RESUMO
Discovered just over 25 years ago in cerebellar Purkinje neurons, resurgent Na current was originally described operationally as a component of voltage-gated Na current that flows upon repolarization from relatively depolarized potentials and speeds recovery from inactivation, increasing excitability. Its presence in many excitable cells and absence from others has raised questions regarding its biophysical and molecular mechanisms. Early studies proposed that Na channels capable of generating resurgent current are subject to a rapid open-channel block by an endogenous blocking protein, which binds upon depolarization and unblocks upon repolarization. Since the time that this mechanism was suggested, many physiological and structural studies of both Na and K channels have revealed aspects of gating and conformational states that provide insights into resurgent current. These include descriptions of domain movements for activation and inactivation, solution of cryo-EM structures with pore-blocking compounds, and identification of native blocking domains, proteins, and modulatory subunits. Such results not only allow the open-channel block hypothesis to be refined but also link it more clearly to research that preceded it. This review considers possible mechanisms for resurgent Na current in the context of earlier and later studies of ion channels and suggests a framework for future research.
Assuntos
Canais de Potássio , Animais , Humanos , Canais de Potássio/metabolismo , Canais de Potássio/química , Canais de Sódio/metabolismo , Canais de Sódio/química , Ativação do Canal IônicoRESUMO
Cyclic nucleotide-gated (CNG) channels are expressed in rod photoreceptors and open in response to direct binding of cyclic nucleotides. We have previously shown that potentiation of CNGA1 channels by transition metals requires a histidine in the A' helix following the S6 transmembrane segment. Here, we used transition metal ion FRET and patch clamp fluorometry with a fluorescent, noncanonical amino acid (3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap)) to show that the potentiating transition metal Co(2+) binds in or near the A' helix. Adding high-affinity metal-binding sites to the membrane (stearoyl-nitrilotriacetic acid (C18-NTA)) increased potentiation for low Co(2+) concentrations, indicating that the membrane can coordinate metal ions with the A' helix. These results suggest that restraining the A' helix to the plasma membrane potentiates CNGA1 channel opening. Similar interactions between the A' helix and the plasma membrane may underlie regulation of structurally related hyperpolarization-activated cyclic nucleotide-gated (HCN) and voltage-gated potassium subfamily H (KCNH) channels by plasma membrane components.
Assuntos
Membrana Celular/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ativação do Canal Iônico/fisiologia , Animais , Bovinos , Membrana Celular/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Ativação do Canal Iônico/efeitos dos fármacos , Oócitos/metabolismo , Propionatos/farmacologia , Estrutura Secundária de Proteína , Xenopus laevisRESUMO
The NMDA-type glutamate receptor (NMDAR) is essential for synaptogenesis, synaptic plasticity, and higher cognitive function. Emerging evidence indicates that NMDAR Ca(2+) permeability is under the control of cAMP/protein kinase A (PKA) signaling. Whereas the functional impact of PKA on NMDAR-dependent Ca(2+) signaling is well established, the molecular target remains unknown. Here we identify serine residue 1166 (Ser1166) in the carboxy-terminal tail of the NMDAR subunit GluN2B to be a direct molecular and functional target of PKA phosphorylation critical to NMDAR-dependent Ca(2+) permeation and Ca(2+) signaling in spines. Activation of ß-adrenergic and D1/D5-dopamine receptors induces Ser1166 phosphorylation. Loss of this single phosphorylation site abolishes PKA-dependent potentiation of NMDAR Ca(2+) permeation, synaptic currents, and Ca(2+) rises in dendritic spines. We further show that adverse experience in the form of forced swim, but not exposure to fox urine, elicits striking phosphorylation of Ser1166 in vivo, indicating differential impact of different forms of stress. Our data identify a novel molecular and functional target of PKA essential to NMDAR-mediated Ca(2+) signaling at synapses and regulated by the emotional response to stress.
Assuntos
Sinalização do Cálcio/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Espinhas Dendríticas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismo , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/genética , Espinhas Dendríticas/genética , Raposas , Células HEK293 , Hipocampo/metabolismo , Humanos , Inibição Neural/fisiologia , Fosforilação/fisiologia , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/fisiologia , Serina/genética , Estresse Psicológico/genética , Estresse Psicológico/metabolismoRESUMO
Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca(2+) flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca(2+) permeability (PCa/PNa) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca(2+) conductance, because neither Na(+) conductance nor Ca(2+)-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca(2+) permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca(2+) permeability.
