RESUMO
At a synapse, fast synchronous neurotransmitter release requires localization of Ca(2+) channels to presynaptic active zones. How Ca(2+) channels are recruited to active zones, however, remains unknown. Using unbiased yeast two-hybrid screens, we here identify a direct interaction of the central PDZ domain of the active-zone protein RIM with the C termini of presynaptic N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels. To test the physiological significance of this interaction, we generated conditional knockout mice lacking all multidomain RIM isoforms. Deletion of RIM proteins ablated most neurotransmitter release by simultaneously impairing the priming of synaptic vesicles and by decreasing the presynaptic localization of Ca(2+) channels. Strikingly, rescue of the decreased Ca(2+)-channel localization required the RIM PDZ domain, whereas rescue of vesicle priming required the RIM N terminus. We propose that RIMs tether N- and P/Q-type Ca(2+) channels to presynaptic active zones via a direct PDZ-domain-mediated interaction, thereby enabling fast, synchronous triggering of neurotransmitter release at a synapse.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Animais , Cálcio/metabolismo , Canais de Cálcio/química , Proteínas de Ligação ao GTP/química , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/química , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Técnicas do Sistema de Duplo-HíbridoRESUMO
Strong human genetic evidence points to an essential contribution of the voltage-gated sodium channel Nav1.7 to pain sensation: loss of Nav1.7 function leads to congenital insensitivity to pain, whereas gain-of-function mutations in the SCN9A gene that encodes Nav1.7 cause painful neuropathies, such as inherited erythromelalgia, a syndrome characterized by episodic spontaneous pain. Selective Nav1.7 channel blockers thus hold promise as potential painkillers with improved safety and reduced unwanted side effects compared with existing therapeutics. To determine the maximum effect of a theoretically perfectly selective Nav1.7 inhibitor, we generated a tamoxifen-inducible KO mouse model enabling genetic deletion of Nav1.7 from adult mice. Electrophysiological recordings of sensory neurons from these mice following tamoxifen injection demonstrated the loss of Nav1.7 channel current and the resulting decrease in neuronal excitability of small-diameter neurons. We found that behavioral responses to most, but surprisingly not all, modalities of noxious stimulus are abolished following adult deletion of Nav1.7, pointing toward indications where Nav1.7 blockade should be efficacious. Furthermore, we demonstrate that isoform-selective acylsulfonamide Nav1.7 inhibitors show robust analgesic and antinociceptive activity acutely after a single dose in mouse pain models shown to be Nav1.7-dependent. All experiments were done with both male and female mice. Collectively, these data expand the depth of knowledge surrounding Nav1.7 biology as it relates to pain, and provide preclinical proof of efficacy that lays a clear path toward translation for the therapeutic use of Nav1.7-selective inhibitors in humans.SIGNIFICANCE STATEMENT Loss-of-function mutations in the sodium channel Nav1.7 cause congenital insensitivity to pain in humans, making Nav1.7 a top target for novel pain drugs. Targeting Nav1.7 selectively has been challenging, however, in part due to uncertainties in which rodent pain models are dependent on Nav1.7. We have developed and characterized an adult-onset Nav1.7 KO mouse model that allows us to determine the expected effects of a theoretically perfect Nav1.7 blocker. Importantly, many commonly used pain models, such as mechanical allodynia after nerve injury, appear to not be dependent on Nav1.7 in the adult. By defining which models are Nav1.7 dependent, we demonstrate that selective Nav1.7 inhibitors can approximate the effects of genetic loss of function, which previously has not been directly established.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/deficiência , Insensibilidade Congênita à Dor/metabolismo , Percepção da Dor/fisiologia , Dor/metabolismo , Bloqueadores dos Canais de Sódio/uso terapêutico , Animais , Células Cultivadas , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Dor/tratamento farmacológico , Dor/genética , Insensibilidade Congênita à Dor/tratamento farmacológico , Insensibilidade Congênita à Dor/genética , Percepção da Dor/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
