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1.
Adv Physiol Educ ; 45(4): 702-708, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34498935

RESUMO

Patch-clamp electrophysiological recordings of neuronal activity require a large amount of space and equipment. The technique is difficult to master and not conducive to demonstration to more than a few medical students. Therefore, neurophysiological education is mostly limited to classroom-based pedagogies such as lectures. However, the demonstration of concepts such as changes in membrane potential and ion channel activity is best achieved with hands-on approaches. This article details an in silico activity suitable for large groups of medical students that demonstrates the key concepts in neurophysiology using the LabAXON simulation software. Learning activities in our practical include 1) measurements of voltage and time parameters of the neuronal action potential and its relationship to the Nernst potentials of Na+ and K+; 2) determination of the stimulus threshold to evoke action potentials; 3) demonstration of the refractory period of an action potential; and 4) voltage-clamp experiments to determine the current-voltage relationship of voltage-gated Na+ and K+ channels and the voltage dependence of, and recovery from, inactivation of voltage-gated Na+ channels. We emphasized the accuracy of quantitative measurements as well as the correct use of units. The level of difficulty of the activity can be altered through different multiple choice questions relating to material introduced in the associated lectures. This practical activity is suitable for different class sizes and is adaptable for delivery with online platforms. Student feedback showed that the students felt the activity helped them consolidate their understanding of the lecture material.


Assuntos
Neurofisiologia , Estudantes de Medicina , Potenciais de Ação , Humanos , Potenciais da Membrana , Sódio
2.
Int J Mol Sci ; 22(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34502229

RESUMO

The two-pore domain K+ (K2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABAAR and GABABR) reduces cellular excitability through Cl- influx and K+ efflux in neurons. Relatively little is known about the link between GABAAR and the K+ channel. The present study was performed to identify the effect of GABAR agonists on K2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABAAR and GABABR agonists. In particular, muscimol, a GABAAR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABAAR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl- influx in GABAergic neurons.


Assuntos
Agonistas de Receptores de GABA-A/farmacologia , Neurônios GABAérgicos/metabolismo , Potenciais da Membrana , Muscimol/farmacologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Receptores de GABA/química , Animais , Células Cultivadas , Neurônios GABAérgicos/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Canais de Potássio de Domínios Poros em Tandem/genética , Ratos
3.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi ; 38(4): 695-702, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34459169

RESUMO

Patch clamp is a technique that can measure weak current in the level of picoampere (pA). It has been widely used for cellular electrophysiological recording in fundamental medical researches, such as membrane potential and ion channel currents recording, etc. In order to obtain accurate measurement results, both the resistance and capacitance of the pipette are required to be compensated. Capacitance compensations are composed of slow and fast capacitance compensation. The slow compensation is determined by the lipid bilayer of cell membrane, and its magnitude usually ranges from a few picofarads (pF) to a few microfarads (µF), depending on the cell size. The fast capacitance is formed by the distributed capacitance of the glass pipette, wires and solution, mostly ranging in a few picofarads. After the pipette sucks the cells in the solution, the positions of the glass pipette and wire have been determined, and only taking once compensation for slow and fast capacitance will meet the recording requirements. However, when the study needs to deal with the temperature characteristics, it is still necessary to make a recognition on the temperature characteristic of the capacitance. We found that the time constant of fast capacitance discharge changed with increasing temperature of bath solution when we studied the photothermal effect on cell membrane by patch clamp. Based on this phenomenon, we proposed an equivalent circuit to calculate the temperature-dependent parameters. Experimental results showed that the fast capacitance increased in a positive rate of 0.04 pF/℃, while the pipette resistance decreased. The fine data analysis demonstrated that the temperature rises of bath solution determined the kinetics of the fast capacitance mainly by changing the inner solution resistance of the glass pipette. This result will provide a good reference for the fine temperature characteristic study related to cellular electrophysiology based on patch clamp technique.


