After the initial studies based on the introduction of point mutations in the ion channel to demonstrate point direct interaction between the ion channel and the PUFA (Xiao et al., 2001a,b), another solid evidence showing how PUFAs differently modify ion channels raised from the observation that the potency of the n-3 PUFA docosahexaenoic acid (DHA) on Shaker voltage dependence changes with local pH (Borjesson et al., 2008). At physiological pH, DHA increased Shaker K⁺ current by shifting the midpoint of activation [G(V) curve] toward hyperpolarized potentials. It is known that different ion channels have different local pH values depending on their local set of surface charges structure (Elinder et al., 1996). To explore the influence of the surface charges and local pH on Shaker modulation by DHA, Borjesson and colleagues used a mutated Shaker channel where residues A419, F425, and V451 were made positive (Borjesson et al., 2008). The presence of these three positive residues in the extracellular loops connecting transmembrane segments S5 and S6 of Shaker K⁺ channels resulted in a local pH change of 0.3. Under these conditions, DHA induced a negative shift of the midpoint of activation in the triple mutant about twice as large as the shift induced in the WT Shaker K⁺ channel. These results clearly indicate that the PUFA-induced effect is channel specific and depends on the channel-specific set of surface charges (Borjesson et al., 2008).

The ultimate proof for the specific PUFAs effects on different ion channels came with the identification of the PUFAs binding site on Shaker channels. Because of its lipophilic character and modulatory effects it was proposed that the PUFAs binding site on Shaker K⁺ channels should be in the vicinity of the gating charges in the voltage sensor (S4 segment) and near residues facing the lipid bilayer (Borjesson et al., 2008). The confirmation for the proposed binding site came quickly. A cysteine scan analysis covering the lipophilic surfaces of the extracellular halves of S3–S6 segment showed that residues I325C and T329C located in the carboxyl end of helix S3, and I360C at S4 were insensitive to DHA. On the contrary, the L366C mutation increased DHA sensitivity of Shaker K⁺ channels (Borjesson and Elinder, 2011). To further define the PUFAs binding site, positives charges were introduced to each of the above-mentioned residues in the S3–S4 regions by using MTSEA⁺ reagent. Consistently with the cysteine scan data, a positive charge at residues I325; T329 and A359; I360 of the S3 and S4 respectively resulted in an increased sensitivity to DHA effects (Borjesson and Elinder, 2011). In addition to the experimental data, a structural model built to predict 3D interactions suggested that a negative charge at R1 (R362) would reduce the PUFA effect. Consistently with the model, when the charge of R362C mutant was modified negatively with MTES⁻ reagent the G(V) shift induced by DHA was smaller than that induced in WT Shaker K⁺ channels. In contrast, R362C⁺ (exposed to MTSET⁺) restored PUFA sensitivity. Some K_V channels such as K_V1.2 have an additional gating charge R0 at the top of the S4 segment. An homology model of the Shaker K⁺ channel based on the K_V1.2/2.1 chimera predicts that a positive residue at that position (A359) could strengthen the interaction between the PUFA head group and the ion channel (Borjesson and Elinder, 2011). When attaching MTSET⁺ to A359C, the DHA-induced G(V) shift was greater. Experiments in which the charge of R0 and R1 was changed support the proposed localization of the PUFA binding site and suggested that different PUFAs should have very different effects in modulating different ion channels depending on the presence of a charge at positions R0 and/or R1 (Borjesson and Elinder, 2011). Together, the data showed that high impact residues for DHA on Shaker K⁺ channels are clustered in a small region of the lipid facing S3–S4 corner of the voltage sensor domain but distant from the pore region (Figure 2). Positive charges located close to the PUFAs binding site increase PUFA potency.

