The K_V7 channel activator retigabine suppresses mouse urinary bladder afferent nerve activity without affecting detrusor smooth muscle K⁺ channel currents

Abstract

Key points

• K_V7 channels are a family of voltage‐dependent K⁺ channels expressed in many cell types, which open in response to membrane depolarization to regulate cell excitability.

• Drugs that target K_V7 channels are used clinically to treat epilepsy. Interestingly, these drugs also cause urinary retention, but it was unclear how.

• In this study, we focused on two possible mechanisms by which retigabine could cause urinary retention: by decreasing smooth muscle excitability, or by decreasing sensory nerve outflow.

• Urinary bladder smooth muscle had no measurable K_V7 channel currents. However, the K_V7 channel agonist retigabine nearly abolished sensory nerve outflow from the urinary bladder during bladder filling.

• We conclude that K_V7 channel activation likely affects urinary bladder function by blocking afferent nerve outflow to the brain, which is key to sensing bladder fullness.

Abstract

K_V7 channels are voltage‐dependent K⁺ channels that open in response to membrane depolarization to regulate cell excitability. K_V7 activators, such as retigabine, were used to treat epilepsy but caused urinary retention. Using electrophysiological recordings from freshly isolated mouse urinary bladder smooth muscle (UBSM) cells, isometric contractility of bladder strips, and ex vivo measurements of bladder afferent activity, we explored the role of K_V7 channels as regulators of murine urinary bladder function. The K_V7 activator retigabine (10 μM) had no effect on voltage‐dependent K⁺ currents or resting membrane potential of UBSM cells, suggesting that these cells lacked retigabine‐sensitive K_V7 channels. The K_V7 inhibitor XE‐991 (10 μM) inhibited UBSM K⁺ currents; the properties of these currents, however, were typical of K_V2 channels and not K_V7 channels. Retigabine inhibited voltage‐dependent Ca²⁺ channel (VDCC) currents and reduced steady‐state contractions to 60 mM KCl in bladder strips, suggesting that reduction in VDCC current was sufficient to directly affect UBSM function. To determine if retigabine altered ex vivo bladder sensory outflow, we measured afferent activity during simulated transient contractions (TCs) of the bladder wall. Simulated TCs caused bursts of afferent activity that were nearly abolished by retigabine. The effects of retigabine were blocked by co‐incubation with XE‐991, suggesting specific activation of K_V7 channels on afferent nerves. These results indicate that retigabine primarily affects urinary bladder function by inhibiting TC generation and afferent nerve activity, which are key to sensing bladder fullness. Any direct inhibition of UBSM contractility is likely to be from non‐specific effects on VDCCs and K_V2 channels.

Key points

• K_V7 channels are a family of voltage‐dependent K⁺ channels expressed in many cell types, which open in response to membrane depolarization to regulate cell excitability.

• Drugs that target K_V7 channels are used clinically to treat epilepsy. Interestingly, these drugs also cause urinary retention, but it was unclear how.

• In this study, we focused on two possible mechanisms by which retigabine could cause urinary retention: by decreasing smooth muscle excitability, or by decreasing sensory nerve outflow.

• Urinary bladder smooth muscle had no measurable K_V7 channel currents. However, the K_V7 channel agonist retigabine nearly abolished sensory nerve outflow from the urinary bladder during bladder filling.

• We conclude that K_V7 channel activation likely affects urinary bladder function by blocking afferent nerve outflow to the brain, which is key to sensing bladder fullness.

Introduction

The urinary bladder performs two seemingly simple functions: to store urine and to void it when necessary. However, this seeming simplicity belies the complexity of the underlying process, which requires integration of afferent sensory nerve signals that communicate bladder fullness during filling with efferent autonomic nerve signals that drive coordinated contraction of urinary bladder smooth muscle (UBSM) cells in the bladder wall during voiding (Satchell & Vaughan, 1989; Yoshimura et al. 1996). During filling, the urinary bladder exhibits transient contractions (TCs) caused by excitation of a small area of the bladder wall, which increases intravesical pressure rapidly without generating enough pressure to cause voiding (Drake et al. 2017). These TCs also activate bursts of sensory afferent nerve activity, which may convey to the central nervous system both a sense of bladder fullness and an indication that wall tension is optimal for efficient voiding (de Groat & Yoshimura, 2009; Heppner et al. 2016). This makes afferent activity associated with TCs an attractive strategy for regulating bladder function; however, it is unclear what generates TCs and how TCs activate sensory nerves.

