Sodium and Calcium Currents in Dispersed Mammalian Septal Neurons

Voltage-gated Na+ and Ca2+ conductances of freshly dissociated septal neurons were studied in the whole-cell configuration of the patch-clamp technique. All cells exhibited a large Na+ current with characteristic fast activation and inactivation time courses. Half-time to peak current at -20 mV was 0.44 +/- 0.18 ms and maximal activation of Na+ conductance occurred at 0 mV or more positive membrane potentials. The average value was 91 +/- 32 nS (approximately 11 mS cm-2). At all membrane voltages inactivation was well fitted by a single exponential that had a time constant of 0.44 +/- 0.09 ms at 0 mV. Recovery from inactivation was complete in approximately 900 ms at -80 mV but in only 50 ms at -120 mV. The decay of Na+ tail currents had a single time constant that at -80 mV was faster than 100 microseconds. Depolarization of septal neurons also elicited a Ca2+ current that peaked in approximately 6-8 ms. Maximal peak Ca2+ current was obtained at 20 mV, and with 10 mM external Ca2+ the amplitude was 0.35 +/- 0.22 nA. During a maintained depolarization this current partially inactivated in the course of 200-300 ms. The Ca2+ current was due to the activity of two types of conductances with different deactivation kinetics. At -80 mV the closing time constants of slow (SD) and fast (FD) deactivating channels were, respectively, 1.99 +/- 0.2 and 0.11 +/- 0.03 ms (25 degrees C). The two kinds of channels also differed in their activation voltage, inactivation time course, slope of the conductance-voltage curve, and resistance to intracellular dialysis. The proportion of SD and FD channels varied from cell to cell, which may explain the differential electrophysiological responses of intracellularly recorded septal neurons

Thyrotropin-releasing-hormone (TRH) and its physiological metabolite TRH-OH inhibit Na+ channel activity in mammalian septal neurons

The interaction of thyrotropin-releasing hormone (TRH) and its physiological metabolite TRH-OH with Na+ channels was studied in enzymatically dissociated guinea pig septal neurons by using the whole-cell variant of the patch-clamp technique. In about 60% of the cells tested, the neuropeptides at concentrations between 0.01 and 2.5 ,uM produced a dose-dependent reversible attenuation of Na+ currents. With 2 ,uM TRH-OH, peak Na+ current amplitude was reduced by 20-50% (27 ± 8%, mean ± SD; n = 16), whereas at the same concentration TRH was approximately half as effective as TRH-OH. In the presence of the tripeptides, the voltage-dependent parameters of the Na+ current were unaltered. TRH-induced reduction of Na+ current amplitude was transient and recovered almost completely during maintained exposure to the peptides. In addition, the response to either TRH-OH or TRH decreased with repeated treatment. Our results demonstrate that neuronal Na+ channels can be modulated by naturally occurring neuropeptides

A Small Domain in the N Terminus of the Regulatory a-Subunit Kv2.3 Modulates Kv2.1 Potassium Channel Gating

Recent work has demonstrated the existence of regulatory K1 channel a-subunits that are electrically silent but capable of forming heterotetramers with other pore-forming subunits to modify their function. We have investigated the molecular determinant of the modulatory effects of Kv2.3, a silent K1 channel a-subunit specific of brain. This subunit induces on Kv2.1 channels a marked deceleration of activation, inactivation, and closing kinetics. We constructed chimeras of the Kv2.1 and Kv2.3 proteins and analyzed the K1 currents resulting from the coexpression of the chimeras with Kv2.1. The data indicate that a region of 59 amino acids in the N terminus, adjacent to the first transmembrane segment, is the major structural element responsible for the regulatory function of Kv2.3. The sequence of this domain of Kv2.3 is highly divergent compared with the same region in the other channels of the Kv2 family. Replacement of the regulatory fragment of Kv2.3 by the equivalent of Kv2.1 leads to loss of modulatory function, whereas gain of modulatory function is observed when the Kv2.3 fragment is transferred to Kv2.1. Thus, this study identifies a N-terminus domain involved in Kv2.1 channel gating and in the modulation of this channel by a regulatory a-subunit