As shown for Shaker K⁺ channels, it has been reported that PUFAs target the voltage sensor domain of many other K_V channels such as K_V7.1 channels (Liin et al., 2015). In K_V7.1 channels, PUFAs interact with extracellular positively charged residues of the S4 segment to promote channel opening. Neutralization of the most external gating charge (R1) of the S4 of K_V7.1 channels, R228Q, made K_V7.1 channels insensitive to DHA, to DHA-glycine (a permanently deprotonated DHA analog at physiological pH due to its lower pKa value) and to N-arachidonoyl taurine (with a permanent negative charge at pH = 7.4). Besides the S4 gating charge residues, other amino acids located in the outer halves of S3 and S4 had a big impact in the PUFA modulatory effect. Again a cysteine scan analysis showed that residues K218C and G219C, located in the S3–S4 extracellular linker of K_V7.1 reduced the PUFA effect by a factor of 3, suggesting the existence of PUFA binding site equivalent in other K_V channels (Liin et al., 2015).

Although in general different potassium channels show high similarity regarding structural domains and gating mechanisms, some important differences exist between different K_V families. One proof of that is the different location of the PUFA binding site on different K_V families as demonstrated for hK_Ca1.1 channels (or large conductance Ca²⁺- activated K⁺ channels).

In a study by Hoshi and colleagues it was shown that DHA increased hK_Ca1.1 channels by 2.5-fold and induced a shift of the G(V) curve into the negative direction (Hoshi et al., 2013c). Regardless the potentiating effects of DHA on hK_Ca1.1 channels K_Ca1.1 channels from Drosophila were insensitive to DHA. In order to determine the molecular determinants within hK_Ca1.1 channels critical for the stimulatory effect, chimeric constructions of Human/Drosophila hK_Ca1.1 channels were done. The chimeric constructions revealed that the PUFA binding site is located in the pore domain (S5, P-loop and S6 segments) of hK_Ca1.1 channels. Furthermore, the introduction of Human-to-Drosophila point mutations in the pore domain of hK_Ca1.1 highlighted the residue Y318S (located in the ion pore) as a crucial determinant of the differential sensitivity of hK_Ca1.1 channels compared to dK_Ca1.1 to PUFAs (Hoshi et al., 2013c).

Besides the identification of the PUFA binding site in K_Ca1.1 channels, additional evidence in favor of a selective action of PUFAs in hK_Ca1.1 channels raises from the different potency shown by the fatty acids in hK_Ca1.1 channels co-expressed with different modulatory subunits. In different tissues, hK_Ca1.1 channels are co-assembled with β1, β2, β4, or Υ subunits (Wu and Marx, 2010). Another study performed by Hoshi and colleagues, showed a 2.5-fold current density increase induced by DHA on hK_Ca1.1 channels (expressed in the absence of modulatory subunits), while it increased hK_Ca1.1+β1 and hK_Ca1.1+β4 by up to 20-fold and shifted the G(V) by −60 mV. On the contrary, DHA was much less effective in hK_Ca1.1+β2 and hK_Ca1.1+Υ1 channels (Figure 3). These differential effects are very noteworthy because all β subunits share a similar amino acid sequence and structural organization. Chimeric experiments determined that the N-terminus and the first transmembrane domain of β1 and β4 subunits are crucial for DHA sensitivity. Finally, point mutations experiments in which amino acids in β1 or β4 were substituted with that in β2 at the corresponding location revealed that R11/E12 and C18/R19 are crucial for DHA sensitivity in β1/β4 respectively. In addition, β2–β1 or β4 substitutions at the equivalent positions were made which showed indistinguishable sensitivity to DHA compared to β1 and β4 subunits (Hoshi et al., 2013a). All these results may suggest that PUFAs binding site resides in both the α and in the β1 and β4 subunits of the hK_Ca1.1 ion channel complex.

