RESUMEN

At the neuromuscular junction (NMJ), changes to the size of the postsynaptic potential induce homeostatic compensation. At the Drosophila NMJ, increased glutamate release causes a compensatory decrease in quantal content, but it is unknown if this mechanism operates at the cholinergic mammalian NMJ. We addressed this question by recording endplate potentials (EPP) and muscle contraction in 3-month and 24-month ChAT-ChR2-EYFP mice that overexpress vesicular acetylcholine transporter and release more acetylcholine per vesicle. At 3 months, the quantal content of EPPs from ChAT-ChR2-EYFP mice were not different from WT controls, however tetanic depression was greater, and quantal size during high-frequency stimulation and the size of the readily releasable pool (RRP) were decreased. At 24 months of age, quantal content was reduced in ChAT-ChR2-EYFP mice, which normalized synaptic depression despite smaller RRP. The effect of pancuronium on indirect evoked muscle twitch was not different between groups. These results indicate that an increase in the amount of acetylcholine per vesicle induces two distinct age-dependent homeostatic mechanisms compensating excessive acetylcholine release.

Asunto(s)

Acetilcolina/metabolismo , Envejecimiento/metabolismo , Envejecimiento/fisiología , Homeostasis/fisiología , Unión Neuromuscular/metabolismo , Transmisión Sináptica/fisiología , Animales , Potenciales Postsinápticos Excitadores/fisiología , Expresión Génica , Ratones , Contracción Muscular/fisiología , Potenciales Sinápticos/fisiología , Proteínas de Transporte Vesicular de Acetilcolina/genética , Proteínas de Transporte Vesicular de Acetilcolina/metabolismo

RESUMEN

Antimicrobial peptides (AMPs) represent a large and ubiquitous group of peptides. The current crisis in antibiotic therapy has led to an intensified search for new antimicrobial agents. In this regard, scorpion venom constitutes a rich source of biologically active peptides including AMPs. In the present study, the purification of a novel peptide with antimicrobial activity against the Gram-negative bacteria Klebsiella pneumoniae is described. This antimicrobial peptide, named Cm38, was purified from Centruroides margaritatus scorpion venom using a two-step chromatographic strategy using C8 and C18 columns. This toxin inhibits the proliferation of the Gram-negative bacteria Klebsiella pneumoniae with a Minimal Inhibitory Concentration (MIC) of 64 µM. An analysis of the N-terminal sequence of Cm38 revealed a close structural relationship to Cn11, a Na+-channel modulator toxin previously isolated from Centruroides noxius scorpion venom. Therefore, to test Cm38 for effects on ion channels, we measured its effects on action potential firing in cultured dorsal root ganglion neurons. Cm38 depolarized and increased action potential firing in a subset of neurons tested. The present work reports a new peptide related to Cn11 with antimicrobial properties that is also active in neurons.

Asunto(s)

Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Ganglios Espinales/efectos de los fármacos , Klebsiella pneumoniae/efectos de los fármacos , Neuronas/efectos de los fármacos , Venenos de Escorpión/química , Secuencia de Aminoácidos , Animales , Antiinfecciosos/química , Antiinfecciosos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Electrofisiología , Ganglios Espinales/citología , Hemólisis/efectos de los fármacos , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Neuronas/citología , Ratas , Ratas Wistar , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

Antimicrobial peptides (AMPs) represent a large and ubiquitous group of peptides. The current crisis in antibiotic therapy has led to an intensified search for new antimicrobial agents. In this regard, scorpion venom constitutes a rich source of biologically active peptides including AMPs. In the present study, the purification of a novel peptide with antimicrobial activity against the Gram-negative bacteria Klebsiella pneumoniae is described. This antimicrobial peptide, named Cm38, was purified from Centruroides margaritatus scorpion venom using a two-step chromatographic strategy using C-8 and C-18 columns. This toxin inhibits the proliferation of the Gram-negative bacteria Klebsiella pneumoniae with a Minimal Inhibitory Concentration (MIC) of 64 mu M. An analysis of the N-terminal sequence of Cm38 revealed a close structural relationship to Cn11, a Na+-channel modulator toxin previously isolated from Centruroides noxius scorpion venom. Therefore, to test Cm38 for effects on ion channels, we measured its effects on action potential firing in cultured dorsal root ganglion neurons. Cm38 depolarized and increased action potential firing in a subset of neurons tested. The present work reports a new peptide related to Cn11 with antimicrobial properties that is also active in neurons.