GluN2B subunit containing NMDARs (GluN2B-NMDARs) mediate pathophysiological effects of acutely applied amyloid beta (Aß), including impaired long-term potentiation (LTP). However, in transgenic Alzheimer's disease (AD) mouse models which feature gradual Aß accumulation, the function of GluN2B-NMDARs and their contribution to synaptic plasticity are unknown. Therefore, we examined the role of GluN2B-NMDARs in synaptic function and plasticity in the hippocampus of PS2APP transgenic mice. Although LTP induced by theta burst stimulation (TBS) was normal in PS2APP mice, it was significantly reduced by the selective GluN2B-NMDAR antagonist Ro25-6981 (Ro25) in PS2APP mice, but not wild type (wt) mice. While NMDARs activated by single synaptic stimuli were not blocked by Ro25, NMDARs recruited during burst stimulation showed larger blockade by Ro25 in PS2APP mice. Thus, the unusual dependence of LTP on GluN2B-NMDARs in PS2APP mice suggests that non-synaptic GluN2B-NMDARs are activated by glutamate that spills out of synaptic cleft during the burst stimulation used to induce LTP. While long-term depression (LTD) was normal in PS2APP mice, and Ro25 had no impact on LTD in wt mice, Ro25 impaired LTD in PS2APP mice, again demonstrating aberrant GluN2B-NMDAR function during plasticity. Together these results demonstrate altered GluN2B-NMDAR function in a model of early AD pathology that has implications for the therapeutic targeting of NMDARs in AD.
Assuntos
Doença de Alzheimer/fisiopatologia , Hipocampo/fisiopatologia , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Doença de Alzheimer/patologia , Animais , Western Blotting , Modelos Animais de Doenças , Estimulação Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/ultraestrutura , Potenciação de Longa Duração/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Masculino , Microscopia Eletrônica de Transmissão , Fenóis/farmacologia , Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Técnicas de Cultura de TecidosRESUMO
Tight coupling of Ca(2+) channels to the presynaptic active zone is critical for fast synchronous neurotransmitter release. RIMs are multidomain proteins that tether Ca(2+) channels to active zones, dock and prime synaptic vesicles for release, and mediate presynaptic plasticity. Here, we use conditional knockout mice targeting all RIM isoforms expressed by the Rims1 and Rims2 genes to examine the contributions and mechanism of action of different RIMs in neurotransmitter release. We show that acute single deletions of each Rims gene decreased release and impaired vesicle priming but did not alter the extracellular Ca(2+)-responsiveness of release (which for Rims gene mutants is a measure of presynaptic Ca(2+) influx). Moreover, single deletions did not affect the synchronization of release (which depends on the close proximity of Ca(2+) channels to release sites). In contrast, deletion of both Rims genes severely impaired the Ca(2+) responsiveness and synchronization of release. RIM proteins may act on Ca(2+) channels in two modes: They tether Ca(2+) channels to active zones, and they directly modulate Ca(2+)-channel inactivation. The first mechanism is essential for localizing presynaptic Ca(2+) influx to nerve terminals, but the role of the second mechanism remains unknown. Strikingly, we find that although the RIM2 C(2)B domain by itself significantly decreased Ca(2+)-channel inactivation in transfected HEK293 cells, it did not rescue any aspect of the RIM knockout phenotype in cultured neurons. Thus, RIMs primarily act in release as physical Ca(2+)-channel tethers and not as Ca(2+)-channel modulators. Different RIM proteins compensate for each other in recruiting Ca(2+) channels to active zones, but contribute independently and incrementally to vesicle priming.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Animais , Potenciais Evocados/fisiologia , Proteínas de Ligação ao GTP/genética , Células HEK293 , Hipocampo/citologia , Humanos , Lipídeos , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Proteínas rab3 de Ligação ao GTP/genéticaRESUMO
