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. 2024 Apr;389(1):118–127. doi: 10.1124/jpet.123.001959

Selective KCNQ2/3 Potassium Channel Opener ICA-069673 Inhibits Excitability in Mouse Vagal Sensory Neurons

Hui Sun 1,✉, Bradley J Undem 1
PMCID: PMC10949160  PMID: 38290975

Abstract

Heightened excitability of vagal sensory neurons in inflammatory visceral diseases contributes to unproductive and difficult-to-treat neuronally based symptoms such as visceral pain and dysfunction. Identification of targets and modulators capable of regulating the excitability of vagal sensory neurons may lead to novel therapeutic options. KCNQ1–KCNQ5 genes encode KV7.1-7.5 potassium channel α-subunits. Homotetrameric or heterotetrameric KV7.2-7.5 channels can generate the so-called M-current (IM) known to decrease the excitability of neurons including visceral sensory neurons. This study aimed to address the hypothesis that KV7.2/7.3 channels are key regulators of vagal sensory neuron excitability by evaluating the effects of KCNQ2/3-selective activator, ICA-069673, on IM in mouse nodose neurons and determining its effects on excitability and action potential firings using patch clamp technique. The results showed that ICA-069673 enhanced IM density, accelerated the activation, and delayed the deactivation of M-channels in a concentration-dependent manner. ICA-069673 negatively shifted the voltage-dependent activation of IM and increased the maximal conductance. Consistent with its effects on IM, ICA-069673 induced a marked hyperpolarization of resting potential and reduced the input resistance. The hyperpolarizing effect was more pronounced in partially depolarized neurons. Moreover, ICA-069673 caused a 3-fold increase in the minimal amount of depolarizing current needed to evoke an action potential, and significantly limited the action potential firings in response to sustained suprathreshold stimulations. ICA-069673 had no effect on membrane currents when Kcnq2 and Kcnq3 were deleted. These results indicate that opening KCNQ2/3-mediated M-channels is sufficient to suppress the excitability and enhance spike accommodation in vagal visceral sensory neurons.

SIGNIFICANCE STATEMENT

This study supports the hypothesis that selectively activating KCNQ2/3-mediated M-channels is sufficient to suppress the excitability and action potential firings in vagal sensory neurons. These results provide evidence in support of further investigations into the treatment of various visceral disorders that involve nociceptor hyperexcitability with selective KCNQ2/3 M-channel openers.

Introduction

Vagal sensory nerves, derived from vagal sensory neurons situated in the jugular and nodose ganglia, innervate visceral organs in the respiratory, cardiovascular, digestive and endocrine systems, sensing and transmitting the sensory information about body’s internal environment to the central nervous system (Prescott and Liberles, 2022; Zhao et al., 2022). The normal function of vagal sensory nerves is essential for maintaining the optimal physiologic activities of the viscera, and for initiating the host defense in response to inhaled or ingested harmful chemicals and pathogens. In inflammatory visceral diseases, however, the vagal sensory nerves are sensitized to become hyper-excitable under the sustained stimulation by pathologic factors, leading to unproductive coughing, diffusive pain, dysphagia, arrhythmias, excessive secretions, and abnormal digestive functions, etc., depending on the affected organ (Undem and Taylor-Clark, 2014; Gebhart and Bielefeldt, 2016). Currently, treatment of visceral pain and other vagal afferent nerve abnormalities remains challenging. Identification of targets and their modulators capable of normalizing the excitability of vagal sensory nerves may help develop novel therapeutic options to relieve the suffering of patients afflicted with these neurologic symptoms.

Neuronal KV7 channels, also known as M-channels or KCNQ channels, are encoded by KCNQ2–KCNQ5 genes. KCNQ2, KCNQ3, and KCNQ5, coding for KCNQ2/KV7.2, KCNQ3/KV7.3, and KCNQ5/KV7.5 α-subunits respectively, are widely expressed across the nervous system whereas the KCNQ4 expression is more restricted (Miceli et al., 2018; Nappi et al., 2020). The voltage-gated M-channel generates a low threshold-activated, slowly activating and non-inactivating potassium current, the M-current (IM), that contributes to the negative resting potential and controls action potential firings (Brown and Adams, 1980; Sun et al., 2019). These unique biophysical properties, along with the fact that IM is subject to modulation by numbers of neurotransmitters and inflammatory mediators (Delmas and Brown, 2005; Abbott, 2020), make the M-channel a key regulator of neuronal excitability in health and in diseases. Indeed, mutations in KCNQ2, KCNQ3, or KCNQ5 genes cause epilepsy in humans, which can be recapitulated in Kcnq2-knockout, Kcnq2- or Kcnq3-knockin, and Kcnq5 loss-of-function mouse models (Nappi et al., 2020; Brun et al., 2022; Wei et al., 2022). Mutations in KCNQ2 also cause a hyperexcitability in peripheral motor neurons (Wuttke et al., 2007), and genetic or pharmacological inhibition of M-currents in peripheral sensory pathway increases nociceptor activity and pain sensitivity (Du et al., 2018). On the other hand, activation of M-channels suppresses the electrically or chemically induced seizure and attenuates both neuropathic and inflammatory pain in laboratory animals (Blackburn-Munro et al., 2005; Du et al., 2018). These findings strongly support the concept that the M-channel is a valid therapeutic target for neuronal hyperexcitability disorders. Notably, the pan-KV7.2–KV7.5 channel opener retigabine significantly reduced the frequency of seizures in patients with partial-onset epilepsy, leading to its approval by the Food and Drug Administration for clinical use (Sikandar and Dickenson, 2012).