Assuntos
Temperatura , Membrana Celular , Capacitância Elétrica , Potenciais da Membrana , Técnicas de Patch-Clamp
4.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361018

RESUMO

H+-ATP-ases, which support proton efflux through the plasma membrane, are key molecular transporters for electrogenesis in cells of higher plants. Initial activities of the transporters can influence the thresholds of generation of electrical responses induced by stressors and modify other parameters of these responses. Previously, it was theoretically shown that the stochastic heterogeneity of individual cell thresholds for electrical responses in a system of electrically connected neuronal cells can decrease the total threshold of the system ("diversity-induced resonance", DIR). In the current work, we tested a hypothesis about decreasing the thresholds of generation of cooling-induced electrical responses in a system of electrically connected plant cells with increasing stochastic spatial heterogeny in the initial activities of H+-ATP-ases in these cells. A two-dimensional model of the system of electrically connected excitable cells (simple imitation of plant leaf), which was based on a model previously developed in our works, was used for the present investigation. Simulation showed that increasing dispersion in the distribution of initial activities of H+-ATP-ases between cells decreased the thresholds of generation of cooling-induced electrical responses. In addition, the increasing weakly influenced the amplitudes of electrical responses. Additional analysis showed two different mechanisms of the revealed effect. The increasing spatial heterogeneity in activities of H+-ATP-ases induced a weak positive shift of the membrane potential at rest. The shift decreased the threshold of electrical response generation. However, the decreased threshold induced by increasing the H+-ATP-ase activity heterogeneity was also observed after the elimination of the positive shift. The result showed that the "DIR-like" mechanism also participated in the revealed effect. Finally, we showed that the standard deviation of the membrane potentials before the induction of action potentials could be used for the estimation of thresholds of cooling-induced plant electrical responses. Thus, spatial heterogeneity in the initial activities of H+-ATP-ases can be a new regulatory mechanism influencing the generation of electrical responses in plants under actions of stressors.


Assuntos
Potenciais da Membrana , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Bombas de Próton/metabolismo , Temperatura Baixa , Células Vegetais/fisiologia
5.
Nat Commun ; 12(1): 4963, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400625

RESUMO

We have shown that calcium-activated potassium (KCa)-channels regulate fundamental progenitor-cell functions, including proliferation, but their contribution to cell-therapy effectiveness is unknown. Here, we test the participation of KCa-channels in human heart explant-derived cell (EDC) physiology and therapeutic potential. TRAM34-sensitive KCa3.1-channels, encoded by the KCNN4 gene, are exclusively expressed in therapeutically bioactive EDC subfractions and maintain a strongly polarized resting potential; whereas therapeutically inert EDCs lack KCa3.1 channels and exhibit depolarized resting potentials. Somatic gene transfer of KCNN4 results in membrane hyperpolarization and increases intracellular [Ca2+], which boosts cell-proliferation and the production of pro-healing cytokines/nanoparticles. Intramyocardial injection of EDCs after KCNN4-gene overexpression markedly increases the salutary effects of EDCs on cardiac function, viable myocardium and peri-infarct neovascularization in a well-established murine model of ischemic cardiomyopathy. Thus, electrophysiological engineering provides a potentially valuable strategy to improve the therapeutic value of progenitor cells for cardioprotection and possibly other indications.


Assuntos
Cálcio/metabolismo , Terapia Baseada em Transplante de Células e Tecidos/métodos , Fenômenos Eletrofisiológicos , Coração , Canais de Potássio Cálcio-Ativados/metabolismo , Potássio/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Citocinas , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Isquemia , Potenciais da Membrana/fisiologia , Camundongos , Miocárdio/metabolismo , Nanopartículas , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Cálcio-Ativados/genética , Células-Tronco
6.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445364

RESUMO

A normally functioning nervous system requires normal extracellular potassium ion concentration ([K]o). Throughout the nervous system, several processes, including those of an astrocytic nature, are involved in [K]o regulation. In this study we investigated the effect of astrocytic photostimulation on [K]o. We hypothesized that in vivo photostimulation of eNpHR-expressing astrocytes leads to a decreased [K]o. Using optogenetic and electrophysiological techniques we showed that stimulation of eNpHR-expressing astrocytes resulted in a significantly decreased resting [K]o and evoked K responses. The amplitude of the concomitant spreading depolarization-like events also decreased. Our results imply that astrocytic membrane potential modification could be a potential tool for adjusting the [K]o.