Asunto(s)

Bioquímica , Genética , Microbiología

RESUMEN

Thiamine deficiency during embryonic or early postnatal development causes deficits in cerebellum-dependent activities including motor control and procedural memory. Here, we give a detailed description of the changes to A-type current in cultured cerebellar granule neurons exposed to thiamine deficiency in vitro. A-type current in treated neurons was reduced to 51% of that in controls. The remaining A-type current in treated neurons exhibited normal activation kinetics and voltage dependence whereas inactivation was markedly faster. These effects were selective because the delayed-rectifier potassium current density and kinetics were unchanged in thiamine-deficient neurons. A computational model of the cerebellar granule neuron was used to test the impact of these alterations and predicts an increase in excitability that is especially pronounced for synaptic activation. Our results suggest that the loss of A-type potassium conductance leads to hyperactivity in cerebellar granule neurons and may contribute to cell death observed in the granule layer of cerebellum during thiamine-deficiency in vivo.

Asunto(s)

Fenómenos Biofísicos/efectos de los fármacos , Neuronas/efectos de los fármacos , Canales de Potasio/fisiología , Tiamina/metabolismo , Tiamina/farmacología , Animales , Animales Recién Nacidos , Biofisica , Cerebelo/citología , Simulación por Computador , Estimulación Eléctrica , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Modelos Neurológicos , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Ratas , Ratas Wistar

RESUMEN

Epilepsy is a neurological disorder associated with excitatory and inhibitory imbalance within the underlying neural network. This study evaluated inhibitory γ-amino-butyric acid (GABA)ergic modulation in the CA1 region of the hippocampus of male Wistar rats and Wistar audiogenic rats (aged 90 ± 3 days), a strain of inbred animals susceptible to audiogenic seizures. Field excitatory postsynaptic potentials and population spike complexes in response to Schaffer collateral fiber stimulation were recorded in hippocampal slices before and during application of picrotoxin (50 µM, 60 min), a GABA A antagonist, and the size of the population spike was quantified by measuring its amplitude and slope. In control audiogenic-resistant Wistar rats (N = 9), picrotoxin significantly increased both the amplitude of the population spike by 51 ± 19 percent and its maximum slope by 73 ± 21 percent. In contrast, in slices from Wistar audiogenic rats (N = 6), picrotoxin caused no statistically significant change in population spike amplitude (33 ± 46 percent) or slope (11 ± 29 percent). Data are reported as means ± SEM. This result indicates a functional reduction of GABAergic neurotransmission in hippocampal slices from Wistar audiogenic rats.

Asunto(s)

Animales , Masculino , Ratas , Región CA1 Hipocampal/efectos de los fármacos , Epilepsia/metabolismo , Antagonistas del GABA/farmacología , Picrotoxina/farmacología , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismo , Región CA1 Hipocampal/metabolismo , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Ratas Wistar , Sinapsis/efectos de los fármacos , Sinapsis/fisiología

RESUMEN

Epilepsy is a neurological disorder associated with excitatory and inhibitory imbalance within the underlying neural network. This study evaluated inhibitory γ-amino-butyric acid (GABA)ergic modulation in the CA1 region of the hippocampus of male Wistar rats and Wistar audiogenic rats (aged 90 ± 3 days), a strain of inbred animals susceptible to audiogenic seizures. Field excitatory postsynaptic potentials and population spike complexes in response to Schaffer collateral fiber stimulation were recorded in hippocampal slices before and during application of picrotoxin (50 µM, 60 min), a GABA A antagonist, and the size of the population spike was quantified by measuring its amplitude and slope. In control audiogenic-resistant Wistar rats (N = 9), picrotoxin significantly increased both the amplitude of the population spike by 51 ± 19% and its maximum slope by 73 ± 21%. In contrast, in slices from Wistar audiogenic rats (N = 6), picrotoxin caused no statistically significant change in population spike amplitude (33 ± 46%) or slope (11 ± 29%). Data are reported as means ± SEM. This result indicates a functional reduction of GABAergic neurotransmission in hippocampal slices from Wistar audiogenic rats.