Retigabine was later withdrawn from the market due to its compound structure-related toxicity (retinal pigmentation and skin discoloration) (Liu et al., 2021), as well as other side effects that are at least partially related to its poor selectivity for distinct KV7 subunits. Although KV7.2 and KV7.3 subunits are mainly expressed in the nervous system, KV7.4 and KV7.5 are also abundantly expressed in non-neuronal tissues. In fact, KV7.4 and KV7.5 are the major KV7 subunits in smooth muscles of vasculature and hollow visceral organs where they regulate the smooth muscle contractility and vascular tone (Jepps et al., 2013; Malysz and Petkov, 2020) and in skeletal muscle where they regulate the proliferation, differentiation, and muscle force development (Schroeder et al., 2000; Iannotti et al., 2013; Zagorchev et al., 2016). Opening KV7.4 or KV7.4/7.5 channels in these tissues likely contributes to the common side effects of retigabine, such as urinary retention, asthenia, and symptomatic hypotension. Hence, searching for safer, more potent and more subtype-selective KCNQ/KV7 channel activators has been ongoing for the last 15 years. This effort has led to the identification of several M-channel openers that have improved selectivity for KV7.2/7.3 over KV7.4/7.5 and KV7.3/7.5 channels, including one of the benzamides ICA-069673 (Amato et al., 2011).

We have previously reported that mouse nodose neurons expressed functional M-channels, as well as Kcnq2, Kcnq3, and Kcnq5 mRNA. Activation of IM by pan-KV7.2-7.5 activator retigabine significantly reduced the excitability of nodose neurons at the level of the cell body and C-fiber terminals within the airways (Sun et al., 2019). This study aimed to address the hypothesis that KV7.2/7.3 channels are key regulators of vagal sensory neuron excitability by evaluating the effects of KCNQ2/3-selective activator, ICA-069673, on IM in mouse nodose neurons and determining its effects on excitability and action potential firings.

Materials and Methods

The experiments in mice reported in this study have been carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health, and were approved by The Johns Hopkins Animal Care and Use Committee.

Isolation of Mouse Nodose Neurons.

Eight- to 12-week-old male wild-type C57BL/6 mice (The Jackson Laboratory), as well as Kcnq2/3-knockout mice (Pirt-Cre-Kcnq2/3fl/fl) and their littermate controls (Kcnq2/3fl/f) at C57BL/6 background were used in this study. The nodose neurons were isolated as previously described (Sun, 2021). Briefly, mice were euthanized by CO2 inhalation and subsequent exsanguinations. The lower two-thirds of the jugular/nodose complex (free of jugular component) (Nassenstein et al., 2010) from both sides were dissected and placed into 1 ml of Hank’s balanced salt solution containing 1.5–2 mg type 1 collagenase and 2 mg dispase II. The enzymatic digestion proceeded at 37°C for 60 minutes, and the ganglion tissue was gently triturated for a few times at 30, 45, and 60 minutes of digestion. The 1-ml enzymatic solution containing the dissociated neurons was then transferred to 10 ml of pre-warmed Leibovitz’s L-15 medium supplemented with 10% of FBS and centrifuged at 600 × g for 2 minutes. After one more wash, the pellet was re-suspended in 200 µl of L-15 medium, then pipetted onto six poly-D-lysine and laminin-treated cover glasses. After the neurons attached to the cover glass (2 hours at 37°C), 2 ml of fresh FBS-supplemented L-15 medium was added to the cover glass, each placed in one 35-mm Petri dish. The isolated neurons were maintained at 37°C overnight and used for recordings within 24 hours after isolation.

Patch Clamp Recordings.

The membrane currents and potential were recorded with amphotericin B-perforated whole-cell patch clamp technique using an Axopatch 200B amplifier interfaced with Axon Digidata 1550A and driven by pCLAMP 10 software (Molecular Devices, Sunnyvale, CA). The signals were sampled at 10 kHz and filtered at 2 kHz. The recording pipettes had a resistance between 1.4 to 1.8 MΩ when filing with the pipette solution containing (in mM) 30 KCl, 115 K-gluconate, and 10 HEPES (pH adjusted to 7.2 with KOH). Bath solution contained (mM): NaCl 136, KCl 5.4, MgCl2 1, CaCl2 1.5, HEPES 10, and glucose 10 with pH adjusted to 7.35 with NaOH. The amphotericin B stock was prepared in DMSO (3%) on the day of recording and sonicated for 10 minutes. The amphotericin B stock was then diluted by 100 times with the pipette solution and used within 2 hours. The liquid junction potential (–13.2 mV estimated using Clampex calculator) was corrected offline. The patch potential was unknown and not corrected. The recordings were made after an adequate whole-cell was established, which usually takes 5–10 minutes. An access resistance ≤25 MΩ for current-clamp and <15 MΩ for voltage-clamp experiments was considered as adequate whole-cell access. The voltage-clamp and current-clamp protocols are given in detail in the Results where appropriate. The membrane potential was recorded on I-clamp normal mode. All recordings were carried out at room temperature.

The current responses to 5-mV depolarization or hyperpolarization pulses from a holding potential of –65 mV were used to estimate the whole-cell capacitance and input resistance (Rinput). To determine the rheobase, a series of 25-millisecond current steps with an increment of 5–10 pA was applied. At the end of some experiments, the responsiveness of neurons to 1 µM capsaicin was examined. Capsaicin usually generates a large inward current in capsaicin-sensitive neurons held at –70 mV.

Data Analyses.

