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1.
Proc Natl Acad Sci U S A ; 117(33): 20171-20179, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747561

RESUMO

Extracellular electron transfer (EET) allows microorganisms to gain energy by linking intracellular reactions to external surfaces ranging from natural minerals to the electrodes of bioelectrochemical renewable energy technologies. In the past two decades, electrochemical techniques have been used to investigate EET in a wide range of microbes, with emphasis on dissimilatory metal-reducing bacteria, such as Shewanella oneidensis MR-1, as model organisms. However, due to the typically bulk nature of these techniques, they are unable to reveal the subpopulation variation in EET or link the observed electrochemical currents to energy gain by individual cells, thus overlooking the potentially complex spatial patterns of activity in bioelectrochemical systems. Here, to address these limitations, we use the cell membrane potential as a bioenergetic indicator of EET by S. oneidensis MR-1 cells. Using a fluorescent membrane potential indicator during in vivo single-cell-level fluorescence microscopy in a bioelectrochemical reactor, we demonstrate that membrane potential strongly correlates with EET. Increasing electrode potential and associated EET current leads to more negative membrane potential. This EET-induced membrane hyperpolarization is spatially limited to cells in contact with the electrode and within a near-electrode zone (<30 µm) where the hyperpolarization decays with increasing cell-electrode distance. The high spatial and temporal resolution of the reported technique can be used to study the single-cell-level dynamics of EET not only on electrode surfaces, but also during respiration of other solid-phase electron acceptors.


Assuntos
Membrana Externa Bacteriana/fisiologia , Transporte de Elétrons/fisiologia , Potenciais da Membrana/fisiologia , Shewanella/fisiologia , Benzotiazóis/metabolismo , Fenômenos Eletrofisiológicos , Corantes Fluorescentes , Análise de Célula Única/métodos , Gravação em Vídeo
2.
PLoS One ; 15(8): e0237347, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785272

RESUMO

Here we examine a class of neurons that have been recently explored, the somatosensory neuronal subclass of cold thermosensors. We create a mathematical model of a cold sensing neuron that has been formulated to understand the variety of ionic channels involved. In particular this model showcases the role of TRPM8 and voltage gated potassium channels in setting the temperature dependent activation and inactivation threshold level. Bifurcation analysis of the model demonstrates that a Hodgkin-Huxley type model with additional TRPM8 channels is sufficient to replicate observable experimental features of when different threshold level cold thermosensors turn on. Additionally, our analysis gives insight into what is happening at the temperature levels at which these neurons shut off and the role sodium and leak currents may have in this. This type of model construction and analysis provides a framework moving forward that will help tackle less well understood neuronal classes and their important ionic channels.


Assuntos
Temperatura Baixa , Modelos Neurológicos , Canais de Cátion TRPM/metabolismo , Termorreceptores/fisiologia , Sensação Térmica/fisiologia , Animais , Potenciais da Membrana/fisiologia , Potássio/metabolismo , Sódio/metabolismo
3.
Life Sci ; 254: 117797, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32417371

RESUMO

AIMS: Atrial remodeling, including structural and electrical remodeling, is considered as the substrate in the development of atrial fibrillation (AF). Structural remodeling mainly involves atrial fibrosis, and electrical remodeling is closely related to the changes of ion channels in atrial myocytes. In this study, we aimed to investigate the changes of ion channels in atrial remodeling induced by CIH in rats, which provide the explication for the mechanisms of AF. MATERIALS AND METHODS: 80 male Sprague-Dawley rats were randomized into two groups: Control and CIH group (n = 40). CIH rats were subjected to CIH 8 h/d for 30 days. Atrial epicardial conduction velocity, conduction inhomogeneity and AF inducibility were examined. Masson's trichrome staining was used to evaluate the extent of atrial fibrosis, and the expression levels of ion channel subunits were measured by RT-qPCR, Western blot, and IHC, respectively. The remaining 40 rats were used for whole-cell patch clamp experiments. Action potential, INa, ICa-L, Ito were recorded and compared between two groups. KEY FINDINGS: CIH rats showed increased AF inducibility, atrial interstitial collagen deposition, APD, expression levels of RyR2, p-RyR2, CaMKII, p-CaMKII, and decreased atrial epicardial conduction velocity, expression levels of Nav1.5, Cav1.2, Kv1.5, Kv4.2, Kv4.3 compared to the Control rats, and the current density of INa, ICa-L, Ito were significantly decreased in CIH group. SIGNIFICANCE: We observed significant atrial remodeling induced by CIH in our rat model, which was characterized by changes in ion channels. These changes may be the mechanisms of CIH promoting AF.