Histone deacetylase 2 (HDAC2) negatively regulates excitatory synapse number and memory performance. However, whether HDAC2 regulation of excitatory synapses occurs in a cell-autonomous manner and whether HDAC2 regulates inhibitory synaptic functions are not well understood. To examine these aspects of HDAC2 function, we used sparse transfection of rat hippocampal slice cultures and whole-cell recordings in pyramidal neurons. HDAC2 knockdown (KD) in single postsynaptic pyramidal neurons enhanced, whereas HDAC2 overexpression (OE) reduced, excitatory synaptic transmission. Postsynaptic KD of HDAC2 also facilitated expression of long-term potentiation induced by subthreshold induction stimuli, without altering long-term depression. In contrast, HDAC2 KD reduced, whereas HDAC2 OE enhanced, inhibitory synaptic transmission. Alterations of postsynaptic GABA(A) receptors (GABA(A)Rs) likely underlie the impact of HDAC2 on inhibitory transmission. Consistent with this, we observed reduced transcript and protein levels of the GABA(A)R γ2 subunit and reduced surface expression of the α2 subunit after HDAC2 KD. Furthermore, we observed a reduction in synaptic but not tonic GABA(A)R currents by HDAC2 KD, suggesting that HDAC2 selectively affects synaptic abundance of functional GABA(A)Rs. Immunostaining for postsynaptic GABA(A)Rs confirmed that HDAC2 KD and OE can regulate the synaptic abundance of these receptors. Together, these results highlight a role for HDAC2 in suppressing synaptic excitation and enhancing synaptic inhibition of hippocampal neurons. Therefore, a shift in the balance of synaptic excitation versus inhibition favoring excitation could contribute to the beneficial effects of reducing HDAC2 function in wild-type mice or of inhibiting HDACs in models of cognitive impairment.
Assuntos
Região CA1 Hipocampal/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Histona Desacetilase 2/metabolismo , Potenciais Pós-Sinápticos Inibidores/fisiologia , Animais , Animais Recém-Nascidos , Linhagem Celular Transformada , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Humanos , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/genética , Masculino , Neurônios , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Transfecção , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
The voltage-gated sodium (NaV) channel NaV1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available NaV channel-blocking drugs are not selective among the nine NaV channel subtypes, NaV1.1-NaV1.9. Moreover, the two currently known classes of NaV1.7 subtype-selective inhibitors (aryl- and acylsulfonamides) have undesirable characteristics that may limit their development. To this point understanding of the structure-activity relationships of the acylsulfonamide class of NaV1.7 inhibitors, exemplified by the clinical development candidate GDC-0310, has been based solely on a single co-crystal structure of an arylsulfonamide inhibitor bound to voltage-sensing domain 4 (VSD4). To advance inhibitor design targeting the NaV1.7 channel, we pursued high-resolution ligand-bound NaV1.7-VSD4 structures using cryogenic electron microscopy (cryo-EM). Here, we report that GDC-0310 engages the NaV1.7-VSD4 through an unexpected binding mode orthogonal to the arylsulfonamide inhibitor class binding pose, which identifies a previously unknown ligand binding site in NaV channels. This finding enabled the design of a novel hybrid inhibitor series that bridges the aryl- and acylsulfonamide binding pockets and allows for the generation of molecules with substantially differentiated structures and properties. Overall, our study highlights the power of cryo-EM methods to pursue challenging drug targets using iterative and high-resolution structure-guided inhibitor design. This work also underscores an important role of the membrane bilayer in the optimization of selective NaV channel modulators targeting VSD4.
Assuntos
Microscopia Crioeletrônica , Humanos , Ligantes , Domínios Proteicos , Sítios de Ligação , Relação Estrutura-AtividadeRESUMO
Loss-of-function mutations in Nav1.7, a voltage-gated sodium channel, cause congenital insensitivity to pain (CIP) in humans, demonstrating that Nav1.7 is essential for the perception of pain. However, the mechanism by which loss of Nav1.7 results in insensitivity to pain is not entirely clear. It has been suggested that loss of Nav1.7 induces overexpression of enkephalin, an endogenous opioid receptor agonist, leading to opioid-dependent analgesia. Using behavioral pharmacology and single-cell RNA-seq analysis, we find that overexpression of enkephalin occurs only in cLTMR neurons, a subclass of sensory neurons involved in low-threshold touch detection, and that this overexpression does not play a role in the analgesia observed following genetic removal of Nav1.7. Furthermore, we demonstrate using laser speckle contrast imaging (LSCI) and in vivo electrophysiology that Nav1.7 function is required for the initiation of C-fiber action potentials (APs), which explains the observed insensitivity to pain following genetic removal or inhibition of Nav1.7.