Transient contractions are caused, in part, by local increases in UBSM excitability that can spread through a small number of electrically coupled muscle bundles (Hashitani et al. 2001). The upstroke of UBSM action potential is driven by calcium entry through voltage‐dependent calcium channels (VDCCs), which also delivers Ca²⁺ ions for contraction. Conversely, K⁺ channels are responsible for decreasing UBSM excitability by repolarizing UBSM membrane potential and maintaining a prolonged afterhyperpolarization (Heppner et al. 1997). Multiple types of K⁺ channels are expressed in UBSM, each of which are responsible for exerting a hyperpolarizing influence on UBSM membrane potential under different circumstances. During Ca²⁺‐driven action potentials, large‐conductance Ca²⁺‐activated K⁺ (BK) channels and voltage‐dependent K⁺ channels (K_V2.1/5.1) are responsible for repolarization and afterhyperpolarization (Heppner et al. 1997; Thorneloe & Nelson, 2003; Herrera & Nelson, 2004). Activation of ATP‐sensitive K⁺ (K_ATP) channels causes membrane potential hyperpolarization, which decreases action potential frequency (Heppner et al. 1996; Petkov et al. 2001). K⁺ channel‐opening drugs, particularly those that activate K_ATP and BK channels, have been explored as therapeutic agents to treat overactive bladder by decreasing UBSM excitability (Gopalakrishnan & Shieh, 2004). The approach of targeting UBSM has been uniformly unsuccessful, however, largely due to the off‐target effects on other types of smooth muscle including vascular smooth muscle (Jackson, 2000). Interestingly, the K_V7 family of voltage‐dependent K⁺ channels has recently garnered considerable attention as potential regulators of bladder function by virtue of the intriguing observation that retigabine, an activator of this channel that is used clinically to treat epilepsy, causes urinary retention (Brickel et al. 2012). A number of reports support a possible role of K_V7 channels in UBSM and vascular smooth muscle (Robbins, 2001; Mackie & Byron, 2008), but retigabine does not affect blood pressure in humans (Jepps et al. 2013). However, if urinary retention caused by retigabine were due to a direct effect on UBSM, then it is unclear why blood pressure would be unaffected, given that both types of smooth muscle reportedly contain K_V7 channels.

K_V7 channels, also known as KCNQ channels or ‘M channels’, are a subfamily of K_V channels that activate at membrane potentials positive to −60 mV, show little or no inactivation over time, and have distinct electrophysiological characteristics (Table 1; Robbins, 2001; Anderson et al. 2013). These channels have been well‐characterized in nerves and in the heart, where they regulate neuronal resting membrane potential and cardiac myocyte repolarization, respectively (Wulff et al. 2009). K_V7 channels have a prominent role in both the central and peripheral nervous systems, including sensory nerves that innervate the urinary bladder (Brown & Passmore, 2009; Aizawa et al. 2017). K_V7.1–7.4 transcripts have been detected in whole‐bladder extracts from guinea pig and rat, whereas primarily K_V7.2 and K_V7.3 are reported in guinea pig UBSM (Svalø et al. 2012; Anderson et al. 2013; Provence et al. 2015, 2018). Expressed K_V7.2 and K_V7.3 channels exhibit a half‐activation voltage of about −40 mV (Table 1), which is roughly the normal resting potential of UBSM as measured with microelectrodes in intact tissue (Heppner et al. 1997; Takagi & Hashitani, 2016). K_V channel currents in murine UBSM exhibit a half‐activation voltage of about 0 mV, and prominent inactivation, consistent with the properties of a heteromultimeric channel composed of K_V2.1 and K_V5.1 and/or K_V6 subunits (Thorneloe & Nelson, 2003). Although K_V channel currents in UBSM do not have biophysical characteristics of Kv7 channels, it is possible that K_V7 channel currents are a minor component of the K_V channel currents.

