RESUMEN
GABAA receptors (GABAARs) are crucial in mediating inhibition in the adult mammalian brain. Although the kinetics of this receptor has been extensively studied, the molecular picture of interactions occurring at various channel conformations remains elusive. While electrophysiology combined with mutagenesis sheds light on the role of specific residues, ultrastructural studies reveal static structures which, in the case of GABAARs, are limited to the ß3 homomer. To take advantage of the newest crystal structures of cys-loop receptors, a homology model of α1ß2γ2 GABAAR in the unbound closed state was built using a template of the homomeric glycine receptor in the closed state. The template model contained strychnine molecules at the binding sites which were removed and molecular dynamics was used to study the system relaxation. The modeled GABAAR preserved the closed conformation. Two interfaces forming orthosteric binding sites (ß2/α1) exhibited opening due to the outward shift of loop C. Similar movement, although less pronounced, was observed at the α1/γ2 (modulatory) interface. In contrast, interfaces α1/ß2 and γ2/ß2 remained closed. The former one, due to interactions mediated mainly by loops C and F, affected the neighboring ß2/α1 interface leading to asymmetry between the orthosteric binding sites. Such interactions were not observed at the ß2/α1 interface preceded by a γ2 subunit. As expected, in the channel pore, the conserved leucine gate and selectivity filter were present. However, an additional constriction was found at the top of the pore which differed from a typical hydrophobic channel gate as it consisted of charged residues. Interestingly, this site showed a capacity to trap chloride ions and to undergo conformation transition-like expansion, suggesting an impact on pore properties. In conclusion, our homology model faithfully reproduced major features of heteromeric GABAARs offering insight into the underlying mechanisms of stabilizing the shut conformation and chloride ion interaction with the channel pore.
Asunto(s)
Cloruros/metabolismo , Receptores de GABA-A/metabolismo , Sitios de Unión , Cloruros/química , Humanos , Modelos Químicos , Simulación de Dinámica Molecular , Unión Proteica , Receptores de GABA-A/química , Estricnina/químicaRESUMEN
Metalloproteinases (MMPs) have been shown to play a crucial role in synaptic plasticity and cognitive processes. We recently reported that in the mossy fiber - CA3 hippocampal pathway, LTP maintenance required fine-tuned MMP-9 activity, as both MMP-9 excess and absence impaired LTP. Here we used acute brain slices from transgenic (TG) rats overexpressing MMP-9 to investigate the impact of excessive MMP-9 activity on the excitatory synaptic transmission in the CA3-CA1 projection. Using field potential recordings, we have demonstrated that MMP-9 overexpression increased the strength of basal synaptic transmission but had no effect on the short-term plasticity in comparison to the wild-type (WT) group. In attempt to shed light on mechanisms underlying this observation, miniature excitatory postsynaptic potentials (mEPSCs) were recorded from pyramidal CA1 neurons. We found that mEPSCs in the TG group had a significantly slower decaying phase than in WT but amplitudes and frequencies were similar. The lack of differences in mEPSC frequency and short-term plasticity between TG and WT groups suggests that MMP-9 overexpression effect on fEPSPs was mainly postsynaptic. Additionally, we have found that excess of MMP-9 in TG rats was associated with impaired late-phase of LTP in the considered pathway. It seems thus that augmented synaptic strength in TG rats occurred in expense of impaired long-term plasticity induced by tetanization. In conclusion, overexpression of MMP-9 leads to increase in the strength of basal excitatory synaptic transmission and impairs of LTP maintenance phase in the CA3-CA1 pathway in vitro.
Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Plasticidad Neuronal/fisiología , Células Piramidales/metabolismo , Animales , Animales Modificados Genéticamente/metabolismo , Animales Modificados Genéticamente/fisiología , Hipocampo/fisiología , Masculino , Células Piramidales/fisiología , Ratas , Ratas Wistar , Transmisión Sináptica/fisiologíaRESUMEN
Acute hippocampal slices and primary neuronal cultures are often used with a tacit assumption that basic characteristics of the two models closely resemble each other. The use of the cell cultures, however, may raise controversies because of non-physiological conditions resulting from e.g. glial cells deficit, random neuronal sprouting, lack of specificity in the synaptic connections, impaired homeostasis, etc. Importantly, alteration in neuronal environment, especially when occurring over a prolonged period of time, may give rise to a profound homeostatic modulation. In the present study we have compared the properties of GABAergic and glutamatergic (non-NMDA) currents in pyramidal neurons from hippocampal slices and neuronal cell culture. We show that, most strikingly, amplitude ratio of currents elicited by ultrafast applications of saturating GABA and glutamate was nearly one order of magnitude larger in cultured neurons than that in slices. Miniature IPSCs and EPSCs also showed substantial differences between these two models. In particular, mEPSC amplitudes were larger and more frequent in cultured neurons but their time duration was longer in slices. Miniature IPSCs did not show differences in amplitude when comparing slices and cultures but their time duration was faster and occurrence more frequent in slices. In conclusion, we provide evidence that expression pattern of GABA(A) and glutamate receptors as well as synaptic current properties in the neuronal cell culture show profound differences with respect to that in the physiological conditions.
Asunto(s)
Ácido Glutámico/fisiología , Hipocampo/fisiología , Homeostasis/fisiología , Neuronas/fisiología , Receptores de GABA-A/fisiología , Receptores de Glutamato/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/citología , Potenciales Postsinápticos Inhibidores/fisiología , Técnicas de Cultivo de Órganos , Ratas , Ratas WistarRESUMEN
The organomercurial, thimerosal, is at the center of medical controversy as a suspected factor contributing to neurodevelopmental disorders in children. Many neurotoxic effects of thimerosal have been described, but its interaction with principal excitatory and inhibitory neurotransmiter systems is not known. We examined, using electrophysiological recordings, thimerosal effects on GABA and NMDA-evoked currents in cultured hippocampal neurons. After brief (3 to 10 min) exposure to thimerosal at concentrations up to 100 µM, there was no significant effect on GABA or NMDA-evoked currents. However, following exposure for 60-90 min to 1 or 10 µM thimerosal, there was a significant decrease in NMDA-induced currents (p<0.05) and GABAergic currents (p<0.05). Thimerosal was also neurotoxic, damaging a significant proportion of neurons after 60-90 min exposure; recordings were always conducted in the healthiest looking neurons. Mercuric chloride, at concentrations 1 µM and above, was even more toxic, killing a large proportion of cells after just a few minutes of exposure. Recordings from a few sturdy cells revealed that micromolar mercuric chloride markedly potentiated the GABAergic currents (p<0.05), but reduced NMDA-evoked currents (p<0.05). The results reveal complex interactions of thimerosal and mercuric ions with the GABA(A) and NMDA receptors. Mercuric chloride act rapidly, decreasing electrophysiological responses to NMDA but enhancing responses to GABA, while thimerosal works slowly, reducing both NMDA and GABA responses. The neurotoxic effects of both mercurials are interwoven with their modulatory actions on GABA(A) and NMDA receptors, which most likely involve binding to these macromolecules.
Asunto(s)
Hipocampo/efectos de los fármacos , Cloruro de Mercurio/toxicidad , N-Metilaspartato/metabolismo , Neuronas/efectos de los fármacos , Timerosal/toxicidad , Ácido gamma-Aminobutírico/metabolismo , Animales , Células Cultivadas , Fenómenos Electrofisiológicos/efectos de los fármacos , Hipocampo/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Neuronas/metabolismo , Neurotransmisores/antagonistas & inhibidores , Neurotransmisores/metabolismo , Unión Proteica/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Factores de TiempoRESUMEN
Gephyrin is a tubulin-binding protein that acts as a scaffold for clustering glycine and GABA(A) receptors at postsynaptic sites. In this study, the role of gephyrin on GABA(A) receptor function was assessed at the post-translational level, using gephyrin-specific single chain antibody fragments (scFv-gephyrin). When expressed in cultured rat hippocampal neurons as a fusion protein containing a nuclear localization signal, scFv-gephyrin were able to remove endogenous gephyrin from GABA(A) receptor clusters. Immunocytochemical experiments revealed a significant reduction in the number of synaptic gamma2-subunit containing GABA(A) receptors and a significant decrease in the density of the GABAergic presynaptic marker vesicular GABA transporter (VGAT). These effects were associated with a slow down of the onset kinetics, a reduction in the amplitude and in the frequency of miniature inhibitory postsynaptic currents (mIPSCs). The quantitative analysis of current responses to ultrafast application of GABA suggested that changes in onset kinetics resulted from modifications in the microscopic gating of GABA(A) receptors and in particular from a reduced entry into the desensitized state. In addition, hampering gephyrin function with scFv-gephyrin induced a significant reduction in GABA(A) receptor-mediated tonic conductance. This effect was probably dependent on the decrease in GABAergic innervation and in GABA release from presynaptic nerve terminals. These results indicate that gephyrin is essential not only for maintaining synaptic GABA(A) receptor clusters in the right position but also for regulating both phasic and tonic inhibition.
