Activation of TREK currents by the neuroprotective agent riluzole in mouse sympathetic neurons.

Background K2P channels play a key role in stabilizing the resting membrane potential, thereby modulating cell excitability in the central and peripheral somatic nervous system. Whole-cell experiments revealed a riluzole-activated current (IRIL), transported by potassium, in mouse superior cervical ganglion (mSCG) neurons. The activation of this current by riluzole, linoleic acid, membrane stretch, and internal acidification, its open rectification and insensitivity to most classic potassium channel blockers, indicated that IRIL flows through channels of the TREK [two-pore domain weak inwardly rectifying K channel (TWIK)-related K channel] subfamily. Whole-ganglia and single-cell reverse transcription-PCR demonstrated the presence of TREK-1, TREK-2, and TRAAK (TWIK-related arachidonic acid-activated K+ channel) mRNA, and the expression of these three proteins was confirmed by immunocytochemistry in mSCG neurons. IRIL was enhanced by zinc, inhibited by barium and fluoxetine, but unaffected by quinine and ruthenium red, strongly suggesting that it was carried through TREK-1/2 channels. Consistently, a channel with properties identical with the heterologously expressed TREK-2 was recorded in most (75%) cell-attached patches. These results provide the first evidence for the expression of K2P channels in the mammalian autonomic nervous system, and they extend the impact of these channels to the entire nervous system.Publicado

Activation of TREK currents by riluzole in three subgroups of cultured mouse nodose ganglion neurons

Two-pore domain potassium channels (K2P) constitute major candidates for the regulation of background potassium currents in mammalian cells. Channels of the TREK subfamily are also well positioned to play an important role in sensory transduction due to their sensitivity to a large number of physiological and physical stimuli (pH, mechanical, temperature). Following our previous report describing the molecular expression of different K2P channels in the vagal sensory system, here we confirm that TREK channels are functionally expressed in neurons from the mouse nodose ganglion (mNG). Neurons were subdivided into three groups (A, Ah and C) based on their response to tetrodotoxin and capsaicin. Application of the TREK subfamily activator riluzole to isolated mNG neurons evoked a concentration-dependent outward current in the majority of cells from all the three subtypes studied. Riluzole increased membrane conductance and hyperpolarized the membrane potential by approximately 10 mV when applied to resting neurons. The resting potential was similar in all three groups, but C cells were clearly less excitable and showed smaller hyperpolarization-activated currents at -100 mV and smaller sustained currents at -30 mV. Our results indicate that the TREK subfamily of K2P channels might play an important role in the maintenance of the resting membrane potential in sensory neurons of the autonomic nervous system, suggesting its participation in the modulation of vagal reflexes

Caracterización de los canales de la subfamilia TREK (K2P) en neuronas del ganglio cervical superior en cultivo

En neuronas simpáticas del ganglio cervical superior (GCS) el potencial de reposo está controlado por un mecanismo complejo en el que intervienen varias corrientes voltaje dependientes, la bomba de Na+/K+ y una corriente de fuga principalmente de K+ cuyo sustrato molecular era desconocido. El descubrimiento en 1996 de los canales K2P y su participación en el mantenimiento del potencial de reposo en varios tipos celulares, los reveló como los principales candidatos para transportar la corriente de fuga. Esta familia de canales está compuesta de seis subfamilias y 15 subunidades que se encuentran ampliamente distribuidas en todo el sistema nervioso somático central y periférico; sin embargo, hasta la realización de este trabajo su expresión en el sistema nervioso autónomo era desconocida. Los resultados obtenidos muestran que las neuronas del GCS expresan mRNA de los tres miembros de la subfamilia TREK (TREK-1, TREK-2 y TRAAK) con un predominio de TREK-2 cuantificado mediante RT-qPCR, el marcaje con anticuerpos específicos muestra también la presencia de los tres miembros de la subfamilia. La aplicación de riluzol, activador de canales de la subfamilia TREK, en experimentos de patch perforado en neuronas en cultivo activa una corriente transitoria resistente a bloqueantes clásicos de potasio, que se incrementa con Zn2+ y se bloquea por fluoxetina. El perfil farmacológico y la cuantificación de los resultados de canal individual indican que la mayor parte de la corriente activada por riluzol fluye a través de TREK-2. A nivel funcional se observa que los canales TREK-2 contribuyen al mantenimiento del potencial de reposo e influyen en el patrón de disparo evocado por estímulos eléctricos, son por lo tanto un regulador importante de la excitabilidad de las neuronas del GCS

Activation of TREK currents by riluzole in three subgroups of cultured mouse nodose ganglion neurons

Two-pore domain potassium channels (K2P) constitute major candidates for the regulation of background potassium currents in mammalian cells. Channels of the TREK subfamily are also well positioned to play an important role in sensory transduction due to their sensitivity to a large number of physiological and physical stimuli (pH, mechanical, temperature). Following our previous report describing the molecular expression of different K2P channels in the vagal sensory system, here we confirm that TREK channels are functionally expressed in neurons from the mouse nodose ganglion (mNG). Neurons were subdivided into three groups (A, Ah and C) based on their response to tetrodotoxin and capsaicin. Application of the TREK subfamily activator riluzole to isolated mNG neurons evoked a concentration-dependent outward current in the majority of cells from all the three subtypes studied. Riluzole increased membrane conductance and hyperpolarized the membrane potential by approximately 10 mV when applied to resting neurons. The resting potential was similar in all three groups, but C cells were clearly less excitable and showed smaller hyperpolarization-activated currents at -100 mV and smaller sustained currents at -30 mV. Our results indicate that the TREK subfamily of K2P channels might play an important role in the maintenance of the resting membrane potential in sensory neurons of the autonomic nervous system, suggesting its participation in the modulation of vagal reflexes.Ministerio de Economía y Competitividad | Ref. BFU2014-58999-PXunta de Galicia | Ref. GPC2015/02