Assuntos
Analgésicos Opioides , Nociceptores , Camundongos , Humanos , Animais , Analgésicos Opioides/farmacologia , Potenciais de Ação , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Dor/genética , Células Receptoras Sensoriais , Peptídeos Opioides , Encefalinas , Gânglios EspinaisRESUMO
At a synapse, presynaptic terminals form a specialized area of the plasma membrane called the active zone that mediates neurotransmitter release. RIM1alpha is a multidomain protein that constitutes a central component of the active zone by binding to other active zone proteins such as Munc13 s, alpha-liprins, and ELKS, and to synaptic vesicle proteins such as Rab3 and synaptotagmin-1. In mice, knockout of RIM1alpha significantly impairs synaptic vesicle priming and presynaptic long-term plasticity, but is not lethal. We now find that the RIM1 gene encodes a second, previously unknown RIM1 isoform called RIM1beta that is upregulated in RIM1alpha knock-out mice. RIM1beta is identical to RIM1alpha except for the N terminus where RIM1beta lacks the N-terminal Rab3-binding sequence of RIM1alpha. Using newly generated knock-out mice lacking both RIM1alpha and RIM1beta, we demonstrate that different from the deletion of only RIM1alpha, deletion of both RIM1alpha and RIM1beta severely impairs mouse survival. Electrophysiological analyses show that the RIM1alphabeta deletion abolishes long-term presynaptic plasticity, as does RIM1alpha deletion alone. In contrast, the impairment in synaptic strength and short-term synaptic plasticity that is caused by the RIM1alpha deletion is aggravated by the deletion of both RIM1alpha and RIM1beta. Thus, our data indicate that the RIM1 gene encodes two different isoforms that perform overlapping but distinct functions in neurotransmitter release.
Assuntos
Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Hipocampo/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Camundongos , Camundongos Knockout , Neurotransmissores/metabolismo , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vesículas Sinápticas/metabolismoRESUMO
Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Nav1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Nav1.7. Compound 24 showed a robust PK/PD response in a Nav1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/química , Sulfonamidas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Analgésicos/química , Analgésicos/metabolismo , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dor Crônica/tratamento farmacológico , Dor Crônica/patologia , Cães , Meia-Vida , Humanos , Ligantes , Masculino , Camundongos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Ratos , Relação Estrutura-Atividade , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Bloqueadores do Canal de Sódio Disparado por Voltagem/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
Herein, we report the discovery and optimization of a series of orally bioavailable acyl sulfonamide NaV1.7 inhibitors that are selective for NaV1.7 over NaV1.5 and highly efficacious in in vivo models of pain and hNaV1.7 target engagement. An analysis of the physicochemical properties of literature NaV1.7 inhibitors suggested that acyl sulfonamides with high fsp3 could overcome some of the pharmacokinetic (PK) and efficacy challenges seen with existing series. Parallel library syntheses lead to the identification of analogue 7, which exhibited moderate potency against NaV1.7 and an acceptable PK profile in rodents, but relatively poor stability in human liver microsomes. Further, design strategy then focused on the optimization of potency against hNaV1.7 and improvement of human metabolic stability, utilizing induced fit docking in our previously disclosed X-ray cocrystal of the NaV1.7 voltage sensing domain. These investigations culminated in the discovery of tool compound 33, one of the most potent and efficacious NaV1.7 inhibitors reported to date.