Assuntos
Remodelamento Atrial/fisiologia , Hipóxia/fisiopatologia , Canais Iônicos/fisiologia , Potenciais de Ação/fisiologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/biossíntese , Doença Crônica , Fibrose/complicações , Fibrose/patologia , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Átrios do Coração/fisiopatologia , Hipóxia/complicações , Hipóxia/metabolismo , Canais Iônicos/biossíntese , Masculino , Potenciais da Membrana/fisiologia , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/biossíntese
4.
Nat Commun ; 11(1): 2395, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32409656

RESUMO

Pollen tubes are highly polarized tip-growing cells that depend on cytosolic pH gradients for signaling and growth. Autoinhibited plasma membrane proton (H+) ATPases (AHAs) have been proposed to energize pollen tube growth and underlie cell polarity, however, mechanistic evidence for this is lacking. Here we report that the combined loss of AHA6, AHA8, and AHA9 in Arabidopsis thaliana delays pollen germination and causes pollen tube growth defects, leading to drastically reduced fertility. Pollen tubes of aha mutants had reduced extracellular proton (H+) and anion fluxes, reduced cytosolic pH, reduced tip-to-shank proton gradients, and defects in actin organization. Furthermore, mutant pollen tubes had less negative membrane potentials, substantiating a mechanistic role for AHAs in pollen tube growth through plasma membrane hyperpolarization. Our findings define AHAs as energy transducers that sustain the ionic circuit defining the spatial and temporal profiles of cytosolic pH, thereby controlling downstream pH-dependent mechanisms essential for pollen tube elongation, and thus plant fertility.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Tubo Polínico/crescimento & desenvolvimento , Polinização/fisiologia , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Citosol/metabolismo , Técnicas de Silenciamento de Genes , Germinação/fisiologia , Concentração de Íons de Hidrogênio , Potenciais da Membrana/fisiologia , Mutação , Plantas Geneticamente Modificadas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ATPases Translocadoras de Prótons/genética , Análise Espaço-Temporal
5.
Nat Neurosci ; 23(7): 881-891, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32451487

RESUMO

As animals navigate, they must identify features within context. In the mammalian brain, the hippocampus has the ability to separately encode different environmental contexts, even when they share some prominent features. To do so, neurons respond to sensory features in a context-dependent manner; however, it is not known how this encoding emerges. To examine this, we performed electrical recordings in the hippocampus as mice navigated in two distinct virtual environments. In CA1, both synaptic input to single neurons and population activity strongly tracked visual cues in one environment, whereas responses were almost completely absent when the same cue was presented in a second environment. A very similar, highly context-dependent pattern of cue-driven spiking was also observed in CA3. These results indicate that CA1 inherits a complex spatial code from upstream regions, including CA3, that have already computed a context-dependent representation of environmental features.


Assuntos
Hipocampo/fisiologia , Potenciais da Membrana/fisiologia , Navegação Espacial/fisiologia , Animais , Sinais (Psicologia) , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia
6.
J Neurosci ; 40(23): 4512-4524, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32332120