Clampfit 10 and SigmaPlot were used for patch clamp data analyses. The whole-cell capacitance was calculated by dividing the integral of capacitance transient by the voltage step amplitude. The amplitude of IM, measured as the current irreversibly blocked by 10 μm XE991, was normalized to the cell capacitance and expressed as current density (pA/pF). The steady-state activation curves (i.e., conductance–voltage relationships) of IM were constructed by plotting the amplitude of tail currents (Itail) density elicited at –60 mV following long voltage steps to different potentials against the prepulse voltages. The amplitude of Itail was measured at the peak. To obtain the activation parameters, data points were fitted in SigmaPlot to the Boltzmann function for each cell: Itail = Gmax/(1 + exp(–(Vm – V0.5)/k)), where Gmax is maximal conductance, Vm is membrane potential, V0.5 is voltage at which 50% of activation occurs, and k is the slope factor. To establish the concentration–response relationship of ICA-069673, the amplitude of Itail (elicited by voltage step from –30 to –60 mV) was measured at baseline and in the presence of different concentrations of ICA-069673. The increases in Itail amplitude by ICA-069673 at different concentrations with respect to that at baseline were then calculated, normalized to the maximal value at saturation, and plotted against the concentration of the opener at log scale. The EC50 was obtained by fitting the mean data points to the logistic function in SigmaPlot.

The Rinput was calculated by dividing the voltage step (from –65 to –60 mV) by sustained current measured at the end of a 10-millisecond voltage pulse. The rheobase was measured as the lowest amount of depolarizing current (25 milliseconds) needed to evoke a single action potential (AP). The AP threshold (APthreshold) was measured by differentiating the action potential with respect to time (dV/dt) and defined as the voltage at which the deflection for dV/dt is greater than zero.

Chemicals and Reagents.

Hank’s balanced salt solution, Leibovitz’s L-15 medium, and heat-inactivated FBS were purchased from Gibco/Life Technologies. Dispase II was from Roche Diagnostics. Type 1 collagenase, amphotericin-B, and capsaicin were from Sigma. ICA-069673 and XE 991 dihydrochloride were purchased from Tocris. ICA-069673 stock (3 × 10−2 M) was prepared in DMSO. XE 991 stock (10−2 M) was prepared in Millipore water. Capsaicin stock (10−2 M) was prepared in ethanol. All stocks were stored at –20°C.

Statistical Analyses.

SigmaPlot software was used to perform the statistical analyses of the results. Pooled data are expressed as mean ± S.D. The statistical significance of differences between two means was determined by using either paired or unpaired Student’s t test. Wilcoxon signed rank test or Mann-Whitney rank sum test was used in the cases that the normality test failed. The significance of differences between multiple means was evaluated by one-way repeated measures ANOVA. The statistical significance of differences between two means at multiple test voltages or current injections was determined by two-way ANOVA. Holm-Sidak test as a post hoc analysis was performed for multiple pairwise comparisons.

Results

Concentration-Dependent Potentiating Effects of ICA-069673 on M-Currents in Nodose Neurons.

In this study, the M-current (IM) in mouse nodose neurons was recorded as the potassium current irreversibly blocked by the M-channel inhibitor XE991 as we previously described (Sun et al., 2019). Figure 1A presents an example. The isolated neuron was held at –30 mV to inactivate the Na+, Ca2+, and most voltage-dependent K+ channels. Step from the holding potential to –60 mV evoked an outward tail current (Itail) that progressively deactivated during the 1-second test pulse. Stepping back to –30 mV elicited a slowly activating current. Bath perfusion of ICA-069673 increased both the current at –30 mV and the tail current. Subsequent addition of XE991 at 10 μm, a concentration that causes the maximal inhibition of IM (by 98.2%) (Romero et al., 2004), blocked most of these currents. Because XE991 also blocks the high-threshold KV2 channels reversibly in addition to its irreversible effects on M-channels (Wladyka and Kunze, 2006; Sun et al., 2019), the recordings were repeated during washout of both ICA-069673 and XE991. The current recorded at 2 minutes post-washout is shown in Fig. 1A and used to obtain the irreversible XE991-sensitive current (i.e., IM) by digital subtractions (Fig. 1A, lower panel). ICA-069673 at 10 μm substantially enhanced the IM amplitude, slowed down the deactivation, and accelerated the activation of the M-channels.

Fig. 1.

Fig. 1.

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Effects of different concentrations of ICA-069673 on M-currents in mouse nodose neurons. (A) Voltage-clamp protocol and representative recordings illustrating how the IM tail currents were recorded in this study. Top: Tail currents recorded before (Control) and after bath application of 10 μm ICA-069673, after subsequent addition of 10 μm XE991 in the presence of ICA-069673, and at 2 minutes post-washout of both compounds. Bottom: Irreversible XE991-sensitive tail currents, measured as IM, in the absence and presence of ICA-069673 obtained by digital subtractions of the current trace recorded post-washout of XE991and ICA-069673 from those recorded at control and after bath application of ICA-069673, respectively. (B) IM recorded as irreversible XE991-sensitive Itail using the same method and protocol shown in (A) in the absence and presence of different concentrations of ICA-069673. The arrows indicate the peak of Itail (Ipeak) and the sustained level of Itail at the end of 1-second pulse (ISS). (C) Concentration–response curve of IM potentiation by ICA-069673. The opener-induced increase in the peak of Itail was normalized to that obtained at 10 μm of ICA-069673 (concentration with maximal effect). The numbers in the parentheses indicate the number of neurons tested. (D) Bar graph showing the averaged ratio of Iss over Ipeak of tail currents (mean ± S.D., n = 8), as an index of M-channel deactivation rate at –60 mV, measured at different concentrations of ICA-069673. The filled circles present the results obtained from individual neurons. (E) Bar graph showing the mean ± S.D. of M-channel activation time constants (τactivation) measured at –30 mV in the absence and presence of different concentrations of ICA-069673 (n = 8). The filled circles give the values obtained from each of eight neurons. The P values shown in (D) and (E) were obtained using one-way repeated measures ANOVA with Holm-Sidak test as a post hoc analysis for all pairwise comparisons.

