PIP2 Mediated Inhibition of TREK Potassium Currents by Bradykinin in Mouse Sympathetic Neurons.

Bradykinin (BK), a hormone inducing pain and inflammation, is known to inhibit potassium M-currents (IM) and to increase the excitability of the superior cervical ganglion (SCG) neurons by activating the Ca2+-calmodulin pathway. M-current is also reduced by muscarinic agonists through the depletion of membrane phosphatidylinositol 4,5-biphosphate (PIP2). Similarly, the activation of muscarinic receptors inhibits the current through two-pore domain potassium channels (K2P) of the "Tandem of pore-domains in a Weakly Inward rectifying K+ channel (TWIK)-related channels" (TREK) subfamily by reducing PIP2 in mouse SCG neurons (mSCG). The aim of this work was to test and characterize the modulation of TREK channels by bradykinin. We used the perforated-patch technique to investigate riluzole (RIL) activated currents in voltage- and current-clamp experiments. RIL is a drug used in the palliative treatment of amyotrophic lateral sclerosis and, in addition to blocking voltage-dependent sodium channels, it also selectively activates the K2P channels of the TREK subfamily. A cell-attached patch-clamp was also used to investigate TREK-2 single channel currents. We report here that BK reduces spike frequency adaptation (SFA), inhibits the riluzole-activated current (IRIL), which flows mainly through TREK-2 channels, by about 45%, and reduces the open probability of identified single TREK-2 channels in cultured mSCG cells. The effect of BK on IRIL was precluded by the bradykinin receptor (B2R) antagonist HOE-140 (d-Arg-[Hyp3, Thi5, d-Tic7, Oic8]BK) but also by diC8PIP2 which prevents PIP2 depletion when phospholipase C (PLC) is activated. On the contrary, antagonizing inositol triphosphate receptors (IP3R) using 2-aminoethoxydiphenylborane (2-APB) or inhibiting protein kinase C (PKC) with bisindolylmaleimide did not affect the inhibition of IRIL by BK. In conclusion, bradykinin inhibits TREK-2 channels through the activation of B2Rs resulting in PIP2 depletion, much like we have demonstrated for muscarinic agonists. This mechanism implies that TREK channels must be relevant for the capture of information about pain and visceral inflammation.

Ion Channels and Thermosensitivity: TRP, TREK, or Both?

Controlling body temperature is a matter of life or death for most animals, and in mammals the complex thermoregulatory system is comprised of thermoreceptors, thermosensors, and effectors. The activity of thermoreceptors and thermoeffectors has been studied for many years, yet only recently have we begun to obtain a clear picture of the thermosensors and the molecular mechanisms involved in thermosensory reception. An important step in this direction was the discovery of the thermosensitive transient receptor potential (TRP) cationic channels, some of which are activated by increases in temperature and others by a drop in temperature, potentially converting the cells in which they are expressed into heat and cold receptors. More recently, the TWIK-related potassium (TREK) channels were seen to be strongly activated by increases in temperature. Hence, in this review we want to assess the hypothesis that both these groups of channels can collaborate, possibly along with other channels, to generate the wide range of thermal sensations that the nervous system is capable of handling.

Tandem pore TWIK-related potassium channels and neuroprotection.

TWIK-related potassium channels (TREK) belong to a subfamily of the two-pore domain potassium channels family with three members, TREK1, TREK2 and TWIK-related arachidonic acid-activated potassium channels. The two-pore domain potassium channels is the last big family of channels being discovered, therefore it is not surprising that most of the information we know about TREK channels predominantly comes from the study of heterologously expressed channels. Notwithstanding, in this review we pay special attention to the limited amount of information available on native TREK-like channels and real neurons in relation to neuroprotection. Mainly we focus on the role of free fatty acids, lysophospholipids and other neuroprotective agents like riluzole in the modulation of TREK channels, emphasizing on how important this modulation may be for the development of new therapies against neuropathic pain, depression, schizophrenia, epilepsy, ischemia and cardiac complications.

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.

Lidocaine effects on acetylcholine-elicited currents from mouse superior cervical ganglion neurons.

Lidocaine is a commonly used local anaesthetic that, besides blocking voltage-dependent Na(+) channels, has multiple inhibitory effects on muscle-type nicotinic acetylcholine (ACh) receptors (nAChRs). In the present study, we have investigated the effects of lidocaine on ACh-elicited currents (IAChs) from cultured mouse superior cervical ganglion (SCG) neurons, which mainly express heteromeric α3ß4 nAChRs. Neurons were voltage-clamped by using the perforated-patch method and IAChs were elicited by fast application of ACh (100-300µM), either alone or in presence of lidocaine at different concentrations. IAChs were reversibly blocked by lidocaine in a concentration-dependent way (IC50=41µM; nH close to 1) and the inhibition was, at least partially, voltage-dependent, indicating an open-channel blockade. Besides, lidocaine blocked resting (closed) nAChRs, as evidenced by the increased inhibition caused by a 12s lidocaine application just before its co-application with the agonist, and also enhanced IAChs desensitisation, at concentrations close to the IC50. These results indicate that lidocaine has diverse inhibitory actions on neuronal heteromeric nAChRs resembling those previously reported for Torpedo (muscle-type) nAChRs (Alberola-Die et al., 2011). The similarity of lidocaine actions on different subtypes of heteromeric nAChRs differs with the specific effects of other compounds, restricted to particular subtypes of nAChRs.

