RESUMO
Neurotransmitter receptor density is a major variable in regulating synaptic strength. Receptors rapidly exchange between synapses and intracellular storage pools through endocytic recycling. In addition, lateral diffusion and confinement exchanges surface membrane receptors between synaptic and extrasynaptic sites. However, the signals that regulate this transition are currently unknown. GABAA receptors containing α5-subunits (GABAAR-α5) concentrate extrasynaptically through radixin (Rdx)-mediated anchorage at the actin cytoskeleton. Here we report a novel mechanism that regulates adjustable plasma membrane receptor pools in the control of synaptic receptor density. RhoA/ROCK signalling regulates an activity-dependent Rdx phosphorylation switch that uncouples GABAAR-α5 from its extrasynaptic anchor, thereby enriching synaptic receptor numbers. Thus, the unphosphorylated form of Rdx alters mIPSCs. Rdx gene knockout impairs reversal learning and short-term memory, and Rdx phosphorylation in wild-type mice exhibits experience-dependent changes when exposed to novel environments. Our data suggest an additional mode of synaptic plasticity, in which extrasynaptic receptor reservoirs supply synaptic GABAARs.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Aprendizagem/fisiologia , Proteínas de Membrana/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/fisiologia , Animais , Proteínas do Citoesqueleto/genética , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica/fisiologia , Hipocampo/citologia , Hipocampo/fisiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Receptores de GABA-A/genéticaRESUMO
We investigated the subthreshold properties of an erg (ether-à-go-go-related gene) K(+) current in Purkinje cells of neonatal mice. Action potentials recorded from Purkinje cells in cerebellar slices exhibited a decreased threshold potential and increased frequency of spontaneous and repetitive activity following application of the specific erg channel blocker E-4031. Accommodation was absent before and after drug application. The erg current of these Purkinje cells activated at membrane potentials near -60 mV and exhibited fast gating kinetics. The functional importance of fast gating subthreshold erg channels in Purkinje cells was corroborated by comparing the results of action potential clamp experiments with erg1a, erg1b, erg2, and erg3 currents heterologously expressed in HEK cells. Computer simulations based on a NEURON model of Purkinje cells only reproduced the effects of the native erg current when an erg channel conductance like that of erg3 was included. Experiments with subunit-sensitive toxins (BeKm-1, APETx1) indicated that erg channels in Purkinje cells are presumably mediated by heteromeric erg1/erg3 or modified erg1 channels. Following mGluR1 activation, the native erg current was reduced by â¼70%, brought about by reduction of the maximal erg current and a shift of the activation curve to more positive potentials. The Purkinje cell erg current contributed to the sustained current component of the biphasic mGluR1 response. Activation of mGluR1 by the agonist 3,4-dihydroxyphenylglycol increased Purkinje cell excitability, similar to that induced by E-4031. The results indicated that erg currents can be modulated and may contribute to the mGluR1-induced plasticity changes in Purkinje cells.
Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Potenciais da Membrana/fisiologia , Células de Purkinje/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Potenciais de Ação/fisiologia , Animais , Cerebelo/metabolismo , Venenos de Cnidários/farmacologia , Simulação por Computador , Agonistas de Aminoácidos Excitatórios/farmacologia , Células HEK293 , Humanos , Masculino , Camundongos , Modelos Neurológicos , Receptores de Glutamato Metabotrópico/agonistas , Venenos de Escorpião/farmacologia , Sesquiterpenos/farmacologia , Sesquiterpenos de GuaianoRESUMO
Secretion of LH from gonadotropes is initiated by a GnRH-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). This increase in [Ca(2+)](i) is the result of Ca(2+) release from intracellular stores and Ca(2+) influx through voltage-dependent Ca(2+) channels. Here we describe an ether-à-go-go-related gene (erg) K(+) current in primary mouse gonadotropes and its possible function in the control of Ca(2+) influx. To detect gonadotropes, we used a knock-in mouse strain, in which GnRH receptor-expressing cells are fluorescently labeled. Erg K(+) currents were recorded in 80-90% of gonadotropes. Blockage of erg currents by E-4031 depolarized the resting potential by 5-8 mV and led to an increase in [Ca(2+)](i), which was abolished by nifedipine. GnRH inhibited erg currents by a reduction of the maximal erg current and in some cells additionally by a shift of the activation curve to more positive potentials. In conclusion, the erg current contributes to the maintenance of the resting potential in gonadotropes, thereby securing a low [Ca(2+)](i) by restricting Ca(2+) influx. In addition, the erg channels are modulated by GnRH by an as-yet unknown signal cascade.