Using the same strategy and voltage-clamp protocol, we evaluated the concentration-dependent potentiating effects of ICA-069673 on IM in mouse nodose neurons. As shown in Fig. 1B, ICA-069673 increased the IM tail current, reduced the deactivation rate, and increased the activation kinetics in a dose-dependent manner. The increase in the peak of tail currents by ICA-069673 reached the maximum at 10 μm at –30 mV. ICA-069673 at 30 μm caused a slight reduction in the peak of Itail. The concentration-response relationship was constructed by plotting the increase in the peak of Itail, normalized to the one obtained at 10 μm of ICA-069673, against the concentration of the opener (Fig. 1C), which generated an EC50 of 0.52 μm. In a total of 48 nodose neurons isolated from wild-type mice and studied on voltage-clamp mode, ICA-069673 enhanced IM in 41 neurons (∼85%).

The concentration-dependent effects of ICA-069673 on the deactivation and activation kinetics of M-channels were analyzed in eight nodose neurons where the five concentrations of the opener were tested. The deactivation rate of M-channels was quantified by calculating the ratio of sustained tail current at the end of 1-second test pulse (ISS) over the peak of Itail (Ipeak). As shown in Fig. 1D, ICA-069673 dose-dependently slowed down the closing of M-channels on repolarization, and this effect reached the maximum at concentrations around 3 μm. The activation of IM, induced by stepping from –60 mV back to the holding potential (Fig. 1, A and B), fits well to a monoexponential function. The activation time constants (τactivation) of IM recorded in the absence and in the presence of different concentrations of ICA-069673 are shown in Fig. 1E. ICA-069673 significantly reduced the τactivation at all concentrations tested. The effect was more pronounced at higher concentrations (10 and 30 μm).

Effects of ICA-069673 on Voltage-Dependent Activation of IM in Nodose Neurons.

The effects of ICA-069673 (10 μM) on the voltage-dependent activation properties of IM in mouse nodose neurons were evaluated. Figure 2A shows the voltage-clamp protocol and families of current recorded from one nodose neuron at baseline (Control, a), in the presence of 10 μm ICA-069673 (b), and after subsequent addition of 10 μm XE991 followed by 2-minute washout of both compounds (c). The neuron was held at –30 mV, and the membrane potential was clamped to different levels between –90 to –10 mV for 1 second (prepulses) followed by a step to –60 mV for 1 second (test pulse) to measure the tail currents. XE991 irreversibly attenuated the currents elicited by the prepulses and almost abolished the slow tail currents. The families of IM obtained at control and in the presence of ICA-069673 are displayed in Fig. 2B as the irreversible XE991-sensitive currents derived from the original recordings shown in Fig. 2A by digital subtractions (a-c and b-c, respectively). ICA-069673 augmented the tail currents and the currents elicited by prepulses, particularly at more negative voltages. The slower relaxation of Itail and accelerated activation rate of IM (revealed by voltage steps from –60 mV back to the holding potential) in the presence of ICA-069673 compared with control are also evident in these recordings.

Fig. 2.

Fig. 2.

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Effects of ICA-069673 on voltage-dependent activation of IM in mouse nodose neurons. (A) Families of membrane currents elicited from one nodose neuron under control condition (a), after bath application of 10 μm ICA-069673 (b), and after subsequent addition of 10 μm XE991 and washout of both compounds (c) by using the voltage-clamp protocol illustrated on (a). (B) M-currents, measured as the irreversible XE991-sensitive currents, in the absence and presence of 10 μm ICA-069673 are obtained by digital subtraction of currents recorded after a 2-minute washout of XE991 and ICA-069673 (Ac) from those recorded before (Aa) and after bath application of ICA-069673 (Ab), respectively. The dotted lines indicate the zero current level. The arrows indicate the instantaneous tail currents (peak of Itail). (C) Conductance-voltage relationships in the absence (Control) and presence of 10 μm ICA-069673 were constructed by plotting the peak amplitude of irreversible XE991-sensitive Itail (mean ± S.D., n = 5) against the prepulse voltages. The solid lines represent the fits of data points to the Boltzmann function. (D) Conductance-voltage curves normalized to the maximal Itail recorded in the absence of ICA-069673 (Control Itail.Max). ICA-069673 increased IM at all voltages. The statistical significance (P < 0.001) was determined by two-way ANOVA followed by all pairwise multiple comparisons using Holm-Sidak method.

The amplitude of the instantaneous tail currents (i.e., the peak of Itail, indicated by arrows in Fig. 2B) irreversibly blocked by XE991 reflects the fraction of the M-channels open at the end of the prepulses. Thus, the effects of ICA-069673 on the voltage dependence of M-channel activation were determined in five nodose neurons by plotting the amplitude of irreversible XE991-sensitive tail currents recorded in the absence and presence of 10 μm ICA-069673, respectively, against the prepulse voltages (Fig. 2C). The solid curves represent the fits of data points to the Boltzmann function, which gave rise to the maximal conductance (Gmax), the voltage at which 50% of activation occurs (V0.5), and the slope factor (k). ICA-069673 significantly increased the Gmax (from 8.1 ± 4.0 to 12.6 ± 7.4 nS/pF, P = 0.031), shifted the V0.5 in hyperpolarizing direction (from –46.7 ± 1.7 to –72.2 ± 4.4 mV, P < 0.001), and increased the slop factor k (from 6.7 ± 0.5 to 13.1 ± 5.0, P = 0.04). For better comparison, the conductance-voltage curves in the absence and presence of ICA-069673 are normalized to the maximal Itail obtained in the absence of the opener for each of five nodose neurons and displayed as mean ± S.D. on Fig. 2D. In addition to a large hyperpolarizing shift of voltage-dependent activation, ICA-069673 increased IM at all voltages from –90 to –10 mV. The M-channel conductance in the presence of ICA-069673 is ∼1.5-fold of the control value at voltages where the channel activation reached the saturation (–20 to –10 mV).

