Neuropathic pain caused by peripheral nerve injury is a debilitating neurological condition of high clinical relevance. On the cellular level, the elevated pain sensitivity is induced by plasticity of neuronal function along the pain pathway. Changes in cortical areas involved in pain processing contribute to the development of neuropathic pain. Yet, it remains elusive which plasticity mechanisms occur in cortical circuits. We investigated the properties of neural networks in the anterior cingulate cortex (ACC), a brain region mediating affective responses to noxious stimuli. We performed multiple whole-cell recordings from neurons in layer 5 (L5) of the ACC of adult mice after chronic constriction injury of the sciatic nerve of the left hindpaw and observed a striking loss of connections between excitatory and inhibitory neurons in both directions. In contrast, no significant changes in synaptic efficacy in the remaining connected pairs were found. These changes were reflected on the network level by a decrease in the mEPSC and mIPSC frequency. Additionally, nerve injury resulted in a potentiation of the intrinsic excitability of pyramidal neurons, whereas the cellular properties of interneurons were unchanged. Our set of experimental parameters allowed constructing a neuronal network model of L5 in the ACC, revealing that the modification of inhibitory connectivity had the most profound effect on increased network activity. Thus, our combined experimental and modeling approach suggests that cortical disinhibition is a fundamental pathological modification associated with peripheral nerve damage. These changes at the cortical network level might therefore contribute to the neuropathic pain condition.
BackgroundActivation of voltage-gated potassium channels of the Kv7 (KCNQ) family reduces cellular excitability. These channels are therefore attractive targets for treatment of diseases characterized by hyperexcitability, such as epilepsy, migraine and neuropathic pain. Retigabine, which opens Kv7.2-5, is now in clinical trial phase III for the treatment of partial onset seizures. One of the main obstacles in developing Kv7 channel active drugs has been to identify compounds that can discriminate between the neuronal subtypes, a feature that could help diminish side effects and increase the potential of drugs for particular indications.Methodology/Principal FindingsIn the present study we have made a thorough investigation of the Bristol-Myers Squibb compound (S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1], [4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide [(S)-2] on human Kv7.1-5 channels expressed in Xenopus laevis oocytes. We found that the compound was a weak inhibitor of Kv7.1. In contrast, (S)-2 efficiently opened Kv7.2-5 by producing hyperpolarizing shifts in the voltage-dependence of activation and enhancing the maximal current amplitude. Further, it reduced inactivation, accelerated activation kinetics and slowed deactivation kinetics. The mechanisms of action varied between the subtypes. The enhancing effects of (S)-2 were critically dependent on a tryptophan residue in S5 also known to be crucial for the effects of retigabine, (S)-1 and BMS-204352. However, while (S)-2 did not at all affect a mutant Kv7.4 with a leucine in this position (Kv7.4-W242L), a Kv7.2 with the same mutation (Kv7.2-W236L) was inhibited by the compound, showing that (S)-2 displays a subtype-selective interaction with in the Kv7 family.Conclusions/SignificanceThese results offer further insight into pharmacological activation of Kv7 channels, add to the understanding of small molecule interactions with the channels and may contribute to the design of subtype selective modulators.
Background/Aims: The neuronal KV7 family members (KV7.2–5) are important regulators of neuronal excitability. KV7 channel openers are therefore attractive drug candidates for the treatment of several hyperexcitability disorders. While most described KV7 channel openers discriminate poorly between KV7.2–5, Icagen’s N-(6-chloropyridin- 3-yl)-3,4-difluorobenzamide (ICA-27243) is more potent at KV7.2/3 than at KV7.4 and KV7.3/5 and offers some progress towards subtype selectivity. We have investigated its mode of action on KV7.2 and KV7.4, compared its effect to that of retigabine and studied the combinatorial effect of retigabine and ICA-27243, as these two compounds recognize different binding sites in the channels. Methods: The effects of ICA-27243 and retigabine were studied using voltage-clamp electrophysiology in Xenopus laevis oocytes and rubidium flux in Chinese hamster ovary cells. Results: We found that in contrast to retigabine’s voltage-dependent action on KV7.2, ICA-27243 induced a voltage-independent current on this subtype, which was not observed on KV7.4. Additionally, the combined treatment of KV7.2 and KV7.4 with retigabine and ICA-27243 revealed that the effect of ICA-27243 on KV7.2 dominates that of retigabine, while the compounds act additively and synergistically on KV7.4. Conclusions: These results offer further detailed insight into pharmacological activation of KV7 channels and offer evidence of differential functional and subtype-specific effects by activation of different binding sites in the KV7 channels.
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