Asunto(s)
Proteínas Portadoras/metabolismo , Hipocampo/fisiología , Proteínas de la Membrana/metabolismo , Inhibición Neural/fisiología , Neuronas/fisiología , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Células Cultivadas , Inmunohistoquímica , Potenciales Postsinápticos Inhibidores/fisiología , Cinética , Potenciales de la Membrana/fisiología , Microscopía Confocal , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Anticuerpos de Cadena Única/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Benzodiazepines (BDZs) are widely used in clinical practice and are known as positive modulators of GABAergic currents. BDZs increase binding affinity and recently they were found to affect GABA(A) receptor gating, including desensitization. Binding and desensitization are also strongly modulated by extracellular pH, a factor that may be severely altered in a pathological brain. It is thus of interest to examine the combined action of BDZ and protons. EXPERIMENTAL APPROACH: Pharmacokinetic analysis was based on patch clamp recordings of miniature IPSCs (mIPSCs) and current responses to GABA applications in rat cultured hippocampal neurons. High temporal resolution of currents evoked by exogenous GABA was achieved by using an ultrafast perfusion system (exchange time ca. 80 micros). KEY RESULTS: At acidic pH, flurazepam produced a stronger enhancement of mIPSC amplitudes than at physiological pH. At low GABA concentrations, flurazepam markedly enhanced current amplitudes both at normal and acidic pH, but at the latter, the relative effect was larger. In contrast, at saturating GABA concentrations, flurazepam reduced current amplitudes at both pH 7.2 and 6.0. The slowing of deactivation kinetics by flurazepam decreased with GABA concentration, but at pH 6.0, this trend was shifted toward a higher GABA concentration. CONCLUSIONS AND IMPLICATIONS: Acidification of extracellular medium may significantly affect the susceptibility of phasic and tonic components of GABAergic currents to modulation by BDZs. Quantitative analysis and model simulations indicate that protons and flurazepam additively affect binding and desensitization of GABA(A) receptors.
Asunto(s)
Espacio Extracelular/metabolismo , Flurazepam/farmacología , Moduladores del GABA/farmacología , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/fisiología , Animales , Electrofisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Modelos Neurológicos , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
Memantine, a clinically used N-methyl-D-aspartate (NMDA)-receptor antagonist, has been shown to prevent apoptotic neuronal damage connected with the over-activity of NMDA receptors. In the present study, we examined the effect of memantine on staurosporine-, salsolinol- and doxorubicin-induced apoptosis in the SH-SY5Y cell line which does not possess functional NMDA receptors. Electrophysiological recordings and toxicity studies showed no response to NMDA-evoked currents in this cell line, irrespective of the stage of its neuronal differentiation. Memantine (0.1-2 microM) attenuated staurosporine-induced apoptosis as evidenced by reversal of the changes in mitochondrial membrane potential (DeltaPsi(m)) and decreased caspase-3 activity, lactate dehydrogenase (LDH) release and DNA fragmentation. Wortmannin (10 nM) and LY 294002 (10 microM) (inhibitors of phosphatidylinositol-3-kinase, PI3-K) reversed the inhibitory effect of memantine on the staurosporine-induced LDH release, suggesting that the PI3-K/Akt prosurvival pathway is a possible target for antiapoptotic action of memantine. Memantine at low micromolar concentrations also attenuated salsolinol- and doxorubicin-induced LDH release and DNA fragmentation, but only in the case of salsolinol was this effect accompanied by a decrease in caspase-3 activity. The present data indicate that memantine attenuates the toxic effects of various proapoptotic agents and the cytoprotective effect of memantine does not seem to be connected with its action on NMDA receptor but rather with its influence on intracellular pathways engaged in cellular survival/apoptotic processes.