Effects of ICA-069673 on M-Currents in Nodose Neurons Lacking Kcnq2 and Kcnq3.

The results presented above revealed that the potentiating effect of ICA-069673 on membrane currents was irreversibly blocked by XE991, indicating that ICA-069673 selectively acts on M-channels. Given that mouse nodose neurons primarily express Kcnq3 and Kcnq2(Sun et al., 2019), these results also indicate that ICA-069673 opens the KCNQ2/3 channels in nodose neurons. To confirm the specificity of action of ICA-069673, we examined the effects of ICA-069673 on IM tail currents in nodose neurons derived from Pirt-driven Kcnq2/3 double knockout mice (Pirt-Cre/Kcnq2/3fl/fl) and their littermate controls (Kcnq2/3fl/fl) that we have recently generated. The effective inactivation of both Kcnq2 and Kcnq3 in nodose neurons of our Kcnq2/3-KO mice has been validated using single-neuron reverse transcription polymerase chain reaction (RT-PCR) and patch clamp techniques (Sun et al., 2023). The results presented in Fig. 3 show that ICA-069673 increased both the sustained current at –30 mV and the tail currents elicited by repolarization from –30 mV to –60 mV in the littermate control nodose neuron (top and middle panels). Despite the large variability in the baseline IM tail current density, ICA-069673 augmented the tail current in each of eight tested littermate control nodose neurons. On average, it caused a 1.63-fold increase in IM tail current density (from 5.72 ± 5.3 to 9.9 ± 9.6 pA/pF, P = 0.008), which is comparable to the 1.65-fold increase in the IM tail current density caused by 10 µM ICA-069673 in nodose neurons of wild-type mice (from 7.7 ± 6.6 to 12.7 ± 10.7 pA/pF, n = 41). On the other hand, the irreversible XE991-sensitive current is largely absent or very small in Kcnq2/3-KO nodose neurons (n = 8). Importantly, application of 10 µM ICA-069673 did not alter the tail currents, the holding currents at –30 mV or the currents elicited by stepping back to the holding potential from –60 mV. These results further confirm that ICA-069673 selectively acts on M-channels in mouse nodose neurons under our experimental conditions, and provide direct evidence in support of KCNQ2/3 channels as targets for the opener.

Fig. 3.

Fig. 3.

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Effects of ICA-069673 on M-currents in nodose neurons of littermate control and Pirt-driven Kcnq2/3-KO mice. Top: Representative recordings of tail currents elicited by voltage steps from –30 mV (holding potential) to –60 mV from a littermate control and a Kcnq2/3-KO nodose neurons at baseline (Control), after bath application of 10 µm ICA-069673 (ICA), after subsequent addition of 10 µM XE991 in the presence of ICA-069673, and after a 2-minute washout of both compounds. Middle: Irreversible XE991-sensitive currents, measured as IM, obtained from the recordings shown on the top panels by digital subtraction. Bottom: Current densities of instantaneous IM tail currents measured from each of eight littermate control and eight Kcnq2/3-KO nodose neurons in the absence (Control) and presence of ICA-069673 (ICA). Each set of interconnected symbols presents data obtained from one nodose neuron. P values were determined by Wilcoxon signed rank test. Please note the different scales of y axes between left and right panels that present data obtained from littermate control and Kcnq2/3-KO nodose neurons, respectively.

Effects of ICA-069673 on Resting Membrane Potential in Mouse Nodose Neurons.

In a total of 27 nodose neurons examined under current clamp conditions, we found that ICA-069673 at 10 μM caused a significant hyperpolarization of membrane potentials in 24 neurons (from –61.2 ± 4.3 mV to –69.2 ± 5.0 mV, P < 0.001). In the remaining three neurons, ICA-069673 had no effect on the resting membrane potential (RP). Further voltage-clamp studies on these neurons revealed that one neuron expressed a substantial IM (13 pA/pF), and the other two had little IM (≤0.5 pA/pF). The ICA-069673-induced hyperpolarization was completely antagonized by the subsequent addition of 10 μm XE991 to the bath solution containing ICA-069673 (Fig. 4A, n = 5), suggesting that the hyperpolarizing effect of ICA-069673 was mediated via its action on M-channels. The ICA-069673-induced hyperpolarization was fully reversible following washout of the opener (Fig. 4B, n = 13).

Fig. 4.

Fig. 4.

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Effects of ICA-069673 on resting membrane potential in mouse nodose neurons. (A) Representative recordings (left) and the corresponding group data (mean ± S.D., n = 5, right) of RP before and after bath application of 10 μm ICA-069673 (ICA), and after subsequent addition of 10 μm XE991. Data obtained from each of five neurons are also displayed with different interconnected symbols on the right panel. (B) Representative recordings (left) and box-and-whisker plots of corresponding group data (n = 13, right) showing RP in the absence (Control) and presence of ICA-069673 (ICA), and after washout of the opener. Horizontal lines of boxes represent 25th percentile, median, and 75th percentile. Whiskers represent 5th/95th percentile. (C) ICA-069673-induced hyperpolarization in seven nodose neurons at baseline (Control) and at slightly depolarized state. The filled circles and error bars represent mean ± S.D. Each pair of interconnected symbols show the results obtained from each individual neuron. (D) Bar graph showing the averaged amplitude of ICA-069673-induced hyperpolarization (mean ± S.D.) obtained from seven nodose neurons at control and at depolarized states. The filled circles give the values obtained from individual neurons. The statistical significance in (A) and (B) was determined by one-way repeated measures ANOVA with Holm-Sidak test as a post hoc analysis for multiple pairwise comparisons. The P values in (C) and (D) were determined by paired t test.