Assuntos
Analgésicos/química , Canal de Sódio Disparado por Voltagem NAV1.7/química , Sulfonamidas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Analgésicos/metabolismo , Analgésicos/uso terapêutico , Animais , Sítios de Ligação , Desenho de Fármacos , Meia-Vida , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor/induzido quimicamente , Dor/tratamento farmacológico , Dor/patologia , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/metabolismo , Sulfonamidas/uso terapêutico , Bloqueadores do Canal de Sódio Disparado por Voltagem/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
Sequential formation of GABAergic and glutamatergic synapses is thought to be crucial for constructing the stereotypic neural networks during brain development. However, why GABAergic synapses are formed earlier than glutamatergic synapses in the developing brain is not well understood. We used electrophysiology and fluorescence imaging to study GABAergic and glutamatergic synaptogenesis in embryonic hypothalamic cultures, which contain approximately 40% GABAergic and approximately 60% glutamatergic neurons. The newly dissociated embryonic hypothalamic neurons contained a significant pool of functional GABA(A) receptors but a very low level of glutamate receptors. Within the first week of culture, the time course of GABAergic synaptogenesis in embryonic neurons coincided with that of presynaptic vesicle cycling, but both measurements lagged behind the detection of functional GABA(A) receptors. Remarkably, the GABA(A) receptors of newly dissociated embryonic neurons can be rapidly clustered into postsynaptic apparatus and generate functional synaptic currents within 4-6 h when cocultured with mature neurons. Consistent with earlier expression of GABA(A) receptors in immature neurons, synaptic GABAergic events were always detected before the onset of glutamatergic events in both purely embryonic and heterochronic cultures. Interestingly, overexpression of glutamate receptors in embryonic neurons not only increased whole-cell glutamate currents but also significantly increased the frequency of excitatory synaptic events. We conclude that the sequential formation of GABAergic and glutamatergic synapses in immature neurons is likely governed by a sequential expression of GABA(A) and glutamate receptors during neuronal development.
Assuntos
Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Ácido Glutâmico/metabolismo , Hipotálamo/citologia , Neurônios/citologia , Sinapses/metabolismo , Sinapses/fisiologia , Ácido gama-Aminobutírico/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Bicuculina/farmacologia , Células Cultivadas , Técnicas de Cocultura/métodos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas GABAérgicos/farmacologia , Glutamato Descarboxilase/metabolismo , Potenciais Pós-Sinápticos Inibidores/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos , Receptores de GABA-A/metabolismo , Receptores de Glutamato/metabolismo , Fatores de TempoRESUMO
Golgi-specific DHHC (Asp-His-His-Cys) zinc finger protein (GODZ) is a DHHC family palmitoyl acyltransferase that is implicated in palmitoylation and regulated trafficking of diverse substrates that function either at inhibitory or excitatory synapses. Of particular interest is the gamma2 subunit of GABA(A) receptors, which is required for targeting these receptors to inhibitory synapses. Here, we report that GODZ and, to a lesser extent, its close paralog sertoli cell gene with a zinc finger domain-beta (SERZ-beta) are the main members of the DHHC family of enzymes that are able to palmitoylate the gamma2 subunit in heterologous cells. Yeast two-hybrid and colocalization assays in human embryonic kidney 293T (HEK293T) cells indicate that GODZ and SERZ-beta show indistinguishable palmitoylation-dependent interaction with the gamma2 subunit. After coexpression in HEK293T cells, they form homomultimers and heteromultimers, as shown by coimmunoprecipitation and in vivo cross-linking experiments. Analyses in neurons transfected with dominant-negative GODZ (GODZ(C157S)) or plasmid-based GODZ-specific RNAi indicate that GODZ is required for normal accumulation of GABA(A) receptors at synapses, for normal whole-cell and synaptic GABAergic inhibitory function and, indirectly, for GABAergic innervation. Unexpectedly, GODZ was found to be dispensable for normal postsynaptic AMPA receptor-mediated glutamatergic transmission. We conclude that GODZ-mediated palmitoylation of GABA(A) receptors and possibly other substrates contributes selectively to the formation and normal function of GABAergic inhibitory synapses.
