Potentiation of Nicotine-Induced Currents by QO58, a Kv7 Channel Opener, in Intracardiac Ganglion Neurons of Rats

Abstract

QO58 (5-(2,6-dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl)-1H-[1,5-a] pyrimidin-7-one) is currently used as a specific activator of the Kv7 (KCNQ) family of K⁺ channels. Here, we report an unexpected potentiating effect of this drug on nicotinic acetylcholine receptors. We recorded the whole-cell responses to the rapid application of nicotine with the Cs⁺-based pipette solution in intracardiac ganglion neurons freshly dissociated from the rat heart. Nicotine-induced inward currents were concentration-dependently blocked by mecamylamine, but not by 1 μM atropine at a holding potential of −60 mV. While the application of QO58 per se evoked a persistent inward current at this holding potential, 10 μM QO58 potentiated the peak amplitude of the nicotine-induced current. The QO58-induced inward currents were inhibited by the Kv7 channel blockers XE991 and Ba²⁺, but not by mecamylamine. On the other hand, the nicotine-induced current potentiated by QO58 was fully inhibited by mecamylamine. The facilitatory action of QO58 on the nicotinic response was unaffected by Ba²⁺. QO58 did not affect the reversal potential of the nicotine-induced current. QO58 apparently shifted the concentration–response curve of nicotine to the left. The half-maximal effective concentrations for nicotine in the absence and presence of 10 μM QO58 were 10.2 and 4.3 μM, respectively. These results suggest that QO58 acts as a positive allosteric modulator of nicotinic acetylcholine receptors. Given the prevalence of nicotinic receptor signaling, the present observations should be considered in future studies on the roles of Kv7 channels in the function of neural circuits and diseases.

INTRODUCTION

Nicotinic acetylcholine receptor (nAChR) is a member of the Cys-loop family of ligand-gated ion channels, which also includes cationic type 3 serotonin receptor and anionic glycine and type A γ-aminobutyric acid (GABA) receptors.^1–3) These receptors have an amino acid loop in the large extracellular domain linked by a disulfide bond. nAChRs respond to nerve-released acetylcholine to mediate excitatory synaptic transmission through the central and peripheral nervous systems. In the autonomic ganglia, nAChRs mediate cholinergic transmission from preganglionic fibers to postganglionic neurons. Sixteen different nAChR subunits are currently known, and a (α1–7, α9, and α10), β (β1–4), γ, δ, and ε subunits have been identified.¹⁾ Neuronal nAChRs contain α2–6 subunits that are usually expressed as heteropentamers in combination with β2–4 subunits and are found in the central and peripheral nervous systems. The α3β4 receptor, which appears to predominate in sensory and autonomic ganglia, is sometimes called “ganglionic nAChR.”³⁾ The intracardiac neurons primarily express nAChRs containing α3β2 and α3β4 receptors.⁴⁾

Kv7 (known as KCNQ) channels are voltage-dependent K⁺ channels composed of homomeric and heteromeric complexes of 5 different subunits in the neuronal plasma membrane.⁵⁾ Kv7 channels are responsible for the M-current, which decreases neuronal excitability and prevents overexcitation of neurons. Loss-of-function mutations in Kv7 channels cause epilepsy and arrhythmia.⁶⁾ In addition, transcriptional downregulation of these channels in sensory nerves leads to chronic pain.⁷⁾ Interestingly, nicotine is reported to enhance the firing activity of cortical neurons through the inhibition of Kv7 channels.⁸⁾ Conversely, activators of Kv7 channels exhibit antiepileptic or analgesic activities.^9,10) Neuronal Kv7 channels are important targets for various drugs, including channel openers and blockers.¹¹⁾ Therefore, studies on the development of new and selective Kv7 channel openers are necessary.

The modulators of Kv7 channels possess pharmacological features that are not restricted to their effects on Kv7 K⁺ channels. Linopirdine, an inhibitor of Kv7 channels, inhibits nAChR and GABA receptors.¹²⁾ The Kv7 channel activator, retigabine, modulates GABA_A receptors containing δ subunits.¹³⁾ XE991, an inhibitor of Kv7 channels, inhibits the nAChR-mediated response in acutely isolated intracardiac ganglion neurons of rats.¹⁴⁾ QO58 (5-(2,6-dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl)-1H-pyrazolol[1,5-a]pyrimidin-7-one) is a potent activator of Kv7 channels with analgesic properties.^15,16) QO58 is also reported to activate large-conductance Ca²⁺-activated K⁺ channels.¹⁷⁾ However, whether this compound modulates other types of membrane ion channels in native cells has not been thoroughly investigated. Therefore, in the present study, we investigated the effect of QO58 on nicotine-induced currents in acutely isolated intracardiac ganglion neurons with voltage–clamp recordings using the patch-clamp technique.

