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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
Elife ; 102021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34190043

RESUMO

The expression of motivated behaviors depends on both external and internally arising neural stimuli, yet the intrinsic releasing mechanisms for such variably occurring behaviors remain elusive. In isolated nervous system preparations of Aplysia, we have found that irregularly expressed cycles of motor output underlying food-seeking behavior arise from regular membrane potential oscillations of varying magnitude in an identified pair of interneurons (B63) in the bilateral buccal ganglia. This rhythmic signal, which is specific to the B63 cells, is generated by organelle-derived intracellular calcium fluxes that activate voltage-independent plasma membrane channels. The resulting voltage oscillation spreads throughout a subset of gap junction-coupled buccal network neurons and by triggering plateau potential-mediated bursts in B63, can initiate motor output driving food-seeking action. Thus, an atypical neuronal pacemaker mechanism, based on rhythmic intracellular calcium store release and intercellular propagation, can act as an autonomous intrinsic releaser for the occurrence of a motivated behavior.


Assuntos
Aplysia/fisiologia , Cálcio/fisiologia , Gânglios dos Invertebrados/fisiologia , Potenciais da Membrana/fisiologia , Organelas/fisiologia , Animais , Interneurônios/fisiologia
10.
Nat Commun ; 12(1): 3630, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131136

RESUMO

Voltage-sensitive dye imaging (VSDI) is a powerful technique for interrogating membrane potential dynamics in assemblies of cortical neurons, but with effective resolution limits that confound interpretation. To address this limitation, we developed an in silico model of VSDI in a biologically faithful digital reconstruction of rodent neocortical microcircuitry. Using this model, we extend previous experimental observations regarding the cellular origins of VSDI, finding that the signal is driven primarily by neurons in layers 2/3 and 5, and that VSDI measurements do not capture individual spikes. Furthermore, we test the capacity of VSD image sequences to discriminate between afferent thalamic inputs at various spatial locations to estimate a lower bound on the functional resolution of VSDI. Our approach underscores the power of a bottom-up computational approach for relating scales of cortical processing.


Assuntos
Simulação por Computador , Potenciais Evocados Visuais/fisiologia , Neurônios/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Eletrofisiologia/métodos , Potenciais da Membrana/fisiologia , Córtex Visual/fisiologia , Imagens com Corantes Sensíveis à Voltagem/instrumentação
11.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067824

RESUMO

Pyramidal neurons in the medial prefrontal cortical layer 2/3 are an essential contributor to the cellular basis of working memory; thus, changes in their intrinsic excitability critically affect medial prefrontal cortex (mPFC) functional properties. Transient Receptor Potential Melastatin 4 (TRPM4), a calcium-activated nonselective cation channel (CAN), regulates the membrane potential in a calcium-dependent manner. In this study, we uncovered the role of TRPM4 in regulating the intrinsic excitability plasticity of pyramidal neurons in the mouse mPFC layer of 2/3 using a combination of conventional and nystatin perforated whole-cell recordings. Interestingly, we found that TRPM4 is open at resting membrane potential, and its inhibition increases input resistance and hyperpolarizes membrane potential. After high-frequency stimulation, pyramidal neurons increase a calcium-activated non-selective cation current, increase the action potential firing, and the amplitude of the afterdepolarization, these effects depend on intracellular calcium. Furthermore, pharmacological inhibition or genetic silencing of TRPM4 reduces the firing rate and the afterdepolarization after high frequency stimulation. Together, these results show that TRPM4 plays a significant role in the excitability of mPFC layer 2/3 pyramidal neurons by modulating neuronal excitability in a calcium-dependent manner.


Assuntos
Córtex Pré-Frontal/metabolismo , Células Piramidais/metabolismo , Canais de Cátion TRPM/metabolismo , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Córtex Pré-Frontal/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Células Piramidais/patologia , Canais de Cátion TRPM/fisiologia
12.
Nat Commun ; 12(1): 3855, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158477

