M‐channels and n‐3 polyunsaturated fatty acids: role in pain and epilepsy

Voltage-gated K+ channels (KV) ‘sense’ voltage differences across the cell membrane and open or close in response to changes in the membrane potential. To date, twelve different subfamilies of KV channels have been described (KV1–12) (Alexander et al. 2015). KV channels are formed by co-assembly of four α subunits, each containing six α-helical TM segments (S1–S6), with both N- and C-termini lying on the cytoplasmic side of the membrane. Each subunit comprises two membrane-integrated functionally distinct modules, one forming the voltage sensor (S1–S4) and the other the K+ selective pore (S5-P-S6). In the nervous system, KV7.1–KV7.5 channels assemble as heterotetramers to generate the ‘M-current’, a K current that is regulated by muscarinic receptor signalling (primarily via PIP2), thus regulating neuronal excitability. Among the KV7 subfamily, KV7.2 and KV7.3 channels play a crucial role in the genesis of M-current. Neuronal hyperexcitability underlies different diseases, including epilepsy and chronic pain. In these pathologies, the activation of neuronal K+ currents in excitatory neurones leads to hyperpolarization of the resting membrane potential and to a reduction in neuronal excitability, whereas channel closure has opposite effects. In fact, it is known that KV7 channels are expressed in dorsal root ganglion (DRG) neurones and in peripheral nerve endings. Moreover, benign familial neonatal convulsions, an autosomal dominant epilepsy of infancy, are caused by mutations in KCNQ2 or KCNQ3 potassium channel genes, which codify KV7.2 and KV7.3 respectively. Thus, KV7 channels represent a potential therapeutic target for both epilepsy and chronic pain. In a study published in this journal Liin et al. (2016) demonstrate that n-3 polyunsaturated fatty acids (PUFAs) increase the opening of the KV7.2/3 channel (M-channel) when expressed in Xenopus oocytes by shifting the activation curve towards negative membrane potentials. N-3 PUFAs present in nature belong to two main classes: n-6 class, mostly present in vegetable oils (arachidonic acid, AA); and n-3 class, mostly coming from fish (eicosapentaenoic acid, EPA, and docosahexaenoic acid, DHA) with the exception of α-linolenic acid (ALA). All are essential as they are necessary for an optimal health status and cannot be synthesized de novo. In fact, there are experimental evidences supporting the beneficial effects of an increased intake of n-3 PUFAs in the prevention and/or treatment of very different pathologies, from cardiovascular diseases, through antidepression and metabolic disorders (Dyall 2015). Two randomized placebo-controlled trials of n-3 PUFAs in patients with drug-resistant epilepsy have been performed and already published. The largest one was reported in 2005 (Yuen et al. 2005) and was the first randomized trial of n-3 PUFAs in epilepsy. The study included 57 patients affected with drug-resistant epilepsy. They were randomized to 1700 mg of EPA and DHA and they were followed up to 12 weeks. After the first 6 weeks, a reduction in seizures was observed; however, this effect was not maintained over the duration of the 12-week treatment period. In the second randomized-controlled trial (Bromfield et al. 2008), a higher dose of n-3 PUFAs (2200 mg) was used to treat patients suffering drug-resistant epilepsy. Following a 4-week prospective baseline and 1-week titration, subjects entered a 12-week treatment period, followed by an optional 4-week open-label phase. No benefit of n-3 PUFAs was observed for the 12-week randomized treatment period. In contrast to the double-blind study, analysis of open-label n-3 PUFAs’ administration among volunteers shows that the 78.9% of them exhibited fewer seizures than during baseline. N-3 PUFAs modulate mostly all voltage-dependent potassium channels. In fact, they inhibit KV1.5, KV KV1.1, KV4.3, KV11.1 whereas they increase KV7.1/KCNE1 (Honoré et al. 1994, Moreno et al. 2012, 2015) without producing any effect on KV7.1 channels alone after expressing these subunits in mammalian cells (Moreno et al. 2015). However, it has been also described that n-3 PUFAs increase KV7.1 current when these channels are expressed in the absence of KCNE1 in Xenopus oocytes (Liin et al. 2015). The authors explain these results by a KCNE1-induced protonation of DHA close to KV7.1 that eliminates the electrostatic effect of DHA on the KV7.1 channel. Also, it has been reported a very complex pattern of effects produced by n-3 PUFAs on ion channels, which can be due to different actions because n-3 PUFAs: (i) produce a direct effect on the channel (Liin et al. 2015); (ii) incorporate into the membrane phospholipids, thus altering the order of the cell membrane (Moreno et al. 2012); and (iii) are able to rupture lipid rafts where KV7.1/KCNE1 channels are located (Moreno et al. 2015). In an article of this journal Liin et al. (2016) demonstrate that several n-3 PUFAs increase the opening of the KV7.2/3 channel (M-channel) when expressed in Xenopus oocytes by shifting the activation curve towards negative membrane