Shaker-Related Potassium Channels in the Central Medial Nucleus of the Thalamus Are Important Molecular Targets for Arousal Suppression by Volatile General Anesthetics

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

Direct interaction of serotonin type 3 receptor ligands with recombinant and native α9α10-containing nicotinic cholinergic receptors

In the present work, we characterized the effects of serotonin type 3 receptor ligands on recombinant and native α9α10-containing nicotinic acetylcholine receptors (nAChRs). Our results indicate that the recombinant α9α10 nAChR shares striking pharmacological properties with 5-HT3 ligand-gated ion channels. Thus, 5-HT3 receptor antagonists block ACh-evoked currents in α9α10-injected Xenopus laevis oocytes with a rank order of potency of tropisetron (IC50, 70.1 ± 0.9 nM) > ondansetron (IC50, 0.6 ± 0.1 μM) = MDL 72222 (IC50, 0.7 ± 0.1 μM). Although serotonin does not elicit responses in α9α10-injected oocytes, it blocks recombinant α9α10 receptors in a noncompetitive and voltage-dependent manner (IC50, 5.4 ± 0.6 μM). On the other hand, we demonstrate an in vivo correlate of these properties of the recombinant receptor, with those of the α9α10-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.Fil: Rothlin, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Lioudyno, Maria I.. Tulane University; Estados UnidosFil: Silbering, Ana F.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Plazas, Paola Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gomez Casati, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Katz, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad Nacional de Córdoba. Facultad de Cs.exactas Físicas y Naturales. Departamento de Biología; ArgentinaFil: Guth, Paul S.. Tulane University; Estados UnidosFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

STIM1, an Essential and Conserved Component of Store-Operated Ca\u3csup\u3e2+\u3c/sup\u3e Channel Function

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+(CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels

Orai1 and STIM1 Move to the Immunological Synapse and Are Up-Regulated during T Cell Activation

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 inDrosophila 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

Shaker-related potassium channels in the central medial nucleus of the thalamus are important molecular targets for arousal suppression by volatile general anesthetics.

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

Orai1 and STIM1 Move to the Immunological Synapse and Are Up-Regulated during T Cell Activation

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 inDrosophila 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

Shaker-related potassium channels in the central medial nucleus of the thalamus are important molecular targets for arousal suppression by volatile general anesthetics.

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