as with the ensemble of K+, Ca2+ and Na+ channels. In both the single subunit of the K+ channels and the individual repeats of the Ca2+ and Na+ channels, the analyses suggest the occurrence of at least two tandemly arranged modules corresponding to the predicted voltage-sensor domain and the pore domain. The phylogenetic analyses reveal strict clustering of segments according to cation-selectivity and repeat unit. We surmise that the pore module of the prokaryotic K+ channel was the primordial polypeptide upon which other modules were superimposed during evolution in order to generate phenotypic diversity. These observations may prove applicable to all members of the VGC family yet to be discovered throughout the prokaryotic and eukaryotic kingdoms.
Asunto(s)
Canales de Calcio/clasificación , Canales de Potasio/clasificación , Canales de Sodio/clasificación , Secuencia de Aminoácidos , Animales , Canales de Calcio/análisis , Humanos , Activación del Canal Iónico , Datos de Secuencia Molecular , Filogenia , Canales de Potasio/análisis , Análisis de Secuencia , Canales de Sodio/análisisRESUMEN
The assembly of target (t-SNARE) and vesicle-associated SNAP receptor (v-SNARE) proteins is a critical step for the docking of synaptic vesicles to the plasma membrane. Syntaxin-1A, SNAP-25, and synaptobrevin-2 (also known as vesicle-associated membrane protein, or VAMP-2) bind to each other with high affinity, and their binding regions are predicted to form a trimeric coiled-coil. Here, we have designed three peptides, which correspond to sequences located in the syntaxin-1A H3 domain, the C-terminal domain of SNAP-25, and a conserved central domain of synaptobrevin-2, that exhibit a high propensity to form a minimal trimeric coiled-coil. The peptides were synthesized by solid phase methods, and their interactions were studied by CD spectroscopy. In aqueous solution, the peptides were unstructured and showed no interactions with each other. In contrast, upon the addition of moderate amounts of trifluoroethanol (30%), the peptides adopted an alpha-helical structure and displayed both homomeric and heteromeric interactions. The interactions observed in ternary mixtures induce a stabilization of peptide structure that is greater than that predicted from individual binary interactions, suggesting the formation of a higher order structure compatible with the assembly of a trimeric coiled-coil.
Asunto(s)
Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Química Encefálica , Dicroismo Circular , Humanos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Qa-SNARE , Proteínas R-SNARE , Espectrofotometría Ultravioleta , Proteína 25 Asociada a Sinaptosomas , Sintaxina 1RESUMEN
Botulinum neurotoxin E (BoNT E) cleaves SNAP-25 at the C-terminal domain releasing a 26-mer peptide. This peptide product may act as an excitation-secretion uncoupling peptide (ESUP) to inhibit vesicle fusion and thus contribute to the efficacy of BoNT E in disabling neurosecretion. We have addressed this question using a synthetic 26-mer peptide which mimics the amino acid sequence of the naturally released peptide, and is hereafter denoted as ESUP E. This synthetic peptide is a potent inhibitor of Ca2+-evoked exocytosis in permeabilized chromaffin cells and reduces neurotransmitter release from identified cholinergic synapses in in vitro buccal ganglia of Aplysia californica. In chromaffin cells, both ESUP E and BoNT E abrogate the slow component of secretion without affecting the fast, Ca2+-mediated fusion event. Analysis of immunoprecipitates of the synaptic ternary complex involving SNAP-25, VAMP and syntaxin demonstrates that ESUP E interferes with the assembly of the docking complex. Thus, the efficacy of BoNTs as inhibitors of neurosecretion may arise from the synergistic action of cleaving the substrate and releasing peptide products that disable the fusion process by blocking specific steps of the exocytotic cascade.
Asunto(s)
Toxinas Botulínicas/metabolismo , Vesículas Cubiertas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/fisiología , Secuencia de Aminoácidos , Animales , Aplysia , Bovinos , Células Cultivadas , Células Cromafines , Vesículas Cubiertas/efectos de los fármacos , Exocitosis/efectos de los fármacos , Sustancias Macromoleculares , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/fisiología , Péptidos/síntesis química , Péptidos/farmacología , Proteínas Qa-SNARE , Proteínas R-SNARE , Ratas , Proteína 25 Asociada a SinaptosomasRESUMEN
This review focuses on two questions: the role of mitochondria in excitotoxic neuronal death and the connection of mitochondria with the apoptotic death cascade. The goal is to highlight the regulatory role of mitochondrial channels on the mitochondrial membrane potential, Deltapsi, and their involvement in determining neuronal survival or death. A hypothesis is developed centered on the notion that protein-protein interactions between members of the Bcl-2 family of death suppressor and promoter proteins lead to the selective elimination of depolarizing currents that, in turn, collapse Deltapsi and set in motion the irreversible pathway of cell death. The model considers the remarkable propensity of Bcl-2 family proteins to dimerize or oligomerize and thereby restrict the localization of partner molecules to mitochondrial membrane contact sites. The fundamental principle invoked here is that through a concerted set of protein-protein interactions, information is exchanged by specific heterodimers, one of the partners acting as a toxic protein and the second as its antidote. The review concludes with the elaboration of a speculative model about cellular mechanisms for the prevention of cell destruction as triggered by extracellular signals which may be conserved in its molecular design from bacteria to eukaryotes.