The effect of ICA-069673 on RP was also evaluated in partially depolarized nodose neurons. A depolarization of 6.6 ± 1.9 mV (–65.3 ± 4.2 to –58.7 ± 3.3 mV) was induced in seven neurons by injecting a small constant depolarizing current, which was expected to increase the fraction of open M-channels from ∼6% to ∼15% according to the voltage dependence of M-channel activation found in these neurons (Fig. 2D). Three of these neurons were sensitive to capsaicin. As shown in Fig. 4C, ICA-069673 evoked a steeper hyperpolarization in the same neurons at depolarized state compared with the control state (without current injection). The amplitude of hyperpolarization was significantly greater when the neurons were partially depolarized (12.0 ± 3.1 vs. 6.9 ± 2.6 mV, P < 0.001) (Fig. 4D), although the absolute values of RP in the presence of ICA-069673 were slightly less negative under the influence of injected currents (–70.7 ± 2.7 vs. –72.2 ± 3.2 mV, P = 0.012). This observation suggests that the hyperpolarizing effect of ICA-069673 may also be more pronounced when the nodose neurons are partially depolarized by noxious stimuli such as sensitization by inflammatory mediators.

Effects of ICA-069673 on Input Resistance and Excitability in Mouse Nodose Neurons.

Consistent with an increase in the M-channel conductance, the input resistance of nodose neurons around RP was significantly reduced by ICA-069673 (Fig. 5A). To determine the effects of ICA-069673 on the excitability of nodose neuron, we measured the minimal amount of depolarizing current needed to evoke a single AP (rheobase), the lowest voltage at which the AP is fired (APthreshold), and AP discharges in response to suprathreshold stimulations in nodose neurons in the absence and presence of 10 µm ICA-069673. As shown in Fig. 5, B and C, ICA-069673 increased the rheobase by 3-fold without affecting the APthreshold. Some nodose neurons fired multiple APs in response to increasing step depolarization whereas others fired only one AP regardless the stimulation intensities. In this study, we evaluated the effects of ICA-069673 on nodose neurons that fired multiple APs in response to the 600-millisecond depolarizing steps of increasing intensity. Representative recordings and results obtained from seven neurons are presented in Fig. 5, D and E. ICA-069673 significantly reduced the number of APs elicited by 100–500 pA depolarizing steps.

Fig. 5.

Fig. 5.

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Effects of ICA-069673 on input resistance, excitability, and action potential firings in mouse nodose neurons. (A) Box and whisker plot of input resistance obtained from nine nodose neurons in the absence (Control) and presence of 10 μM ICA-069673 (ICA). The P value was determined by paired t test. (B) (a) Representative recordings of membrane potentials showing the minimal amount of 25-millisecond depolarizing current needed to evoke an action potential (the rheobase) at baseline (control) and after bath application of 10 μm ICA-069673 (ICA). The current-clamp protocols used to determine the rheobase are shown below the recordings. Note that only a few traces are shown for clarity. (b) Box and whisker plot of rheobase obtained from 13 nodose neurons at baseline and in the presence of ICA-069673. The statistical significance was determined by paired t test. (C) Box and whisker plot of voltage threshold for AP generation obtained from 13 nodose neurons at baseline and after application of ICA-069673. Paired t test was used to evaluate the statistical significance. (D) Representative recordings of action potential firing recorded from one nodose neuron in response to 600-millisecond depolarizing current steps of indicated intensities at baseline and after bath application of 10 μm ICA-069673. (E) Averaged numbers (mean ± S.D.) of AP obtained from seven nodose neurons in response to 600-millisecond depolarizing current injections at baseline and in the presence of ICA-069673 are plotted against the intensity of injected currents. The P value was determined by two-way ANOVA. Box and whisker plots in (A), (B) (b), and (C): Horizontal lines of boxes represent 25th percentile, median, and 75th percentile. Whiskers represent 5th/95th percentile.

Effects of ICA-069673 on IM and Resting Potential in Capsaicin-Sensitive and Capsaicin-Insensitive Nodose Neurons.

Based on their responsiveness to the TRPV1 agonist capsaicin, nodose neurons can be classified into capsaicin-sensitive and capsaicin-insensitive neurons. Capsaicin-sensitive nodose neurons are nociceptive C-fiber neurons that can be activated by chemicals and inflammatory mediators. Whether the effects of ICA-069673 on IM and resting potential are different in these two groups of nodose neurons were examined. The amplitude of IM determined by using the tail current protocol shown in Fig. 1A varied considerably in both capsaicin-sensitive and capsaicin-insensitive neurons, ranging from 0.8 to 23.6 pA/pF and from 1.3 to 22.8 pA/pF, respectively. The current densities at baseline are not statistically different between two groups of neurons (7.0 ± 7.0 pA/pF, n = 10 vs. 9.3 ± 7.1 pA/pF, n = 24. P = 0.416). ICA-069673 significantly augmented the IM densities in both groups of nodose neurons (Fig. 6A) with similar degrees of potentiation (Fig. 6B).

Fig. 6.

Fig. 6.