Assuntos
Astrócitos/fisiologia , Halobacteriaceae/metabolismo , Halorrodopsinas/genética , Neocórtex/química , Potássio/metabolismo , Animais , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Membrana Celular , Halobacteriaceae/genética , Halorrodopsinas/metabolismo , Potenciais da Membrana , Camundongos , Optogenética
7.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445768

RESUMO

The two-pore domain K2P subunits form background (leak) potassium channels, which are characterized by constitutive, although not necessarily constant activity, at all membrane potential values. Among the fifteen pore-forming K2P subunits encoded by the KCNK genes, the three members of the TREK subfamily, TREK-1, TREK-2, and TRAAK are mechanosensitive ion channels. Mechanically induced opening of these channels generally results in outward K+ current under physiological conditions, with consequent hyperpolarization and inhibition of membrane potential-dependent cellular functions. In the past decade, great advances have been made in the investigation of the molecular determinants of mechanosensation, and members of the TREK subfamily have emerged among the best-understood examples of mammalian ion channels directly influenced by the tension of the phospholipid bilayer. In parallel, the crucial contribution of mechano-gated TREK channels to the regulation of membrane potential in several cell types has been reported. In this review, we summarize the general principles underlying the mechanical activation of K2P channels, and focus on the physiological roles of mechanically induced hyperpolarization.


Assuntos
Potenciais da Membrana/fisiologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Membrana Celular/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Fenômenos Físicos
8.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445219

RESUMO

In the heart, TRPM4 is most abundantly distributed in the conduction system. Previously, a single mutation, 'E7K', was identified in its distal N-terminus to cause conduction disorder because of enhanced cell-surface expression. It remains, however, unclear how this expression increase leads to conduction failure rather than abnormally enhanced cardiac excitability. To address this issue theoretically, we mathematically formulated the gating kinetics of the E7K-mutant TRPM4 channel by a combined use of voltage jump analysis and ionomycin-perforated cell-attached recording technique and incorporated the resultant rate constants of opening and closing into a human Purkinje fiber single-cell action potential (AP) model (Trovato model) to perform 1D-cable simulations. The results from TRPM4 expressing HEK293 cells showed that as compared with the wild-type, the open state is much preferred in the E7K mutant with increased voltage-and Ca2+-sensitivities. These theoretical predictions were confirmed by power spectrum and single channel analyses of expressed wild-type and E7K-mutant TRPM4 channels. In our modified Trovato model, the facilitated opening of the E7K mutant channel markedly prolonged AP duration with concomitant depolarizing shifts of the resting membrane potential in a manner dependent on the channel density (or maximal activity). This was, however, little evident in the wild-type TRPM4 channel. Moreover, 1D-cable simulations with the modified Trovato model revealed that increasing the density of E7K (but not of wild-type) TRPM4 channels progressively reduced AP conduction velocity eventually culminating in complete conduction block. These results clearly suggest the brady-arrhythmogenicity of the E7K mutant channel which likely results from its pathologically enhanced activity.