Assuntos
Proteínas de Membrana/fisiologia , Inibição Neural/fisiologia , Palmitatos/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Humanos , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Inibição Neural/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Receptores de GABA-A/genética , Sinapses/metabolismo , Transfecção , Ácido gama-Aminobutírico/genética , Ácido gama-Aminobutírico/fisiologiaRESUMO
Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission and are key nervous system drug targets. While diverse pharmacological tools have yielded insight into iGluR extracellular domain function, less is known about molecular mechanisms underlying the ion conduction gating process within the transmembrane domain (TMD). We have discovered a novel NMDAR positive allosteric modulator (PAM), GNE-9278, with a unique binding site on the extracellular surface of the TMD. Mutation of a single residue near the Lurcher motif on GluN1 M3 can convert GNE-9278 modulation from positive to negative, and replacing three AMPAR pre-M1 residues with corresponding NMDAR residues can confer GNE-9278 sensitivity to AMPARs. Modulation by GNE-9278 is state-dependent and significantly alters extracellular domain pharmacology. The unique properties and structural determinants of GNE-9278 reveal new modulatory potential of the iGluR TMD.
Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/fisiologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Doxiciclina/farmacologia , Estimulação Elétrica , Fármacos Atuantes sobre Aminoácidos Excitatórios/química , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , Glicina/metabolismo , Células HEK293 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Técnicas de Patch-Clamp , Domínios Proteicos/efeitos dos fármacos , Domínios Proteicos/genética , Pirimidinonas/química , Pirimidinonas/farmacologia , Receptores de N-Metil-D-Aspartato/genética , Sulfonamidas/química , Sulfonamidas/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , TransfecçãoRESUMO
Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. Selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Targeting the human Nav1.7 channel involved in pain perception, we present a protein-engineering strategy that has allowed us to determine crystal structures of a novel receptor site in complex with isoform-selective antagonists. GX-936 and related inhibitors bind to the activated state of voltage-sensor domain IV (VSD4), where their anionic aryl sulfonamide warhead engages the fourth arginine gating charge on the S4 helix. By opposing VSD4 deactivation, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism, likely by stabilizing inactivated states of the channel. Residues from the S2 and S3 helices are key determinants of isoform selectivity, and bound phospholipids implicate the membrane as a modulator of channel function and pharmacology. Our results help to elucidate the molecular basis of voltage sensing and establish structural blueprints to design selective Nav channel antagonists.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/química , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Tiadiazóis/química , Tiadiazóis/farmacologia , Sequência de Aminoácidos , Membrana Celular/química , Cristalização/métodos , Cristalografia por Raios X , Análise Mutacional de DNA , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Percepção da Dor/efeitos dos fármacos , Engenharia de Proteínas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Although antagonists to GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) have been widely considered to be neuroprotective under certain pathological conditions, their immediate and lasting impacts on synaptic, circuit, and cognitive functions are poorly understood. In hippocampal slices, we found that the GluN2B-selective antagonist Ro25-6981 (Ro25) reduced synaptic NMDAR responses and consequently neuronal output in a subpopulation of GABAergic interneurons, but not pyramidal neurons. Consistent with these effects, Ro25 reduced GABAergic responses in pyramidal neurons and hence could affect circuit functions by altering the excitation/inhibition balance in the brain. In slices from Ts65Dn mice, a Down syndrome model with excess inhibition and cognitive impairment, acutely applied Ro25-rescued long-term potentiation (LTP) and gamma oscillation deficits, whereas prolonged dosing induced persistent rescue of LTP. In contrast, Ro25 did not impact LTP in wild-type (wt) mice but reduced gamma oscillations both acutely and following prolonged treatment. Although acute Ro25 treatment impaired memory performance in wt mice, memory deficits in Ts65Dn mice were unchanged. Thus, GluN2B-NMDARs contribute to the excitation/inhibition balance via impacts on interneurons, and blocking GluN2B-NMDARs can alter functions that depend on this balance, including synaptic plasticity, gamma oscillations, and memory. That prolonged GluN2B antagonism leads to persistent changes in synaptic and circuit functions, and that the influence of GluN2B antagonism differs between wt and disease model mice, provide critical insight into the therapeutic potential and possible liabilities of GluN2B antagonists.