Assuntos
Cálcio/metabolismo , Canais de Potássio Éter-A-Go-Go/metabolismo , Gonadotrofos/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Animais , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Líquido Extracelular/metabolismo , Hormônio Liberador de Gonadotropina/farmacologia , Líquido Intracelular/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Different erg (ether-à-go-go-related gene; Kv11) K+ channel subunits are expressed throughout the brain. Especially mitral cells of the olfactory bulb are stained intensely by erg1a, erg1b, erg2, and erg3 antibodies. This led us to study the erg current in mitral/tufted (M/T) neurons from mouse olfactory bulb in primary culture. M/T neurons were identified by their morphology and presence of mGluR1 receptors, and RT-PCR demonstrated the expression of all erg subunits in cultured M/T neurons. Using an elevated external K+ concentration, a relatively uniform erg current was recorded in the majority of M/T cells and isolated with the erg channel blocker E-4031. With 4-s depolarizations, the erg current started to activate at -65 mV and exhibited half maximal activation at -51 mV. An increase in the external K+ concentration resulted in an increase in erg whole-cell conductance. The specific group 1 mGluR agonist, DHPG, which depolarizes mitral cells, reduced erg channel availability. DHPG accelerated erg current deactivation, reduced the maximum current amplitude, and shifted availability and activation curves to more depolarized potentials. A pharmacological block of erg channels depolarized the resting potential of M/T cells and clearly demonstrated the involvement of erg channels in the control of mitral cell excitability.
Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Neurônios/metabolismo , Bulbo Olfatório/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Antiarrítmicos/farmacologia , Células Cultivadas , Canal de Potássio ERG1 , Imuno-Histoquímica , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Bulbo Olfatório/efeitos dos fármacos , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Piridinas/farmacologia , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Gonadotropes are crucial in the control of reproduction but difficult to isolate for functional analysis due to their scattered distribution in the anterior pituitary gland. We devised a binary genetic approach, and describe a new mouse model that allows visualization and manipulation of gonadotrope cells. Using gene targeting in embryonic stem cells, we generated mice in which Cre recombinase is coexpressed with the GnRH receptor, which is expressed in gonadotrope cells. We show that we can direct Cre-mediated recombination of a yellow fluorescent protein reporter allele specifically in gonadotropes within the anterior pituitary of these knock-in mice. More than 99% of gonadotropin-containing cells were labeled by yellow fluorescent protein fluorescence and readily identifiable in dissociated pituitary cell culture, allowing potentially unbiased sampling from the gonadotrope population. Using electrophysiology, calcium imaging, and the study of secretion on the single-cell level, the functional properties of gonadotropes isolated from male mice were analyzed. Our studies demonstrate a significant heterogeneity in the resting properties of gonadotropes and their responses to GnRH. About 50% of gonadotropes do not exhibit secretion of LH or FSH. Application of GnRH induced a broad range of both electrophysiological responses and increases in the intracellular calcium concentration. Our mouse model will also be able to direct expression of other Cre recombination-dependent reporter genes to gonadotropes and, therefore, represents a versatile new tool in the understanding of gonadotrope biology.