Assuntos
Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação do Canal Iônico , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/química , Serina/metabolismoRESUMO
Na channels that generate resurgent current express an intracellular endogenous open-channel blocking protein, whose rapid binding upon depolarization and unbinding upon repolarization minimizes fast and slow inactivation. Na channels also bind exogenous compounds, such as lidocaine, which functionally stabilize inactivation. Like the endogenous blocking protein, these use-dependent inhibitors bind most effectively at depolarized potentials, raising the question of how lidocaine-like compounds affect neurons with resurgent Na current. We therefore recorded lidocaine inhibition of voltage-clamped, tetrodotoxin-sensitive Na currents in mouse Purkinje neurons, which express a native blocking protein, and in mouse hippocampal CA3 pyramidal neurons with and without a peptide from the cytoplasmic tail of NaVß4 (the ß4 peptide), which mimics endogenous open-channel block. To control channel states during drug exposure, lidocaine was applied with rapid-solution exchange techniques during steps to specific voltages. Inhibition of Na currents by lidocaine was diminished by either the ß4 peptide or the native blocking protein. In peptide-free CA3 cells, prolonging channel opening with a site-3 toxin, anemone toxin II, reduced lidocaine inhibition; this effect was largely occluded by open-channel blockers, suggesting that lidocaine binding is favored by inactivation but prevented by open-channel block. In constant 100 µm lidocaine, current-clamped Purkinje cells continued to fire spontaneously. Similarly, the ß4 peptide reduced lidocaine-dependent suppression of spiking in CA3 neurons in slices. Thus, the open-channel blocking protein responsible for resurgent current acts as a natural antagonist of lidocaine. Neurons with resurgent current may therefore be less susceptible to use-dependent Na channel inhibitors used as local anesthetic, antiarrhythmic, and anticonvulsant drugs.
Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Lidocaína/farmacologia , Neurônios/efeitos dos fármacos , Canais de Sódio/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Fenômenos Biofísicos/efeitos dos fármacos , Região CA1 Hipocampal/citologia , Venenos de Cnidários/farmacologia , Estimulação Elétrica , Feminino , Técnicas In Vitro , Masculino , Camundongos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Subunidade beta-4 do Canal de Sódio Disparado por Voltagem/químicaRESUMO
BACKGROUND AND PURPOSE: Select neuroactive steroids tune neural activity by modulating excitatory and inhibitory neurotransmission, including the endogenous cholesterol metabolite 24(S)-hydroxycholesterol (24(S)-HC), which is an N-methyl-d-aspartate (NMDA) receptor positive allosteric modulator (PAM). NMDA receptor PAMs are potentially an effective pharmacotherapeutic strategy to treat conditions associated with NMDA receptor hypofunction. EXPERIMENTAL APPROACH: Using in vitro and in vivo electrophysiological recording experiments and behavioural approaches, we evaluated the effect of SAGE-718, a novel neuroactive steroid NMDA receptor PAM currently in clinical development for the treatment of cognitive impairment, on NMDA receptor function and endpoints that are altered by NMDA receptor hypoactivity and assessed its safety profile. KEY RESULTS: SAGE-718 potentiated GluN1/GluN2A-D NMDA receptors with equipotency and increased NMDA receptor excitatory postsynaptic potential (EPSP) amplitude without affecting decay kinetics in striatal medium spiny neurons. SAGE-718 increased the rate of unblock of the NMDA receptor open channel blocker ketamine on GluN1/GluN2A in vitro and accelerated the rate of return on the ketamine-evoked increase in gamma frequency band power, as measured with electroencephalogram (EEG), suggesting that PAM activity is driven by increased channel open probability. SAGE-718 ameliorated deficits due to NMDA receptor hypofunction, including social deficits induced by subchronic administration of phencyclidine, and behavioural and electrophysiological deficits from cholesterol and 24(S)-HC depletion caused by 7-dehydrocholesterol reductase inhibition. Finally, SAGE-718 did not produce epileptiform activity in a seizure model or neurodegeneration following chronic dosing. CONCLUSIONS AND IMPLICATIONS: These findings provide strong evidence that SAGE-718 is a neuroactive steroid NMDA receptor PAM with a mechanism that is well suited as a treatment for conditions associated with NMDA receptor hypofunction.