RESUMO

Principal neurons in rodent medial entorhinal cortex (MEC) generate high-frequency bursts during natural behavior. While in vitro studies point to potential mechanisms that could support such burst sequences, it remains unclear whether these mechanisms are effective under in vivo conditions. In this study, we focused on the membrane-potential dynamics immediately following action potentials (APs), as measured in whole-cell recordings from male mice running in virtual corridors (Domnisoru et al., 2013). These afterpotentials consisted either of a hyperpolarization, an extended ramp-like shoulder, or a depolarization reminiscent of depolarizing afterpotentials (DAPs) recorded in vitro in MEC principal neurons. Next, we correlated the afterpotentials with the cells' propensity to fire bursts. All DAP cells with known location resided in Layer II, generated bursts, and their interspike intervals (ISIs) were typically between 5 and 15 ms. The ISI distributions of Layer-II cells without DAPs peaked sharply at around 4 ms and varied only minimally across that group. This dichotomy in burst behavior is explained by cell-group-specific DAP dynamics. The same two groups of bursting neurons also emerged when we clustered extracellular spike-train autocorrelations measured in real 2D arenas (Latuske et al., 2015). Apart from slight variations in grid spacing, no difference in the spatial coding properties of the grid cells across all three groups was discernible. Layer III neurons were only sparsely bursting (SB) and had no DAPs. As various mechanisms for modulating ion-channels underlying DAPs exist, our results suggest that temporal features of MEC activity can be altered while maintaining the cells' overall spatial tuning characteristics.SIGNIFICANCE STATEMENT Depolarizing afterpotentials (DAPs) are frequently observed in principal neurons from slice preparations of rodent medial entorhinal cortex (MEC), but their functional role in vivo is unknown. Analyzing whole-cell data from mice running on virtual tracks, we show that DAPs do occur during behavior. Cells with prominent DAPs are found in Layer II; their interspike intervals (ISIs) reflect DAP time-scales. In contrast, neither the rarely bursting cells in Layer III, nor the high-frequency bursters in Layer II, have a DAP. Extracellular recordings from mice exploring real 2D arenas demonstrate that grid cells within these three groups have similar spatial coding properties. We conclude that DAPs shape the temporal response characteristics of principal neurons in MEC with little effect on spatial properties.


Assuntos
Potenciais de Ação/fisiologia , Córtex Entorrinal/citologia , Córtex Entorrinal/fisiologia , Células de Grade/fisiologia , Animais , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Componente Principal/métodos
7.
Muscle Nerve ; 61(6): 808-814, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32129495

RESUMO

INTRODUCTION: Mutations of the voltage-gated sodium channel gene (SCN4A), which encodes Nav1.4, cause nondystrophic myotonia that occasionally is associated with severe apnea and laryngospasm. There are case reports of nondystrophic myotonia due to mutations in the C-terminal tail (CTerm) of Nav1.4, but the functional analysis is scarce. METHODS: We present two families with nondystrophic myotonia harboring a novel heterozygous mutation (E1702del) and a known heterozygous mutation (E1702K). RESULTS: The proband with E1702K exhibited repeated rhabdomyolysis, and the daughter showed laryngospasm and cyanosis. Functional analysis of the two mutations as well as another known heterozygous mutation (T1700_E1703del), all located on EF hand-like motif in CTerm, was conducted with whole-cell recording of heterologously expressed channel. All mutations displayed impaired fast inactivation. DISCUSSION: The CTerm of Nav1.4 is vital for regulating fast inactivation. The study highlights the importance of accumulating pathological mutations of Nav1.4 and their functional analysis data.


Assuntos
Motivos EF Hand/genética , Potenciais da Membrana/fisiologia , Mutação/genética , Transtornos Miotônicos/diagnóstico , Transtornos Miotônicos/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Pré-Escolar , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Miotônicos/fisiopatologia , Adulto Jovem
8.
Brain ; 143(3): 891-905, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32129831

RESUMO

Epilepsy is a major health burden, calling for new mechanistic insights and therapies. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronziation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof-of-principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Disfunção Cognitiva/genética , Disfunção Cognitiva/prevenção & controle , Epilepsia do Lobo Temporal/prevenção & controle , Edição de Genes/métodos , Canal de Potássio Kv1.1/biossíntese , Adenoviridae , Animais , Eletroencefalografia , Epilepsia do Lobo Temporal/complicações , Feminino , Hipocampo/metabolismo , Masculino , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Camundongos , Neurônios/fisiologia , Cultura Primária de Células , Transfecção , Regulação para Cima
9.
PLoS Comput Biol ; 16(3): e1007605, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32119665

RESUMO

Intracellular calcium ([Ca2+]i) is a basic and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of [Ca2+]i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelope and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation produced by an Upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to intraflagellar pH (pHi), voltage and [Ca2+]i fluctuations. The Upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelope and the progressive spacing of the spikes. In the first module, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-dependent CatSper dynamics articulated with voltage-dependent neutral sodium-proton exchanger (NHE). We analysed the dynamics of these two modules alone and in mixed scenarios. We show that the [Ca2+]i dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper and NHE module but not by those including the pH-independent CaV and BK module or proportionate mixed scenarios. We conclude in favour of the module containing CatSper and NHE and highlight experimentally testable predictions that would corroborate this conclusion.