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Effects of ICA-069673 on IM density and RP in capsaicin-sensitive and capsaicin-insensitive nodose neurons. (A) Box and whisker plot of IM tail current density measured in the absence (BL) and presence of 10 μm ICA-069673 (ICA) from capsaicin-sensitive (n = 10) and capsaicin-insensitive nodose neurons (n = 24). (B) Box and whisker plot showing the percentage increase in IM tail current density by ICA-069673 in capsaicin-sensitive and capsaicin-insensitive nodose neurons, derived from data shown in (A). (C) Box and whisker plot of RP recorded in the absence (BL) and presence of 10 μm ICA-069673 (ICA) in capsaicin-sensitive (n = 8) and capsaicin-insensitive (n = 15) nodose neurons. (D) Box and whisker plot of ICA-069673-induced hyperpolarization (ΔRP) in capsaicin-sensitive (n = 8) and capsaicin-insensitive nodose neurons (n = 15), derived from data shown in (C) Cap: capsaicin. Box and whisker plots in (A)–(D): Horizontal lines of boxes represent 25th percentile, median, and 75th percentile. Whiskers represent 5th/95th percentile. The black dots outside the boxes and whiskers are outliers. The P values in (A) and (C) were determined by paired t test. The P values in (B) and (D) were determined by unpaired t test.

The effect of ICA-069673 on RP was examined in eight capsaicin-sensitive neurons and 15 capsaicin-insensitive neurons. The baseline RP values were similar between two groups (–60.2 ± 5.6 mV, n = 8 vs. –61.3 ± 3.4 mV, n = 15, P = 0.567). ICA-069673 at 10 μm hyperpolarized the capsaicin-sensitive neurons by 7.0 ± 2.9 mV and capsaicin-insensitive neurons by 8.7 ± 3.3 mV. The amplitudes of hyperpolarization were not significantly different (Fig. 6, C and D).

Discussion

Because KCNQ/KV7 channels have been increasingly recognized as promising therapeutic targets for an array of disorders (Miceli et al., 2008; Large et al., 2012; Barrese et al., 2018; Du et al., 2018; Vigil et al., 2020), considerable effort has been directed toward developing small molecule modulators of KV7 channels during the last two decades. Hundreds of newly synthesized, repurposed, or naturally occurred KV7 channel activators (openers) have been reported (Du et al., 2018; Miceli et al., 2018; Borgini et al., 2021). Many of these structurally different compounds have been evaluated for KV7 subunit selectivity in the heterologous expression systems. Currently available M-channel openers that are relatively selective for KCNQ2/3 channels over other KCNQ subunit compositions can be categorized into two classes: 1) the retigabine derivatives that binds in the channel pore domain, including RL-81 (Kumar et al., 2016), RL-56 (Hernandez et al., 2022), and SF-0034 (Kalappa et al., 2015); and 2) the benzamides that binds to the voltage sensor domain, including ICA-27243, ICA-069673, and ICA-110381. ICA-27243 is the first selective KCNQ2/3 channel opener reported in the literature. It is 20 times more potent as an activator of KV7.2/7.3 channels than homomeric KV7.4 channels, has an even weaker potentiating effect on KV7.3/7.5 channels, and does not activate homomeric KV7.3 channels (Wickenden et al., 2008; Padilla et al., 2009). ICA-27243 exhibits the antiepileptic activity in a board range of rodent seizure models, but failed the toxicity test (Roeloffs et al., 2008; Amato et al., 2011). ICA-069673, a pirimidine analog that has a less toxicity and an in vitro selectivity comparable to that of ICA-27243, was then identified and advanced into a phase I clinical trial (Amato et al., 2011). In addition to its antiepileptic effects, ICA-069673 has been shown to attenuate the spinal nociceptive transmission, hyperpolarize the spinal motor neurons, and reduce the excitability and burst duration in spinal locomotors-related interneurons (Vicente-Baz et al., 2016; Verneuil et al., 2020). Here, we report that ICA-069673 is a strong activator of M-channels in both capsaicin-sensitive and capsaicin-insensitive vagal sensory neurons. By enhancing the KV7.2/7.3-mediated IM in nodose neurons, ICA-069673 significantly suppressed their excitability and reduced the repetitive action potential firings in response to sustained depolarization.

ICA-069673 was first described as a potent KCNQ2/3 channel opener, being 20-fold selective for KCNQ2/3 over KCNQ3/5 channels and >150-fold over KCNQ1 channels (Amato et al., 2011). Later studies on the molecular mechanism of action revealed that ICA-069673 interacts with the voltage sensor domain (S1–S4) of KCNQ2 subunit. The KCNQ2 residues F168 and A181 in the S3 segment are essential determinants of ICA-069673 subunit specificity (Wang et al., 2017). In this study, we found that ICA-069673 enhanced M-currents in the vast majority of mouse nodose neurons (∼85%). The EC50 of IM potentiating effect was found to be 0.52 μm, similar to the 0.69 μm determined by rubidium efflux assay in CHO cells stably expressing the KCNQ2/KCNQ3 channels (Amato et al., 2011). Moreover, the effects of ICA-069673 on IM density and membrane potential were not different between capsaicin-sensitive and capsaicin-insensitive nodose neurons. These findings are consistent with our previous report that Kcnq2 and Kcnq3 are the major Kcnq genes expressed in mouse nodose neurons, and their expression profiles are similar in Trpv1-positive and Trpv1-negative nodose neurons (Sun et al., 2019). Importantly, the present study reveals that ICA-069673 has no effects on membrane currents in nodose neurons when the Kcnq2 and Kcnq3 genes are deleted. These results provide direct evidence that ICA-069673 specifically opens the KCNQ2/3 channels and enhance M-currents in mouse nodose neurons.