RESUMO

Human voltage-gated proton channels (hHv1) extrude protons from cells to compensate for charge and osmotic imbalances due metabolism, normalizing intracellular pH and regulating protein function. Human albumin (Alb), present at various levels throughout the body, regulates oncotic pressure and transports ligands. Here, we report Alb is required to activate hHv1 in sperm and neutrophils. Dose-response studies reveal the concentration of Alb in semen is too low to activate hHv1 in sperm whereas the higher level in uterine fluid yields proton efflux, allowing capacitation, the acrosomal reaction, and oocyte fertilization. Likewise, Alb activation of hHv1 in neutrophils is required to sustain production and release of reactive oxygen species during the immune respiratory burst. One Alb binds to both voltage sensor domains (VSDs) in hHv1, enhancing open probability and increasing proton current. A computational model of the Alb-hHv1 complex, validated by experiments, identifies two sites in Alb domain II that interact with the VSDs, suggesting an electrostatic gating modification mechanism favoring the active "up" sensor conformation. This report shows how sperm are triggered to fertilize, resolving how hHv1 opens at negative membrane potentials in sperm, and describes a role for Alb in physiology that will operate in the many tissues expressing hHv1.


Assuntos
Albuminas/metabolismo , Mediadores da Inflamação/metabolismo , Canais Iônicos/metabolismo , Neutrófilos/metabolismo , Capacitação Espermática/fisiologia , Reação Acrossômica/fisiologia , Albuminas/química , Sequência de Aminoácidos , Fertilização/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/química , Canais Iônicos/genética , Masculino , Potenciais da Membrana/fisiologia , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Prótons , Sêmen/citologia , Sêmen/metabolismo , Homologia de Sequência de Aminoácidos , Espermatozoides/fisiologia , Eletricidade Estática
13.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063769

RESUMO

Maturation of the cardiovascular system is associated with crucial structural and functional remodeling. Thickening of the arterial wall, maturation of the sympathetic innervation, and switching of the mechanisms of arterial contraction from calcium-independent to calcium-dependent occur during postnatal development. All these processes promote an almost doubling of blood pressure from the moment of birth to reaching adulthood. This review focuses on the developmental alterations of potassium channels functioning as key smooth muscle membrane potential determinants and, consequently, vascular tone regulators. We present evidence that the pattern of potassium channel contribution to vascular control changes from Kir2, Kv1, Kv7 and TASK-1 channels to BKCa channels with maturation. The differences in the contribution of potassium channels to vasomotor tone at different stages of postnatal life should be considered in treatment strategies of cardiovascular diseases associated with potassium channel malfunction.


Assuntos
Artérias/metabolismo , Miócitos de Músculo Liso/metabolismo , Canais de Potássio/metabolismo , Animais , Pressão Sanguínea/fisiologia , Cálcio/metabolismo , Potenciais da Membrana/fisiologia , Músculo Liso Vascular/metabolismo , Cuidado Pós-Natal/métodos
14.
Am J Physiol Cell Physiol ; 321(1): C158-C175, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34038243

RESUMO

In whole cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two-pore (K2P) leak-type and a G protein-coupled, inwardly rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly rectifying current was activated by arachidonic acid and inhibited by angiotensin II (ANG II), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by ANG II, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by ANG II or ACTH and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by ANG II and ACTH is likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.


Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Angiotensina II/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canal de Potássio Kv1.4/genética , Proteínas do Tecido Nervoso/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Zona Glomerulosa/metabolismo , Adolescente , Adulto , Aldosterona/biossíntese , Ácido Araquidônico/farmacologia , Autopsia , Criança , Colforsina/farmacologia , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Expressão Gênica , Humanos , Canal de Potássio Kv1.4/antagonistas & inibidores , Canal de Potássio Kv1.4/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Cultura Primária de Células , Zona Glomerulosa/citologia , Zona Glomerulosa/efeitos dos fármacos
15.
Exp Neurol ; 342: 113758, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33991525