PUFAs such as DHA and arachidonic acid (AA) are negatively charged at physiological pH 7.4. In order to test the effect of the charge of the carboxyl group, Borjesson et al. (2008) compared the effects of DHA (negatively charged), DHA methyl ester (non-charged transesterified DHA), and a charged lipolytic toxin GsMTx4⁺ (structurally similar to PUFAs but with four positive charges) on Shaker channels. DHA shifted the G(V) curve by 10 mV toward the negative potentials. DHA methyl ester had no effect on the voltage dependence. Conversely, the GsMTx4⁺ toxin shifted the G(V) curve 5.7 mV into the positive direction. In the same line of evidence it was shown that the potency of DHA changes with pH. A higher pH potentiates DHA effects because a larger fraction of the PUFA is deprotonated (Borjesson et al., 2008). In another study, the pH-dependence of the modulation of Shaker by arachidonoyl amine (AA⁺) was analyzed (Borjesson et al., 2010). AA⁺ shifted the G(V) curve of Shaker K⁺ channels toward positive potentials and decreased the maximum conductance. AA methyl ester had no effect. Following a similar reasoning, if there is a pH-dependence for the amine effect AA⁺ should be more effective at low pH. As expected, AA⁺ was more potent at a lower pH when a larger fraction of AA⁺ is protonated (Borjesson et al., 2010).

The importance of a negative charge on the head group of DHA is applicable to many other PUFAs but also to many other potassium channels. In a recent study by Liin et al. (2015), it was shown that DHA (negatively charged) shifted the G(V) by ~ −9 mV of K_V7.1 channels. On the contrary, DHA⁺ and AA⁺ shifted the G(V) by ~ +9 mV. Together, these results demonstrate that the modulation of the voltage dependence of activation of K⁺ channels toward hyperpolarized or depolarized potentials is determined by the valence of the charge in the carboxyl head group. A negative charge shifts the G(V) curve toward more negative potentials and vice versa.

Borjesson and colleagues tested ten fatty acids or fatty acid-like compounds with different acyl chain lengths and different numbers, position, and geometry of double bonds to identify structural features on the acyl tail important for the PUFA modulation of Shaker K⁺ channels. Fatty acids with the most structural similarity to DHA by having two or more methylene-interrupted double bonds at either the n-3 or the n-6 position showed similar shifts of the G(V) curves in Shaker channels (Borjesson et al., 2008). The rest of the compounds were not effective.

Recently, Yazdi et al. (2016) studied how the saturation levels in the carbon tail affect the structure of the lipids in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane patches. From their molecular dynamics simulations the following conclusions were extracted: (i) the order parameters of PUFA and saturated fatty acids decreased from the carboxyl end located in the bilayer interface to the end of the acyl chain tail in the bilayer center. (ii) The order parameters displayed a different overall shape reflecting the positions of the cis double-bonds of PUFAs. (iii) There are differences in the shape and packing properties by measuring the radius of gyration of the carbon tail and head-to-tail length. Compared to other fatty acids, the PUFA molecule tends to twirl and curl up. All together, these results indicate that the PUFA molecule has greater conformational mobility compared to saturated fatty acids. The predominant saturated fatty acid conformation is more extended resulting in a decreased flexibility and mobility of phospholipids of the cell membrane (Yazdi et al., 2016).

Contrary to the importance of a negatively charged carboxyl group for the PUFA effect and the presence of two or more double bonds, the impact of the length of the acyl tail for the PUFAs effects is not so obvious. In Shaker K⁺ channels the presence of two or more double bonds and its location in cis geometry (more flexible) seem to be more important than the number of carbons of the tail for the PUFA induced G(V) shift (Borjesson et al., 2008). Similar findings were shown by Liin et al. in K_V7.1 channels (Liin et al., 2015). Two fatty acids: EPA and oleic acid (OA), poly- and mono-unsaturated fatty acids respectively with similar acyl tail length showed very different effects on K_V7.1 currents. OA had no effect on K_V7.1 currents while EPA showed a similar potency increasing K_V7.1 compared to DHA (Liin et al., 2015). On the other hand, a study by Hoshi et al. (2013c) in which the effects of three n-3 PUFAs: DHA, EPA and α-linolenic acid (ALA) were examined in hK_Ca1.1 channels revealed that DHA had the largest stimulatory effect on hK_Ca1.1 currents followed by EPA and finally ALA. These results suggest the importance of the length of the acyl might be channel specific and that the longer the acyl tail, the larger the effect of the PUFA will be.