Assuntos
Ketamina , Neuroesteroides , Receptores de N-Metil-D-Aspartato/metabolismo , Ketamina/farmacologia , Hidroxicolesteróis/farmacologia , Colesterol , Regulação AlostéricaRESUMO
NMDA receptors (NRs) are glutamate-gated calcium-permeable channels that are essential for normal synaptic transmssion and contribute to neurodegeneration. Tetrameric proteins consist of two obligatory GluN1 (N1) and two GluN2 (N2) subunits, of which GluN2A (2A) and GluN2B (2B) are prevalent in adult brain. The intracellularly located C-terminal domains (CTDs) make a significant portion of mass of the receptors and are essential for plasticity and excitotoxicity, but their functions are incompletely defined. Recent evidence shows that truncation of the N2 CTD alters channel kinetics; however, the mechanism by which this occurs is unclear. Here we recorded activity from individual NRs lacking the CTDs of N1, 2A, or 2B and determined the gating mechanisms of these receptors. Receptors lacking the N1 CTDs had larger unitary conductance and faster deactivation kinetics, receptors lacking the 2A or 2B CTDs had longer openings and longer desensitized intervals, and the first 100 amino acids of the N2 CTD were essential for these changes. In addition, receptors lacking the CTDs of either 2A or 2B maintained isoform-specific kinetic differences and swapping CTDs between 2A and 2B had no effect on single-channel properties. Based on these results, we suggest that perturbations in the CTD can modify the NR-mediated signal in a subunit-dependent manner, in 2A these effects are most likely mediated by membrane-proximal residues, and the isoform-specific biophysical properties conferred by 2A and 2B are CTD-independent. The kinetic mechanisms we developed afford a quantitative approach to understanding how the intracellular domains of NR subunits can modulate the responses of the receptor.
Assuntos
Ativação do Canal Iônico/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Células HEK293 , Humanos , Cinética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Ratos , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
Voltage-gated Na channels of cerebellar Purkinje neurons express an endogenous open-channel blocking protein. This blocker binds channels at positive potentials and unbinds at negative potentials, generating a resurgent Na current and permitting rapid firing. The macroscopic voltage dependence of resurgent current raises the question of whether the blocker directly senses membrane potential or whether voltage dependence is conferred indirectly. Because we previously found that inwardly permeating Na ions facilitate dissociation of the blocker, we measured voltage-clamped currents in different Na gradients to test the role of permeating ions in generating the voltage dependence of unblock. In reverse gradients, outward resurgent currents were tiny or absent, suggesting that unblock normally requires "knockoff" by Na. Inward resurgent currents at strongly negative potentials, however, were larger in reverse than in control gradients. Moreover, occupancy of the blocked state was prolonged both in reverse gradients and in control gradients with reduced Na concentrations, indicating that block is more stable when inward currents are small. Accordingly, reverse gradients shifted the voltage dependence of block, such that resurgent currents were evoked even after conditioning at negative potentials. Additionally, in control gradients, peak resurgent currents decreased linearly with driving force during the conditioning step, suggesting that the stability of block varies directly with inward Na current amplitude. Thus, the voltage dependence of blocker unbinding results almost entirely from repulsion by Na ions occupying the external pore. The lack of voltage sensitivity of the blocking protein suggests that the blocker's binding site lies outside the membrane field, in the permeation pathway.
Assuntos
Cerebelo/metabolismo , Proteínas de Membrana/metabolismo , Células de Purkinje/metabolismo , Canais de Sódio/metabolismo , Sódio/metabolismo , Animais , Permeabilidade da Membrana Celular/fisiologia , Cerebelo/citologia , Ativação do Canal Iônico/fisiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Bloqueadores dos Canais de Sódio/metabolismoRESUMO
Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET, patch-clamp fluorometry and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removing cAMP produced a largely rigid-body rotation of the C-linker relative to the transmembrane domain, bringing the A' helix of the C-linker in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not the presence of cAMP. These results suggest that - in contrast to electromechanical coupling for channel activation - the A' helix serves to couple the S4-helix movement for channel inactivation, which is likely a conserved mechanism for CNBD-family channels.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Animais , AMP Cíclico , Canais de Cátion Regulados por Nucleotídeos Cíclicos/antagonistas & inibidores , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Ativação do Canal Iônico , Masculino , Mecanotransdução Celular , Potenciais da Membrana , Modelos Moleculares , Oócitos/metabolismo , Técnicas de Patch-Clamp , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Ouriços-do-Mar/metabolismo , Espermatozoides/metabolismo , Xenopus/metabolismoRESUMO
The beta subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming alpha subunits, as well as their trafficking and localization. In heterologous expression systems, beta1, beta2, and beta3 subunits influence inactivation and persistent current in different ways. To test how the beta4 protein regulates Na channel gating, we transfected beta4 into HEK (human embryonic kidney) cells stably expressing Na(V)1.1. Unlike a free peptide with a sequence from the beta4 cytoplasmic domain, the full-length beta4 protein did not block open channels. Instead, beta4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of beta1 or chimeric subunits including the beta1 extracellular domain, however, favored inactivation. Coexpressing Na(V)1.1 and beta4 with beta1 produced tiny persistent currents, indicating that beta1 overcomes the effects of beta4 in heterotrimeric channels. In contrast, beta1(C121W), which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by beta4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with beta4, persistent current was slightly but significantly increased. Moreover, in beta4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that beta1 and beta4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted beta1 subunits.