Asunto(s)
Antibióticos Antineoplásicos/antagonistas & inhibidores , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Doxorrubicina/antagonistas & inhibidores , Doxorrubicina/toxicidad , Inhibidores Enzimáticos/toxicidad , Agonistas de Aminoácidos Excitadores/farmacología , Isoquinolinas/antagonistas & inhibidores , Isoquinolinas/toxicidad , Memantina/farmacología , Estaurosporina/antagonistas & inhibidores , Estaurosporina/toxicidad , Androstadienos/farmacología , Caspasa 3/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromonas/farmacología , Electrofisiología , Humanos , L-Lactato Deshidrogenasa/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Morfolinas/farmacología , N-Metilaspartato/toxicidad , Técnicas de Placa-Clamp , Tretinoina/farmacología , WortmaninaRESUMEN
We applied the whole-cell patch-clamp technique to study the influence of zinc ions (Zn(2+)) and extracellular protons at acidic pH (pH(o)) on voltage-gated potassium currents in cultured rat hippocampal neurons. The first goal of the study was to estimate whether Kv1.3 currents significantly contributed to voltage-gated potassium currents in examined cells. Then, the influence of both ions on the activity of other voltage-gated potassium currents in the neurons was examined. We examined both the total current and the delayed - rectifier component. Results obtained in both cases were not significantly different from each other. Available data argued against any significant contribution of Kv1.3 currents to the recorded currents. Nevertheless, application of Zn(2+) in the concentration range from 100 microM to 5 mM reversibly modulated the recorded currents. The activation midpoint was shifted by about 40 mV (total current) and 30 mV (delayed-rectifier current) towards positive membrane potentials and the activation kinetics were slowed significantly (2 - 3 fold) upon application of Zn(2+). The inactivation midpoint was also shifted towards positive membrane potentials, but less significantly (about 14 mV). The current amplitudes were reduced in a concentration-dependent manner to about 0.5 of the control value. The effects of Zn(2+) were saturated at the concentration of 1 mM. Raising extracellular proton concentration by lowering the pH(o) from 7.35 to 6.4 did not affect significantly the currents. Possible mechanisms underlying the observed phenomena and their possible physiological significance are discussed.
Asunto(s)
Hipocampo/citología , Neuronas/fisiología , Canales de Potasio con Entrada de Voltaje/fisiología , Zinc/fisiología , Animales , Cationes Bivalentes , Células Cultivadas , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Activación del Canal Iónico , Canal de Potasio Kv1.3/fisiología , Potenciales de la Membrana , Neuronas/citología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Protones , Ratas , Ratas Wistar , Zinc/farmacologíaRESUMEN
We applied the whole-cell patch-clamp technique to study the inhibitory effect of copper ions (Cu) on the activity of Kv1.3 channels expressed in human lymphocytes. Application of Cu reversibly inhibited the currents to about 10% of the control value in a concentration-dependent manner with the half blocking concentration of 5.28+/-0.5 microM and the Hill's coefficient of 3.83+/-0.18. The inhibitory effect was saturated at 10 microM concentration. The inhibition was time-dependent and it was correlated in time with a significant slowing of the current activation rate. In contrast the voltage dependence of activation was not changed by Cu as well as the inactivation kinetics. The inhibitory effect of Cu was voltage-independent. It was also unaffected by changing the extracellular pH in the range from 6.4 to 8.4, raising the extracellular potassium concentration to 150 mM and by changing the holding potential from -90 to -60 mV. The inhibitory effect of Cu was not changed in the presence of an equivalent concentration of Zn. Altogether, obtained data suggest that Cu inhibits Kv1.3 channels by a different mechanism than Zn and that Cu and Zn act on different binding sites. The inhibitory effect of Cu was probably due to a specific binding of Cu on binding sites on the channels. Possible physiological significance of the Cu-induced inhibition of Kv1.3 channels is discussed.