RESUMO

To get insight into the mechanism of action of carbonic anhydrase inhibitors (CAI) in neuromuscular disorders, we investigated effects of dichlorphenamide (DCP) and acetazolamide (ACTZ) on ClC-1 chloride channels and skeletal muscle excitability. We performed patch-clamp experiments to test drugs on chloride currents in HEK293T cells transfected with hClC-1. Using the two-intracellular microelectrode technique in current-clamp mode, we measured the effects of drugs on the resting chloride conductance and action potential properties of sarcolemma in rat and mouse skeletal muscle fibers. Using BCECF dye fluorometry, we measured the effects of ACTZ on intracellular pH in single rat muscle fibers. Similarly to ACTZ, DCP (100 µM) increased hClC-1 chloride currents in HEK cells, because of the negative shift of the open probability voltage dependence and the slowing of deactivation kinetics. Bendroflumethiazide (BFT, 100 µM), structurally related to DCP but lacking activity on carbonic anhydrase, had little effects on chloride currents. In isolated rat muscle fibers, 50-100 µM of ACTZ or DCP, but not BFT, induced a ~ 20% increase of the resting chloride conductance. ACTZ reduced action potential firing in mouse muscle fibers. ACTZ (100 µM) reduced intracellular pH to 6.8 in rat muscle fibers. These results suggest that carbonic anhydrase inhibitors can reduce muscle excitability by increasing ClC-1 channel activity, probably through intracellular acidification. Such a mechanism may contribute in part to the clinical effects of these drugs in myotonia and other muscle excitability disorders.


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Canais de Cloreto/metabolismo , Doenças Musculares/metabolismo , Sarcolema/metabolismo , Animais , Inibidores da Anidrase Carbônica/uso terapêutico , Diclorofenamida/farmacologia , Diclorofenamida/uso terapêutico , Células HEK293 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Doenças Musculares/tratamento farmacológico , Ratos , Ratos Wistar , Sarcolema/efeitos dos fármacos
16.
Development ; 148(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33999994

RESUMO

It is well known that electrical signals are deeply associated with living entities. Much of our understanding of excitable tissues is derived from studies of specialized cells of neurons or myocytes. However, electric potential is present in all cell types and results from the differential partitioning of ions across membranes. This electrical potential correlates with cell behavior and tissue organization. In recent years, there has been exciting, and broadly unexpected, evidence linking the regulation of development to bioelectric signals. However, experimental modulation of electrical potential can have multifaceted and pleiotropic effects, which makes dissecting the role of electrical signals in development difficult. Here, I review evidence that bioelectric cues play defined instructional roles in orchestrating development and regeneration, and further outline key areas in which to refine our understanding of this signaling mechanism.


Assuntos
Comunicação Celular/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Desenvolvimento Embrionário/fisiologia , Potenciais da Membrana/fisiologia , Regeneração/fisiologia , Animais , Transporte Biológico/fisiologia , Padronização Corporal/fisiologia , Humanos , Canais Iônicos/fisiologia , Transdução de Sinais/fisiologia
17.
Elife ; 102021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33821794

RESUMO

Whether ion channel gating is independent of ion permeation has been an enduring, unresolved question. Here, applying single channel recording to the archetypal muscle nicotinic receptor, we unmask coupling between channel gating and ion permeation by structural perturbation of a conserved intramembrane salt bridge. A charge-neutralizing mutation suppresses channel gating, reduces unitary current amplitude, and increases fluctuations of the open channel current. Power spectra of the current fluctuations exhibit low- and high-frequency Lorentzian components, which increase in charge-neutralized mutant receptors. After aligning channel openings and closings at the time of transition, the average unitary current exhibits asymmetric relaxations just after channel opening and before channel closing. A theory in which structural motions contribute jointly to channel gating and ion conduction describes both the power spectrum and the current relaxations. Coupling manifests as a transient increase in the open channel current upon channel opening and a decrease upon channel closing.


Assuntos
Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Músculos/metabolismo , Receptores Nicotínicos/metabolismo , Células HEK293 , Humanos
18.
Philos Trans A Math Phys Eng Sci ; 379(2198): 20200237, 2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-33840215

RESUMO

Chaotic resonance (CR) is a new phenomenon induced by an intermediate level of chaotic signal intensity in neuronal systems. In the current study, we investigated the effects of autapse on the CR phenomenon in single neurons and small-world (SW) neuronal networks. In single neurons, we assume that the neuron has only one autapse modelled as electrical, excitatory chemical and inhibitory chemical synapse, respectively. Then, we analysed the effects of each one on the CR, separately. Obtained results revealed that, regardless of its type, autapse significantly increases the chaotic resonance of the appropriate autaptic parameter's values. It is also observed that, at the optimal chaotic current intensity, the multiple CR emerges depending on autaptic time delay for all the autapse types when the autaptic delay time or its integer multiples match the half period or period of the weak signal. In SW networks, we investigated the effects of chaotic activity on the prorogation of pacemaker activity, where pacemaker neurons have different kinds of autapse as considered in single neuron cases. Obtained results revealed that excitatory and electrical autapses prominently increase the prorogation of pacemaker activity, whereas inhibitory autapse reduces or does not change it. Also, the best propagation was obtained when the autapse was excitatory. This article is part of the theme issue 'Vibrational and stochastic resonance in driven nonlinear systems (part 2)'.