Identification and Functional Characterization of a K1 Channel a- Subunit with Regulatory Properties Specific to Brain

The physiological diversity of K1 channels mainly depends on the expression of several genes encoding different a-subunits. We have cloned a new K1 channel a-subunit (Kv2.3r) that is unable to form functional channels on its own but that has a major regulatory function. Kv2.3r can coassemble selectively with other a-subunits to form functional heteromultimeric K1 channels with kinetic properties that differ from those of the parent channels. Kv2.3r is expressed exclusively in the brain, being concentrated particularly in neocortical neurons. The functional expression of this regulatory a-subunit represents a novel mechanism without precedents in voltage-gated channels, which might contribute to further increase the functional diversity of K1 channels necessary to specify the intrinsic electrical properties of individual neurons

Orai1 and TRPC1 Proteins Co-localize with CaV1.2 Channels to Form a Signal Complex in Vascular Smooth Muscle Cells

Voltage-dependent CaV1.2 L-type Ca2 channels (LTCC) are the main route for calcium entry in vascular smooth muscle cells (VSMC). Several studies have also determined the relevant role of store-operated Ca2 channels (SOCC) in vascular tone regulation. Nevertheless, the role of Orai1- and TRPC1-dependent SOCC in vascular tone regulation and their possible interaction with CaV1.2 are still unknown. The current study sought to characterize the co-activation of SOCC and LTCC upon stimulation by agonists, and to determine the possible crosstalk between Orai1, TRPC1, and CaV1.2. Aorta rings and isolated VSMCobtained from wild type or smooth muscle-selective conditional CaV1.2 knock-out (CaV1.2KO) mice were used to study vascular contractility, intracellular Ca2 mobilization, and distribution of ion channels. We found that serotonin (5-HT) or store depletion with thapsigargin (TG) enhanced intracellular free Ca2 concentration ([Ca2 ]i) and stimulated aorta contraction. These responses were sensitive to LTCC and SOCC inhibitors. Also, 5-HT- and TG-induced responses were significantly attenuated in CaV1.2KO mice. Furthermore, hyperpolarization induced with cromakalim or valinomycin significantly reduced both 5-HT and TG responses, whereas these responses were enhanced with LTCC agonist Bay-K-8644. Interestingly, in situ proximity ligation assay revealed that CaV1.2 interacts with Orai1 and TRPC1 in untreated VSMC. These interactions enhanced significantly after stimulation of cells with 5-HT and TG. Therefore, these data indicate for the first time a functional interaction between Orai1, TRPC1, and CaV1.2 channels in VSMC, confirming that upon agonist stimulation, vessel contraction involves Ca2 entry due to co-activation of Orai1- and TRPC1-dependent SOCC and LTCC.Ministerio de Economía y Competitividad BFU2013-45564-C2-1-PMinisterio de Economía y Competitividad BFU2013-45564-C2-2-PInstituto de Salud Carlos III RD12/0042/ 0041Cardiovascular Network “RIC” PI12/00941Junta de Andalucía PI-0108-2012Junta de Andalucía P12- CTS-196

Canales iónicos de neuronas septales aisladas: Caracterización electrofisiológica y modulación por TRH y LHRH

