RESUMO
NaChBac, the first bacterial voltage-gated Na+ (Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Nav channels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Nav channels in lipid bilayers. To study GMTs in mammalian Nav channels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV-bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from â¼6 to â¼4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Nav channels in a membrane environment.
Assuntos
Proteínas de Bactérias/química , Microscopia Crioeletrônica/métodos , Nanoestruturas/química , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Proteínas de Bactérias/genética , Humanos , Bicamadas Lipídicas/química , Modelos Moleculares , Conformação Proteica , Canais de Sódio , Venenos de Aranha/química , Canais de Sódio Disparados por Voltagem/genéticaRESUMO
Calcium (Ca2+) influx into the mitochondrial matrix stimulates ATP synthesis. Here, we investigate whether mitochondrial Ca2+ transport pathways are altered in the setting of deficient mitochondrial energy synthesis, as increased matrix Ca2+ may provide a stimulatory boost. We focused on mitochondrial cardiomyopathies, which feature such dysfunction of oxidative phosphorylation. We study a mouse model where the main transcription factor for mitochondrial DNA (transcription factor A, mitochondrial, Tfam) has been disrupted selectively in cardiomyocytes. By the second postnatal week (10-15day old mice), these mice have developed a dilated cardiomyopathy associated with impaired oxidative phosphorylation. We find evidence of increased mitochondrial Ca2+ during this period using imaging, electrophysiology, and biochemistry. The mitochondrial Ca2+ uniporter, the main portal for Ca2+ entry, displays enhanced activity, whereas the mitochondrial sodium-calcium (Na+-Ca2+) exchanger, the main portal for Ca2+ efflux, is inhibited. These changes in activity reflect changes in protein expression of the corresponding transporter subunits. While decreased transcription of Nclx, the gene encoding the Na+-Ca2+ exchanger, explains diminished Na+-Ca2+ exchange, the mechanism for enhanced uniporter expression appears to be post-transcriptional. Notably, such changes allow cardiac mitochondria from Tfam knockout animals to be far more sensitive to Ca2+-induced increases in respiration. In the absence of Ca2+, oxygen consumption declines to less than half of control values in these animals, but rebounds to control levels when incubated with Ca2+. Thus, we demonstrate a phenotype of enhanced mitochondrial Ca2+ in a mitochondrial cardiomyopathy model, and show that such Ca2+ accumulation is capable of rescuing deficits in energy synthesis capacity in vitro.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Cardiomiopatias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Fosforilação Oxidativa , Sódio/metabolismo , Trocador de Sódio e Cálcio/metabolismoRESUMO
AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.
Assuntos
Actinas , Dendritos , Hipocampo , Plasticidade Neuronal , Polimerização , Receptores de AMPA , Animais , Receptores de AMPA/metabolismo , Actinas/metabolismo , Ratos , Plasticidade Neuronal/fisiologia , Dendritos/metabolismo , Hipocampo/metabolismo , Hipocampo/citologia , Transporte Proteico , Neurônios/metabolismo , Células Cultivadas , ExocitoseRESUMO
The central processes of primary nociceptors form synaptic connections with the second-order nociceptive neurons located in the dorsal horn of the spinal cord. These synapses gate the flow of nociceptive information from the periphery to the CNS, and plasticity at these synapses contributes to centrally mediated hyperalgesia and allodynia. Although exocytosis and synaptic plasticity are controlled by Ca(2+) at the release sites, the mechanisms underlying presynaptic Ca(2+) signalling at the nociceptive synapses are not well characterized. We examined the presynaptic mechanisms regulating Ca(2+) clearance following electrical stimulation in capsaicin-sensitive nociceptors using a dorsal root ganglion (DRG)/spinal cord neuron co-culture system. Cytosolic Ca(2+) concentration ([Ca(2+)]i) recovery following electrical stimulation was well