The Acrylamide (S)-2 As a Positive and Negative Modulator of Kv7 Channels Expressed in Xenopus laevis Oocytes

BACKGROUND: Activation 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 FINDINGS: In 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/SIGNIFICANCE: These 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

Sensitivity of heteromeric Kv7.4/Kv7.4-W242L channels to (S)-2.

<p>(A) Current-voltage (I-V) relationship of Kv7.4 and Kv7.4/Kv7.4-W242L in the absence and presence of 15 µM (S)-2. The steady state peak current measured at potentials between –100 and +40 mV were normalized against the current at +40 mV in control recordings and plotted against the test potential. (B) Effect of (S)-2 on the voltage-dependence of activation of Kv7.4 and Kv7.4/Kv7.4-W242L. Tail currents measured after stepping back to –120 mV from potentials between –100 and +60 mV in the absence and presence of 15 µM (S)-2 were normalized and plotted against the preceding potential. The tail current-voltage relationship was then fitted to the Boltzmann equation to yield half-activation potentials (V_0.5): Kv7.4 (5.1±1.3 mV); Kv7.4+(S)-2 (−30.5±1.6 mV); Kv7.4/Kv7.4-W242L (4.1±1.6 mV); Kv7.4/Kv7.4-W242L+(S)-2 (−15.0±1.0 mV). (C) Dose-response relationship of (S)-2 on Kv7.4 and Kv7.4-W242L. The steady state peak currents elicited by a 5 s step to 0 mV in response to increasing concentrations of (S)-2 were normalized to the current in the absence of compound and plotted as a function of the concentration of (S)-2. The values were then analyzed by non-linear regression to fit a sigmoidal curve. The EC₅₀ values were determined to 0.46 µM and 0.72 µM and the Hill coefficients to 1.32±0.17 and 1.24±0.25 for Kv7.4 and Kv7.4/Kv7.4-W242L, respectively. Bars represent S.E.M. and <i>n</i> = 6–10.</p

Nerve Injury-Induced Neuropathic Pain Causes Disinhibition of the Anterior Cingulate Cortex

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

Effect of (S)-2 on the deactivation kinetics of Kv7.2 and Kv7.4.

<p>Normalized tail current traces for Kv7.2 (A) and Kv7.4 (E) obtained at the indicated potentials in the absence and presence of (S)-2 illustrating the pronounced effect of the compound on the deactivation kinetics. Deactivation kinetics for Kv7.2 were determined by fitting the tail currents measured at potentials between –110 mV and –90 mV after an activating step to +40 mV to a double exponential function. The time constants τ_fast (B) and τ_slow (C) were plotted against the potential. (D) Relative contribution of the slow component of the deactivation kinetics for Kv7.2. Tail currents measured between −110 and −80 mV for Kv7.4 were fitted to a double exponential function and τ_fast (F) and τ_slow (G) were plotted against the potential. (H) Relative contribution of the slow component of the deactivation kinetics for Kv7.4. Asterisks indicate statistical significant difference between absence and presence of (S)-2 determined by two-way ANOVA followed by Bonferroni post-test. * P<0.05, ** P<0.01 and *** P<0.001. Bars represent S.E.M. and <i>n</i> = 5–14.</p

Potency of (S)-2 on Kv7.2 and Kv7.4.

<p>Dose-response relationship for the effect of (S)-2 on (A) Kv7.2 (<i>n</i> = 6) and (B) Kv7.4 (<i>n</i> = 8) measured using an ⁸⁶Rb-flux assay.</p

Activation of Kv7 channels by (S)-2.

<p>Representative two-electrode voltage-clamp current traces in the absence (left) and presence (middle) of 10 µM (S)-2 and effect of (S)-2 on current-voltage (I-V) relationship (right) of Kv7.1 (A), Kv7.2 (B), Kv7.2/Kv7.3 (C), Kv7.4 (D) and Kv7.5 (E) channels expressed in <i>Xenopus laevis</i> oocytes. The channels were activated by 5 s voltage steps from −80 mV to potentials ranging from –100 to +40 mV in 10 mV increments followed by a 2 S step to –120 mV. The steady state peak current amplitudes in the absence and presence of 10 µM (S)-2 were normalized against the current at +40 mV in control recordings and plotted against the test potential to obtain I-V curves (left). Bars represent S.E.M. and <i>n</i> = 6-17. Please note that in some instances the S.E.M. is so small that the error bars are not visible.</p