Assuntos
Gonadotrofos/fisiologia , Gonadotropinas/genética , Receptores LHRH/genética , Animais , Cálcio/fisiologia , Eletrofisiologia , Éxons , Humanos , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Adeno-Hipófise/fisiologia , Potássio/fisiologiaRESUMO
We have further tested the hypothesis that receptor-mediated modulation of KCNQ channels involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC). We used four parallel assays to characterize the agonist-induced PLC response of cells (tsA or CHO cells) expressing M1 muscarinic receptors: translocation of two fluorescent probes for membrane lipids, release of calcium from intracellular stores, and chemical measurement of acidic lipids. Occupation of M1 receptors activates PLC and consumes cellular PIP2 in less than a minute and also partially depletes mono- and unphosphorylated phosphoinositides. KCNQ current is simultaneously suppressed. Two inhibitors of PLC, U73122 and edelfosine (ET-18-OCH3), can block the muscarinic actions completely, including suppression of KCNQ current. However, U73122 also had many side effects that were attributable to alkylation of various proteins. These were mimicked or occluded by prior reaction with the alkylating agent N-ethylmaleimide and included block of pertussis toxin-sensitive G proteins and effects that resembled a weak activation of PLC or an inhibition of lipid kinases. By our functional criteria, the putative PLC activator m-3M3FBS did stimulate PLC, but with a delay and an irregular time course. It also suppressed KCNQ current. The M1 receptor-mediated activation of PLC and suppression of KCNQ current were stopped by lowering intracellular calcium well below resting levels and were slowed by not allowing intracellular calcium to rise in response to PLC activation. Thus calcium release induced by PLC activation feeds back immediately on PLC, accelerating it during muscarinic stimulation in strong positive feedback. These experiments clarify important properties of receptor-coupled PLC responses and their inhibition in the context of the living cell. In each test, the suppression of KCNQ current closely paralleled the expected fall of PIP2. The results are described by a kinetic model.
Assuntos
Cálcio/farmacologia , Fosfatidilinositol Diacilglicerol-Liase/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Receptor Muscarínico M1/metabolismo , Alquilação , Animais , Células CHO , Cricetinae , Ativação Enzimática/efeitos dos fármacos , Estrenos/farmacologia , Etilmaleimida/farmacologia , Humanos , Fosfatos de Inositol/metabolismo , Canais de Potássio KCNQ , Canal de Potássio KCNQ1 , Potenciais da Membrana/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Oxotremorina/análogos & derivados , Oxotremorina/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol Diacilglicerol-Liase/antagonistas & inibidores , Éteres Fosfolipídicos/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Proteína Quinase C/genética , Pirrolidinonas/farmacologia , Receptor Muscarínico M1/efeitos dos fármacos , Receptor Muscarínico M1/genética , Sulfonamidas/farmacologia , Fatores de Tempo , TransfecçãoRESUMO
Ether-á-go-go-related gene (erg) channels form one subfamily of the ether-á-go-go (EAG) K(+) channels and all three erg channels (erg1-3) are expressed in the brain. In the present study we characterize a fast erg current in neurones in primary culture derived from the median part of rat embryonic rhombencephala (E15-16). The relatively uniform erg current was regularly found in large multipolar serotonergic neurones, and occurred also in other less well characterized neurones. The erg current was blocked by the antiarrhythmic substance E-4031. Single-cell RT-PCR revealed the expression of erg1a, erg1b, erg2 and erg3 mRNA in different combinations in large multipolar neurones. These cells also contained neuronal tryptophan hydroxylase, a key enzyme for serotonin production. To characterize the molecular properties of the channels mediating the native erg current, we compared the voltage and time dependence of activation and deactivation of the neuronal erg current to erg1a, erg1b, erg2 and erg3 currents heterologously expressed in CHO cells. The biophysical properties of the neuronal erg current were well within the range displayed by the different heterologously expressed erg currents. Activation and deactivation kinetics of the neuronal erg current were fast and resembled those of erg3 currents. Our data suggest that the erg channels in rat embryonic rhombencephalon neurones are heteromultimers formed by different erg channel subunits.