Assuntos
Canais de Cálcio/metabolismo , Ouriços-do-Mar/metabolismo , Espermatozoides/fisiologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Quimiotaxia/fisiologia , Biologia Computacional/métodos , Íons/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Modelos Teóricos , Transdução de Sinais , Motilidade Espermática/fisiologia
10.
Nat Commun ; 11(1): 1073, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103010

RESUMO

Denervation of skeletal muscles induces severe muscle atrophy, which is preceded by cellular alterations such as increased plasma membrane permeability, reduced resting membrane potential and accelerated protein catabolism. The factors that induce these changes remain unknown. Conversely, functional recovery following denervation depends on successful reinnervation. Here, we show that activation of nicotinic acetylcholine receptors (nAChRs) by quantal release of acetylcholine (ACh) from motoneurons is sufficient to prevent changes induced by denervation. Using in vitro assays, ACh and non-hydrolysable ACh analogs repressed the expression of connexin43 and connexin45 hemichannels, which promote muscle atrophy. In co-culture studies, connexin43/45 hemichannel knockout or knockdown increased innervation of muscle fibers by dorsal root ganglion neurons. Our results show that ACh released by motoneurons exerts a hitherto unknown function independent of myofiber contraction. nAChRs and connexin hemichannels are potential molecular targets for therapeutic intervention in a variety of pathological conditions with reduced synaptic neuromuscular transmission.


Assuntos
Acetilcolina/metabolismo , Gânglios Espinais/crescimento & desenvolvimento , Músculo Esquelético/inervação , Atrofia Muscular/patologia , Receptores Nicotínicos/metabolismo , Acetilcolina/análogos & derivados , Acetilcolina/farmacologia , Animais , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Conexina 43/metabolismo , Conexinas/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/metabolismo
11.
Nat Biotechnol ; 38(4): 439-447, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32042166

RESUMO

Antidepressants are widely prescribed, but their efficacy relative to placebo is modest, in part because the clinical diagnosis of major depression encompasses biologically heterogeneous conditions. Here, we sought to identify a neurobiological signature of response to antidepressant treatment as compared to placebo. We designed a latent-space machine-learning algorithm tailored for resting-state electroencephalography (EEG) and applied it to data from the largest imaging-coupled, placebo-controlled antidepressant study (n = 309). Symptom improvement was robustly predicted in a manner both specific for the antidepressant sertraline (versus placebo) and generalizable across different study sites and EEG equipment. This sertraline-predictive EEG signature generalized to two depression samples, wherein it reflected general antidepressant medication responsivity and related differentially to a repetitive transcranial magnetic stimulation treatment outcome. Furthermore, we found that the sertraline resting-state EEG signature indexed prefrontal neural responsivity, as measured by concurrent transcranial magnetic stimulation and EEG. Our findings advance the neurobiological understanding of antidepressant treatment through an EEG-tailored computational model and provide a clinical avenue for personalized treatment of depression.


Assuntos
Antidepressivos/uso terapêutico , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Maior/fisiopatologia , Eletroencefalografia , Modelos Neurológicos , Transtorno Depressivo Maior/terapia , Método Duplo-Cego , Humanos , Aprendizado de Máquina , Potenciais da Membrana/fisiologia , Valor Preditivo dos Testes , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/fisiologia , Reprodutibilidade dos Testes , Sertralina/uso terapêutico , Estimulação Magnética Transcraniana , Resultado do Tratamento
12.
Invest Ophthalmol Vis Sci ; 61(2): 4, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32031576

RESUMO

Purpose: Oxidative stress affects the retinal pigment epithelium (RPE) leading to development of vascular eye diseases. Cholecalciferol (VIT-D) is a known modulator of oxidative stress and angiogenesis. This in vitro study was carried out to evaluate the protective role of VIT-D on RPE cells incubated under hyperoxic conditions. Methods: Cadaver primary RPE (PRPE) cells were cultured in hyperoxia (40% O2) with or without VIT-D (α-1, 25(OH) 2D3). The functional and physiological effects of PRPE cells with VIT-D treatment were analyzed using molecular and biochemical tools. Results: Vascular signaling modulators, such as vascular endothelial growth factor (VEGF) and Notch, were reduced in hyperoxic conditions but significantly upregulated in the presence of VIT-D. Additionally, PRPE conditioned medium with VIT-D induced the tubulogenesis in primary human umbilical vein endothelial cells (HUVEC) cells. VIT-D supplementation restored phagocytosis and transmembrane potential in PRPE cells cultured under hyperoxia. Conclusions: VIT-D protects RPE cells and promotes angiogenesis under hyperoxic insult. These findings may give impetus to the potential of VIT-D as a therapeutic agent in hyperoxia induced retinal vascular diseases.