Table 1.

Electrophysiological properties of K_V7 channel subtypes

Channel	Conductance (pS)	V 0.5 activation (mV)	Slope, k (mV)	τ (ms) (holding potential)	References
KV7.1	3.2	−23.0	10.0	70.0 (+20 mV)	Tristani‐Firouzi & Sanguinetti (1998), Selyanko et al. (2000), Werry et al. (2013)
KV7.2	6.2	−40.0 to −37.0	12.1	132.0 (−40 mV)	Biervert et al. (1998), Selyanko et al. (2000), Soldovieri et al. (2007)
KV7.3	8.5	−37.0 to −29.0	5.5	64.4 (+40 mV)	Selyanko et al. (2000), Li et al. (2004)
KV7.4	2.1	−13.0 to −28.0	9.8	163.0 (+40 mV)	Selyanko et al. (2000), Gamper et al. (2003)
KV7.5	2.2	−48.0 to −41.0	9.5	119.0 (+40 mV)	Lerche et al. (2000), Huang & Trussell (2011)

Data are reported values in expression systems (Xenopus oocytes or CHO cells).

In this study, we comprehensively explored the possible role of K_V7 channels as regulators of sensory outflow or UBSM excitability in mice. We found no electrophysiological evidence to support the presence of functional K_V7 channels in freshly dissociated UBSM cells; however, patch‐clamp experiments revealed that the K_V7 activator retigabine inhibited VDCCs in these cells. Interestingly, in absence of smooth muscle activity, retigabine did inhibit baseline afferent nerve activity during filling of ex vivo whole bladders and eliminated afferent bursts of activity in response to mechanical pinches of the bladder to simulate TCs. Although retigabine did not directly affect UBSM cell K_V currents, it did inhibit TCs and associated bursts of bladder afferent nerve activity in an ex vivo bladder preparation. We conclude that K_V7 channel activation likely affects urinary bladder function by inhibiting the generation of TCs and afferent nerve activity, both of which are key to sensing bladder fullness.

Methods

Ethical approval

The animal experiments were conducted in accordance with the animal care protocols approved by the Institute of Animal Care and Use Committee of the University of Vermont (animal welfare assurance number A3301‐01), and conform to the principles and regulations as of The Journal of Physiology (Grundy, 2015).

Animal care and use

Male C57BL/6 mice (3–4 months of age) were euthanized by intraperitoneal injection of sodium pentobarbital (150 mg/kg) followed by decapitation.

Urinary bladder smooth muscle cell isolation

Single UBSM cells were obtained from mouse detrusor muscle using a modified enzymatic digestion technique, as described previously (Thorneloe & Nelson, 2003). Briefly, UBSM was isolated, cut into 1–2 mm‐wide strips, and placed in dissociation solution containing (mM): 6 KCl, 55 NaCl, 80 sodium glutamate, 2 MgCl₂, 10 HEPES and 7 glucose (pH 7.3). Strips were first digested for 18 min at 37°C in 1.5 ml of dissociation solution containing 0.5 mg/ml papain (Worthington Biochemical, Lakewood, NJ, USA) and 0.5 mg/ml dithioerythreitol. Strips were then digested for 10 min at 37°C in dissociation solution containing 1 mg/ml collagenase II and 0.1 mM CaCl₂. After washout in dissociation solution, cells were obtained by gentle trituration with a wide‐bore pipette.

