BACKGROUND AND PURPOSEFlupirtine is a non-opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). Nevertheless, its mechanisms of action remain controversial and are the purpose of this study. EXPERIMENTAL APPROACHEffects of flupirtine on native and recombinant voltage-and ligand-gated ion channels were explored in patch-clamp experiments using the following experimental systems: recombinant KIR3 and KV7 channels and a3b4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage-gated Na, and TRPV1 channels, as well as GABAA, glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons. KEY RESULTSTherapeutic flupirtine concentrations (Յ10 mM) did not affect voltage-gated Na + or Ca 2+ channels, inward rectifier K + channels, nicotinic acetylcholine receptors, glycine or ionotropic glutamate receptors. Flupirtine shifted the gating of KV7 K + channels to more negative potentials and the gating of GABAA receptors to lower GABA concentrations. These latter effects were more pronounced in dorsal root ganglion and dorsal horn neurons than in hippocampal neurons. In dorsal root ganglion and dorsal horn neurons, the facilitatory effect of therapeutic flupirtine concentrations on KV7 channels and GABAA receptors was comparable, whereas in hippocampal neurons the effects on KV7 channels were more pronounced. CONCLUSIONS AND IMPLICATIONSThese results indicate that flupirtine exerts its analgesic action by acting on both GABAA receptors and KV7 channels. AbbreviationsBMI, bicuculline methiodide; CNQX, cyano-2,3-dihydroxy-7-nitroquinoxaline; DRG, dorsal root ganglion; NBQX, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide; SCG, superior cervical ganglion; SNEPCO, selective neuronal potassium channel opener

Key points Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a key regulator of many membrane proteins, including voltage‐gated Kv7.2 channels.In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2‐binding domains in Kv7.2.Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2.Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors.Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2, thereby ensuring the tight regulation of the channel via G protein‐coupled receptors. AbstractThe function of numerous ion channels is tightly controlled by G protein‐coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol‐4,5‐bisphosphate (PIP2). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP2 and through phosphorylation. Using liquid chromatography‐coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2‐binding domains. To evaluate the effect of phosphorylation on PIP2‐mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A5 mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP2 depletion via the voltage‐sensitive phosphatase Dr‐VSP than were wild‐type channels. In vitro phosphorylation assays with the purified C‐terminus of Kv7.2 revealed that CDK5, p38 MAPK, CaMKIIα and PKA were able to phosphorylate the five serines. Inhibition of these protein kinases reduced the sensitivity of wild‐type but not mutant Kv7.2 channels towards PIP2 depletion via Dr‐VSP. In superior cervical ganglion neurons, the protein kinase inhibitors attenuated Kv7 current regulation via M1 receptors, but left unaltered the control by B2 receptors. Our results revealed that the phosphorylation status of serines located within a putative PIP2‐binding domain determined the phospholipid sensitivity of Kv7.2 channels and supported GPCR‐mediated channel regulation.

Amphetamine abuse is a major public health concern for which there is currently no effective treatment. To develop effective treatments, the mechanisms by which amphetamine produces its abuse-related effects need to be fully understood. It is well known that amphetamine exerts its actions by targeting high-affinity transporters for monoamines, in particular the cocaine-sensitive dopamine transporter. Organic cation transporter 3 (OCT3) has recently been found to play an important role in regulating monoamine signaling. However, whether OCT3 contributes to the actions of amphetamine is unclear. We found that OCT3 is expressed in dopamine neurons. Then, applying a combination of in vivo, ex vivo, and in vitro approaches, we revealed that a substantial component of amphetamine’s actions is OCT3-dependent and cocaine insensitive. Our findings support OCT3 as a new player in the actions of amphetamine and encourage investigation of this transporter as a potential new target for the treatment of psychostimulant abuse.

The prime task of nociceptors is the transformation of noxious stimuli into action potentials that are propagated along the neurites of nociceptive neurons from the periphery to the spinal cord. This function of nociceptors relies on the coordinated operation of a variety of ion channels. In this review, we summarize how members of nine different families of ion channels expressed in sensory neurons contribute to nociception. Furthermore, data on 35 different types of G protein coupled receptors are presented, activation of which controls the gating of the aforementioned ion channels. These receptors are not only targeted by more than 20 separate endogenous modulators, but can also be affected by pharmacotherapeutic agents. Thereby, this review provides information on how ion channel modulation via G protein coupled receptors in nociceptors can be exploited to provide improved analgesic therapy.

Serotonin (5HT) is a constituent of the so-called “inflammatory soup” that sensitizes nociceptors during inflammation. Nevertheless, receptors and signaling mechanisms that mediate an excitation of dorsal root ganglion (DRG) neurons by 5HT remained controversial. Therefore, capsaicin-sensitive nociceptive neurons dissociated from rat DRGs were used to investigate effects of 5HT on membrane excitability and currents through ligand- as well as voltage-gated ion channels. In 58% of the neurons tested, 5HT increased action potential firing, an effect that was abolished by the 5HT2 receptor antagonist ritanserin, but not by the 5HT3 antagonist tropisetron. Unlike other algogenic mediators, such as PGE2 and bradykinin, 5HT did not affect currents through TTX-resistant Na+ channels or Kv7 K+ channels. In all neurons investigated, 5HT potentiated capsaicin-evoked currents through TRPV1 channels, an effect that was attenuated by antagonists at 5HT2A (4 F 4 PP), 5HT2B (SB 204741), as well as 5HT2C (RS 102221) receptors. 5HT triggered slowly arising inward Cl− currents in 53% of the neurons. This effect was antagonized by the 5HT2C receptor blocker only, and the current was prevented by an inhibitor of Ca2+-activated chloride channels (CaCC). The 5HT-induced increase in action potential firing was also abolished by this CaCC blocker and by the TRPV1 inhibitor capsazepine. Amongst the subtype selective 5HT2 antagonists, only RS 102221 (5HT2C-selectively) counteracted the rise in action potential firing elicited by 5HT. These results show that 5HT excites DRG neurons mainly via 5HT2C receptors which concomitantly mediate a sensitization of TRPV1 channels and an opening of CaCCs.