Asunto(s)

Región CA1 Hipocampal/efectos de los fármacos , Epilepsia/metabolismo , Antagonistas del GABA/farmacología , Picrotoxina/farmacología , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismo , Animales , Región CA1 Hipocampal/metabolismo , Masculino , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Ratas , Ratas Wistar , Sinapsis/efectos de los fármacos , Sinapsis/fisiología

RESUMEN

Neurotoxicity is a major symptom of envenomation caused by Brazilian coral snake Micrurus frontalis. Due to the small amount of material that can be collected, no neurotoxin has been fully sequenced from this venom. In this work we report six new three-finger like toxins isolated from the venom of the coral snake M. frontalis which we named Frontoxin (FTx) I-VI. Toxins were purified using multiple steps of RP-HPLC. Molecular masses were determined by MALDI-TOF and ESI ion-trap mass spectrometry. The complete amino acid sequence of FTx II, III, IV and V were determined by sequencing of overlapping proteolytic fragments by Edman degradation and by de novo sequencing. The amino acid sequences of FTx I, II, III and VI predict 4 conserved disulphide bonds and structural similarity to previously reported short-chain alpha-neurotoxins. FTx IV and V each contained 10 conserved cysteines and share high similarity with long-chain alpha-neurotoxins. At the frog neuromuscular junction FTx II, III and IV reduced miniature endplate potential amplitudes in a time-and concentration-dependent manner suggesting Frontoxins block nicotinic acetylcholine receptors.

Asunto(s)

Venenos Elapídicos/química , Elapidae , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Placa Motora/efectos de los fármacos , Neurotoxinas/toxicidad , Proteínas de Reptiles/toxicidad , Alquilación , Secuencia de Aminoácidos , Animales , Fraccionamiento Químico , Cisteína/análisis , Venenos Elapídicos/toxicidad , Técnicas In Vitro , Datos de Secuencia Molecular , Peso Molecular , Placa Motora/fisiología , Neurotoxinas/química , Neurotoxinas/aislamiento & purificación , Neurotoxinas/metabolismo , Concentración Osmolar , Oxidación-Reducción , Músculos Pectorales/efectos de los fármacos , Músculos Pectorales/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/toxicidad , Rana catesbeiana , Proteínas de Reptiles/química , Proteínas de Reptiles/aislamiento & purificación , Proteínas de Reptiles/metabolismo , Alineación de Secuencia

RESUMEN

PnTx3-1 is a peptide isolated from the venom of the spider Phoneutria nigriventer that specifically inhibits A-type K(+) currents (I(A)) in GH(3) cells. Here we used a bacterial expression system to produce an NH(2)-extended mutant of PnTx3-1 (ISEF-PnTx3-1) and tested whether the toxin is functional. The recombinant toxin was purified from bacterial extracts by a combination of affinity and ion-exchange chromatography. The recombinant toxin blocked A-type K(+) currents in GH(3) cells in a fashion similar to that observed with the wild-type toxin purified from the spider venom. These results suggest that recombinant cDNA methods provide a novel source for the production of functional Phoneutria toxins. The recombinant ISEF-PnTx3-1 should be useful for further understanding of the role of A-type K(+) currents in biological processes.