El presente trabajo continua con una de las líneas de investigación de nuestro grupo que trata del estudio de las propiedades electrofisiológicas de neuronas del sistema nervioso central de mamíferos. Este estudio se inició en diferentes estructuras cerebrales, entre ellas el área septal, mediante la técnica de registro intracelular in vitro, lo que permitió una clasificación de los distintos tipos celulares y una caracterización preliminar de las conductancias iónicas responsables de las propiedades funcionales intrínsecas de los elementos neuronales. Uno de los objetivos generales de este trabajo ha sido profundizar más en el conocimiento de la fisiología de neuronas individuales registrando las corrientes iónicas de forma directa y con una metodología (“patch-clamp”) que permite cuantificar los parámetros característicos de las conductancias iónicas de la membrana. Este estudio es de interés no solo porque complementa experimentos previos de nuestro grupo, sino porque existe muy poca electrofisiología cuantitativa realizada en neuronas centrales. Para llevar a cabo el trabajo se ha puesto a punto una preparación de neuronas septales en cultivo primario que por su estabilidad, geometría y alta densidad de canales iónicos con la calidad necesaria para el estudio cuantitativo indicado anteriormente. El otro objetivo general del trabajo ha sido el estudio de la modulación de los canales dependientes del voltaje por neuropéptidos. El mecanismo de acción de los péptidos cerebrales, de gran trascendencia fisiológica y farmacológica, está siendo muy estudiado en los últimos años pero existen muy pocos datos sobre su posible efecto a nivel de las conductancias iónicas de la membrana. Este trabajo se centra en los péptidos, TRH y LHRH, que se encuentran en alta concentración en el septum, pero cuyo papel fisiológico y mecanismos de acción se desconocen. CONCLUSIONES 1. En este trabajo se ha puesto a punto una preparación de neuronas del SNC de mamíferos adecuada para la realización de un estudio biofísico cuantitativo utilizando la configuración de registro en “célula completa” (“whole-cell”) de la técnica de “patch-clamp”. 2. Las neuronas septales dispersas son excitables y mantienen funcionales, y aparentemente inalterados, los canales iónicos dependientes del potencial de membrana. 3. En todas las células la despolarización induce la génesis de una corriente de sodio. Esta corriente alcanza la amplitud máxima en 0,4-0,5 ms a 0 mV y se inactiva de forma rápida en pocos milisegundos. La corriente de sodio es la responsable de la génesis de los potenciales de acción rápidos de neuronas septales y es muy parecida a la descrita por Hodgkin y Huxley en el axón gigante de calamar. 4. Las neuronas septales poseen, al menos, dos tipos de corrientes de calcio: una medida por canales con cinética de desactivación lenta o SD y otra que se debe a la actividad de canales con cinética de desactivación rápida o FD. La contribución relativa a la corriente “total” de cada tipo de canal iónico varia de célula a célula. Es, sin embargo, importante destacar que los canales FD se observan, en mayor o menor medida, en todas las células, mientras que más del 20% delas neuronas no tienen canales SD. 5. Los canales SD, tipo T o de bajo umbral, se activan a un potencial de membrana cercano a -40 mV y se inactivan con pulsos de larga duración. La corriente producida por la actividad de estos canales, aunque en todos los casos de menor amplitud que la mediada por los canales FD, es posiblemente la que genera las “espigas de calcio de bajo umbral” típica de las neuronas del septum intermedio y responsable del cambio del patrón de disparo tónico al fásico. 6. Los canales FD, tipo L o de alto umbral, se activan a un potencial de membrana alrededor de -20 mV, y apenas se inactivan. La corriente producida por la actividad de estos canales, que como nuestro estudio muestra se puede generar tanto en el soma como en las dendritas, es posiblemente la que produce las “espigas de calcio de alto umbral”. Estas espigas de calcio se observan en todas las neuronas septales (y en general en todas las neuronas del SNC de mamíferos) tras bloquear la conductancia a los iones de sodio con TTX. 7. En el conjunto de la población de células estudiadas se han identificado tres tipos distintos de canales de potasio. Dos de estos canales (IK y IK(Ca)) tienen una cinética de activación lenta y solo se inactivan parcialmente con pulsos de larga duración. El tercer tipo de canal de potasio (It) se activa rápidamente y se inactiva en menos de 50 ms. 8. La corriente lenta de potasio (IK) se ha registrado en todas las células. La activación de esta corriente sigue un curso temporal sigmoideo y alcanza un máximo en unos 15 – 20 ms. La amplitud de la corriente lenta de potasio activada por calcio (IK(Ca)) varía de célula a célula. Esta corriente no se ha estudiado con detalle debido a que la mayoría de los experimentos se realizaron tras el “lavado” de los canales de calcio. Ambas corrientes de potasio determinan la amplitud y duración de la posthiperpolarización que sigue a un tren de potenciales de acción y quizás contribuyan a la fase final de la repolarización de los potenciales de acción. 9. La corriente de potasio rápida y transitoria (It), se ha observado solamente en el 27% de las neuronas septales. Esta corriente se activa en 3-4 ms y a -80 mV el 17% de los canales responsables de ella se encuentran inactivados. Los canales que producen la corriente It son posiblemente característicos de las neuronas del septum medial y al limitar la excitabilidad de la membrana, quizás sean los mayores responsables del disparo fásico típico de estas células. 10. La configuración “whole-cell” de la técnica de “patch-clamp” aplicada a neuronas septales permite el registro de forma estable de corrientes iónicas durante más de 30 minutos. Sin embargo, tanto en los canales de sodio como en los canales de calcio del tipo FD se observan modificaciones producidas por la diálisis del medio intracelular. En el caso de los canales de sodio la alteración más aparente es un desplazamiento de 5 – 10 mV hacia potenciales más negativos en la relación corriente-voltaje. La corriente debida a la actividad de canales de calcio FD decrece en amplitud a lo largo del tiempo, aunque no se modifican sus características cinéticas. La aparición de ambos fenómenos se retarda por la adición de ATP al medio intracelular. 11. La hormona liberadora de la Tirotropina (TRH) produce, en un porcentaje de células, un aumento reversible en la amplitud de la corriente de potasio con cinética de activación lenta. Este incremento se debe a la activación de la corriente de potasio dependiente de calcio producido a través de un incremento en los niveles de Ca2+ intracelular. 12. La forma ácida de la TRH produce, en el 35% de las neuronas estudiadas, una disminución reversible de la amplitud de la corriente de sodio. El porcentaje medio de reducción de la amplitud máxima de la corriente de sodio en presencia de 2,75 μM TRH es del 22,8% del valor control. Un efecto similar al de la TRHa lo produce la adición al medio externo de hormona liberadora de la hormona luteinizante (LHRH) a concentraciones micromolares. 13. La observación de los efectos de la TRH y LHRH solamente en un porcentaje de células sugiere que su mecanismo de acción implica la unión de las hormonas a receptores específicos. De hecho, la exposición repetida a TRHa produce un efecto menor, lo que podría explicarse por “desensbilización” del receptor o de algunos de los procesos que acoplan el receptor a los canales de sodio. 14. La inhibición reversible de la corriente de sodio por TRH y LHRH es una observación sin precedentes en la literatura. Esta bien demostrado que los canales de potasio y calcio pueden ser modulados por neurotransmisores y hormonas, sin embargo se ha asumido tradicionalmente que los canales de sodio dependientes del potencial de membrana son solamente sensibles a cambios en el campo eléctrico y por lo tanto no susceptibles de modulación