The slow cholinergic transmission in autonomic ganglia is known to be mediated by an inhibition of Kv7 channels via M1 muscarinic acetylcholine receptors. However, in the present experiments using primary cultures of rat superior cervical ganglion neurons, the extent of depolarisation caused by the M1 receptor agonist oxotremorine M did not correlate with the extent of Kv7 channel inhibition in the very same neuron. This observation triggered a search for additional mechanisms. As the activation of M1 receptors leads to a boost in protein kinase C (PKC) activity in sympathetic neurons, various PKC enzymes were inhibited by different means. Interference with classical PKC isoforms led to reductions in depolarisations and in noradrenaline release elicited by oxotremorine M, but left the Kv7 channel inhibition by the muscarinic agonist unchanged. M1 receptor-induced depolarisations were also altered when extra- or intracellular Cl− concentrations were changed, as were depolarising responses to γ-aminobutyric acid. Depolarisations and noradrenaline release triggered by oxotremorine M were reduced by the non-selective Cl− channel blockers 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid and niflumic acid. Oxotremorine M induced slowly rising inward currents at negative membrane potentials that were blocked by inhibitors of Ca2+-activated Cl− and TMEM16A channels and attenuated by PKC inhibitors. These channel blockers also reduced oxotremorine M-evoked noradrenaline release. Together, these results reveal that slow cholinergic excitation of sympathetic neurons involves the activation of classical PKCs and of Ca2+-activated Cl− channels in addition to the well-known inhibition of Kv7 channels.

Subunit-selective modulation of γ-aminobutyric acid type A receptors (GABA A R) is considered to exert fewer side effects compared to unselective clinically used drugs. Here, the β2/3 subunit-selective GABA A R modulators valerenic acid (VA) and loreclezole (LOR) guided the synthesis of novel subunit-selective ligands with simplified structures. We studied their effects on GABA A Rs expressed in Xenopus laevis oocytes using twomicroelectrode voltage clamp technique. Five compounds showed significantly more efficacious modulation of GABA-evoked currents than VA and LOR with retained potency and selectivity. Compound 18 [(E)-2− Cyano-3-(2,4-dichlorophenyl)but-2-enamide] induced the highest maximal modulation of GABA-induced chloride currents (E max : 3114 ± 242%), while 12 [(Z)-3-(2,4-dichlorophenyl)but-2-enenitrile] displayed the highest potency (EC 50 : 13 ± 2 μM). Furthermore, in hippocampal neurons 12 facilitated phasic and tonic GABAergic inhibition, and in vivo studies revealed significantly more potent protection against pentylenetetrazole (PTZ)-induced seizures compared to VA and LOR. Collectively, compound 12 constitutes a novel, simplified, and subunit-selective GABA A R modulator with low-dose anticonvulsant activity.

Background and PurposeThe Kv7 channel activator flupirtine is a clinical analgesic characterized as ‘selective neuronal potassium channel opener’. Flupirtine was found to exert comparable actions at GABAA receptors and Kv7 channels in neurons of pain pathways, but not in hippocampus.Experimental ApproachExpression patterns of GABAA receptors were explored in immunoblots of rat dorsal root ganglia, dorsal horns and hippocampi using antibodies for 10 different subunits. Effects of flupirtine on recombinant and native GABAA receptors were investigated in patch clamp experiments and compared with the actions on Kv7 channels.Key ResultsImmunoblots pointed towards α2, α3, β3 and γ2 subunits as targets, but in all γ2-containing receptors the effects of flupirtine were alike: leftward shift of GABA concentration-response curves and diminished maximal amplitudes. After replacement of γ2S by δ, flupirtine increased maximal amplitudes. Currents through α1β2δ receptors were more enhanced than those through Kv7 channels. In hippocampal neurons, flupirtine prolonged inhibitory postsynaptic currents, left miniature inhibitory postsynaptic currents (mIPSCs) unaltered and increased bicuculline-sensitive tonic currents; penicillin abolished mIPSCs, but not tonic currents; concentration-response curves for GABA-induced currents were shifted to the left by flupirtine without changes in maximal amplitudes; in the presence of penicillin, maximal amplitudes were increased; GABA-induced currents in the presence of penicillin were more sensitive towards flupirtine than K+ currents. In dorsal horn neurons, currents evoked by the δ-preferring agonist THIP (gaboxadol) were more sensitive towards flupirtine than K+ currents.Conclusions and ImplicationsFlupirtine prefers δ-containing GABAA receptors over γ-containing ones and over Kv7 channels.