RESUMO
Spontaneously opening, chloride-selective channels that showed outward rectification were recorded in ripped-off patches from rat cultured hippocampal neurons and in cell-attached patches from rat hippocampal CA1 pyramidal neurons in slices. In both preparations, channels had multiple conductance states and the most common single-channel conductance varied. In the outside-out patches it ranged from 12 to 70 pS (Vp=40 mV) whereas in the cell-attached patches it ranged from 56 to 85 pS (-Vp=80 mV). Application of GABA to a patch showing spontaneous channel activity evoked a rapid, synchronous activation of channels. During prolonged exposure to either 5 or 100 microM GABA, the open probability of channels decreased. Application of GABA appeared to have no immediate effect on single-channel conductance. Exposure of the patches to 100 microM bicuculline caused a gradual decrease on the single-channel conductance of the spontaneous channels. The time for complete inhibition to take place was slower in the outside-out than in the cell-attached patches. Application of 100 microM pentobarbital or 1 microM diazepam caused 2 - 4 fold increase in the maximum channel conductance of low conductance (<40 pS) spontaneously active channels. The observation of spontaneously opening GABA(A) channels in cell-attached patches on neurons in slices suggests that they may have a role in neurons in vivo and could be an important site of action for some drugs such as benzodiazepines, barbiturates and general anaesthetics.
Assuntos
Bicuculina/farmacologia , Diazepam/farmacologia , Moduladores GABAérgicos/farmacologia , Hipocampo/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Pentobarbital/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Animais , Células Cultivadas , Hipocampo/fisiologia , Ratos , Ácido gama-Aminobutírico/farmacologiaRESUMO
Spontaneous, single channel, chloride currents were recorded in 48% of cell-attached patches on neurones in the CA1 region of rat hippocampal slices. In some patches, there was more than 1 channel active. They showed outward rectification: both channel conductance and open probability were greater at depolarized than at hyperpolarized potentials. Channels activated by gamma-aminobutyric acid (GABA) in silent patches on the same neurones had similar conductance and outward rectification. The spontaneous currents were inhibited by bicuculline and potentiated by diazepam. It was concluded that the spontaneously opening channels were constitutively active, nonsynaptic GABA(A) channels. Such spontaneously opening GABA(A) channels may provide a tonic inhibitory mechanism in these cells and perhaps in other cells that have GABA(A) receptors although not having a GABA(A) synaptic input. They may also be a target for clinically useful drugs such as the benzodiazepines.
Assuntos
Canais de Cloreto/fisiologia , Hipocampo/fisiologia , Potenciais da Membrana/fisiologia , Células Piramidais/fisiologia , Receptores de GABA-A/fisiologia , Ácido gama-Aminobutírico/farmacologia , Animais , Bicuculina/farmacologia , Canais de Cloreto/efeitos dos fármacos , Diazepam/farmacologia , Condutividade Elétrica , Hipocampo/citologia , Técnicas In Vitro , Ativação do Canal Iônico/fisiologia , Cinética , Técnicas de Patch-Clamp , Células Piramidais/efeitos dos fármacos , Ratos , Receptores de GABA-A/efeitos dos fármacosRESUMO
Although there is a high degree of homology in the M2 transmembrane segments of alpha1 and beta1 subunits, subunit-specific effects were observed in alpha1beta1 GABA(A) receptors expressed in Spodoptera frugipedra (Sf9) cells when the conserved 13' threonine residue in the M2 transmembrane region was mutated to alanine. When threonine 263 (13') was mutated to alanine in the beta1 subunit, high-affinity muscimol binding and the response to GABA were abolished. This did not occur when the threonine 263 (13') was mutated to alanine in the alpha1 subunit, but the rate of desensitisation increased and the effect of bicuculline, a competitive inhibitor, was reduced. The results show differential effects of subunits on receptor function and support a role for M2 in desensitisation.
Assuntos
Alanina/genética , Regulação da Expressão Gênica , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Treonina/química , Potenciais de Ação , Animais , Membrana Celular/química , Humanos , Técnicas In Vitro , Muscimol/metabolismo , Mutação , Ligação Proteica , Receptores de GABA-A/genética , Receptores de GABA-B/genética , Spodoptera/fisiologiaRESUMO
Single channel currents were activated by GABA (0.5 to 5 microM) in cell-attached and inside-out patches from cells in the dentate gyrus of rat hippocampal slices. The currents reversed at the chloride equilibrium potential and were blocked by bicuculline (100 microM). Several different kinds of channel were seen: high conductance and low conductance, rectifying and "nonrectifying." Channels had multiple conductance states. The open probability (Po) of channels was greater at depolarized than at hyperpolarized potentials and the relationship between Po and potential could be fitted with a Boltzmann equation with equivalent valency (z) of 1. The combination of outward rectification and potential-dependent open probability gave very little chloride current at hyperpolarized potentials but steeply increasing current with depolarization, useful properties for a tonic inhibitory mechanism.