Based on the following effects of DHA on Shaker K⁺ channels: (i) shift of the G(V) and of the steady state inactivation curve to the negative direction, and (ii) acceleration of the activation kinetics and slower deactivation kinetics; Borjesson and colleagues formulated in 2008 the “Lipoelectric hypothesis” to explain the PUFAs-dependent modulation of K⁺ channels mechanism. All these findings are in agreement with DHA and other PUFAs causing electrostatic effect on the voltage sensor of ion channels (Borjesson et al., 2008). The lipoelectric hypothesis proposes that the lipophilic PUFAs bind to a hydrophobic environment (the lipid bilayer or bilayer/channel interface close to the S3-S4 segment) from where they act electrostatically on the voltage sensor changing the voltage dependence of the ion channel (Borjesson et al., 2008).

It is known that the activation of Shaker K⁺ channels occur in at least two steps. In the early transitions the four Shaker subunits go through transitions between the closed states (C0 to C2) and, then the channel goes through other two transitions to the final closed and the open states (C2 to O). The transitions between the first closed states are sequential and represent the activation of the voltage sensor, whereas those between C2 to O are concerted and couple the movement of the main voltage sensors to channel opening (Schoppa et al., 1992; Bezanilla et al., 1994; Zagotta et al., 1994; Schoppa and Sigworth, 1998).     α0    α1       α1     ​ α1       α2      α3      α4C0↔C1↔C11↔C12↔C2↔C3↔C4↔O  β0   β1  β1  β1    β2    β3   β4 To explore which activation step is affected by DHA, gating currents were measured in two mutated Shaker K⁺ channel constructions. The final transitions (from close C2 to open O) were studied in the ILT mutant Shaker channel where three hydrophobic residues in S4 are substituted for three other hydrophobic residues (V369I, I372L, and S376T). In the ILT mutant the voltage sensor activation and pore domain opening steps are energetically separated allowing the study of the late transitions during the channel activation in isolation The initial steps of activation (C0 to C2) were studied in the non-conducting channel resulting from the ILT mutant plus the pore mutation W343F. DHA significantly shifted the G(V) curve in the negative direction along the voltage axis. On the contrary, DHA had a much smaller effect on the gating currents Q(V) of the ILT/W343F Shaker mutant. The larger effect of DHA seen on the G(V) compared to the Q(V) curve suggests that the opening step (the second gating component) is more affected than the early transition by the PUFA (Borjesson et al., 2008). Despite DHA had a much smaller effect on the gating currents, this small effect indicates that the activation of independent voltage sensors is also affected. A gating model of Shaker K⁺ channels predicted a DHA-dependent shift of 30 and 5 mV in the G(V) and Q(V) respectively corroborating the experimental data (Borjesson and Elinder, 2011).

PUFAs interaction sites in the open and closed state were determined by steered molecular dynamics simulations of Shaker K⁺ channels in POPC bilayers (Yazdi et al., 2016). PUFAs form clusters around the voltage sensor domain regions (VSD) rather than the pore helices S5–S6. In general, significantly higher contact frequencies were identified between the PUFA and the protein in the open state. PUFA acyl tail-ion channel protein contacts are predominantly non-specific: hydrophobic and distributed across the VSD. PUFA heads-protein interactions are more frequent in the open vs. closed state and are more specific: hydrogen bonds between charged or polar residues of the extracellular halves of S3 and S4 and PUFA head groups. Saturated fatty acids established significantly fewer interactions in closed vs. open state simulations and fewer contacts in the open state compared to PUFAs. In summary, specific protein residues involved in PUFAs vs. saturated fatty acids interactions are largely different. While PUFAs establish predominantly contacts with S3–S4 residues, there is a shift to the S1–S2 in saturated fatty acids (Yazdi et al., 2016).