Electrophysiology

For outward current (K_V channel) measurements, current measurements were performed using the conventional whole cell (CWC) configuration of the patch‐clamp technique. The bath solution contained (mM): 6 KCl, 134 NaCl, 0.1 MgCl₂, 0.1 CaCl₂, 10 HEPES and 7 glucose (pH 7.4; 25°C). Paxilline (1 μM) was also added to the bath solution to inhibit BK channel currents. The pipette solution contained (mM): 134 KCl, 6 KOH, 10 NaOH, 1 MgCl₂, 5 EGTA, 10 HEPES, 1.866 CaCl₂ and 2 Mg·ATP (pH 7.2). This resulted in a pipette solution containing 100 nM free Ca²⁺, as calculated using online Max‐Chelator software (Chris Patton, Stanford University, CA, USA). K_V currents were elicited by 500 ms steps from −60 to +20 mV (10 mV increments, 10 s pause between steps). For experiments in 95 mM K⁺, current measurements were performed using the perforated patch‐clamp technique. Bath solutions were prepared as above, except with equimolar replacement of NaCl with KCl. Current was then recorded with membrane potential clamped at −40 mV. For Ca²⁺ channel measurements, dissociated cells were placed in the recording chamber and superfused with solution containing (mM) 10 BaCl₂, 6 KCl, 122 NaCl, 1 MgCl₂, 10 HEPES, and 7 glucose (pH 7.4; 25°C). The pipette solution contained (mM): 137.4 CsCl, 12.6 CsOH, 1 MgCl₂, 5 EGTA, 10 HEPES and 2 Mg·ATP (pH 7.2, adjusted with CsOH). Inward Ca²⁺ currents in response to 200 ms steps (from −60 mV to +10 mV) were recorded every 30–60 s.

Membrane potential measurements were performed using the perforated patch‐clamp technique in current clamp mode (I = 0). The bath solution contained (mM): 6 KCl, 134 NaCl, 1 mM MgCl₂, 0.1 CaCl₂, 10 HEPES and 7 glucose (pH 7.4; 25°C). The pipette solution contained (mM) 30 KCl, 110 potassium aspartate, 10 NaCl, 1 MgCl₂, 10 HEPES and 200 μg/ml amphotericin B (pH 7.2; 25°C). Currents or membrane potentials were filtered at 2 kHz and digitized at 20 kHz using an Axopatch 200B amplifier and Digidata 1322A Data Acquisition System (Molecular Devices, Sunnyvale, CA, USA). All currents were recorded and analysed using ClampEx 9 software (Molecular Devices).

Isometric contractility

Isometric contractility was measured as described previously (Herrera et al. 2003; Heppner et al. 2009). Briefly, bladders were dissected, placed in Ca²⁺‐free HEPES‐buffered physiological saline solution (HEPES‐PSS), and cut into strips (2–3 mm  ×  5–7 mm). Isometric contractility was then measured using an 820MS Muscle Strip Myography System (Danish Myo Technology, Aarhus, Denmark). Tissues were warmed to 37°C in aerated (20% O₂, 5% CO₂, balance N₂) PSS, and passive tension of 1 g was applied. For high‐K⁺ experiments, isosmotic replacement of KCl for NaCl was used to achieve a concentration of 60 mM K⁺ in the tissue bath. Contractions elicited by 60 mM K⁺ were allowed to plateau for 5 min prior to addition of agonist, antagonist, and vehicle control solutions. Data were analysed using LabChart 7 (ADInstruments) and GraphPad Prism 6 (GraphPad Software).

Ex vivo bladder preparation

Ex vivo bladder experiments were conducted as described previously (Heppner et al. 2016). Briefly, the urinary bladder, with associated ureters, urethra, major pelvic ganglia and pelvic nerves, was removed and placed in ice‐cold HEPES‐buffered PSS consisting of (mM): 134 NaCl, 6 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES and 7 glucose (pH 7.4). The ureters were tied adjacent to the bladder wall, and the pelvic nerves were exposed and cleaned of connective tissue. The bladder preparation was then placed in the recording chamber and superfused with bicarbonate‐buffered PSS consisting of (mM): 118.5 NaCl, 4.6 KCl, 1.2 KH₂PO₄, 1.2 MgCl₂, 2 CaCl₂, 24 NaHCO₃ and 7 glucose; the pH of the solution was maintained at 7.4 by bubbling with biological atmosphere gas (20% O₂, 5% CO₂, balance N₂). All experiments were performed at 37°C. The urethra was cannulated and attached to a syringe pump for continuous filling with PSS at 1.8 ml/h. At a pressure of 25 mmHg, filling was stopped, and the bladder was emptied. Bladder pressure was measured using a pressure transducer and a PS‐200 Pressure Servo Controller (Living Systems Instrumentation, St Albans, VT, USA). One of the pelvic nerves distal to the pelvic ganglia was attached to a suction electrode for electrophysiological recordings. All drugs were added directly to the recirculating bath.