Assuntos
Potenciais de Ação/genética , Encéfalo/metabolismo , Membrana Celular/metabolismo , Ativação do Canal Iônico/genética , Neurônios/metabolismo , Canais de Sódio/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.1 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Sódio/metabolismo , Canais de Sódio/química , Canais de Sódio/genética , Transfecção , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem , Subunidade beta-4 do Canal de Sódio Disparado por VoltagemRESUMO
Voltage-gated ion channels (VGICs) contain positively charged residues within the S4 helix of the voltage-sensing domain (VSD) that are displaced in response to changes in transmembrane voltage, promoting conformational changes that open the pore. Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are unique among VGICs because their open probability is increased by membrane hyperpolarization rather than depolarization. Here we measured the precise movement of the S4 helix of a sea urchin HCN channel using transition metal ion fluorescence resonance energy transfer (tmFRET). We show that the S4 undergoes a substantial (~10 Å) downward movement in response to membrane hyperpolarization. Furthermore, by applying distance constraints determined from tmFRET experiments to Rosetta modeling, we reveal that the carboxy-terminal part of the S4 helix exhibits an unexpected tilting motion during hyperpolarization activation. These data provide a long-sought glimpse of the hyperpolarized state of a functioning VSD and also a framework for understanding the dynamics of reverse gating in HCN channels.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Sequência de Aminoácidos , Animais , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana , Modelos Moleculares , Movimento (Física) , Técnicas de Patch-Clamp , Mutação Puntual , Potássio/metabolismo , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Strongylocentrotus purpuratus/químicaRESUMO
Biological membranes are complex assemblies of lipids and proteins that serve as platforms for cell signaling. We have developed a novel method for measuring the structure and dynamics of the membrane based on fluorescence resonance energy transfer (FRET). The method marries four technologies: (1) unroofing cells to isolate and access the cytoplasmic leaflet of the plasma membrane; (2) patch-clamp fluorometry (PCF) to measure currents and fluorescence simultaneously from a membrane patch; (3) a synthetic lipid with a metal-chelating head group to decorate the membrane with metal-binding sites; and (4) transition metal ion FRET (tmFRET) to measure short distances between a fluorescent probe and a transition metal ion on the membrane. We applied this method to measure the density and affinity of native and introduced metal-binding sites in the membrane. These experiments pave the way for measuring structural rearrangements of membrane proteins relative to the membrane.
Assuntos
Membrana Celular/metabolismo , Metais/metabolismo , Animais , Sítios de Ligação/fisiologia , Linhagem Celular , Citoplasma/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica/fisiologia , RatosRESUMO
Purkinje and cerebellar nuclear neurons both have Na currents with resurgent kinetics. Previous observations, however, suggest that their Na channels differ in their susceptibility to entering long-lived inactivated states. To compare fast inactivation, slow inactivation, and open-channel block, we recorded voltage-clamped, tetrodotoxin-sensitive Na currents in Purkinje and nuclear neurons acutely isolated from mouse cerebellum. In nuclear neurons, recovery from all inactivated states was slower, and open-channel unblock was less voltage-dependent than in Purkinje cells. To test whether specific subunits contributed to this differential stability of inactivation, experiments were repeated in Na(V)1.6-null (med) mice. In med Purkinje cells, recovery times were prolonged and the voltage dependence of open-channel block was reduced relative to control cells, suggesting that availability of Na(V)1.6 is quickly restored at negative potentials. In med nuclear cells, however, currents were unchanged, suggesting that Na(V)1.6 contributes little to wild-type nuclear cells. Extracellular Na(+) prevented slow inactivation more effectively in Purkinje than in nuclear neurons, consistent with a resilience of Na(V)1.6 to slow inactivation. The tendency of nuclear Na channels to inactivate produced a low availability during trains of spike-like depolarization. Hyperpolarizations that approximated synaptic inhibition effectively recovered channels, suggesting that increases in Na channel availability promote rebound firing after inhibition.