According to the lipoelectric hypothesis, the PUFA molecule act electrostatically on positive residues of the voltage sensor domain among which arginine 1 (R1) seems to have a crucial role. Shifting the R1 from position 362–361 eliminated the enhancing DHA effect. Moving the charge 2 steps further potentiated the PUFA effect (Ottosson et al., 2014). In the same line of evidence it was found that the introduction of a negative charge (substitution to glutamic acid) in certain positions of the S4 such as A359E, had an opposite effects on the G(V) curve (smaller hyperpolarizing shift compared to WT Shaker K⁺ channels) to the potentiated effect of arginine (Ottosson et al., 2014). Even more interestingly, the application of AA⁺ that was previously shown to decrease WT Shaker K⁺ current now it up-regulated A359E channels and shifted the G(V) curve −8 mV strongly indicating an electrostatic interaction. AA⁺ did not up-regulated A361E showing again amino acid side facing dependence. A positive charge at 359 or a negative at 361 promotes channel opening and vice versa (Ottosson et al., 2014).

According to the structural prediction by Borjesson and colleagues, Shaker K⁺ channels, with all S4s in an activated but non-conducting position, R1 and R2 are exposed to the extracellular solution with its charges at a distance of 16 Å away from the approximate PUFA position in the lipid bilayer adjacent to S3 and S4 (Borjesson and Elinder, 2011). Assuming that the final position of S4 is according to the K_V1.2/2.1 chimera crystal structure (Long et al., 2007), the charge of R1 moves to a position only 6 Å away from the PUFA when the channel is open. A modified Coulomb law was used to estimate the distance between the PUFA carboxyl charge and the S4 mediated change in surface as a function of distance from a charge. The predicted 5 mV effect on the first four independent transition steps suggests that the PUFA charge is located 15.2 Å away from the position where the positive charges emerge on the channel protein's surface. This calculation was based on the assumption that the channel surface is smooth and that the top S4 charges turn up on the surface of the channel leaving the inner part of the ion pore with positive gating charges pairing with negative counter charges. This is very close to the structural prediction of 16 Å. The 30 mV effect on the opening step suggests that the PUFA charge is located 6.3 Å away from R1 in the open state, which again is almost identical to their prediction from the structural model.

Collectively the data strongly suggest that the late transitions (from C2 to O) are the more sensitive steps to DHA. In the last closed state (C4), R1 and R2 are exposed to the extracellular solution 16 Å away from the PUFA head group. In the opening step, R1 moves along the longitudinal axis of S4 and rotate toward the lipid bilayer, in the vicinity of the negatively charged bound PUFA (6 Å; Figure 2). DHA electrostatically affects this rotation promoting Shaker K⁺ channel activation (Borjesson and Elinder, 2011; Ottosson et al., 2014).

Contrary to the DHA effects on Shaker K⁺ channels, hK_Ca1.1 channels do not require voltage sensor activation for the PUFA effect. DHA augmented single channel open probability at hyperpolarized potentials where the voltage sensors are predominantly at rest (Hoshi et al., 2013b). In hK_Ca1.1 channels, opening probability at hyperpolarizing potentials is mainly driven by the opening and closing transitions of the activation gate located near or within the selectivity filter (Seibold et al., 2008). The closure of the activation gate controls the deactivation kinetics and the opening of the gate controls the macroscopic activation kinetics. DHA did not affect the deactivation kinetics of hK_Ca1.1 channels at hyperpolarized potentials, suggesting that the PUFA accelerates the opening of the activation gate and increases open probability (Hoshi et al., 2013b).