Assuntos
Ondas Encefálicas/fisiologia , Interneurônios/fisiologia , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Ondas Encefálicas/efeitos dos fármacos , Síndrome de Down/tratamento farmacológico , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Interneurônios/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória/efeitos dos fármacos , Camundongos , Camundongos Mutantes Neurológicos , Plasticidade Neuronal/efeitos dos fármacos , Fenóis , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidoresRESUMO
At a synapse, the presynaptic active zone mediates synaptic vesicle exocytosis. RIM proteins are active zone scaffolding molecules that--among others--mediate vesicle priming and directly or indirectly interact with most other essential presynaptic proteins. In particular, the Zn²+ finger domain of RIMs binds to the C2A domain of the priming factor Munc13, which forms a homodimer in the absence of RIM but a heterodimer with it. Here, we show that RIMs mediate vesicle priming not by coupling Munc13 to other active zone proteins as thought but by directly activating Munc13. Specifically, we found that the isolated Zn²+ finger domain of RIMs autonomously promoted vesicle priming by binding to Munc13, thereby relieving Munc13 homodimerization. Strikingly, constitutively monomeric mutants of Munc13 rescued priming in RIM-deficient synapses, whereas wild-type Munc13 did not. Both mutant and wild-type Munc13, however, rescued priming in Munc13-deficient synapses. Thus, homodimerization of Munc13 inhibits its priming function, and RIMs activate priming by disrupting Munc13 homodimerization.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Vesículas Sinápticas/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Animais , Dimerização , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/ultraestrutura , Dedos de Zinco , Proteínas rab3 de Ligação ao GTP/genéticaRESUMO
The presynaptic active zone is composed of a protein network that contains ELKS2alpha (a.k.a. CAST) as a central component. Here we demonstrate that in mice, deletion of ELKS2alpha caused a large increase in inhibitory, but not excitatory, neurotransmitter release, and potentiated the size, but not the properties, of the readily-releasable pool of vesicles at inhibitory synapses. Quantitative electron microscopy revealed that the ELKS2alpha deletion did not change the number of docked vesicles or other ultrastructural parameters of synapses, except for a small decrease in synaptic vesicle numbers. The ELKS2alpha deletion did, however, alter the excitatory/inhibitory balance and exploratory behaviors, possibly as a result of the increased synaptic inhibition. Thus, as opposed to previous studies indicating that ELKS2alpha is essential for mediating neurotransmitter release, our results suggest that ELKS2alpha normally restricts release and limits the size of the readily-releasable pool of synaptic vesicles at the active zone of inhibitory synapses.
Assuntos
Deleção de Genes , Potenciais Pós-Sinápticos Inibidores/genética , Proteínas do Tecido Nervoso/deficiência , Neurotransmissores/metabolismo , Sinapses/metabolismo , Animais , Animais Recém-Nascidos , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Cálcio/metabolismo , Proteínas de Transporte/genética , Células Cultivadas , Estimulação Elétrica/métodos , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Hipocampo/fisiologia , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/fisiologia , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Modelos Neurológicos , Movimento/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Neurônios/ultraestrutura , Técnicas de Patch-Clamp/métodos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sinapses/ultraestrutura , Sinaptossomos/metabolismo , Sinaptossomos/ultraestrutura , Proteínas rab de Ligação ao GTPRESUMO
Ionotropic glutamate and gamma-aminobutyric acid type A (GABAA) receptors mediate critical excitatory and inhibitory actions in the brain. Cyclothiazide (CTZ) is well known for its effect of enhancing glutamatergic transmission and is widely used as a blocker for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor desensitization. Here, we report that in addition to its action on AMPA receptors, CTZ also exerts a powerful but opposite effect on GABAA receptors. We found that CTZ reversibly inhibited both evoked and spontaneous inhibitory postsynaptic currents, as well as GABA application-induced membrane currents, in a dose-dependent manner. Single-channel analyses revealed further that CTZ greatly reduced the open probability of GABAA receptor channels. These results demonstrate that CTZ interacts with both glutamate and GABAA receptors and shifts the excitation-inhibition balance in the brain by two independent mechanisms. Understanding the molecular mechanism of this double-faceted drug-receptor interaction may help in designing new therapies for neurological diseases.