Asunto(s)
Cobre/farmacología , Canal de Potasio Kv1.3/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Linfocitos T/efectos de los fármacos , Cationes/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Concentración de Iones de Hidrógeno , Canal de Potasio Kv1.3/fisiología , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Linfocitos T/citología , Linfocitos T/fisiología , Factores de Tiempo , Zinc/farmacologíaRESUMEN
In the present study we applied the whole-cell patch-clamp technique to study the influence of extracellular pH (pH(o)) on the modulatory effect exerted by zinc ions (Zn(2+)) on voltage-gated potassium channels Kv1.3 expressed in human lymphocytes. Obtained data provide evidence that lowering of pH(o) from the 7.35 to 6.4 slowed significantly the current activation rate, shifted the activation midpoint by about 16 mV towards positive membrane potentials and reduced the current amplitude to about 0.55 of the control value. In contrast, raising the pH(o) from 7.35 to 8.4 did not affect significantly the activation midpoint and current amplitude. Application of Zn(2+) in the concentration range from 100 microM to 1 mM at pH(o)=6.4 slowed additionally the activation rate, shifted the activation midpoint by about 20 mV towards positive membrane potentials and reduced the current amplitude in a concentration-dependent manner. The total effect exerted by Zn(2+) and protons at pH(o) = 6.4 was more significant than the effect exerted by Zn alone. Both the magnitude of the shift and the degree of current inhibition by Zn(2+) were independent on pH(o) in the range from 6.4 to 8.4. The data might suggest that the effects exerted by protons and zinc ions occur independently on each other and probably involve different mechanisms. Changing the holding potential from -90 mV to -60 mV at pH(o)=7.35 abolished the Zn(2+)-induced inhibition of the current amplitudes at concentrations below 300 microM. At pH(o)=6.4 the total inhibition caused by Zn(2+) and protons was also diminished, however, a significant reduction was observed at 100 microM concentration. In contrast, changing the holding potential did not change the Zn(2+)- and proton-induced shift of the activation midpoint. Altogether, obtained data suggest that extracellular protons exert the modulatory effects that are additive to the effects exerted by Zn(2+) on the channels. Possible physiological significance of these additive effects is discussed.
Asunto(s)
Canal de Potasio Kv1.3/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Zinc/farmacología , 4-Aminopiridina/farmacología , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.3/fisiología , Potenciales de la Membrana/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Linfocitos T/fisiologíaRESUMEN
In a recent study we have provided evidence that inhibition of native GABA(A) receptors by zinc depends primarily on the allosteric modulation of receptor gating. Both the kinetics and the sensitivity of the GABA(A) receptor to zinc depend on subunit composition, especially on the presence of the gamma(2) subunit. To analyze the mechanism of action of zinc its effects have been tested on recombinant alpha(1)beta(2)gamma(2) and alpha(1)beta(2) receptors expressed in HEK 293 cells. The currents produced by ultrafast application of GABA have been measured to assess the impact of zinc ions on GABA(A) receptor gating with resolution corresponding to the time scale of synaptic currents. While, as expected, zinc markedly reduced the peak amplitude of alpha(1)beta(2)-mediated currents, its effect on kinetics was significantly different from that observed for alpha(1)beta(2)gamma(2). In particular, unlike alpha(1)beta(2)gamma(2), zinc did not affect the onset of alpha(1)beta(2)-mediated responses. Moreover, zinc increased the extent of desensitisation of alpha(1)beta(2)gamma(2) receptors and reduced desensitisation of alpha(1)beta(2) ones. Quantitative analysis suggests that zinc exerts an allosteric modulation on both alpha(1)beta(2)gamma(2) and alpha(1)beta(2) receptors. Zinc effects on alpha(1)beta(2)gamma(2) were qualitatively similar to those reported for native receptors.