Assuntos
Colecalciferol/farmacologia , Hiperóxia/fisiopatologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Vitaminas/farmacologia , Adolescente , Adulto , Cadáver , Células Cultivadas , Criança , Pré-Escolar , Células Endoteliais da Veia Umbilical Humana , Humanos , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Estresse Oxidativo/fisiologia , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Receptores Notch/metabolismo , Regulação para Cima/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto Jovem
13.
Brain ; 143(3): 771-782, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32011655

RESUMO

Small fibre neuropathy is a common pain disorder, which in many cases fails to respond to treatment with existing medications. Gain-of-function mutations of voltage-gated sodium channel Nav1.7 underlie dorsal root ganglion neuronal hyperexcitability and pain in a subset of patients with small fibre neuropathy. Recent clinical studies have demonstrated that lacosamide, which blocks sodium channels in a use-dependent manner, attenuates pain in some patients with Nav1.7 mutations; however, only a subgroup of these patients responded to the drug. Here, we used voltage-clamp recordings to evaluate the effects of lacosamide on five Nav1.7 variants from patients who were responsive or non-responsive to treatment. We show that, at the clinically achievable concentration of 30 µM, lacosamide acts as a potent sodium channel inhibitor of Nav1.7 variants carried by responsive patients, via a hyperpolarizing shift of voltage-dependence of both fast and slow inactivation and enhancement of use-dependent inhibition. By contrast, the effects of lacosamide on slow inactivation and use-dependence in Nav1.7 variants from non-responsive patients were less robust. Importantly, we found that lacosamide selectively enhances fast inactivation only in variants from responders. Taken together, these findings begin to unravel biophysical underpinnings that contribute to responsiveness to lacosamide in patients with small fibre neuropathy carrying select Nav1.7 variants.


Assuntos
Lacosamida/farmacologia , Potenciais da Membrana/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.7/fisiologia , Neuropatia de Pequenas Fibras/fisiopatologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Humanos , Lacosamida/uso terapêutico , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Pessoa de Meia-Idade , Mutação , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Dor/complicações , Dor/tratamento farmacológico , Medição da Dor/efeitos dos fármacos , Técnicas de Patch-Clamp , Neuropatia de Pequenas Fibras/tratamento farmacológico , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores dos Canais de Sódio/uso terapêutico , Resultado do Tratamento , Adulto Jovem
14.
J Neurosci ; 40(12): 2519-2537, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32054675

RESUMO

The bed nucleus of the stria terminalis (BNST) is a forebrain region highly responsive to stress that expresses corticotropin-releasing hormone (CRH) and is implicated in mood disorders, such as anxiety. However, the exact mechanism by which chronic stress induces CRH-mediated dysfunction in BNST and maladaptive behaviors remains unclear. Here, we first confirmed that selective acute optogenetic activation of the oval nucleus BNST (ovBNST) increases maladaptive avoidance behaviors in male mice. Next, we found that a 6 week chronic variable mild stress (CVMS) paradigm resulted in maladaptive behaviors and increased cellular excitability of ovBNST CRH neurons by potentiating mEPSC amplitude, altering the resting membrane potential, and diminishing M-currents (a voltage-gated K+ current that stabilizes membrane potential) in ex vivo slices. CVMS also increased c-fos+ cells in ovBNST following handling. We next investigated potential molecular mechanism underlying the electrophysiological effects and observed that CVMS increased CRH+ and pituitary adenylate cyclase-activating polypeptide+ (PACAP; a CRH upstream regulator) cells but decreased striatal-enriched protein tyrosine phosphatase+ (a STEP CRH inhibitor) cells in ovBNST. Interestingly, the electrophysiological effects of CVMS were reversed by CRHR1-selective antagonist R121919 application. CVMS also activated protein kinase A (PKA) in BNST, and chronic infusion of the PKA-selective antagonist H89 into ovBNST reversed the effects of CVMS. Coadministration of the PKA agonist forskolin prevented the beneficial effects of R121919. Finally, CVMS induced an increase in surface expression of phosphorylated GluR1 (S845) in BNST. Collectively, these findings highlight a novel and indispensable stress-induced role for PKA-dependent CRHR1 signaling in activating BNST CRH neurons and mediating maladaptive behaviors.SIGNIFICANCE STATEMENT Chronic stress and acute activation of oval bed nucleus of the stria terminalis (ovBNST) induces maladaptive behaviors in rodents. However, the precise molecular and electrophysiological mechanisms underlying these effects remain unclear. Here, we demonstrate that chronic variable mild stress activates corticotropin-releasing hormone (CRH)-associated stress signaling and CRH neurons in ovBNST by potentiating mEPSC amplitude and decreasing M-current in male mice. These electrophysiological alterations and maladaptive behaviors were mediated by BNST protein kinase A-dependent CRHR1 signaling. Our results thus highlight the importance of BNST CRH dysfunction in chronic stress-induced disorders.