Prolyl Hydroxylase-dependent Modulation of Eukaryotic Elongation Factor 2 Activity and Protein Translation under Acute Hypoxia

Early adaptive responses to hypoxia are essential for cell survival, but their nature and underlying mechanisms are poorly known. We have studied the post-transcriptional changes in the proteome of mammalian cells elicited by acute hypoxia and found that phosphorylation of eukaryotic elongation factor 2 (eEF2), a ribosomal translocase whose phosphorylation inhibits protein synthesis, is under the precise and reversible control of O(2) tension. Upon exposure to hypoxia, phosphorylation of eEF2 at Thr(56) occurred rapidly (<15 min) and resulted in modest translational arrest, a fundamental homeostatic response to hypoxia that spares ATP and thus facilitates cell survival. Acute inhibitory eEF2 phosphorylation occurred without ATP depletion or AMP kinase activation. Furthermore, eEF2 phosphorylation was mimicked by prolyl hydroxylase (PHD) inhibition with dimethyloxalylglycine or by selective PHD2 siRNA silencing but was independent of hypoxia-inducible factor α stabilization. Moreover, overexpression of PHD2 blocked hypoxic accumulation of phosphorylated eEF2. Therefore, our findings suggest that eEF2 phosphorylation status (and, as a consequence, translation rate) is controlled by PHD2 activity. They unravel a novel pathway for cell adaptation to hypoxia that could have pathophysiologic relevance in tissue ischemia and cancer.Spanish Ministry of Science and HealthAndalusian Governmen