Asunto(s)
Receptores de GABA-A/efectos de los fármacos , Zinc/farmacología , Algoritmos , Línea Celular , Estimulación Eléctrica , Electrofisiología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Cinética , Modelos Neurológicos , Perfusión , Unión Proteica , Proteínas Recombinantes/efectos de los fármacosRESUMEN
The effect of chlorpromazine (CPZ) on deactivation of N-methyl-D-aspartate (NMDA)-induced currents was studied in the whole-cell configuration of the patch-clamp technique in cultured rat hippocampal neurons. We report that CPZ (at 30-1000 microM) strongly slowed down the deactivation process in a dose-dependent manner. At high CPZ concentration (1 mM), the NMDA-elicited currents were insensitive to NMDA removal as long as CPZ was present and deactivated only when both NMDA and CPZ were washed out. CPZ by itself did not activate any current. These data indicate that one of CPZ actions is to stabilise the open conformation of NMDA receptors probably by fixing it in the bound state. This CPZ effect may be important as the synaptic currents represent mainly the deactivation process.
Asunto(s)
Antipsicóticos/farmacología , Clorpromazina/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/fisiología , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Animales , Células Cultivadas , Potenciales Postsinápticos Excitadores , Hipocampo/citología , Neuronas/fisiología , Técnicas de Placa-Clamp , RatasRESUMEN
The effect of a widely used phenothiazine, chlorpromazine (CPZ) on ionotropic glutamate receptors was investigated using the patch-clamp technique in cultured rat hippocampal neurons. The non-N-methyl-D-aspartate (NMDA) receptors were insensitive to CPZ. The current responses to NMDA showed a wide range of variability of both the rate and the extent of desensitization. The responses characterized by fast and profound desensitization were strongly inhibited by 30 microM CPZ. The effectiveness of block of NMDA responses was clearly correlated with the apparent time constant of the desensitization onset. The extent of desenstitization was found to be increased by CPZ. We conclude that CPZ inhibits the NMDA receptors and propose that the sensibility to CPZ may depend both on the subunit composition and on the state of receptor modulation by intracellular factors.
Asunto(s)
Antipsicóticos/farmacología , Clorpromazina/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Animales , Células Cultivadas , Conductividad Eléctrica , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/citología , N-Metilaspartato/farmacología , Técnicas de Placa-Clamp , Ratas , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
Synaptic transmission between neurons plays a key role in signal transduction in the central nervous system (CNS). Postsynaptic currents can be routinely recorded using the patch-clamp technique but mechanisms underlying their kinetics are not entirely clear. Model simulations and experimental evidence indicate that the time course of synaptic agonist is extremely fast and short lasting implying that activation of postsynaptic receptors takes place in conditions of extreme non-equilibrium. For this reason, studies of postsynaptic receptors' kinetics requires ultrafast agonist application techniques able to mimic synaptic conditions, the goal that only recently has been technically achieved. In the present paper, examples of kinetic studies of synaptic currents and current responses to ultrafast agonist applications are presented. Examples are given that pharmacological modulation of synaptic receptors strongly depends on the non-equilibrium conditions of receptors' activation. Application of kinetic studies to determine subtypes of synaptic receptors are also presented and discussed.
Asunto(s)
Transmisión Sináptica/fisiología , Animales , Clorpromazina/farmacología , Electrofisiología , Modelos Neurológicos , Técnicas de Placa-Clamp , Transducción de Señal , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Brain-derived calcium-binding protein S100 induces apoptosis in a significant fraction of rat phaeochromocytoma (PC12) cells. We used single cell techniques (patch clamp, videomicroscopy and immunocytochemistry) to clarify some of the specific aspects of S100-induced apoptosis, the modality(ies) of early intracellular Ca2+ concentration increase and the expression of some classes of genes (c-fos, c-jun, bax, bcl-x, p-15, p-21) known to be implicated in apoptosis of different cells. The results show that S100: (1) causes an increase of [Ca2+]i due to an increased conductance of L-type Ca2+ channels; (2) induces a sustained increase of the Fos levels which is evident since the first time point tested (3 h) and remains elevated until to the last time point (72 h). All these data suggest that S100-derived apoptosis in PC12 cells may be the consequence of a system involving an increase in L-type Ca2+ channel conductance with consequent [Ca2+]i increase which up-regulates, directly or indirectly, the expression of Fos.
Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al Calcio/fisiología , Calcio/fisiología , Genes fos/fisiología , Proteínas S100/fisiología , Animales , Química Encefálica/fisiología , Calcio/metabolismo , Canales de Calcio/fisiología , Señalización del Calcio/genética , Señalización del Calcio/fisiología , Regulación Neoplásica de la Expresión Génica/genética , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Microscopía Fluorescente , Microscopía por Video , Células PC12 , Técnicas de Placa-Clamp , RatasRESUMEN
Zinc is abundantly present in the CNS, and after nerve stimulation is thought to be released in sufficient quantity to modulate the synaptic transmission. Although it is known that this divalent cation inhibits the GABAergic synaptic currents, the underlying mechanisms were not fully elucidated. Here we report that zinc reduced the amplitude, slowed the rise time, and accelerated the decay of mIPSCs in cultured hippocampal neurons. The analysis of current responses to rapid GABA applications and model simulations indicated that these effects on mIPSCs are caused by zinc modulation of GABA(A) receptor gating. In particular, zinc slowed the onset of GABA-evoked currents by decreasing both the binding (k(on)) and the transition rate from closed to open state (beta(2)). Moreover, slower onset and recovery from desensitization as well as an increased unbinding rate (k(off)) were shown to underlie the accelerated deactivation kinetics in the presence of zinc. The nonequilibrium conditions of GABA(A) receptor activation were found to strongly affect zinc modulation of this receptor. In particular, an extremely fast clearance of synaptic GABA is implicated to be responsible for a stronger zinc effect on mIPSCs than on current responses to exogenous GABA. Finally, the analysis of currents evoked by GABA coapplied with zinc indicated that the interaction between zinc and GABA(A) receptors was too slow to explain zinc effects in terms of competitive antagonism. In conclusion, our results provide evidence that inhibition of mIPSCs by zinc is attributable to the allosteric modulation of GABA(A) receptor gating.
Asunto(s)
Regulación Alostérica/fisiología , Activación del Canal Iónico/fisiología , Neuronas/metabolismo , Receptores de GABA-A/metabolismo , Zinc/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Unión Competitiva/efectos de los fármacos , Células Cultivadas , Simulación por Computador , Relación Dosis-Respuesta a Droga , Potenciales Evocados/efectos de los fármacos , Antagonistas del GABA/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Modelos Neurológicos , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Conformación Proteica/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de GABA-A/efectos de los fármacos , Bloqueadores de los Canales de Sodio , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Zinc/farmacología , beta-Alanina/farmacología , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
The whole cell configuration of the patch clamp technique was used to study the effects of chlorpromazine (CPZ), a widely used antipsychotic drug, on miniature GABAA-mediated synaptic currents (mIPSCs) in hippocampal cells in culture. CPZ (10-30 microM) induced a clear dose-dependent increase of mIPSCs frequency that was associated with a decrease in amplitude and with an acceleration of their decay kinetics. When applied in a calcium-free medium, CPZ was less effective in enhancing mIPSCs frequency, suggesting that this effect was partially calcium dependent. While a low (10 microM) CPZ concentration induced a 2-fold increase in the total charge transfer a higher (30 microM) dose of this drug produced no changes, indicating that the presynaptic effect was counterbalanced by the postsynaptic one.
Asunto(s)
Antipsicóticos/farmacología , Clorpromazina/farmacología , Antagonistas de Dopamina/farmacología , Hipocampo/fisiología , Neuronas/fisiología , Terminales Presinápticos/efectos de los fármacos , Ácido gamma-Aminobutírico/fisiología , Animales , Calcio/metabolismo , Células Cultivadas , Conductividad Eléctrica , Espacio Extracelular/metabolismo , Hipocampo/citología , Hipocampo/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/efectos de los fármacos , Terminales Presinápticos/metabolismo , Ratas , Sinapsis/fisiologíaRESUMEN
The scope of this work is to make a brief presentation of the electrophysiological recordings employed in the studies on ionic channel properties in physiological and pathological conditions. A number of examples are made on how electrophysiological recording contributed to understand the pathogenesis of certain diseases such as myasthenia gravis, myothonia congenita, cystic fibrosis and degradation of neurons in CA1 hippocampal region following ischaemia.
