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.

The neuronal serum- and glucocorticoid-regulated kinase 1.1 reduces neuronal excitability and protects against seizures through upregulation of the M-current.

The M-current formed by tetramerization of Kv7.2 and Kv7.3 subunits is a neuronal voltage-gated K(+) conductance that controls resting membrane potential and cell excitability. In Xenopus laevis oocytes, an increase in Kv7.2/3 function by the serum- and glucocorticoid-regulated kinase 1 (SGK1) has been reported previously (Schuetz et al., 2008). We now show that the neuronal isoform of this kinase (SGK1.1), with distinct subcellular localization and modulation, upregulates the Kv7.2/3 current in Xenopus oocytes and mammalian human embryonic kidney HEK293 cells. In contrast to the ubiquitously expressed SGK1, the neuronal isoform SGK1.1 interacts with phosphoinositide-phosphatidylinositol 4,5-bisphosphate (PIP(2)) and is distinctly localized to the plasma membrane (Arteaga et al., 2008). An SGK1.1 mutant with disrupted PIP(2) binding sites produced no effect on Kv7.2/3 current amplitude. SGK1.1 failed to modify the voltage dependence of activation and did not change activation or deactivation kinetics of Kv7.2/3 channels. These results suggest that the kinase increases channel membrane abundance, which was confirmed with flow cytometry assays. To evaluate the effect of the kinase in neuronal excitability, we generated a transgenic mouse (Tg.sgk) expressing a constitutively active form of SGK1.1 (S515D). Superior cervical ganglion (SCG) neurons isolated from Tg.sgk mice showed a significant increase in M-current levels, paralleled by reduced excitability and more negative resting potentials. SGK1.1 effect on M-current in Tg.sgk-SCG neurons was counteracted by muscarinic receptor activation. Transgenic mice with increased SGK1.1 activity also showed diminished sensitivity to kainic acid-induced seizures. Altogether, our results unveil a novel role of SGK1.1 as a physiological regulator of the M-current and neuronal excitability.

Expression of K2P channels in sensory and motor neurons of the autonomic nervous system.

Several types of neurons within the central and peripheral somatic nervous system express two-pore-domain potassium (K2P) channels, providing them with resting potassium conductances. We demonstrate that these channels are also expressed in the autonomic nervous system where they might be important modulators of neuronal excitability. We observed strong mRNA expression of members of the TRESK and TREK subfamilies in both the mouse superior cervical ganglion (mSCG) and the mouse nodose ganglion (mNG). Motor mSCG neurons strongly expressed mRNA transcripts for TRESK and TREK-2 subunits, whereas TASK-1 and TASK-2 subunits were only moderately expressed, with only few or very few transcripts for TREK-1 and TRAAK (TRESK ≈ TREK-2 > TASK-2 ≈ TASK-1 > TREK-1 > TRAAK). Similarly, the TRESK and TREK-1 subunits were the most strongly expressed in sensorial mNG neurons, while TASK-1 and TASK-2 mRNAs were moderately expressed, and fewer TREK-2 and TRAAK transcripts were detected (TRESK ≈ TREK-1 > TASK-1 ≈ TASK-2 > TREK-2 > TRAAK). Moreover, cell-attached single-channel recordings showed a major contribution of TRESK and TREK-1 channels in mNG. As the level of TRESK mRNA expression was not statistically different between the ganglia analysed, the distinct expression of TREK-1 and TREK-2 subunits was the main difference observed between these structures. Our results strongly suggest that TRESK and TREK channels are important modulators of the sensorial and motor information flowing through the autonomic nervous system, probably exerting a strong influence on vagal reflexes.

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 (I(RIL)), 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 I(RIL) 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. I(RIL) 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.