Assuntos
Mutação com Ganho de Função , Modelos Biológicos , Canais de Cátion TRPM , Células HEK293 , Humanos , Potenciais da Membrana , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo
9.
Biomolecules ; 11(8)2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34439844

RESUMO

2,4-Dinitrophenol (DNP) is a classic uncoupler of oxidative phosphorylation in mitochondria which is still used in "diet pills", despite its high toxicity and lack of antidotes. DNP increases the proton current through pure lipid membranes, similar to other chemical uncouplers. However, the molecular mechanism of its action in the mitochondria is far from being understood. The sensitivity of DNP's uncoupling action in mitochondria to carboxyatractyloside, a specific inhibitor of adenine nucleotide translocase (ANT), suggests the involvement of ANT and probably other mitochondrial proton-transporting proteins in the DNP's protonophoric activity. To test this hypothesis, we investigated the contribution of recombinant ANT1 and the uncoupling proteins UCP1-UCP3 to DNP-mediated proton leakage using the well-defined model of planar bilayer lipid membranes. All four proteins significantly enhanced the protonophoric effect of DNP. Notably, only long-chain free fatty acids were previously shown to be co-factors of UCPs and ANT1. Using site-directed mutagenesis and molecular dynamics simulations, we showed that arginine 79 of ANT1 is crucial for the DNP-mediated increase of membrane conductance, implying that this amino acid participates in DNP binding to ANT1.


Assuntos
2,4-Dinitrofenol/farmacologia , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Desacoplamento Mitocondrial/metabolismo , Animais , Camundongos , Ratos
10.
Toxicol Appl Pharmacol ; 428: 115676, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34389319

RESUMO

The insecticide deltamethrin of the pyrethroid class mainly targets voltage-gated sodium channels (Navs). Deltamethrin prolongs the opening of Navs by slowing down fast inactivation and deactivation. Pyrethroids are supposedly safe for humans, however, they have also been linked to the gulf-war syndrome, a neuropathic pain condition that can develop following exposure to certain chemicals. Inherited neuropathic pain conditions have been linked to mutations in the Nav subtypes Nav1.7, Nav1.8, and Nav1.9. Here, we examined the effect of deltamethrin on the human isoforms Nav1.7, Nav1.8, and Nav1.9_C4 (chimera containing the C-terminus of rat Nav1.4) heterologously expressed in HEK293T and ND7/23 cells using whole-cell patch-clamp electrophysiology. For all three Nav subtypes, we observed increased persistent and tail currents that are typical for Nav channels modified by deltamethrin. The most surprising finding was an enhanced slow inactivation induced by deltamethrin in all three Nav subtypes. An enhanced slow inactivation is contrary to the prolonged opening caused by pyrethroids and has not been described for deltamethrin or any other pyrethroid before. Furthermore, we found that the fraction of deltamethrin-modified channels increased use-dependently. However, for Nav1.8, the use-dependent potentiation occurred only when the holding potential was increased to -90 mV, a potential at which the tail currents decay more slowly. This indicates that use-dependent modification is due to an accumulation of tail currents. In summary, our findings support a novel mechanism whereby deltamethrin enhances slow inactivation of voltage-gated sodium channels, which may, depending on the cellular resting membrane potential, reduce neuronal excitability and counteract the well-described pyrethroid effects of prolonging channel opening.


Assuntos
Inseticidas/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.8/fisiologia , Nitrilas/farmacologia , Piretrinas/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.9/fisiologia
11.
Biomolecules ; 11(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34356653