Assuntos
Neurônios/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Canais de Potássio/fisiologia , Serotonina/fisiologia , Potenciais de Ação/fisiologia , Animais , Células CHO , Células Cultivadas , Cricetinae , Canal de Potássio ERG1 , Embrião de Mamíferos , Canais de Potássio Éter-A-Go-Go , Feminino , Neurônios/química , Canais de Potássio/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Gravidez , Ratos , Rombencéfalo/citologia , Rombencéfalo/metabolismo , Rombencéfalo/fisiologia , Serotonina/análiseRESUMO
We studied modulation of current in human embryonic kidney tsA-201 cells coexpressing rat erg1 channels with M(1) muscarinic receptors. Maximal current was inhibited 30% during muscarinic receptor stimulation, with a small positive shift of the midpoint of activation. Inhibition was attenuated by coexpression of the regulator of G-protein signalling RGS2 or of a dominant-negative protein, G(q), but not by N-ethylmaleimide or C3 toxin. Overexpression of a constitutively active form of G(q) (but not of G(13) or of G(s)) abolished the erg current. Hence it is likely that G(q/11), and not G(i/o) or G(13), mediates muscarinic inhibition. Muscarinic suppression of erg was attenuated by chelating intracellular Ca(2+) to < 1 nm free Ca(2+) with 20 mm BAPTA in the pipette, but suppression was normal if internal Ca(2+) was strongly clamped to a 129 nm free Ca(2+) level with a BAPTA buffer and this was combined with numerous other measures to prevent intracellular Ca(2+) transients (pentosan polysulphate, preincubation with thapsigargin, and removal of extracellular Ca(2+)). Hence a minimum amount of Ca(2+) was necessary for the inhibition, but a Ca(2+) elevation was not. The ATP analogue AMP-PCP did not prevent inhibition. The protein kinase C (PKC) blockers staurosporine and bisindolylmaleimide I did not prevent inhibition, and the PKC-activating phorbol ester PMA did not mimic it. Neither the tyrosine kinase inhibitor genistein nor the tyrosine phosphatase inhibitor dephostatin prevented inhibition by oxotremorine-M. Hence protein kinases are not needed. Experiments with a high concentration of wortmannin were consistent with recovery being partially dependent on PIP(2) resynthesis. Wortmannin did not prevent muscarinic inhibition. Our studies of muscarinic inhibition of erg current suggest a role for phospholipase C, but not the classical downstream messengers, such as PKC or a calcium transient.
Assuntos
Canais de Potássio/fisiologia , Receptor Muscarínico M1/fisiologia , Receptor Muscarínico M3/fisiologia , Animais , Linhagem Celular , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Humanos , Agonistas Muscarínicos/farmacologia , Oxotremorina/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Ratos , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inibidores , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M3/antagonistas & inibidoresRESUMO
Receptor-mediated modulation of KCNQ channels regulates neuronal excitability. This study concerns the kinetics and mechanism of M1 muscarinic receptor-mediated regulation of the cloned neuronal M channel, KCNQ2/KCNQ3 (Kv7.2/Kv7.3). Receptors, channels, various mutated G-protein subunits, and an optical probe for phosphatidylinositol 4,5-bisphosphate (PIP2) were coexpressed by transfection in tsA-201 cells, and the cells were studied by whole-cell patch clamp and by confocal microscopy. Constitutively active forms of Galphaq and Galpha11, but not Galpha13, caused a loss of the plasma membrane PIP2 and a total tonic inhibition of the KCNQ current. There were no further changes upon addition of the muscarinic agonist oxotremorine-M (oxo-M). Expression of the regulator of G-protein signaling, RGS2, blocked PIP2 hydrolysis and current suppression by muscarinic stimulation, confirming that the Gq family of G-proteins is necessary. Dialysis with the competitive inhibitor GDPbetaS (1 mM) lengthened the time constant of inhibition sixfold, decreased the suppression of current, and decreased agonist sensitivity. Removal of intracellular Mg2+ slowed both the development and the recovery from muscarinic suppression. When combined with GDPbetaS, low intracellular Mg2+ nearly eliminated muscarinic inhibition. With nonhydrolyzable GTP analogs, current suppression developed spontaneously and muscarinic inhibition was enhanced. Such spontaneous suppression was antagonized by GDPbetaS or GTP or by expression of RGS2. These observations were successfully described by a kinetic model representing biochemical steps of the signaling cascade using published rate constants where available. The model supports the following sequence of events for this Gq-coupled signaling: A classical G-protein cycle, including competition for nucleotide-free G-protein by all nucleotide forms and an activation step requiring Mg2+, followed by G-protein-stimulated phospholipase C and hydrolysis of PIP2, and finally PIP2 dissociation from binding sites for inositol lipid on the channels so that KCNQ current was suppressed. Further experiments will be needed to refine some untested assumptions.