MATERIALS AND METHODS

Neuron Isolation

The present study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals prescribed by the Japanese Physiological Society. All animal experiments were approved by the Kitasato University Institutional Animal Care and Use Committee (Approval Numbers: EiKen19-10-4, 19-10-5, and 24-04-1).

Experiments were performed on intrinsic cardiac neurons freshly dissociated from 2-week-old Wistar rats. The procedure for isolating ganglion neurons was similar to that used in our previous studies.^18,19) Briefly, rats were terminally anesthetized with isoflurane. The heart and lungs were quickly excised, and the ganglia at the outer surface of the atria were rapidly removed.²⁰⁾ Then, the ganglia were treated with a standard external solution containing 0.4% collagenase and 0.4% trypsin for 60 min at 35°C. Ganglion neurons were mechanically dissociated by trituration with fire-polished Pasteur pipettes in a culture dish (Primaria 3801; Becton Dickinson, Rutherford, NJ, U.S.A.). Dissociated neurons adhered to the bottom of the dish within 20 min. Isolated neurons were used 1–6 h after preparation.

Solutions and Chemicals

The ionic composition of the standard external solution was (mM) as follows: NaCl 150, KCl 2.5, MgCl₂ 1, CaCl₂ 2, N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid (HEPES) 10, and glucose 10. pH was adjusted to 7.4 using tris(hydroxymethyl)aminomethane (Trizma-base). The composition of the patch-pipette (internal) solution for perforated patch-clamp recording was (mM): CsCl 70, cesium methanesulfonate 80, HEPES 10. pH was adjusted to 7.3 using Trizma-base. In some experiments, 150 mM Cs⁺ in the pipette solution was replaced with 150 mM K⁺. It should be noted that both Cs⁺-based and K⁺-based pipette solutions contain the same (70 mM) concentration of Cl^–. Amphotericin B was dissolved in dimethyl sulfoxide (DMSO) to prepare a 100 mg·mL^–1 stock solution, which was added to the internal solution at a final concentration of 500 μg·mL^–1 just before use. Test solutions were topically applied using the so-called Y-tube solution exchange device.²¹⁾

Drugs used in the present study were amphotericin B, atropine, collagenase, mecamylamine, nicotine, Trizma-base, trypsin (Sigma-Aldrich, St. Louis, MO, U.S.A.), and QO58 and XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone) (Tocris, Cookson, Avonmouth, U.K.). All the other reagents were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). QO58 was prepared as a 20 mM stock solution in DMSO and diluted with the standard external solution. The final concentration of DMSO did not exceed 0.05%.

Electrophysiological Recordings

Electrical measurements were performed in perforated patch-clamp recording mode using amphotericin B.²²⁾ Patch pipettes were made from borosilicate glass tubes in 2 stages on a vertical pipette puller (PC-10, Narishige, Tokyo, Japan). The resistance between the recording electrode filled with the internal solution and the reference electrode in the normal external solution was 3–8 MΩ. Current signals were amplified using a patch-clamp amplifier (EPC-7plus; List-Medical, Darmstadt, Germany). After the formation of stable perforated patches, the series resistance ranged between 10 and 22 MΩ and was compensated in the same manner as previously described.¹⁸⁾ Before digitization (at a sampling rate of 10 kHz), the signals were filtered at 2 kHz using a 3-pole low-pass Bessel-type filter. The data were stored on the computer hard disk for subsequent analysis using a pClamp 10 system (Axon Instruments, Foster City, CA, U.S.A.). All experiments were performed at room temperature (21–24°C). Cellular capacitance was calculated as the area under capacitive transients divided by the amplitude of a hyperpolarizing test pulse (5–10 mV). When constructing current–voltage (I–V) relationships, the currents were normalized to cell capacitance.

Statistical Analysis

Statistical significance was determined by Student’s t-test for unpaired or paired data, or by one-way ANOVA followed by Dunnett’s multiple comparison test where appropriate. In all instances, p < 0.05 was considered statistically significant. To construct the concentration–response curve, the data were fitted into a modified Michaelis–Menten equation using least-squares fitting with the following formula:

  

$$I=(I_{\max }{\mathrm{C}}^{\mathrm{n}})/({\mathrm{C}}^{\mathrm{n}}+\mathrm{E}{\mathrm{C}}_{50}),$$(1)

where I is the normalized current amplitude; I_max is the maximum response; C is the agonist concentration; EC₅₀ is the concentration at which a half-maximum response occurs; and n is the Hill coefficient. The concentration–inhibition curves were drawn according to the following equation:

  

$$I=1-{\mathrm{C}}^{\mathrm{n}}/({\mathrm{C}}^{\mathrm{n}}+\mathrm{I}{\mathrm{C}}_{50}),$$(2)

where I is the normalized current amplitude, C is the antagonist concentration, IC₅₀ is the antagonist concentration that produced the half-maximal response, and n is the Hill coefficient.