Assuntos
Adaptação Psicológica , Hormônio Liberador da Corticotropina/fisiologia , Núcleos Septais/fisiologia , Transdução de Sinais/fisiologia , Estresse Psicológico/psicologia , Animais , Doença Crônica , Hormônio Liberador da Corticotropina/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fenômenos Eletrofisiológicos/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Genes fos , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Canais de Potássio/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores
15.
Artigo em Inglês | MEDLINE | ID: mdl-32004067

RESUMO

Arterial membrane potential (Vm) is set by an active interplay among ion channels whose principal function is to set contractility through the gating of voltage-operated Ca2+ channels. To garner an understanding of this electrical parameter, the activity of each channel must be established under near-physiological conditions, a significant challenge given their small magnitude. The inward rectifying K+ (KIR) channel is illustrative of the problem, as its outward "physiological" component is almost undetectable. This study describes a stepwise approach to dissect small ionic currents at physiological Vm using endothelial and smooth muscle cells freshly isolated from rat cerebral arteries. We highlight three critical steps, beginning with the voltage clamping of vascular cells bathed in physiological solutions while maintaining a giga-ohm seal. KIR channels are then inhibited (micromolar Ba2+) so that a difference current can be created, once Ba2+ traces are corrected for the changing seal resistance and subtle instrument drift, pulling the reversal potential rightward. The latter is a new procedure and entails the alignment of whole cell current traces at a voltage where KIR is silent and other channels exhibit limited activity. We subsequently introduced corrected and uncorrected currents into computer models of the arterial wall to show how these subtle adjustments markedly impact the importance of KIR in Vm and arterial tone regulation. We argue that this refined approach can be used on an array of vascular ion channels to build a complete picture of how they dynamically interact to set arterial tone in key organs like the brain.NEW & NOTEWORTHY This work describes a stepwise approach to resolve small ionic currents involved in controlling Vm in resistance arteries. Using this new methodology, we particularly resolved the outward component of the KIR current in native vascular cells, voltage clamped in near-physiological conditions. This novel approach can be applied to any other vascular currents and used to better interpret how vascular ion channels cooperate to control arterial tone.


Assuntos
Artérias Cerebrais/fisiologia , Potenciais da Membrana/fisiologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Animais , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Ratos , Ratos Sprague-Dawley
16.
Nat Commun ; 11(1): 547, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992706

RESUMO

TrkH is a bacterial ion channel implicated in K+ uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA. Here we report the structures of the TrkH-TrkA complex in the presence of ADP or ATP. TrkA forms a tetrameric ring when bound to ADP and constrains TrkH to a closed conformation. The TrkA ring splits into two TrkA dimers in the presence of ATP and releases the constraints on TrkH, resulting in an open channel conformation. Functional studies show that both the tetramer-to-dimer conversion of TrkA and the loss of constraints on TrkH are required for channel gating. In addition, deletion of TrkA in Escherichia coli depolarizes the cell, suggesting that the TrkH-TrkA complex couples changes in intracellular nucleotides to membrane potential.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Potenciais da Membrana/fisiologia , Canais de Potássio/química , Canais de Potássio/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Difosfato de Adenosina , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Biológico/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutagênese , Potássio/metabolismo , Canais de Potássio/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência , Vibrio parahaemolyticus/genética , Difração de Raios X
17.
Proc Natl Acad Sci U S A ; 117(2): 977-981, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31889006