RESUMO

Rosmarinic acid, a major component of rosemary, is a polyphenolic compound with potential neuroprotective effects. Asreducing the synaptic release of glutamate is crucial to achieving neuroprotectant's pharmacotherapeutic effects, the effect of rosmarinic acid on glutamate release was investigated in rat cerebrocortical nerve terminals (synaptosomes). Rosmarinic acid depressed the 4-aminopyridine (4-AP)-induced glutamate release in a concentration-dependent manner. The removal of extracellular calcium and the blockade of vesicular transporters prevented the inhibition of glutamate release by rosmarinic acid. Rosmarinic acid reduced 4-AP-induced intrasynaptosomal Ca2+ elevation. The inhibition of N-, P/Q-type Ca2+ channels and the calcium/calmodulin-dependent kinase II (CaMKII) prevented rosmarinic acid from having effects on glutamate release. Rosmarinic acid also reduced the 4-AP-induced activation of CaMKII and the subsequent phosphorylation of synapsin I, the main presynaptic target of CaMKII. In addition, immunocytochemistry confirmed the presence of GABAA receptors. GABAA receptor agonist and antagonist blocked the inhibitory effect of rosmarinic acid on 4-AP-evoked glutamate release. Docking data also revealed that rosmarinic acid formed a hydrogen bond with the amino acid residues of GABAA receptor. These results suggested that rosmarinic acid activates GABAA receptors in cerebrocortical synaptosomes to decrease Ca2+ influx and CaMKII/synapsin I pathway to inhibit the evoked glutamate release.


Assuntos
Cinamatos/farmacologia , Depsídeos/farmacologia , Ácido Glutâmico/metabolismo , Sinaptossomos/efeitos dos fármacos , 4-Aminopiridina/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Cinamatos/química , Depsídeos/química , Agonistas de Receptores de GABA-A/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ratos Sprague-Dawley , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Sinaptossomos/metabolismo
12.
Cell Physiol Biochem ; 55(S3): 157-170, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34318654

RESUMO

BACKGROUND/AIMS: The Amyloid Precursor Protein (APP) is involved in the regulation of multiple cellular functions via protein-protein interactions and has been most studied with respect to Alzheimer's disease (AD). Abnormal processing of the single transmembrane-spanning C99 fragment of APP contributes to the formation of amyloid plaques, which are causally related to AD. Pathological C99 accumulation is thought to associate with early cognitive defects in AD. Here, unexpectedly, sequence analysis revealed that C99 exhibits 24% sequence identity with the KCNE1 voltage-gated potassium (Kv) channel ß subunit, comparable to the identity between KCNE1 and KCNE2-5 (21-30%). This suggested the possibility of C99 regulating Kv channels. METHODS: We quantified the effects of C99 on Kv channel function, using electrophysiological analysis of subunits expressed in Xenopus laevis oocytes, biochemical and immunofluorescence techniques. RESULTS: C99 isoform-selectively inhibited (by 30-80%) activity of a range of Kv channels. Among the KCNQ (Kv7) family, C99 isoform-selectively inhibited, shifted the voltage dependence and/or slowed activation of KCNQ2, KCNQ3, KCNQ2/3 and KCNQ5, with no effects on KCNQ1, KCNQ1-KCNE1 or KCNQ4. C99/APP co-localized with KCNQ2 and KCNQ3 in adult rat sciatic nerve nodes of Ranvier. Both C99 and full-length APP co-immunoprecipitated with KCNQ2 in vitro, yet unlike C99, APP only weakly affected KCNQ2/3 activity. Finally, C99 altered the effects on KCNQ2/3 function of inhibitors tetraethylammounium and XE991, but not openers retigabine and ICA27243. CONCLUSION: Our findings raise the possibility of C99 accumulation early in AD altering cellular excitability by modulating Kv channel activity.


Assuntos
Precursor de Proteína beta-Amiloide/farmacologia , Canais de Potássio KCNQ/genética , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ3/genética , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Antracenos/farmacologia , Expressão Gênica , Humanos , Canais de Potássio KCNQ/metabolismo , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Nós Neurofibrosos/efeitos dos fármacos , Nós Neurofibrosos/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tetraetilamônio/farmacologia , Xenopus laevis
13.
Methods Mol Biol ; 2341: 95-101, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264465

RESUMO

Quantifying fluorescent markers in cell populations using flow cytometry has been a powerful technological advance. Fluorescent properties of cyanine dyes coupled with flow cytometry allow investigators to monitor the membrane potential (MP), an important component of the proton motive force (PMF). MP (or ΔΨ) is the electrical potential across the cell membrane. The other component of the PMF is ΔpH, or the difference in interior and exterior proton concentrations. MP plays a critical role in bacterial physiology. In Staphylococcus aureus, MP is required for generation of ATP, regulating autolytic activity, maintaining ion homeostasis, and resistance to some classes of antibiotics. This protocol exploits unique spectral and physical properties of the cyanine-based molecule diethyloxacarbocyanine iodide, or DiOC, and flow cytometry technology to quantify MP in S. aureus. This assay has been used by researchers to define the electron transport chain of S. aureus as well as determine how intrinsic and extrinsic factors affect MP.