Brugada syndrome masked by complete left bundle branch block: A clinical and functional study of its association with the p.1449Y>H SCN5A variant

SCN5A gene variants are associated with both Brugada syndrome and conduction disturbances, sometimes expressing an overlapping phenotype. Functional consequences of SCN5A variants assessed by patch‐clamp electrophysiology are particularly beneficial for correct pathogenic classification and are related to disease penetrance and severity. Here, we identify a novel SCN5A loss of function variant, p.1449Y>H, which presented with high penetrance and complete left bundle branch block, totally masking the typical findings on the electrocardiogram. We highlight the possibility of this overlap combination that makes impossible an electrocardiographic diagnosis and, through a functional analysis, associate the p.1449Y>H variant to SCN5A pathogenicity.Sociedad Española de Cardiología Ritmo 2017Consejería de Salud of the Junta de Andalucía PI‐0365‐201

PKCα-Mediated Downregulation of RhoA Activity in Depolarized Vascular Smooth Muscle: Synergistic Vasorelaxant Effect of PKCα and ROCK Inhibition

Background/Aims: Protein kinase C (PKC)- and RhoA/Rho-associated kinase (ROCK) play important roles in arterial sustained contraction. Although depolarization-elicited RhoA/ROCK activation is accepted, the role of PKC in depolarized vascular smooth muscle cells (VSMCs) is a subject of controversy. Our aim was to study the role of PKC in arterial contraction and its interaction with RhoA/ROCK. Methods: Mass spectrometry was used to identify the PKC isoenzymes. PKCα levels and RhoA activity were analyzed by western blot and G-LISA, respectively, and isometric force was measured in arterial rings. Results: In depolarized VSMCs RhoA and PKCα were translocated to the plasma membrane, where they colocalize and coimmunoprecipitate. Interestingly, depolarization-induced RhoA activation was downregulated by PKCα, effect reverted by PKCα inhibition. Phorbol 12,13-dibutyrate (PDBu) induced the translocation of PKCα to the plasma membrane, increased the level of RhoA in the cytosol and reduced RhoA/ROCK activity. These effects were reverted when PKC was inhibited. Pharmacological or siRNA inhibition of PKCα synergistically potentiated the vasorelaxant effect of RhoA/ROCK inhibition. Conclusion: The present study provides the first evidence that RhoA activity is downregulated by PKCα in depolarized and PDBu treated freshly isolated VSMCs and arteries, with an important physiological role on arterial contractility

Changes in adhesion and the expression of adhesion molecules in PBMCs after aneurysmal subarachnoid hemorrhage: relation to cerebral vasospasm

Aneurysmal subarachnoid hemorrhage (aSAH) is a neurovascular disease produced by extravasation of blood to the subarachnoid space after rupture of the cerebral vessels. After bleeding, the immune response is activated. The role of peripheral blood mononuclear cells (PBMCs) in this response is a current subject of research. We have analysed the changes in PBMCs of patients with aSAH and their interaction with the endothelium, focusing on their adhesion and the expression of adhesion molecules. Using an in vitro adhesion assay, we observed that the adhesion of PBMCs of patients with aSAH is increased. Flow cytometry analysis shows that monocytes increased signifcantly in patients, especially in those who developed vasospasm (VSP). In aSAH patients, the expression of CD162, CD49d, CD62L and CD11a in T lymphocytes and of CD62L in monocytes increased. However, the expression of CD162, CD43, and CD11a decreased in monocytes. Furthermore, monocytes from patients who developed arteriographic VSP had lower expression of CD62L. In conclusion, our results confrm that after aSAH, monocyte count and adhesion of PBMCs increase, especially in patients with VSP, and that the expression of several adhesion molecules is altered. These observations can help predict VSP and to improve the treatment of this pathology