RESUMO

LacY catalyzes accumulation of galactosides against a concentration gradient by coupling galactoside and H+ transport (i.e., symport). While alternating access of sugar- and H+-binding sites to either side of the membrane is driven by binding and dissociation of sugar, the electrochemical H+ gradient ([Formula: see text]) functions kinetically by decreasing the Km for influx 50- to 100-fold with no change in Kd The affinity of protonated LacY for sugar has an apparent pK (pKapp) of ∼10.5, due specifically to the pKa of Glu325, a residue that plays an irreplaceable role in coupling. In this study, rates of lactose/H+ efflux were measured from pH 5.0 to 9.0 in the absence or presence of a membrane potential (ΔΨ, interior positive), and the effect of the imposed ΔΨ on the kinetics of efflux was also studied in right-side-out membrane vesicles. The findings reveal that [Formula: see text] induces an asymmetry in the transport cycle based on the following observations: 1) the efflux rate of WT LacY exhibits a pKapp of ∼7.2 that is unaffected by the imposed ΔΨ; 2) ΔΨ increases the rate of efflux at all tested pH values, but enhancement is almost 2 orders of magnitude less than observed for influx; 3) mutant Glu325 - Ala does little or no efflux in the absence or presence of ΔΨ, and ambient pH has no effect; and 4) the effect of ΔΨ (interior positive) on the Km for efflux is almost insignificant relative to the 50- to 100-fold decrease in the Km for influx driven by ΔΨ (interior negative).


Assuntos
Galactosídeos/metabolismo , Potenciais da Membrana/fisiologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Prótons , Sítios de Ligação , Transporte Biológico , Concentração de Íons de Hidrogênio , Transporte de Íons , Cinética , Lactose/metabolismo , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares
18.
J Neurosci ; 40(9): 1795-1809, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-31969470

RESUMO

In mouse cerebellar Purkinje neurons (PNs), the climbing fiber (CF) input provides a signal to parallel fiber (PF) synapses, triggering PF synaptic plasticity. This signal is given by supralinear Ca2+ transients, associated with the CF synaptic potential and colocalized with the PF Ca2+ influx, occurring only when PF activity precedes the CF input. Here, we unravel the biophysical determinants of supralinear Ca2+ signals associated with paired PF-CF synaptic activity. We used membrane potential (V m) and Ca2+ imaging to investigate the local CF-associated Ca2+ influx following a train of PF synaptic potentials in two cases: (1) when the dendritic V m is hyperpolarized below the resting V m, and (2) when the dendritic V m is at rest. We found that supralinear Ca2+ signals are mediated by type-1 metabotropic glutamate receptors (mGluR1s) when the CF input is delayed by 100-150 ms from the first PF input in both cases. When the dendrite is hyperpolarized only, however, mGluR1s boost neighboring T-type channels, providing a mechanism for local coincident detection of PF-CF activity. The resulting Ca2+ elevation is locally amplified by saturation of endogenous Ca2+ buffers produced by the PF-associated Ca2+ influx via the mGluR1-mediated nonselective cation conductance. In contrast, when the dendritic V m is at rest, mGluR1s increase dendritic excitability by inactivating A-type K+ channels, but this phenomenon is not restricted to the activated PF synapses. Thus, V m is likely a crucial parameter in determining PF synaptic plasticity, and the occurrence of hyperpolarization episodes is expected to play an important role in motor learning.SIGNIFICANCE STATEMENT In Purkinje neurons, parallel fiber synaptic plasticity, determined by coincident activation of the climbing fiber input, underlies cerebellar learning. We unravel the biophysical mechanisms allowing the CF input to produce a local Ca2+ signal exclusively at the sites of activated parallel fibers. We show that when the membrane potential is hyperpolarized with respect to the resting membrane potential, type-1 metabotropic glutamate receptors locally enhance Ca2+ influx mediated by T-type Ca2+ channels, and that this signal is amplified by saturation of endogenous buffer also mediated by the same receptors. The combination of these two mechanisms is therefore capable of producing a Ca2+ signal at the activated parallel fiber sites, suggesting a role of Purkinje neuron membrane potential in cerebellar learning.