RESULTS

The Nicotine-Induced Currents

To characterize the electrophysiological properties of nicotine response, perforated patch-clamp recordings were conducted on isolated intracardiac ganglion neurons of rats under voltage–clamp conditions using a Cs⁺-based pipette solution. At a holding potential of −60 mV, 10 μM nicotine elicited a fast inward current. The muscarinic receptor antagonist atropine (1 μM) had no effect on the inward current (Fig. 1A). In 6 neurons tested, the nicotine-induced inward currents in the absence and presence of atropine were 453.7 ± 73.1 and 460.2 ± 66.5 pA, respectively (p = 0.68). At this concentration, atropine is reported to completely inhibit the muscarinic response.²³⁾ By contrast, the nicotine-induced current was concentration-dependently inhibited by the nicotinic receptor antagonist, mecamylamine, with an IC₅₀ of 0.57 ± 0.11 μM (n = 5, Fig. 1B). This IC₅₀ value is comparable to that observed for recombinant nicotinic receptors.²⁴⁾ These results suggest that nicotine activates nAChR in intracardiac neurons of rats.

Fig. 1. Nicotine-Induced Currents in Acutely Isolated Intracardiac Ganglion Neurons of Rats

(A) Effect of atropine on the nicotine-induced currents. Each column represents the average value ± S.E.M. from 6 neurons. (B) Concentration-dependent inhibition of the nicotine-induced currents by the nicotinic antagonist mecamylamine. Each point represents the average value ± S.E.M. from 5 neurons. S.E.M.: standard error of the mean.

Potentiation of the Nicotine-Induced Currents by QO58

As shown in Fig. 2A, the nicotine-induced current was enhanced by 10 μM QO58, while QO58 per se produced small and persistent inward currents at a holding potential of –60 mV. This potentiating effect of QO58 was reversed by washout (Fig. 2B). The action of QO58 on nicotinic response was concentration-dependent, and QO58 significantly enhanced the nicotinic response at concentrations ≥3 μM (Fig. 2C). Since 0.1% DMSO is reported to inhibit the nAChR-mediated current with 1-min preincubation,²⁵⁾ the effects of QO58 at concentrations >10 μM were not studied in the present study. As shown in Fig. 3A, the potentiated current in the presence of 10 μM QO58 was largely inhibited by 6 μM mecamylamine (92.9 ± 1.5% inhibition, n = 5). On the other hand, mecamylamine (6 μM) produced no detectable effect on the Q58-induced currents (n = 6, data not shown). The inorganic Kv7 channel inhibitor Ba²⁺ (1 mM) affected neither the nicotine-induced current nor the potentiating effect of QO58 on the nicotinic response (Fig. 3B).

Fig. 2. Potentiation of the Nicotine-Induced Current by QO58

(A) Representative current trace showing the nicotinic response before and during application of QO58. (B) Time course of the effects of 10 μM QO58 and 0.05% DMSO on the nicotine-induced current. Data were normalized to the current amplitude recorded at time zero. Drugs were applied during the period indicated by the bars. Each point represents mean ± S.E.M. determined from 5 neurons. (C) Concentration-dependent effect of QO58. Each point represents the average value ± S.E.M. from 5 to 9 neurons. **p < 0.01, ***p < 0.001 vs. the DMSO-treated group. S.E.M.: standard error of the mean.

Fig. 3. Effects of Mecamylamine and Ba²⁺ on the QO58 Action

(A) Representative current traces illustrating the inhibition of the nicotine-induced current by mecamylamine in the presence of QO58. (B) Effect of the inorganic Kv7 channel blocker Ba²⁺ on the QO58 action. Each column indicates the mean ± S.E.M. from 5 to 9 neurons. n.s. indicates not significant. S.E.M.: standard error of the mean.