As with many other K⁺ channels, K_V7.1 channels and, therefore, I_Ks are modulated by PUFAs. Doolan et al. (2002) demonstrated that EPA increases the magnitude of the I_Ks in Xenopus oocytes. Furthermore, long term PUFA treatment was shown to enhance I_Ks in cardiac myocytes from pigs pigs fed on a PUFA high diet compared to I_Ks from pigs fed with a normal diet (Verkerk et al., 2006). However, the molecular mechanism underlying the PUFAs-dependent modulation of K_V7.1/KCNE1 remained to be elucidated. In 2015, two studies addressed this question (Liin et al., 2015; Moreno et al., 2015).

Moreno et al. (2015) analyzed the effects EPA and DHA in K_V7.1/KCNE1 channels expressed on a heterologous expression system and in I_Ks in guinea pig myocytes. Two aspects of PUFA-dependent modulation of ion channels merit particular attention. (i) Dietary PUFAs incorporate to the cell membrane. Therefore, PUFAs-dependent modulation of ion channel depend on the way of administration: acute vs. chronic [see Moreno et al. (2012) for extensive review]. In fact, Moreno and colleagues reported that acute but not chronic administration of PUFAs increased the magnitude of K_V7.1/KCNE1 channels and shifted the activation curve toward opposite directions (leftward and rightward shift for acute and chronic administration, respectively). In addition, long-term administration of n-3 PUFAs reduced K_V7.1 but not KCNE1 expression due to increased degradation via proteasome. The significant decrease in K_V7.1 expression level might account for the lack of current density increase when n-3 PUFAs were applied chronically (Moreno et al., 2015). (ii) Direct vs. indirect effects. As previously mentioned, there were two trends of opinion to explain the mechanism of action by which PUFAs modify ion channels: direct interaction vs. alteration of the cell membrane fluidity. Evidences in favor of both type of modulation were found simultaneously in the study by Moreno et al. (2015). Chronic administration of EPA and DHA disrupted lipid rafts domains where K_V7.1 channels are normally found (Balijepalli et al., 2007) delocalizing K_V7.1 in the cell membrane. Lipid raft disruption can be mimicked within minutes by the acute application of methyl-β-cyclodextrin (MBCD). The application of MBCD increased the current magnitude of K_V7.1/KCNE1 at potentials positive to +30 mV and shifted the activation curve toward positive potentials emphasizing the impact of the membrane environment on channel properties (indirect effect; Figure 4; Moreno et al., 2015). On the other hand, after lipid raft disruption with MBCD, acute application of EPA produced similar effects than those observed in non-cholesterol-depleted cells: Current density increase and shift of the midpoint in the hyperpolarizing direction. This fast modulation was explained by a direct effect on the ion channel of EPA during acute application. Together these data provided evidence for direct and indirect PUFA dependent modulation of K⁺ channels suggesting that both mechanisms are not mutually exclusive and can happen simultaneously (Moreno et al., 2015).