Assuntos
Sinalização do Cálcio/fisiologia , Cerebelo/fisiologia , Células de Purkinje/fisiologia , Receptores de AMPA/fisiologia , Algoritmos , Animais , Canais de Cálcio Tipo T/fisiologia , Cerebelo/citologia , Simulação por Computador , Dendritos/fisiologia , Potenciais Pós-Sinápticos Excitadores , Feminino , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Plasticidade Neuronal/fisiologia , Canais de Potássio/fisiologia , Sinapses/fisiologia
19.
FASEB J ; 34(1): 1591-1601, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914597

RESUMO

The Slack (KCNT1) gene encodes sodium-activated potassium channels that are abundantly expressed in the central nervous system. Human mutations alter the function of Slack channels, resulting in epilepsy and intellectual disability. Most of the disease-causing mutations are located in the extended cytoplasmic C-terminus of Slack channels and result in increased Slack current. Previous experiments have shown that the C-terminus of Slack channels binds a number of cytoplasmic signaling proteins. One of these is Phactr1, an actin-binding protein that recruits protein phosphatase 1 (PP1) to certain phosphoprotein substrates. Using co-immunoprecipitation, we found that Phactr1 is required to link the channels to actin. Using patch clamp recordings, we found that co-expression of Phactr1 with wild-type Slack channels reduces the current amplitude but has no effect on Slack channels in which a conserved PKC phosphorylation site (S407) that regulates the current amplitude has been mutated. Furthermore, a Phactr1 mutant that disrupts the binding of PP1 but not that of actin fails to alter Slack currents. Our data suggest that Phactr1 regulates the Slack by linking PP1 to the channel. Targeting Slack-Phactr1 interactions may therefore be helpful in developing the novel therapies for brain disorders associated with the malfunction of Slack channels.


Assuntos
Canais de Potássio Ativados por Sódio/metabolismo , Proteína Fosfatase 1/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Células HEK293 , Humanos , Potenciais da Membrana/fisiologia , Camundongos , Mutação/genética , Neurônios/metabolismo , Técnicas de Patch-Clamp/métodos , Ratos , Transdução de Sinais/fisiologia
20.
Int J Mol Sci ; 21(1)2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31948124

RESUMO

Pterostilbene (PTER), a natural dimethylated analog of resveratrol, has been demonstrated to produce anti-neoplastic or neuroprotective actions. However, how and whether this compound can entail any perturbations on ionic currents in electrically excitable cells remains unknown. In whole-cell current recordings, addition of PTER decreased the amplitude of macroscopic Ih during long-lasting hyperpolarization in GH3 cells in a concentration-dependent manner, with an effective IC50 value of 0.84 µM. Its presence also shifted the activation curve of Ih along the voltage axis to a more hyperpolarized potential, by 11 mV. PTER at a concentration greater than 10 µM could also suppress l-type Ca2+ and transient outward K+ currents in GH3 cells. With the addition of PTER, IK(Ca) amplitude was increased, with an EC50 value of 2.23 µM. This increase in IK(Ca) amplitude was attenuated by further addition of verruculogen, but not by tolbutamide or TRAM-39. Neither atropine nor nicotine, in the continued presence of PTER, modified the PTER-stimulated IK(Ca). PTER (10 µM) slightly suppressed the amplitude of l-type Ca2+ current and transient outward K+ current. The presence of PTER (3 µM) was also effective at increasing the open-state probability of large-conductance Ca2+-activated K+ (BKCa) channels identified in hippocampal mHippoE-14 neurons; however, its inability to alter single-channel conductance was detected. Our study highlights evidence to show that PTER has the propensity to perturb ionic currents (e.g., Ih and IK(Ca)), thereby influencing the functional activities of neurons, and neuroendocrine or endocrine cells.


Assuntos
Potenciais da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/fisiologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/fisiologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Potenciais da Membrana/fisiologia , Camundongos , Neurônios/química , Neurônios/metabolismo , Neurônios/fisiologia , Técnicas de Patch-Clamp , Hipófise/efeitos dos fármacos , Hipófise/fisiologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Ratos
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