Properties of the QO58-Induced Currents

To characterize the currents induced by QO58, the current–voltage (I–V) relationship of the QO58-induced currents was investigated. The QO58-induced currents at different holding potentials with an internal solution containing 150 mM Cs⁺ are shown in Fig. 4A. The reversal potential, which was determined by interpolation in each neuron, was –31.7 ± 3.1 mV (n = 5; Fig. 4C). The replacement of Cs⁺ in the pipette solution with K⁺ resulted in a negative shift of the reversal potential to –78.5 ± 3.0 mV (n = 5; Figs. 4B and 4C). These results suggest that the Kv7 channel opener QO58 activated K⁺ channels. Therefore, the effects of Kv7 channel inhibitors on the QO58-induced currents were also examined. As shown in Fig. 5, Ba²⁺ and XE991 markedly inhibited the QO58-induced currents recorded with Cs⁺-based pipette solution at a holding potential of –60 mV.

Fig. 4. The QO58-Induced Currents Measured at Various Holding Potentials

(A, B) Representative current traces are measured at 3 different holding potentials, which are indicated above each trace. Recordings were performed with Cs⁺-based (A) and K⁺-based (B) pipette solutions. (C) Current–voltage relationships of the QO58-induced currents. The solid line indicates the least-squares fit to the experimental data. Each point represents the average value ± S.E.M. from 5 neurons. S.E.M.: standard error of the mean.

Fig. 5. Effects of Kv7 Channel Inhibitors on the QO58-Induced Currents

(A) Representative current traces showing the effects of 1 mM Ba²⁺ and 10 μM XE991 on the currents evoked by 10 μM QO58. (B) Inhibitory actions of 1 mM Ba²⁺ and 10 μM XE991 on the QO58-induced current. Each column is the average of 5 neurons.

Current–Voltage Relationship of the Nicotinic Response

Neuronal nicotinic receptors are reported to display strong inward rectification. These receptors conduct larger inward currents at membrane potentials negative to the reversal potential than outward currents at positive voltages.^23–25) In the present study, as shown in Fig. 6, the nicotine-induced current exhibited inward rectification. The precise reversal potential could not be determined because of rectification. However, the nicotine-induced currents in the absence and presence of QO58 reversed near 0 mV. These results suggest that QO58 potentiated the nicotine-induced current without shifting the reversal potential.

Fig. 6. Current–Voltage Relationship for the Nicotine-Induced Currents

(A) Typical current traces evoked by 10 μM nicotine at holding potentials of +20 and –60 mV. The baseline was adjusted to the level just before applying the agonist. (B) Current–voltage relationships for the nicotine-induced currents in the absence and presence of 10 μM QO58. To further illustrate the current–voltage relationships, line segments were drawn between data points. Each point represents the average value ± S.E.M. from 5 neurons. S.E.M.: standard error of the mean.

Concentration–Response Relationship

The nicotine-induced current increased with increasing agonist concentrations. As presented in Fig. 7, under control conditions, a graph of the normalized concentration–response data for the peaks of nicotine-induced current recordings from 5 cells showed a typical sigmoid curve with an EC₅₀ of 10.4 ± 0.4 μM. QO58 shifted the concentration–response curve for nicotine to the left. The EC₅₀ value in the presence of 10 μM QO58 was 4.4 ± 0.4 μM (n = 5; p < 0.001).

Fig. 7. Effect of QO58 on the Concentration–Response Relationship for Nicotine

Nicotine was applied to the neuron in the absence (closed circle) or presence (open circle) of 10 μM QO58. Each point represents a mean peak response (n = 5) normalized with respect to the control response to 10 μM nicotine alone. Each point represents the average value ± S.E.M. from 5 neurons. S.E.M.: standard error of the mean.

DISCUSSION

The present study clearly showed that QO58 reversibly potentiated the nicotine-induced currents. This drug also induced inward currents when recorded with a Cs⁺-based pipette solution at a holding potential of –60 mV. Since Kv7 channels are reported to be scarcely activated at membrane potentials negative to –60 mV,²⁶⁾ we first suspected that QO58 by itself activated the nAChRs, thereby causing inward currents. However, the QO58-induced inward shift of the holding current was not reversed by the nAChR antagonist mecamylamine (Fig. 3A). Instead, we found that the currents evoked by QO58 were markedly inhibited by Ba²⁺ and XE991, Kv7 channel inhibitors (Fig. 5). QO58 is reported to shift the voltage-dependent activation curve of Kv7 channels in a more negative direction.¹⁶⁾ Furthermore, Zhang et al.¹⁵⁾ reported that QO58 produced significant hyperpolarization from the resting membrane potential of –59 to –78 mV in isolated rat dorsal root ganglion neurons and that this hyperpolarization was fully inhibited by XE991. Intracardiac ganglion neurons certainly express Kv7 channels.¹⁹⁾ Taken together, these observations suggest that the QO58-induced inward currents observed at a holding potential of –60 mV are due to the activation of the Kv7 channels.