approximated by a monoexponential function with a â¼2 s. Inhibition of sarco-endoplasmic reticulum Ca(2+)-ATPase did not affect presynaptic [Ca(2+)]i recovery, and blocking plasmalemmal Na(+)/Ca(2+) exchange produced only a small reduction in the rate of [Ca(2+)]i recovery (â¼12%) that was independent of intracellular K(+). However, [Ca(2+)]i recovery in presynaptic boutons strongly depended on the plasma membrane Ca(2+)-ATPase (PMCA) and mitochondria that accounted for â¼47 and 40%, respectively, of presynaptic Ca(2+) clearance. Measurements using a mitochondria-targeted Ca(2+) indicator, mtPericam, demonstrated that presynaptic mitochondria accumulated Ca(2+) in response to electrical stimulation. Quantitative analysis revealed that the mitochondrial Ca(2+) uptake is highly sensitive to presynaptic [Ca(2+)]i elevations, and occurs at [Ca(2+)]i levels as low as â¼200-300 nm. Using RT-PCR, we detected expression of several putative mitochondrial Ca(2+) transporters in DRG, such as MCU, Letm1 and NCLX. Collectively, this work identifies PMCA and mitochondria as the major regulators of presynaptic Ca(2+) signalling at the first sensory synapse, and underlines the high sensitivity of the mitochondrial Ca(2+) uniporter in neurons to cytosolic Ca(2+).
Assuntos
Sinalização do Cálcio/fisiologia , Mitocôndrias/fisiologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Animais Recém-Nascidos , Cálcio/fisiologia , Capsaicina , Células Cultivadas , Técnicas de Cocultura , Gânglios Espinais/citologia , Dor/fisiopatologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Sinapses/fisiologiaRESUMO
The Ca(2+)/calcineurin-dependent transcription factor NFAT (nuclear factor of activated T-cells) is implicated in regulating dendritic and axonal development, synaptogenesis, and neuronal survival. Despite the increasing appreciation for the importance of NFAT-dependent transcription in the nervous system, the regulation and function of specific NFAT isoforms in neurons are poorly understood. Here, we compare the activation of NFATc3 and NFATc4 in hippocampal and dorsal root ganglion neurons following electrically evoked elevations of intracellular Ca(2+) concentration ([Ca(2+)](i)). We find that NFATc3 undergoes rapid dephosphorylation and nuclear translocation that are essentially complete within 20 min, although NFATc4 remains phosphorylated and localized to the cytosol, only exhibiting nuclear localization following prolonged (1-3 h) depolarization. Knocking down NFATc3, but not NFATc4, strongly diminished NFAT-mediated transcription induced by mild depolarization in neurons. By analyzing NFATc3/NFATc4 chimeras, we find that the region containing the serine-rich region-1 (SRR1) mildly affects initial NFAT translocation, although the region containing the serine-proline repeats is critical for determining the magnitude of NFAT activation and nuclear localization upon depolarization. Knockdown of glycogen synthase kinase 3ß (GSK3ß) significantly increased the depolarization-induced nuclear localization of NFATc4. In contrast, inhibition of p38 or mammalian target of rapamycin (mTOR) kinases had no significant effect on nuclear import of NFATc4. Thus, electrically evoked [Ca(2+)](i) elevation in neurons rapidly and strongly activates NFATc3, whereas activation of NFATc4 requires a coincident increase in [Ca(2+)](i) and suppression of GSK3ß, with differences in the serine-proline-containing region giving rise to these distinct activation properties of NFATc3 and NFATc4.
Assuntos
Cálcio/metabolismo , Fatores de Transcrição NFATC/metabolismo , Neurônios/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Animais Recém-Nascidos , Núcleo Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Estimulação Elétrica , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Immunoblotting , Microscopia Confocal , Fatores de Transcrição NFATC/genética , Células PC12 , Fosforilação , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Transcrição GênicaRESUMO
The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/metabolismo , Endossomos/metabolismo , Canais Iônicos/metabolismoRESUMO
The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.