potentials. The effects of DHA were analysed at different pH values (between 6.5 and 10) to test the hypothesis that the negative charge of the carboxyl head of DHA is crucial to increase the KV7.2/3 channel opening. The authors observed that the negative shift of the activation curve increased at high pH, at which a larger fraction of DHA molecules are deprotonated and negatively charged, whereas at low pH, at which most of DHA are protonated and uncharged, the ability of DHA to shift the activation curve was reduced. Previously, this research group have reported the mechanism of action of n-3 PUFAs on KV7.1 with or without KCNE1, concluding that the binding site was located at the outermost gating charges (including positively charged arginines) of the voltage sensor S4 in KV channels at the interface between the lipid bilayer and the voltage-sensor domain (S1–S4) (Liin et al. 2015). The high similarity between the PUFA-induced modulation of KV7.1 and KV7.2/3 channels implies a general PUFA binding site and mechanism of action for PUFA-induced increase in KV7 channels. The authors have also demonstrate in this article that KCNE1 or KCNE2 do not affect the effects of n-3 PUFAs on KV7.2/3-KCNE currents. Also, in this study Liin et al. (2016) analysed the evoked action potentials of mouse dorsal root ganglion (DRG) neurones in the absence and in the presence of n-3 PUFAs. These experiments showed a hyperpolarization of the membrane resting potential that was inhibited by an M-channel blocker, XE991, indicating that this effect is due to an effect on M-channels. Moreover, it was observed that in the presence of DHA, the threshold current potential needed to evoke an action potential increased by a factor of 1.3 and 2 of 6 neurones were unable to evoke such action potentials. The authors conclude from these experiments that DHA produces a dampening effect on the excitability of these DRG neurones, likely due to an increase in the M-current. In fact Liin et al. (2016) analyse the effects of ALA, EPA and DHA on KV7.2/3 channels demonstrating that all of them increase this current. However, when the effects of DHA methyl ester (DHA-me, lacking of negative charge at physiological pH) or those produced by the monosaturated fatty acid oleic acid (OA, lacking of two of the double bonds present in ALA) were analysed, none effect on KV7.2/3 was observed. These findings are explained by the author suggesting that the possible anti-excitable effects of PUFA-me and monounsaturated fatty acids are not mediated via KV7 channels. These effects could be explained if the binding of n-3 PUFAs to KV7.2/3 channels has two structural requirements: (i) the presence of a negatively charged moiety at the carboxyl group; and, at least, two double bonds in the acyl chain (ALA vs. OA). Liin et al. (2016) provide a valuable contribution to our understanding of how n-3 PUFAs act on KV7.2/3 channels. However, future studies comparing the PUFAs’ binding sites in these channels are required for a molecular understanding of differential PUFA sensitivity in KV7 channels. Also, there are conflicting results on the impact of KCNE coexpression on PUFA sensitivity in KV7 channels, obtained in different studies (Liin et al. 2015, Moreno et al. 2015). Therefore, it will be really interesting to determine the ultimately reason of these discrepancies. A possible explanation may be due to the proper nature of the compound and the different expression system, given to the lipidic yolk of the Xenopus oocytes in contrast to the mammalian cell membrane. In fact, it is known that the yolk in oocytes can absorb lipophilic molecules (such as PUFAs), thus reducing the cytoplasmic concentration. Thus, when studying the effects of lipophilic compounds, the apparent potency decreases by approximately 10 times when using Xenopus oocytes, as an expression system than when using patch-clamp recordings. Also, it is known that n-3 PUFAs: (i) incorporate into the phospholipids of the cell membrane, altering in this way the membrane fluidity, as well as the composition of the lipid rafts, and thus modifying the potassium current (Moreno et al. 2015), and (ii) modify the expression level of different potassium channels (Guizy et al. 2008, Moreno et al. 2012). Therefore, studying all these issues on the basis of the understanding of the mechanism of action of n-3 PUFAs on KV7 channels should be an interesting issue. Finally, it is known that PUFAs can also (i) modify protein kinase C and A activity, (ii) activate Akt pathway in compromised neurones and (iii) inhibit the activation of the inflammatory transcription factor NF-κB within macrophages, among some actions. Thus, all these effects can also be involved in their effects on KV7 channels (Fig. 1). It is exciting that, even though the KV7 channels field has come a long way, so many interesting questions remain. None. This work was supported by SAF2013-45800-R, CSIC 201420E107 and FIS-RIC RD12/0042/0019, funded by the Instituto de Salud Carlos III. The cost of this publication was paid in part by funds from the European Fund for Economic and Regional Development.