Asunto(s)

Neuropéptidos/biosíntesis , Bloqueadores de los Canales de Potasio , Proteínas Recombinantes de Fusión/biosíntesis , Venenos de Araña/genética , Secuencia de Aminoácidos , Animales , Fraccionamiento Químico , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Clonación Molecular , Datos de Secuencia Molecular , Neuropéptidos/genética , Neuropéptidos/farmacología , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Venenos de Araña/farmacología , Células Tumorales Cultivadas

RESUMEN

The present experiments investigated the effect of a neurotoxin purified from the venom of the spider Phoneutria nigriventer. This toxic component, P. nigriventer toxin 3-6 (PnTx3-6), abolished Ca(2+)-dependent glutamate release with an IC(50) of 74.4nM but did not alter Ca(2+)-independent secretion of glutamate when brain cortical synaptosomes were depolarized by KCl (33mM). This effect was most likely due to interference with the entry of calcium through voltage activated calcium channels (VACC), reducing the increase in the intrasynaptosomal free calcium induced by membrane depolarization with an IC(50) of 9.5nM. We compared the alterations induced by PnTx3-6 with the actions of toxins known to block calcium channels coupled to exocytosis. Our results indicate that PnTx3-6 inhibition of glutamate release and intrasynaptosomal calcium involves P/Q type calcium channels and this toxin can be a valuable tool in the investigation of calcium channels.

Asunto(s)

Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Ácido Glutámico/metabolismo , Neuropéptidos/farmacología , Potasio/farmacología , Sinaptosomas/metabolismo , Animales , Calcio/fisiología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Electrofisiología , Femenino , Técnicas In Vitro , Masculino , Neuropéptidos/aislamiento & purificación , Potasio/antagonistas & inhibidores , Ratas , Ratas Wistar , Sinaptosomas/efectos de los fármacos

RESUMEN

The SN56 cell line, a fusion of septal neurons and neuroblastoma cells, has been used as a model for central cholinergic neurons. These cells show increased expression of cholinergic neurochemical features upon differentiation, but little is known about how differentiation affects their electrophysiological properties. We examined the changes in Ca(2+) channel expression that occur as these cells undergo morphological differentiation in response to serum withdrawal and exposure to dibutyryl-cAMP. Undifferentiated cells expressed a T-type current with biophysical and pharmacological properties similar, although not identical, to those reported for the current generated by the alpha(1H) (CaV3.2) Ca(2+) channel subunit. Differentiated cells expressed, in addition to this T-type current, high voltage activated currents which were inhibited 38% by the L-type channel antagonist nifedipine (5 microM), 37% by the N-type channel antagonist omega-conotoxin-GVIA (1 microM), and 15% by the P/Q-type channel antagonist omega-agatoxin-IVA (200 nM). Current resistant to these inhibitors accounted for 15% of the high voltage activated current in differentiated SN56 cells. Our data demonstrate that differentiation increases the expression of neuronal type voltage gated Ca(2+) channels in this cell line, and that the channels expressed are comparable to those reported for native basal forebrain cholinergic neurons. This cell line should thus provide a useful model system to study the relationship between calcium currents and cholinergic function and dysfunction.

Asunto(s)

Acetilcolina/metabolismo , Encéfalo/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Diferenciación Celular/fisiología , Neuronas/metabolismo , Animales , Encéfalo/citología , Bucladesina/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Modelos Biológicos , Neuronas/citología , Técnicas de Placa-Clamp , omega-Conotoxina GVIA/farmacología

RESUMEN

Synaptic vesicle proteins are suggested to travel from the trans-Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP-VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP-VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4-64 and GFP-Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP-VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis-defective GFP-VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin-mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.

Asunto(s)

Acetilcolina/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/farmacocinética , Proteínas de Transporte de Membrana , Neuronas/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis/fisiología , Expresión Génica/fisiología , Proteínas Fluorescentes Verdes , Indicadores y Reactivos/farmacocinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/farmacocinética , Datos de Secuencia Molecular , Mutagénesis/fisiología , Neuronas/citología , Transmisión Sináptica/fisiología , Transfección , Células Tumorales Cultivadas , Proteínas de Transporte Vesicular de Acetilcolina