Assuntos
Potenciais de Ação/fisiologia , Cerebelo/fisiologia , Ativação do Canal Iônico/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Inibição Neural/fisiologia , Neurônios/fisiologia , Canais de Sódio/fisiologia , Animais , Células Cultivadas , Cerebelo/citologia , Camundongos , Camundongos Transgênicos , Canal de Sódio Disparado por Voltagem NAV1.6 , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Canais de Sódio/química , Canais de Sódio/genética , Relação Estrutura-AtividadeRESUMO
The dorsal raphe (DR) receives a prominent dopamine (DA) input that has been suggested to play a key role in the regulation of central serotoninergic transmission. DA is known to directly depolarize DR serotonin neurons, but the underlying mechanisms are not well understood. Here, we show that activation of D2-like dopamine receptors on DR 5-HT neurons elicits a membrane depolarization and an inward current associated with an increase in membrane conductance. The DA-induced inward current (I(DA)) exhibits a linear I-V relationship and reverses polarity at around -15 mV, suggesting the involvement of a mixed cationic conductance. Consistent with this notion, lowering the extracellular concentration of sodium reduces the amplitude of I(DA) and induces a negative shift of its reversal potential to approximately -45 mV. This current is abolished by inhibiting G-protein function with GDPbetaS. Examination of the downstream signaling mechanisms reveals that activation of the nonselective cation current requires the stimulation of phospholipase C but not an increase in intracellular calcium. Thus, pharmacological inhibition of phospholipase C reduces the amplitude of I(DA). In contrast, buffering intracellular calcium has no effect on the amplitude of I(DA). Bath application of transient receptor potential (TRP) channels blockers, 2-aminoethoxydiphenyl borate and SKF96365 [1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole], strongly inhibits I(DA) amplitude, suggesting the involvement of TRP-like conductance. These results reveal previously unsuspected mechanism by which D2-like DA receptors induce membrane depolarization and enhance the excitability of DR 5-HT neurons.
Assuntos
Núcleos da Rafe/fisiologia , Receptores de Dopamina D2/fisiologia , Canais de Cátion TRPC/fisiologia , Animais , Dopamina/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fosfolipases Tipo C/fisiologiaRESUMO
The Scn8a gene encodes the voltage-gated Na channel alpha subunit Na(V)1.6, which is widely expressed throughout the nervous system. Global null mutations that eliminate Scn8a in all cells result in severe motor dysfunction and premature death, precluding analysis of the physiological role of Na(V)1.6 in different neuronal types. To test the effect of cerebellar Na(V)1.6 on motor coordination in mice, we used the Cre-lox system to eliminate Scn8a expression exclusively in Purkinje neurons (Purkinje KO) and/or granule neurons (granule KO). Whereas granule KO mice had only minor behavioral defects, adult Purkinje KO mice exhibited ataxia, tremor, and impaired coordination. These disorders were exacerbated in double mutants lacking Scn8a in both Purkinje and granule cells (double KO). In Purkinje cells isolated from adult Purkinje KO and double KO but not granule KO mice, the ratio of resurgent-to-transient tetrodotoxin- (TTX)-sensitive Na current amplitudes decreased from approximately 15 to approximately 5%. In cerebellar slices, Purkinje cell spontaneous and maximal firing rates were reduced 10-fold and twofold relative to control in Purkinje KO and double KO but not granule KO mice. Additionally, short-term plasticity of high-frequency parallel fiber EPSCs was altered relative to control in Purkinje KO and double KO but not granule KO mice. These data suggest that the specialized kinetics of Purkinje Na channels depend directly on Scn8a expression. The loss of these channels leads to a decrease in Purkinje cell firing rates as well as a modification of the synaptic properties of afferent parallel fibers, with the ultimate consequence of disrupting motor behavior.