The Kv7 channel is reported to be more permeable to K⁺ than to Cs⁺.²⁷⁾ As shown in Fig. 4, the QO58-induced currents in neurons filled with 150 mM Cs⁺ were reversed at –31.7 mV. By contrast, the QO58-induced currents recorded with the K⁺-based pipette solution were reversed at –78.5 mV, which was close to the equilibrium potential of K⁺ calculated from K⁺ concentrations in the present experimental environment. The shift in the reversal potential (DErev) was –46.8 mV. Because the concentrations of monovalent cations (K⁺ or Cs⁺) in the patch–pipette solutions were the same, the permeability of an internal cation, Cs⁺, relative to that of K⁺, is defined by the following relationship from the Nernst–Planck equation:

  

$${\mathrm{P}}_{\mathrm{C}\mathrm{s}}/{\mathrm{P}}_{\mathrm{k}}={\mathrm{e}}^{-(\mathrm{F}\mathrm{\Delta }\mathrm{E}\mathrm{r}\mathrm{e}\mathrm{v}/\mathrm{R}\mathrm{T})},$$(3)

where F is Faraday’s constant; R is the universal gas constant; and T is the absolute temperature. The relative Cs⁺ permeability obtained using this equation was 0.16. This value is in good agreement with that previously reported for the Kv7 K⁺ channels in sympathetic ganglion neurons.²⁷⁾ Therefore, these results also support the idea that QO58-induced currents recorded with the Cs⁺-based pipette solution were generated owing to the activation of Kv7 channels.

The nicotinic receptor has a nonselective cation channel that is permeable to Na⁺, K⁺, Cs⁺, and Ca²⁺. Because the flux of specific ions through ion channels depends on their balance with other permeant ions, the activation of Kv7 K⁺ channels by QO58 might have changed the driving force for cation flux through nicotinic receptors on the plasma membrane, thereby increasing the nicotinic response. However, QO58 did not change the reversal potential of nicotine-induced currents (Fig. 6). Therefore, the action of QO58 on the nicotinic response was not because of an increase in the driving force for cations. Furthermore, the potentiating effect of QO58 on the nicotine-induced current was not inhibited by the inorganic Kv7 channel blocker Ba²⁺ (Fig. 3), suggesting that the QO58 action on the nicotine-induced current is not due to the functional interaction between Kv7 channels and nAChRs.

Neuronal nAChRs are pentameric ligand-gated ion channels. Each nAChR has at least 2 agonist-binding sites located at the interface between 2 subunits.²⁸⁾ In addition to the agonist-binding sites, allosteric-binding sites have also been identified within the extracellular and transmembrane domains of nAChRs. So far, a lot of ligands have been reported to act as allosteric modulators for neuronal nAChRs.²⁹⁾ Positive allosteric modulators are compounds that increase the receptor response induced by an agonist, while negative allosteric modulators decrease the receptor response. Since QO58 did not directly activate nAChRs on its own, QO58 may act as a positive allosteric modulator of nAChRs. As shown in Fig. 7, QO58 produced a leftward shift in the concentration–response curve for nicotine, significantly reducing the EC₅₀ of nicotine. The results suggest that QO58 enhances the agonist binding to the resting state of the receptor. The positive allosteric modulator galantamine is also reported to cause a parallel shift toward the left in the concentration–response curve of ACh.³⁰⁾ The leftward shift of the concentration–response curve was also observed in the positive allosteric modulation of GABA response by benzodiazepines.³¹⁾

Recently, positive allosteric modulators of nAChRs have emerged as promising drugs for neurological disorders such as Alzheimer’s disease, Parkinson’s disease, and schizophrenia.^32,33) While the mechanism of allosteric modulation of nicotinic response by QO58 needs to be further clarified, the enhancement of nicotinic response by QO58 observed in the present study will contribute to the future pharmaceutical development of positive allosteric modulators of nAChRs. On the other hand, the Kv7 voltage-gated potassium channels are important determinants of the excitability of the cardiac and neuronal membranes. The pharmacological activation of neuronal Kv7 channels alleviates chronic pain; therefore, they are considered a potential therapeutic target of analgesics. Therefore, care must be exercised when interpreting the data generated with QO58 as it is not entirely Kv7 channel selective.

Acknowledgments

This research was financially supported by JSPS KAKENHI (Grant Numbers: 19K07287 and 24K14655).

Conflict of Interest

The authors declare no conflict of interest.

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