Assuntos
Membrana Externa Bacteriana/fisiologia , Staphylococcus aureus/fisiologia , Autólise , Carbocianinas/química , Corantes/química , Citometria de Fluxo , Potenciais da Membrana , Força Próton-Motriz
14.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202215

RESUMO

Cytokines are important neuroinflammatory modulators in neurodegenerative brain disorders including traumatic brain injury (TBI) and stroke. However, their temporal effects on the physiological properties of microglia and neurons during the recovery period have been unclear. Here, using an ATP-induced cortical injury model, we characterized selective effects of ATP injection compared to needle-control. In the damaged region, the fluorescent intensity of CX3CR1-GFP (+) cells, as well as the cell density, was increased and the maturation of newborn BrdU (+) cells continued until 28 day-post-injection (dpi) of ATP. The excitability and synaptic E/I balance of neurons and the inward and outward membrane currents of microglia were increased at 3 dpi, when expressions of tumor necrosis factor (TNF)-α/interleukin (IL)-1ß and IL-10/IL-4 were also enhanced. These changes of both cells at 3 dpi were mostly decayed at 7 dpi and were suppressed by any of IL-10, IL-4, suramin (P2 receptor inhibitor) and 4-AP (K+ channel blocker). Acute ATP application alone induced only small effects from both naïve neurons and microglial cells in brain slice. However, TNF-α alone effectively increased the excitability of naïve neurons, which was blocked by suramin or 4-AP. TNF-α and IL-1ß increased and decreased membrane currents of naïve microglia, respectively. Our results suggest that ATP and TNF-α dominantly induce the physiological activities of 3 dpi neurons and microglia, and IL-10 effectively suppresses such changes of both activated cells in K+ channel- and P2 receptor-dependent manner, while IL-4 suppresses neurons preferentially.


Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Potenciais da Membrana , Microglia/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Lesões Encefálicas Traumáticas/diagnóstico , Lesões Encefálicas Traumáticas/etiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Expressão Gênica , Genes Reporter , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Antagonistas Purinérgicos/farmacologia
15.
Biomed Pharmacother ; 139: 111615, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243598

RESUMO

BACKGROUND: Severe acidosis deteriorates cardiac injury. Rat coronary arteries (RCAs) are unusually hypercontractive to extracellular (o) acidosis (EA). TMEM16A-encoded anoctamin 1 (ANO1), a Ca2+-activated chloride channel (CaCC), plays an important role in regulating coronary arterial tension. PURPOSE: We tested the possibility that the activation of CaCCs in the arterial smooth muscle cell (ASMC) contributes to EA-induced RCA constriction. METHODS: ANO1 expression was detected with immunofluorescence staining and Western blot. TMEM16A mRNA was assessed with quantitative Real-Time PCR. Cl- currents and membrane potentials were quantified with a patch clamp. The vascular tension was recorded with a myograph. Intracellular (i) level of Cl- and Ca2+ was measured with fluorescent molecular probes. RESULTS: ANO1 was expressed in all tested arterial myocytes, but was much more abundant in RCA ASMCs as compared with ASMCs isolated from rat cerebral basilar, intrarenal and mesenteric arteries. EA reduced [Cl-]i levels, augmented CaCC currents exclusively in RCA ASMCs and depolarized RCA ASMCs to a greater extent. Cl- deprivation, which depleted [Cl-]i by incubating the arteries or their ASMCs in Cl--free bath solution, decreased EA-induced [Cl-]i reduction, diminished EA-induced CaCC augmentation and time-dependently depressed EA-induced RCA constriction. Inhibitor studies showed that these EA-induced effects including RCA constriction, CaCC current augmentation, [Cl-]i reduction and/or [Ca2+]i elevation were depressed by various Cl- channel blockers, [Ca2+]i release inhibitors and L-type voltage-gated Ca2+ channel inhibitor nifedipine. ANO1 antibody attenuated all observed changes induced by EA in RCA ASMCs. CONCLUSION: The greater activity of RCA ASMC CaCCs complicated with an enhanced Ca2+ mobilization from both [Ca2+]i release and [Ca2+]o influx plays a pivotal role in the distinctive hypercontractility of RCAs to acidosis. Translation of these findings to human beings may lead to a new conception in our understanding and treating cardiac complications in severe acidosis.