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.

A riluzole- and valproate-sensitive persistent sodium current contributes to the resting membrane potential and increases the excitability of sympathetic neurones.

Non-adapting superior cervical ganglion (SCG) neurones with a clustering activity and sub-threshold membrane potential oscillations were occasionally recorded, suggesting the presence of a persistent sodium current (I(NaP)). The perforated-patch technique was used to establish its properties and physiological role. Voltage-clamp experiments demonstrated that all SCG cells have a TTX-sensitive I(NaP) activating at about -60 mV and with half-maximal activation at about -40 mV. The mean maximum I(NaP) amplitude was around -40 pA at -20 mV. Similar results were achieved when voltage steps or voltage ramps were used to construct the current-voltage relationships, and the general I(NaP) properties were comparable in mouse and rat SCG neurons. I(NaP) was inhibited by riluzole and valproate with an IC(50) of 2.7 and 3.8 microM, respectively, while both drugs inhibited the transient sodium current (I (NaT)) with a corresponding IC(50) of 34 and 150 microM. It is worth noting that 30 microM valproate inhibited the I(NaP) by 70% without affecting the I(NaT). In current clamp, valproate (30 microM) hyperpolarised resting SCG membranes by about 2 mV and increased the injected current necessary to evoke an action potential by about 20 pA. Together, these results demonstrate for the first time that a persistent sodium current exists in the membrane of SCG sympathetic neurones which could allow them to oscillate in the sub-threshold range. This current also contributes to the resting membrane potential and increases cellular excitability, so that it is likely to play an important role in neuronal behaviour.

Intrinsic spontaneous activity and subthreshold oscillations in neurones of the rat dorsal column nuclei in culture.

The basis of rhythmic activity observed at the dorsal column nuclei (DCN) is still open to debate. This study has investigated the electrophysiological properties of isolated DCN neurones deprived of any synaptic influence, using the perforated-patch technique. About half of the DCN neurones (64/130) were spontaneously active. More than half of the spontaneous neurones (36/64) showed a low threshold membrane oscillation (LTO) with a mean frequency of 11.4 Hz (range: 4.3-22.1 Hz, n = 20; I = 0). Cells showing LTOs also invariably showed a rhythmic 1.2 Hz clustering activity (groups of 2-5 action potentials separated by silent LTO periods). Also, more than one-third of the silent neurones presented clustering activity, always accompanied by LTOs, when slightly depolarised. The frequency of LTOs was voltage dependent and could be abolished by TTX (0.5 microM) and riluzole (30 microM), suggesting the participation of a sodium current. LTOs were also abolished by TEA (15 mM), which transformed clustering into tonic activity. In voltage clamp, most DCN neurones (85%) showed a TTX-/riluzole-sensitive persistent sodium current (INa,p), which activated at about -60 mV and had a half-maximum activation at -49.8 mV. An M-like, non-inactivating outward current was present in 95% of DCN neurones, and TEA (15 mM) inhibited this current by 73.7 %. The non-inactivating outward current was also inhibited by barium (1 mM) and linopirdine (10 microM), which suggests its M-like nature; both drugs failed to block the LTOs, but induced a reduction in their frequency by 56 and 20%, respectively. These results demonstrate for the first time that DCN neurones have a complex and intrinsically driven clustering discharge pattern, accompanied by subthreshold membrane oscillations. Subthreshold oscillations rely on the interplay of a persistent sodium current and a non-inactivating TEA-sensitive outward current.

Ionic basis of the resting membrane potential in cultured rat sympathetic neurons.

The conductances which determine the resting membrane potential of rat superior cervical ganglia (SCG) neurons were investigated using perforated voltage- and current-clamp whole-cell techniques. The resting potential of SCG cells varied from -47 to -80 mV (-58.3 +/- 0.8 mV, n = 55). Blockade of M and h currents induced a depolarisation (7.4 +/- 0.7 mV, n = 22) and a hyperpolarisation (7.2 +/- 0.7 mV, n = 20) respectively; however, no correlation between the amplitude of these currents and the resting potential was found. The inhibition of the Na/K pump also induced membrane depolarisation (3.2 +/- 0.2 mV, n = 8). Inhibition of voltage-gated currents unmasked a voltage-independent resting conductance reversing at -50 mV. The reversal potential of the voltage-independent conductance, which included the electrogenic contribution of the Na/K pump, was strongly correlated with the resting potential (R = 0.87, p < 0.0001, n = 30). Ionic substitution experiments confirmed the existence of a voltage-independent conductance (leakage) with four components, a main potassium conductance, two minor sodium and chloride conductances and a small contribution of the Na/K pump. It is concluded that the resting potential of SCG cells strongly depends on the reversal potential of the voltage-independent conductance, with voltage-activated M and h currents playing a prominent stabilising role.