RESUMO
The molecular targets and neural circuits that underlie general anesthesia are not fully elucidated. Here, we directly demonstrate that Kv1-family (Shaker-related) delayed rectifier K(+) channels in the central medial thalamic nucleus (CMT) are important targets for volatile anesthetics. The modulation of Kv1 channels by volatiles is network specific as microinfusion of ShK, a potent inhibitor of Kv1.1, Kv1.3, and Kv1.6 channels, into the CMT awakened sevoflurane-anesthetized rodents. In heterologous expression systems, sevoflurane, isoflurane, and desflurane at subsurgical concentrations potentiated delayed rectifier Kv1 channels at low depolarizing potentials. In mouse thalamic brain slices, sevoflurane inhibited firing frequency and delayed the onset of action potentials in CMT neurons, and ShK-186, a Kv1.3-selective inhibitor, prevented these effects. Our findings demonstrate the exquisite sensitivity of delayed rectifier Kv1 channels to modulation by volatile anesthetics and highlight an arousal suppressing role of Kv1 channels in CMT neurons during the process of anesthesia.
Assuntos
Anestésicos Gerais/farmacologia , Nível de Alerta/efeitos dos fármacos , Núcleos Intralaminares do Tálamo/efeitos dos fármacos , Superfamília Shaker de Canais de Potássio/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Desflurano , Núcleos Intralaminares do Tálamo/metabolismo , Isoflurano/análogos & derivados , Isoflurano/farmacologia , Espectroscopia de Ressonância Magnética , Masculino , Éteres Metílicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Sevoflurano , Superfamília Shaker de Canais de Potássio/metabolismo , Compostos Orgânicos Voláteis/farmacologiaRESUMO
For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai in Drosophila cells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca2+ concentration near the interface, indicating Ca2+ influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca2+ signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca2+ influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca2+ signaling during subsequent antigen encounter.
Assuntos
Canais de Cálcio/fisiologia , Ativação Linfocitária , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Linfócitos T/imunologia , Regulação para Cima , Cálcio/metabolismo , Linhagem Celular , Humanos , Transporte de Íons , Proteína ORAI1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Molécula 1 de Interação EstromalRESUMO
Studies of the electrophysiological response to acetylcholine (ACh) in mammalian outer hair cells (OHCs) are hindered by the presence of a large potassium current, I(K,n), most likely mediated by channels containing the KCNQ4 subunit. Since I(K,n) can be blocked by linopirdine, cholinergic effects might be better revealed in the presence of this compound. The aim of the present work was to study the effects of linopirdine on the ACh-evoked responses through alpha9alpha10-containing native and recombinant nicotinic cholinergic receptors. Responses to ACh were blocked by linopirdine in both OHCs and inner hair cells (IHCs) of rats at postnatal days 21-27 (OHCs) and 9-11 (IHCs). In addition, linopirdine blocked responses of recombinant alpha9alpha10 nicotinic cholinergic receptors (nAChRs) in a concentration-dependent manner with an IC(50) of 5.2 microM. Block by linopirdine was readily reversible, voltage independent, and surmountable at high concentrations of ACh, thus suggestive of a competitive type of interaction with the receptor. The present results contribute to the pharmacological characterization of alpha9alpha10-containing nicotinic receptors and indicate that linopirdine should be used with caution when analyzing the cholinergic sensitivity of cochlear hair cells.
Assuntos
Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/fisiologia , Indóis/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Subunidades Proteicas/metabolismo , Piridinas/farmacologia , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Expressão Gênica , Potenciais da Membrana/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Subunidades Proteicas/genética , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
The impact of synthetic amyloid ß (1-42) (Aß(1-42)) oligomers on biophysical properties of voltage-gated potassium channels Kv 1.3 and lipid bilayer membranes (BLMs) was quantified for protocols using hexafluoroisopropanol (HFIP) or sodium hydroxide (NaOH) as solvents prior to initiating the oligomer formation. Regardless of the solvent used Aß(1-42) samples contained oligomers that reacted with the conformation-specific antibodies A11 and OC and had similar size distributions as determined by dynamic light scattering. Patch-clamp recordings of the potassium currents showed that synthetic Aß(1-42) oligomers accelerate the activation and inactivation kinetics of Kv 1.3 current with no significant effect on current amplitude. In contrast to oligomeric samples, freshly prepared, presumably monomeric, Aß(1-42) solutions had no effect on Kv 1.3 channel properties. Aß(1-42) oligomers had no effect on the steady-state current (at -80 mV) recorded from Kv 1.3-expressing cells but increased the conductance of artificial BLMs in a dose-dependent fashion. Formation of amyloid channels, however, was not observed due to conditions of the experiments. To exclude the effects of HFIP (used to dissolve lyophilized Aß(1-42) peptide), and trifluoroacetic acid (TFA) (used during Aß(1-42) synthesis), we determined concentrations of these fluorinated compounds in the stock Aß(1-42) solutions by (19)F NMR. After extensive evaporation, the concentration of HFIP in the 100× stock Aß(1-42) solutions was â¼1.7 µM. The concentration of residual TFA in the 70× stock Aß(1-42) solutions was â¼20 µM. Even at the stock concentrations neither HFIP nor TFA alone had any effect on potassium currents or BLMs. The Aß(1-42) oligomers prepared with HFIP as solvent, however, were more potent in the electrophysiological tests, suggesting that fluorinated compounds, such as HFIP or structurally-related inhalational anesthetics, may affect Aß(1-42) aggregation and potentially enhance ability of oligomers to modulate voltage-gated ion channels and biological membrane properties.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Condutividade Elétrica , Canal de Potássio Kv1.3/metabolismo , Bicamadas Lipídicas/metabolismo , Fragmentos de Peptídeos/farmacologia , Solventes/química , Peptídeos beta-Amiloides/síntese química , Halogenação , Cinética , Luz , Membranas Artificiais , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/síntese química , Propanóis/química , Espalhamento de Radiação , Hidróxido de Sódio/químicaRESUMO
In the present work, we characterized the effects of serotonin type 3 receptor ligands on recombinant and native alpha 9 alpha 10-containing nicotinic acetylcholine receptors (nAChRs). Our results indicate that the recombinant alpha 9 alpha 10 nAChR shares striking pharmacological properties with 5-HT(3) ligand-gated ion channels. Thus, 5-HT(3) receptor antagonists block ACh-evoked currents in alpha 9 alpha 10-injected Xenopus laevis oocytes with a rank order of potency of tropisetron (IC(50), 70.1 +/- 0.9 nM) > ondansetron (IC(50), 0.6 +/- 0.1 microM) = MDL 72222 (IC(50), 0.7 +/- 0.1 microM). Although serotonin does not elicit responses in alpha 9 alpha 10-injected oocytes, it blocks recombinant alpha 9 alpha 10 receptors in a noncompetitive and voltage-dependent manner (IC(50), 5.4 +/- 0.6 microM). On the other hand, we demonstrate an in vivo correlate of these properties of the recombinant receptor, with those of the alpha 9 alpha 10-containing nAChR of frog saccular hair cells. The possibility that the biogenic amine serotonin might act as a neuromodulator of the cholinergic efferent transmission in the vestibular apparatus and in the organ of Corti is discussed.