Assuntos
Modelos Neurológicos , Rede Nervosa/fisiologia , Neurônios/fisiologia , Potenciais de Ação/fisiologia , Animais , Fenômenos Eletrofisiológicos , Humanos , Potenciais da Membrana/fisiologia , Dinâmica não Linear , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Fatores de Tempo
19.
Elife ; 102021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33904400

RESUMO

In addition to the hallmark muscle stiffness, patients with recessive myotonia congenita (Becker disease) experience debilitating bouts of transient weakness that remain poorly understood despite years of study. We performed intracellular recordings from muscle of both genetic and pharmacologic mouse models of Becker disease to identify the mechanism underlying transient weakness. Our recordings reveal transient depolarizations (plateau potentials) of the membrane potential to -25 to -35 mV in the genetic and pharmacologic models of Becker disease. Both Na+ and Ca2+ currents contribute to plateau potentials. Na+ persistent inward current (NaPIC) through NaV1.4 channels is the key trigger of plateau potentials and current through CaV1.1 Ca2+ channels contributes to the duration of the plateau. Inhibiting NaPIC with ranolazine prevents the development of plateau potentials and eliminates transient weakness in vivo. These data suggest that targeting NaPIC may be an effective treatment to prevent transient weakness in myotonia congenita.


Assuntos
Potenciais da Membrana/fisiologia , Miotonia Congênita/fisiopatologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Miotonia Congênita/diagnóstico , Miotonia Congênita/genética , Sódio/fisiologia
20.
Am J Physiol Cell Physiol ; 320(6): C1074-C1087, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33852365

RESUMO

Voltage-gated Kv7 (KCNQ family) K+ channels are expressed in many neuronal populations and play an important role in regulating membrane potential by generating a hyperpolarizing K+ current and decreasing cell excitability. However, the role of KV7 channels in the neural regulation of intestinal epithelial Cl- secretion is not known. Cl- secretion in mouse distal colon was measured as a function of short-circuit current (ISC), and pharmacological approaches were used to test the hypothesis that activation of KV7 channels in enteric neurons would inhibit epithelial Cl- secretion. Flupirtine, a nonselective KV7 activator, inhibited basal Cl- secretion in mouse distal colon and abolished or attenuated the effects of drugs that target various components of enteric neurotransmission, including tetrodotoxin (NaV channel blocker), veratridine (NaV channel activator), nicotine (nicotinic acetylcholine receptor agonist), and hexamethonium (nicotinic antagonist). In contrast, flupritine did not block the response to epithelium-targeted agents VIP (endogenous VPAC receptor ligand) or carbachol (nonselective cholinergic agonist). Flupirtine inhibited Cl- secretion in both full-thickness and seromuscular-stripped distal colon (containing the submucosal, but not myenteric plexus) but generated no response in epithelial T84 cell monolayers. KV7.2 and KV7.3 channel proteins were detected by immunofluorescence in whole mount preparations of the submucosa from mouse distal colon. ICA 110381 (KV7.2/7.3 specific activator) inhibited Cl- secretion comparably to flupirtine. We conclude that KV7 channel activators inhibit neurally driven Cl- secretion in the colonic epithelium and may therefore have therapeutic benefit in treating pathologies associated with hyperexcitable enteric nervous system, such as irritable bowel syndrome with diarrhea (IBS-D).


Assuntos
Cloretos/metabolismo , Colo/metabolismo , Sistema Nervoso Entérico/efeitos dos fármacos , Células Epiteliais/metabolismo , Canais de Potássio KCNQ/metabolismo , Neurônios/metabolismo , Aminopiridinas/farmacologia , Animais , Carbacol/farmacologia , Linhagem Celular Tumoral , Agonistas Colinérgicos/farmacologia , Colo/efeitos dos fármacos , Sistema Nervoso Entérico/metabolismo , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos
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