Expresión de canales K2P en neuronas ganglionares del sistema nervioso periférico

El potencial de reposo de las neuronas del sistema nervioso central y periférico está determinado, al menos en parte, por la presencia de canales de potasio de doble dominio de poro (K2P), que permanecen abiertos a todos los voltajes. En los últimos años se ha observado que las neuronas pueden presentar varios miembros de la familia K2P, lo que les permitiría modular su potencial de reposo de acuerdo a diferentes estímulos. En el presente trabajo nos proponemos estudiar la expresión de canales K2P en neuronas del ganglio cervical superior (SCG) y ganglio nodoso (NG) de ratón. Se utilizaron RT-PCR y qRT-PCR para determinar y cuantificar la presencia del mRNA en ganglio entero, y técnicas inmunocitoquímicas para detectar la presencia de las proteínas en cultivos primarios de neuronas de SCG y NG. Se observó una fuerte expresión del mRNA de las subfamilias TRESK y TREK tanto en SCG como en NG. Las neuronas del SCG expresaron principalmente TRESK y TREK-2, mientras que TASK-1 y TASK-2 solo tuvieron una expresión moderada; finalmente, TREK-1 y TRAAK presentaban unos niveles de expresión más reducidos. Se estudió también la expresión del mRNA de TWIK-1, TWIK-2, THIK-1, THIK-2 Y TALK-1. En el NG los niveles de mRNA más altos correspondieron a TRESK y TREK-1, TASK-1 y TASK-2 presentaron unos niveles moderados, y los más reducidos correspondieron a TREK-2 y TRAAK. Tanto en el SCG como en el NG se detectó la presencia de las proteínas mediante marcaje con anticuerpos en cultivos primarios. Nuestros resultados demuestran la presencia de múltiples subunidades de canales K2P en neuronas del SCG y del NG

Effect of riluzole based on sensitivity to capsaicin.

<p><b>(A)</b> Neuron unresponsive to capsaicin: <b>(A</b>_<b>1</b><b>)</b> Response to 1 μM capsaicin (V_m = -60 mV). <b>(A</b>_<b>2</b><b>)</b> Response of the same cell to 300 μM riluzole in the presence of the cocktail (V_m = -30 mV). <b>(B)</b> Neuron responsive to capsaicin: <b>(B</b>_<b>1</b><b>)</b> Response to 1 μM capsaicin (V_m = -60 mV). <b>(B</b>_<b>2</b><b>)</b> Response of the same cell to 300 μM riluzole in the presence of the cocktail (V_m = -30 mV). <b>(C)</b> Resting membrane potential levels are not dependent on capsaicin sensitivity (left). The steady-state outward current with the membrane clamped at -30 mV (<i>I</i>_-30) is significantly higher in capsaicin-insensitive neurons (***<i>P</i><0.001, right). <b>(D)</b> The number of action potentials in response to increasing one second current injections is smaller in capsaicin-insensitive neurons (*<i>P</i><0.05).</p

Riluzole-activated outward current is mediated by TREK channels.

<p><b>(A)</b> Short (50 ms) voltage steps (to -45 mV) were applied at high frequency (50 Hz) to detect changes of membrane conductance in the presence of 30 μM ML67-33 and the cocktail solution (holding potential -30 mV). Note the clear inhibition of the <i>I</i>_ML by fluoxetine. <b>(B)</b> Short (50 ms) voltage steps (to -45 mV) were applied at high frequency (50 Hz) to detect changes of membrane conductance in the presence of 3 μM BL-1249 and cocktail B (holding potential -30 mV). Note the clear inhibition of the <i>I</i>_BL by fluoxetine. <b>(C)</b> Short (50 ms) voltage steps (to -45 mV) were applied at high frequency (50 Hz) to detect changes of membrane conductance in the presence of 300 μM Riluzole and cocktail B (holding potential -30 mV). Note the clear inhibition of the <i>I</i>_Ril by fluoxetine. <b>(D)</b> After 1.5 hour of incubation in 1 μM spadin, short (50 ms) voltage steps (to -45 mV) were applied at high frequency (50 Hz) to detect changes of membrane conductance in the presence of 300 μM riluzole and cocktail B (holding potential -30 mV). Note that the decrease of <i>I</i>_Ril in comparison to control conditions (C). <b>(E)</b> Summary bars describing the inhibitory effect that 1.5-hour incubation of the TREK-1 inhibitor spadin exert on the current-activated by riluzole.</p

Riluzole (100 μM) is a potent inhibitor of human TRESK channels.

<p><b>(A)</b> Bath application of riluzole (100 μM, black bar) rapidly and reversible inhibited TRESK current measured at -40 mV. <b>(B)</b> Representative traces of TRESK currents in a single cell evoked using the “step ramp” protocol described in the methods in the absence (black) and presence (red) of riluzole (100 μM). <b>(C)</b> Representative current-voltage relationships for TRESK channels in a single cell in the absence (black) and presence (red) of riluzole (100 μM). <b>(D)</b> Inhibition of TRESK channel current at -40 mV by riluzole (100 μM) in 8 individual cells from 3 different recording days (gray dots). Error bars represent the mean ± SEM.</p