Asunto(s)
Electrofisiología/métodos , Canales Iónicos/metabolismo , Transducción de Señal/fisiología , Animales , Fibrosis Quística/fisiopatología , Hipocampo/metabolismo , Humanos , Ataque Isquémico Transitorio/fisiopatología , Miastenia Gravis/fisiopatología , Miotonía Congénita/fisiopatología , Unión Neuromuscular/metabolismoRESUMEN
Recent studies have emphasized that nonequilibrium conditions of postsynaptic GABAA receptor (GABAAR) activation is a key factor in shaping the time course of IPSCs (Puia et al., 1994; Jones and Westbrook, 1995). Such nonequilibrium, resulting from extremely fast agonist time course, may affect the interaction between pharmacological agents and postsynaptic GABAARs. In the present study we found that chlorpromazine (CPZ), a widely used antipsychotic drug known to interfere with several ligand and voltage-gated channels, reduces the amplitude and accelerates the decay of miniature IPSCs (mIPSCs). A good qualitative reproduction of the effects of CPZ on mIPSCs was obtained when mIPSCs were mimicked by responses to ultrafast GABA applications to excised patches. Our experimental data and model simulations indicate that CPZ affects mIPSCs by decreasing the binding (kon) and by increasing the unbinding (koff) rates of GABAARs. Because of reduction of kon by CPZ, the binding reaction becomes rate-limiting, and agonist exposure of GABAARs during mIPSC is too short to activate the receptors to the same extent as in control conditions. The increase in unbinding rate is implicated as the mechanism underlying the acceleration of mIPSC decaying phase. The effect of CPZ on GABAAR binding rate, resulting in slower onset of GABA-evoked currents, provides a tool to estimate the speed of synaptic clearance of GABA. Moreover, the onset kinetics of recorded responses allowed the estimate the peak synaptic GABA concentration.
Asunto(s)
Antipsicóticos/farmacología , Clorpromazina/farmacología , Receptores de GABA-A/efectos de los fármacos , Ácido gamma-Aminobutírico/fisiología , Animales , Células Cultivadas , Potenciales Evocados/efectos de los fármacos , Cinética , Técnicas de Placa-Clamp , Ratas , Receptores de GABA-A/metabolismo , Transmisión Sináptica/efectos de los fármacos , Grabación en VideoRESUMEN
The whole cell configuration of the patch-clamp technique was used to study the effects of different cytosolic calcium concentrations [Ca2+]i on desensitization kinetics of gamma-aminobutyric acid (GABA)-activated receptors in acutely dissociated rat hippocampal neurons. Two different intrapipette concentrations of the calcium chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 11 and 0.9 mM, respectively) were used to yield a low (1.2 x 10(-8) M) or a high (2.2 x 10(-6) M) [Ca2+]i. In low [Ca2+]i, peak values of GABA-evoked currents (20 microM) evoked at -30 mV, were significantly larger than those recorded in high calcium [2,970 +/- 280 (SE) pA vs. 1,870 +/- 150 pA]. The extent of desensitization, assessed from steady-state to peak ratio was significantly higher in high calcium conditions (0.14 +/- 0.007 vs. 0.11 +/- 0.008). Similar effects of -Ca2+-i on desensitization were observed with GABA (100 microM). Recovery from desensitization, measured at 30 s interval with double pulse protocol was significantly slower in high [Ca2+]i than in low [Ca2+]i (54 +/- 3% vs. 68 +/- 2%). The current-voltage relationship of GABA-evoked currents was linear in the potential range between -50 and 50 mV. The kinetics of desensitization process including the rate of onset, extent of desensitization, and recovery were voltage independent. The run down of GABA-evoked currents was faster with the higher intracellular calcium concentration. The run down process was accompanied by changes in desensitization kinetics: in both high and low [Ca2+]i desensitization rate was progressively increasing with time as the slow component of the desensitization onset was converted into the fast one. In excised patches, the desensitization kinetics was much faster and more profound than in the whole cell configuration, indicating the involvement of intracellular factors in regulation of this process. In conclusion, [Ca2+]i affects the desensitization of GABAA receptors possibly by activating calcium-dependent enzymes that regulate their phosphorylation state. This may lead to modifications in cell excitability because of changes in GABA-mediated synaptic currents.