Assuntos
Acidose/metabolismo , Anoctamina-1/metabolismo , Vasos Coronários/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Vasoconstrição/fisiologia , Acidose/tratamento farmacológico , Animais , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Vasos Coronários/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nifedipino/farmacologia , Técnicas de Patch-Clamp/métodos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos
16.
Nat Commun ; 12(1): 4527, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312384

RESUMO

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.


Assuntos
Membrana Celular/fisiologia , Opsinas/metabolismo , Optogenética/métodos , Células Piramidais/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Furões/genética , Furões/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Potenciais da Membrana/fisiologia , Camundongos Transgênicos , Opsinas/genética , Técnicas de Patch-Clamp/métodos , Células Piramidais/citologia , Células Piramidais/metabolismo , Ratos Wistar , Reprodutibilidade dos Testes
17.
Phys Chem Chem Phys ; 23(29): 15784-15795, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34286758

RESUMO

G protein-gated inwardly rectifying potassium (GIRK) channels play essential roles in electrical signaling in neurons and muscle cells. Nonequilibrium environments provide crucial driving forces behind many cellular events. Here, we apply the antiparallel alignment double bilayer model to study GIRK2 in response to the time-dependent membrane potential. Using molecular dynamics and umbrella sampling, we examined the time-dependent environmental impact on the ion conduction, energy basis, and primary motions of GIRK2 in different complex states with phosphatidylinositol-4,5-bisphosphate (PIP2) and G-protein ßγ subunits (Gßγ). The antiparallel alignment double bilayer model enables us to study the transport performance in inward and outward K+ and mixed K+ and Na+. We obtained the recoverable discharge process of GIRK2 complexed with both PIP2 and Gßγ, compared with occasional conduction under PIP2-only regulation. Calculations of potential of mean force suggest different regulation by the helix bundle crossing (HBC) gate and G-loop gate regarding different complex states and under a membrane potential. In a nonequilibrium environment, distinct functional rocking motions of GIRK2 were identified under strengthened correlations between the transmembrane helices and downstream cytoplasmic domains with binding of PIP2, cations, and Gßγ. The findings suggest the potential domain motions and dynamics associated with a nonequilibrium environment and highlight the application of the antiparallel alignment double bilayer model to investigate factors in an asymmetric environment.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Cátions/química , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/química , Potenciais da Membrana , Simulação de Dinâmica Molecular , Fosfatidilinositol 4,5-Difosfato/química , Potássio/química , Conformação Proteica , Sódio/química , Termodinâmica
18.
Nat Commun ; 12(1): 4482, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301959

RESUMO

Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia - histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism.