RESUMEN

The venom of a Brazilian spider, Lasiodora sp (Mygalomorphae, Theraphosidae), was screened for activity against ion channels using Ca2+ imaging and whole-cell patch clamp in GH3 cells. When tetrodotoxin (TTX) was present to block Na+ channels, the venom abolished the Ca2+ oscillations that are normally present in these cells and reduced the basal level of intracellular Ca2+. Under patch clamp, the venom reduced the L-type Ca2+ channel conductance and caused a positive shift in its voltage dependence of activation. In addition to these effects, when applied without TTX, the venom also caused a slow and noisy increase in intracellular Ca2+. The sensitivity of this second effect to TTX suggested an effect on Na+ channels, which was tested using patch clamp. Control Na+ currents inactivated completely as a single exponential. Treatment with the venom did not affect the amplitude of I(Na), but caused it to divide in two slower exponential components plus a sustained component, all of which were suppressed by TTX. The venom also caused a negative shift in the voltage dependence of activation and steady-state inactivation of I(Na). The observed effects of this venom on whole-cell currents explain the changes it causes in intracellular Ca2+ in GH3 cells and demonstrate that the venom of this spider is a source of toxins active against ion channels.

Asunto(s)

Canales de Calcio/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Venenos de Araña/farmacología , Algoritmos , Bario/metabolismo , Canales de Calcio Tipo L/efectos de los fármacos , Línea Celular , Colorantes Fluorescentes , Activación del Canal Iónico/efectos de los fármacos , Cinética , Técnicas de Placa-Clamp , Venenos de Araña/química

RESUMEN

The present experiments investigated the trafficking of the vesicular acetylcholine transporter (VAChT) tagged with the enhanced green fluorescent protein (EGFP) in living cholinergic cells (SN56). The EGFP-VAChT chimera was located in endosomal-like compartments in the soma of SN56 cells, and it was also targeted to varicosities of neurites. In contrast, EGFP alone in cells was soluble in the cytoplasm. The C-terminal cytoplasmic tail of VAChT has been implicated in targeting of VAChT to synaptic vesicles; thus, we have examined the role of the C-terminal region in the trafficking to varicosities. A C-terminal fragment tagged with EGFP appeared to be selectively accumulated in varicosities when expressed in SN56 cells. Interestingly, the protein was not freely soluble in the cytosol, and it presented a punctate pattern of expression. However, EGFP-C terminus did not present this peculiar pattern of expression in a nonneuronal cell line (HEK 293). Moreover, the C-terminal region of VAChT did not seem to be essential for VAChT trafficking, as a construct that lacks the C-terminal tail was, similar to EGFP-VAChT, partially targeted to endocytic organelles in the soma and sorted to varicosities. These experiments visualize VAChT for the first time in living cells and suggest that there might be multiple signals that participate in trafficking of VAChT to sites of synaptic vesicle accumulation.

Asunto(s)

Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Endocitosis/fisiología , Proteínas de Transporte de Membrana , Microscopía Confocal/métodos , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Animales , Técnica del Anticuerpo Fluorescente , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Indicadores y Reactivos/metabolismo , Riñón/citología , Proteínas Luminiscentes/genética , Ratones , Neuroblastoma , Neuronas/citología , Técnicas de Placa-Clamp , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales Cultivadas , Proteínas de Transporte Vesicular de Acetilcolina

RESUMEN

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in CatFr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (pH2O) on the order of 1 micron/sec whereas normal fiber cell membrane pH2O was 17 micron/sec frog, 32 micron/sec rabbit and 43 micron/sec mouse. CatFr mouse lens fiber cell pH2O was reduced by 13 micron/sec for heterozygous and 30 micron/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the pH2O conferred by MIP is not sensitive to Hg2+ whereas that of CHIP28 (AQP1) is blocked by Hg2+. The fiber cell membrane pH2O was also not sensitive to Hg2+ whereas lens epithelial cell pH2O (136 micron/sec in rabbit) was blocked by Hg2+. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous CatFr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses.