Assuntos
Benzotiadiazinas/farmacologia , Potenciais Evocados/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Antagonistas GABAérgicos/farmacologia , Antagonistas de Receptores de GABA-A , Ácido Glutâmico/fisiologia , Hipocampo/fisiologia , Ácido gama-Aminobutírico/farmacologia , Animais , Potenciais Evocados/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Sinapses/efeitos dos fármacos , Sinapses/fisiologiaRESUMO
The large, medium-sized, and small neurons of the dorsal root ganglion (DRG) have different functions in the processing of various senses. Hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) contribute greatly to neuronal excitability. In the present study, which used whole-cell patch clamp techniques and immunohistochemical staining methods, the electrophysiological properties of DRG neurons were systematically compared, and the roles of HCN-1, -2, and -4 were examined. The main results were as follows. 1) The large neurons had significantly higher V0.5 values (membrane potential at which the HCN channels were half-activated) and shorter time constants (tau) than small or medium-sized DRG neurons. However, large DRG neurons had higher Ih density (HCN neuron current). 2) HCN-1 was found predominantly, but not exclusively, in large and medium-sized DRG neurons; HCN-2 was found in all DRG neurons; and HCN-4 was poorly visualized in all DRG neurons. HCN-1 and HCN-2 were colocalized in large and medium-sized neurons with immunostaining of adjacent sections. In the dorsal horn of the spinal cord, HCN-1, HCN-2, and HCN-4 were all expressed in laminae I-IV, although HCN-1 was not detectable in lamina II. 3) Blockade of Ih current in DRG neurons caused a significant decrease in V0.5, resting membrane potential, and repetitive firing number of action potential and a significant increase in time of rising phase of action potential. These results suggest that the different HCN channels in the three types of DRG neurons might contribute to their differential electrophysiological properties.
Assuntos
Potenciais de Ação/fisiologia , Gânglios Espinais/citologia , Canais Iônicos/fisiologia , Neurônios Aferentes/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/efeitos da radiação , Animais , Células Cultivadas , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Estimulação Elétrica/métodos , Imunofluorescência/métodos , Glicoproteínas/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/classificação , Canais Iônicos/metabolismo , Lectinas/metabolismo , Masculino , Proteínas de Neurofilamentos/metabolismo , Neurônios Aferentes/classificação , Neurônios Aferentes/metabolismo , Neurônios Aferentes/efeitos da radiação , Técnicas de Patch-Clamp/métodos , Canais de Potássio , Pirimidinas/farmacologia , Ratos , Ratos Wistar , VersicanasRESUMO
A tight balance between synaptic vesicle exocytosis and endocytosis is fundamental to maintaining synaptic structure and function. Calcium influx through voltage-gated Ca2+ channels is crucial in regulating synaptic vesicle exocytosis. However, much less is known about how Ca2+ regulates vesicle endocytosis or how the endocytic machinery becomes enriched at the nerve terminal. We report here a direct interaction between voltage-gated Ca2+ channels and endophilin, a key regulator of clathrin-mediated synaptic vesicle endocytosis. Formation of the endophlin-Ca2+ channel complex is Ca2+ dependent. The primary Ca2+ binding domain resides within endophilin and regulates both endophilin-Ca2+ channel and endophilin-dynamin complexes. Introduction into hippocampal neurons of a dominant-negative endophilin construct, which constitutively binds to Ca2+ channels, significantly reduces endocytosis-mediated uptake of FM 4-64 dye without abolishing exocytosis. These results suggest an important role for Ca2+ channels in coordinating synaptic vesicle recycling by directly coupling to both exocytotic and endocytic machineries.