The activation of IM in mouse nodose neurons by ICA-069673 is characterized by a hyperpolarizing shift of voltage-dependent activation, an increase in the maximal conductance, an accelerated activation kinetics and a marked slow-down of deactivation, qualitatively similar to the reported effects of ICA-069673 on KCNQ2 homomeric channels and KCNQ2/3 heteromeric channels expressed in the heterologous systems (Wang et al., 2017, 2018). The IM enhancing effect of ICA-069673 differs from that of the channel pore-targeting retigabine in that retigabine does not increase the maximal conductance of the channels (Tatulian et al., 2001; Sun et al., 2019). The molecular mechanisms underlying the ICA-069673-induced channel gating shift and current potentiation remain unknown, but it appears that these two effects could be separated by certain mutations in the KCNQ2 voltage-sensing domain (Wang et al., 2017). Regardless of the mechanisms, the hyperpolarizing shift of M-channel activation curve and the increase in the maximal conductance caused a prominent enhancement of IM from around resting potential to potentials above the action potential firing threshold in nodose neurons. As a result, ICA-069673 induced a pronounced hyperpolarization and a drop of input resistance, which in turn led to an increase in the depolarizing current needed to evoke an action potential, and along with increasing activation of IM by increasing depolarization, limited the repetitive AP firings in response to sustained suprathreshold depolarization.

Another finding of this study is that ICA-069673 caused a more pronounced hyperpolarization in partially depolarized nodose neurons and effectively counteracted against the sustained depolarizing force. It is well known that vagal nociceptive neurons, but generally not non-nociceptive neurons, can be activated (firing action potential) or sensitized (evoking subthreshold generator potentials) by noxious stimuli such as environmental irritants and inflammatory mediators (Mazzone and Undem, 2016; Undem and Sun, 2020). In both cases (activation and sensitization), the membrane potential of nociceptive neurons is depolarized. It is tempting to speculate that the depolarized vagal nociceptive neurons, with heightened excitability, under the influence of various inflammatory mediators in visceral inflammatory diseases may be more sensitive to the inhibitory effects of ICA-069673.

In addition, the increase in subunit selectivity of a compound like ICA-069673 is likely to improve the therapeutic index over older less selective compounds by minimizing the effects on non-neuronal tissues. It should be recognized, however, that KCNQ2 is widely expressed in the central and peripheral nervous systems. To avoid the unwanted central effects, ICA-069673 could be applied locally to treat the peripheral hyperexcitability disorders. For example, in the case of respiratory disorders that likely involve over-activation of vagal C-fibers such as chronic pathologic cough, asthma, and chronic obstructive pulmonary disease (Mazzone and Undem, 2016), the KCNQ2/3 channels in the vagal nociceptive nerves can be reached topically by inhalation of the opener in a manner that would likely reduce unwanted systemic side effects. Our previous finding that inhaled retigabine substantially inhibits irritant gases-induced coughing in mice may be relevant in this regard (Sun et al., 2019). The role of KCNQ channel subtypes in regulating postganglionic autonomic nerves in the airways is unknown, so it remains possible that such a KCNQ opener strategy may also reduce their activity in a manner that would lead to reductions in parasympathetic reflex secretions and bronchoconstriction.

One hallmark feature of M-channels is their susceptibility to the modulation by an array of neurotransmitters and inflammatory mediators. Stimulation of Gq-coupled B2 receptor by bradykinin, protease-activated receptor 2 by proteases and P2Y1 receptor by ATP inhibits IM in nociceptive sensory neurons of dorsal root ganglia (DRG) (Linley et al., 2008; Liu et al., 2010; Yousuf et al., 2011). Nerve injury, tissue inflammation, or prolonged exposure to the mixture of inflammatory mediators has been shown to downregulate the expression of Kcnq2 and Kcnq3 in DRG neurons (Mucha et al., 2010; Rose et al., 2011; Zhang et al., 2019a, 2019b). A downregulation of M-channel expression and/or function may also occur in vagal sensory neurons exposed to inflammatory mediators or in visceral inflammation. In future studies, it would be important to determine whether ICA-069673 is still able to enhance the M-current and rescue the abnormal excitability of vagal sensory nerves under the pathologic conditions that partially inhibit the M-channel expression and function. In this regard, it has been reported that the non-selective M-channel opener retigabine and flupirtine effectively relieved the neuropathic pain and inflammatory pain, in a XE991-sensitive manner, in animal models where the expression of Kcnq2/3 and IM density in DRG neurons were reduced by half (Rose et al., 2011; Zheng et al., 2013; Zhang et al., 2019b). At the cellular level, the presence of bradykinin, that inhibits IM by 63%, did not reduce the efficacy of retigabine or flupirtine to hyperpolarize the DRG neurons (Linley et al., 2012).

In summary, this study demonstrated that ICA-069673 is a potent and effective M-channel opener in vagal sensory neurons. Activating the KCNQ2/3-mediated channels is sufficient to reduce the excitability and repetitive action potential discharges in these neurons. Future studies are warranted to evaluate its function in modulating the vagal nociceptive responses in health and in diseases where fewer side-effects may be predicted relative to that observed with nonselective KCNQ openers.

Acknowledgments

The authors would like to thank Anastasios Tzingounis at the University of Connecticut for providing the Kcnq2/3fl/fl mice, Xinzhong Dong at Johns Hopkins University for providing the Pirt-Cre mice, and Fei Ru for maintaining the Kcnq2/3 knockout mice.

Data Availability

The authors declare all data supporting the findings of this study are contained within the paper.

Abbreviations

AP

action potential

APthreshold

voltage threshold of action potential generation

BL

baseline

Cap

capsaicin

DRG

dorsal root ganglion

Gmax

maximal conductance

IM

M-current

Ipeak

peak of tail current

Iss

sustained level of tail current at the end of 1-s test pulse

Itail

tail current

Rinput

input resistance

RP

resting potential

Vm

membrane potential

Authorship Contributions

Participated in research design: Sun.

Conducted experiments: Sun.

Performed data analysis: Sun.

Wrote or contributed to the writing of the manuscript: Sun, Undem.

Footnotes

This work was supported by National Institutes of Health National Heart, Lung and Blood Institute [Grant R35HL155671].

No author has an actual or perceived conflict of interest with the contents of this article.

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