Asunto(s)

Proteínas del Ojo/farmacología , Canales Iónicos/fisiología , Corteza del Cristalino/fisiología , Animales , Anuros , Acuaporinas , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Conductividad Eléctrica , Células Epiteliales/fisiología , Proteínas del Ojo/genética , Proteínas del Ojo/fisiología , Uniones Comunicantes/efectos de los fármacos , Glicerol/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacología , Glicoproteínas de Membrana/fisiología , Ratones , Mutación/fisiología , Conejos , Agua/metabolismo , Agua/fisiología

RESUMEN

GH3 cells present spontaneous Ca2+ action potentials and oscillations of intracellular Ca2+, which can be modified by altering the activity of K+ or Ca2+ channels. We took advantage of this spontaneous activity to screen for effects of a purified toxin (Tx3-1) from the venom of Phoneutria nigriventer on ion channels. We report that Tx3-1 increases the frequency of Ca2+ oscillations, as do two blockers of potassium channels, 4-aminopyridine and charybdotoxin. Whole-cell patch clamp experiments show that Tx3-1 reversibly inhibits the A-type K+ current (I(A)) but does not block other K+ currents (delayed-rectifying, inward-rectifying, and large-conductance Ca2+-sensitive) or Ca2+ channels (T and L type) in these cells. In addition, we describe the sequence of a full cDNA clone of Tx3-1, which shows that Tx3-1 has no homology to other known blockers of K+ channels and gives insights into the processing of this neurotoxin. We conclude that Tx3-1 is a selective inhibitor of I(A), which can be used to probe the role of this channel in the control of cellular function. Based on the effect of Tx3-1, we suggest that I(A) is an important determinant of the frequency of Ca2+ oscillations in unstimulated GH3 cells.

Asunto(s)

Calcio/metabolismo , Neuropéptidos/farmacología , Periodicidad , Canales de Potasio/fisiología , Venenos de Araña/farmacología , 4-Aminopiridina/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Línea Celular , Caribdotoxina/farmacología , Quelantes/farmacología , ADN Complementario/aislamiento & purificación , Ácido Egtácico/farmacología , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Neuropéptidos/genética , Nifedipino/farmacología , Técnicas de Placa-Clamp , Hipófisis/química , Hipófisis/citología , Bloqueadores de los Canales de Potasio , Venenos de Araña/genética

RESUMEN

The GH3 anterior pituitary cell line has been used as a model to investigate diverse aspects of pituitary cell physiology including Ca2+ homeostasis and secretion. These cells possess muscarinic receptors which, by activating K+ channels and inhibiting Ca2+ channels, should decrease electrical excitability. We measured the effect of carbachol (10 microM) on the frequency of Ca2+ oscillations caused by Ca2+ action potentials in the plasma membrane. Carbachol reduced oscillation frequency by approximately 85% (p < 0.001). This inhibition was reversed by atropine (1 microM), and was prevented by pre-incubation with pertussis toxin (200 ng/ml, 24 h). Since many anterior pituitary cell types secrete acetylcholine, the presence of muscarinic receptors coupled to cell excitability in these cells suggest that ACh could exert a paracrine- or autocrine-like action in GH3 cell cultures. In experiments designed to test this idea, perfusion with 1 microM atropine caused a small but significant increase (p < 0.05) in oscillation frequency when the cells had previously been incubated for 30 min without perfusion. However, this effect was not blocked by either pre-treatment with pertussis toxin or by including atropine during the entire experiment (including the 30-min incubation without perfusion). We conclude that these cells respond to muscarinic agonists by decreasing oscillation frequency but find no evidence for feedback control by endogenous ACh under these conditions.

Asunto(s)

Potenciales de Acción/fisiología , Calcio/metabolismo , Adenohipófisis/metabolismo , Receptores Muscarínicos/metabolismo , Potenciales de Acción/efectos de los fármacos , Atropina/farmacología , Carbacol/farmacología , Células Cultivadas , Agonistas Colinérgicos/farmacología , Proteínas de Unión al GTP/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Antagonistas Muscarínicos/farmacología , Toxina del Pertussis , Adenohipófisis/efectos de los fármacos , Receptores Muscarínicos/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacología

RESUMEN

Lens Major Intrinsic Protein (MIP) is a member of a family of membrane transport proteins including the Aquaporins and bacterial glycerol transporters. When expressed in Xenopus oocytes, MIP increased both glycerol permeability and the activity of glycerol kinase. Glycerol permeability (pGly) was 2.3 +/- 0.23 x 10(-6) cm sec-1 with MIP vs. 0.92 +/- 0.086 x 10(-6) cm sec-1 in control oocytes. The pGly of MIP was independent of concentration from 5 x 10(-5) to 5 x 10(-2) m, had a low temperature dependence, and was inhibited approximately 90%, 80% and 50% by 1.0 mM Hg++, 0.2 mM DIDS (diisothiocyanodisulfonic stilbene), and 0.1 mm Cu++, respectively. MIP-enhanced glycerol phosphorylation, resulting in increased incorporation of glycerol into lipids. This could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based on methods we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase (0.12 +/- 0.03 vs. 0.06 +/- 0.01 pmol min-1 cell-1) without changing the binding of glycerol to the kinase (KM approximately 10 micron).

Asunto(s)

Permeabilidad de la Membrana Celular , Proteínas del Ojo/metabolismo , Glicerol/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Acuaporinas , Proteínas del Ojo/biosíntesis , Femenino , Cinética , Modelos Biológicos , Oocitos/fisiología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Xenopus laevis

RESUMEN

Gamma1- and gamma2-zeathionins (gamma1-Z and gamma2-Z) are members of a family of small and basic peptides involved in plant protection. These plant defensins exhibit remarkable structural similarity to scorpion neurotoxins and insect defensins. In the present report, we used the whole-cell patch clamp technique to investigate the inhibition of the sodium current (I(Na)) by gamma1-Z and gamma2-Z in the GH3 cell line. Both gamma1-Z and gamma2-Z rapidly and reversibly inhibited I(Na) without changing the kinetics or voltage dependence of activation or inactivation. To our knowledge, this is the first example of a plant protein that inhibits the sodium channel. From structural comparisons with the mu-conotoxins, a family of peptides that block the sodium channel, we detected some similar features that could provide the basis of inhibition of sodium channels by gamma-zeathionins.

Asunto(s)

Proteínas de Plantas/farmacología , Proteínas/farmacología , Bloqueadores de los Canales de Sodio , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos , Línea Celular , Defensinas , Relación Dosis-Respuesta a Droga , Electrofisiología , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas/química , Ratas , Homología de Secuencia de Aminoácido , Canales de Sodio/metabolismo , Zea mays

RESUMEN

We have expressed frog (Rana pipiens) lens major intrinsic protein (MIP) in Xenopus oocytes and observed its effect on ion conductance, water permeability and neutral solute transport. SDS-PAGE and immunoblotting demonstrated oocytes injected with MIP mRNA expressed the protein at high levels. Immunolocalization indicated the expressed MIP migrated to the plasma membrane. MIP had no effect on the slope of oocyte I-V relations in the range -50 to +10 mV, although the averaged I-V curve was shifted 10 mV positive to control. MIP increased oocyte water permeability by a factor of 1.9 +/- 0.2, whereas the permeability to sucrose, 2-deoxyglucose, inositol, sorbitol, reduced glutathione or urea was unchanged. Glycerol permeability was enhanced in oocytes expressing MIP. In contrast to control oocytes, 3H-glycerol radioactivity accumulation did not follow first order kinetics. Radioactivity continued to accumulate even after 19 h of uptake and went beyond equilibrium with the bath. The time course of MIP-mediated glycerol uptake was modeled assuming metabolic trapping with good results. Based on this model, MIP increased oocyte glycerol permeability by a factor of 2.7.

Asunto(s)

Proteínas del Ojo/biosíntesis , Glicoproteínas de Membrana , Oocitos/metabolismo , Animales , Acuaporinas , Western Blotting , Permeabilidad de la Membrana Celular , Electroforesis en Gel de Poliacrilamida , Femenino , Glicerol/farmacocinética , Técnicas In Vitro , Transporte Iónico , Rana pipiens , Factores de Tiempo , Agua/metabolismo , Xenopus