Assuntos
Conexinas/metabolismo , Desacetilase 6 de Histona/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Acetilação , Animais , Células Cultivadas , Conexinas/genética , Conexinas/fisiologia , Células HEK293 , Desacetilase 6 de Histona/genética , Humanos , Células Jurkat , Lisina/genética , Lisina/metabolismo , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Técnicas de Patch-Clamp , Receptores Adrenérgicos alfa 1/genética , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
19.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205849

RESUMO

The ability of spermatozoa to swim towards an oocyte and fertilize it depends on precise K+ permeability changes. Kir5.1 is an inwardly-rectifying potassium (Kir) channel with high sensitivity to intracellular H+ (pHi) and extracellular K+ concentration [K+]o, and hence provides a link between pHi and [K+]o changes and membrane potential. The intrinsic pHi sensitivity of Kir5.1 suggests a possible role for this channel in the pHi-dependent processes that take place during fertilization. However, despite the localization of Kir5.1 in murine spermatozoa, and its increased expression with age and sexual maturity, the role of the channel in sperm morphology, maturity, motility, and fertility is unknown. Here, we confirmed the presence of Kir5.1 in spermatozoa and showed strong expression of Kir4.1 channels in smooth muscle and epithelial cells lining the epididymal ducts. In contrast, Kir4.2 expression was not detected in testes. To examine the possible role of Kir5.1 in sperm physiology, we bred mice with a deletion of the Kcnj16 (Kir5.1) gene and observed that 20% of Kir5.1 knock-out male mice were infertile. Furthermore, 50% of knock-out mice older than 3 months were unable to breed. By contrast, 100% of wild-type (WT) mice were fertile. The genetic inactivation of Kcnj16 also resulted in smaller testes and a greater percentage of sperm with folded flagellum compared to WT littermates. Nevertheless, the abnormal sperm from mutant animals displayed increased progressive motility. Thus, ablation of the Kcnj16 gene identifies Kir5.1 channel as an important element contributing to testis development, sperm flagellar morphology, motility, and fertility. These findings are potentially relevant to the understanding of the complex pHi- and [K+]o-dependent interplay between different sperm ion channels, and provide insight into their role in fertilization and infertility.


Assuntos
Infertilidade Masculina/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Espermatozoides/metabolismo , Animais , Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Infertilidade Masculina/patologia , Masculino , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Músculo Liso/metabolismo , Oócitos/crescimento & desenvolvimento , Potássio/metabolismo , Motilidade Espermática/genética , Espermatozoides/crescimento & desenvolvimento , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
20.
Nat Plants ; 7(9): 1229-1238, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34282287

RESUMO

The membrane potential reflects the difference between cytoplasmic and apoplastic electrical potentials and is essential for cellular operation. The application of the phytohormone auxin (3-indoleacetic acid (IAA)) causes instantaneous membrane depolarization in various cell types1-6, making depolarization a hallmark of IAA-induced rapid responses. In root hairs, depolarization requires functional IAA transport and TIR1-AFB signalling5, but its physiological importance is not understood. Specifically in roots, auxin triggers rapid growth inhibition7-9 (RGI), a process required for gravitropic bending. RGI is initiated by the TIR1-AFB co-receptors, with the AFB1 paralogue playing a crucial role10,11. The nature of the underlying rapid signalling is unknown, as well as the molecular machinery executing it. Even though the growth and depolarization responses to auxin show remarkable similarities, the importance of membrane depolarization for root growth inhibition and gravitropism is unclear. Here, by combining the DISBAC2(3) voltage sensor with microfluidics and vertical-stage microscopy, we show that rapid auxin-induced membrane depolarization tightly correlates with RGI. Rapid depolarization and RGI require the AFB1 auxin co-receptor. Finally, AFB1 is essential for the rapid formation of the membrane depolarization gradient across the gravistimulated root. These results clarify the role of AFB1 as the central receptor for rapid auxin responses.


Assuntos
Proteínas de Arabidopsis/metabolismo , Membrana Celular/efeitos dos fármacos , Gravitropismo/efeitos dos fármacos , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Variação Genética , Genótipo , Potenciais da Membrana/fisiologia , Plantas Geneticamente Modificadas/metabolismo
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