The study by Liin et al. (2015) examines the mechanism by which n-3 PUFAs promote K_V7.1 channel opening and how KCNE1 modulated K_V7.1 sensitivity to PUFAs. In addition, they explored the possibility to develop PUFAs analogs as drugs to prevent cardiac arrhythmias. Similarly to what was described for Shaker K⁺ channels, DHA promotes channel opening by acting electrostatically on the VSD of K_V7.1 channels. KCNE1 tunes K_V7.1 sensitivity to PUFAs by lowering the local pH in the vicinity of the PUFA head group which results in DHA protonation. In other words, the PUFA effect is KCNE1-independent as long as the PUFA is negatively charged (Liin et al., 2015). This finding raised the possibility to study the antiarrhythmic potential of n-3 PUFAs analogs as novel therapeutic agents. In this regard, Liin and colleagues tested the effects of two PUFAs analogs in two arrhythmia models: isolated embryonic rat cardiomyocytes and the intact heart of guinea pig. In isolated embryonic rat cardiomyocytes (in which I_Ks but not I_Kr rapidly activating delayed rectifier potassium current is expressed) arrhythmia was induced by applying subsaturating concentrations of chromanol 293B and the effects of N-arachidonoyl taurine were tested. Chromanol 293B increased action potential duration and led to arrhythmic firing. The permanently negatively charged N-arachidonoyl-taurine reversed the effect of chromanol 293B decreasing the action potential duration (APD) and abolishing the arrhythmic firing. The effects of docosahexaenoyl glycine were tested on the intact heart of guinea pig (DHA-Gly has a lower pKa than DHA and therefore is negatively charged at physiological pH even in the presence of KCNE1). To mimic the long QT (syndrome characterized by a long interval between the Q and the T waves of the ECG) setting in this model (APD duration and QT prolongation), the E4031 I_Kr blocker was applied. Perfusion of the guinea pig heart with 10 μM DHA-Gly reversed the effects of E4031 restoring QT interval duration (Liin et al., 2015) demonstrating their antiarrhythmic potential.

Concerning the PUFAs antiarrhythmic mechanism, minor discrepancies can be found through the literature. Overall, in vitro and in vivo experiments and clinical trials support an antiarrhythmic effect of PUFAs. Liin and colleagues claim that the antiarrhythmic effects of DHA cannot be due to effects on I_Ks gating because at physiological pH 7.4, KCNE promotes DHA protonation making DHA ineffective (Liin et al., 2015). Instead they propose that the antiarrhythmic effect of PUFAs in the heart is due to the inhibition of I_Na or I_CaL (Leaf et al., 2003, 2005; Danthi et al., 2005; Boland and Drzewiecki, 2008). However, we found that both EPA and DHA increase I_Ks in guinea pig cardiomyocytes and shifted the G(V) curve toward the hyperpolarizing direction of I_Ks expressed in COS7 cells. Furthermore, action potential simulations showed that the increase of I_Ks induced by acute administration of PUFAs could play a relevant role in action potential shortening when other repolarizing current for instance I_Kr, are compromised. On the other hand, chronic exposure to PUFAs does not affect APD so that the protective role seems lost in this condition.

It has been reported that PUFAs increase both seizure and pain thresholds (Voskuyl et al., 1998; Xiao and Li, 1999; Taha et al., 2010; Bandero et al., 2013) in rats and mice, and reduce neuronal excitability in vitro (Xiao and Li, 1999; Young et al., 2000). As it was shown for the heart, the modulation of neuronal excitability by PUFAs is mediated by effects on ion channels crucial for neuronal excitability (Boland and Drzewiecki, 2008). PUFAs have been suggested to increase the M-current but their effects are complex and not completely understood. In a recent paper, it was shown again that only negatively charged n-3 PUFAs such as DHA, EPA, ALA, can quickly shift the G(V) curve of K_V7.2/K_V7.3 channels expressed on Xenopus oocytes toward more negative voltages facilitating channel opening (Liin et al., 2016; Valenzuela, 2016). More importantly, the anti-excitable effects of PUFAs on the neuronal M-current were studied in dorsal root ganglions from mice. Addition of DHA to dorsal root ganglion neurons hyperpolarized the V_m (by −2.4 mV) increasing the threshold current required to evoke action potentials. Since these effects were reverted by the M-current blocker XE991, the reduced neuronal excitability was attributed to the PUFA mediated augmentation of M-current (Liin et al., 2016). In the same line of evidence, computer simulations predict that equivalent changes around 1–5 mV in the V_m of neurons and in the voltage-dependence of ion channel activation is sufficient to decrease neuronal excitability and prevent epileptic episodes (Tigerholm et al., 2012). In summary, all these results suggest that PUFAs can be used as a blueprint for rational drug design of new M-currents activators more selective and efficient for the treatment of neurological disorders such as epilepsy and pain.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.