Afferent nerve recordings

Afferent nerve‐recording experiments were conducted as described previously (Heppner et al. 2016). Briefly, one pelvic nerve was attached to a fire‐polished glass tip (tip opening, ∼100 μm) of a suction electrode. Action potentials from the pelvic nerve were recorded using a Neurolog headstage (NL100AKS, Digitimer, Welwyn Garden City, UK), amplified with an AC preamplifier (NL104, Digitimer), and band‐pass filtered at 200–4000 Hz (NL125/NL126, Digitimer) to remove noise. Data were collected and stored using a Power 401 analog‐to‐digital interface and Spike2 software (Cambridge Electronic Design, Cambridge, UK). Pressure data were acquired at a rate of 100 Hz, and afferent activity was acquired at a rate of 25,000 Hz. Action potential occurrence was determined by setting the detection threshold to twice the root mean square of the recorded signal in the absence of action potentials. Action potential frequency was calculated using Spike2 software. For comparisons with other published reports (Yeh et al. 2010; Zvara et al. 2010; Daly et al. 2014; Nocchi et al. 2014; Heppner et al. 2016), ‘afferent activity’ was defined as the frequency of action potentials per second (Hz). All data were analysed using LabChart 7 Pro software (ADInstruments, Dunedin, New Zealand).

Afferent activity analysis

Afferent activity was analysed as described previously (Heppner et al. 2016). Briefly, baseline afferent activity was determined for every 2 mmHg change in intravesical pressure. TCs and concomitant bursts of afferent activity were measured and analysed using LabChart 7 Pro software (ADInstruments, Colorado Springs, CO, USA). Only TCs occurring at baseline intravesical pressures between 0 and 12 mmHg were included. The start time, amplitude, duration, leading slope, integral, and trailing slope were recorded for both the TC and the concomitant increase in afferent activity. TC mean frequency was measured by counting the number of TCs occurring during a fixed period (between 1 and 4 min) within the intravesical pressure range of 4–8 mmHg.

Drugs and chemicals

Retigabine was obtained from Alomone Labs (Jerusalem, Israel). Unless otherwise noted, all other chemicals, including pinacidil, diltiazem and XE‐991, were obtained from Sigma‐Aldrich (St. Louis, MO, USA).

Statistical analysis

For comparisons of two samples with equal variance, statistical significance between groups was assessed using two‐tailed, paired or unpaired Student's t test (α = 0.05). For multiple comparisons, an ordinary or repeated‐measures one‐way analysis of variance (ANOVA) was used followed by Bonferroni's post hoc analysis to compare individual means. Calculations were performed using Microsoft Excel, Prism 7 (GraphPad Software Inc., San Diego, CA, USA), or SigmaStat 3.5 (Systat Software, San Jose, CA, USA). Unless otherwise indicated, N represents the number of animals in each group, whereas n represents samplings taken from within these groups. Data are reported as means ± standard error of the mean (SEM).

Results

Retigabine does not affect UBSM K_V currents or membrane potential

Using conventional whole‐cell patch clamp electrophysiology, we measured K_V currents from isolated UBSM cells in the absence or presence of the K_V7 channel activator retigabine or the K_V7 channel inhibitor XE‐991. Retigabine (10 μM) had no effect on outward currents during voltage steps from −60 to +20 mV (Fig. 1 Aa and b). In contrast, XE‐991 significantly reduced K_V currents during the same voltage steps at voltages positive of −10 mV (Fig. 1 Ba and b). However, the XE‐991‐sensitive and ‐insensitive currents exhibit the same activation properties (Fig. 1 Bc; τ = 16.7 ± 2.8 ms, V _0.5 = 3.5 ± 1.5 mV) as the whole cell K_V current (Table 2). These properties are very different from K_V7 channel properties (Table 1) and are in agreement with previous demonstrations that UBSM K_V currents are caused by activation of K_V2.1/5.1 channels.

Figure 1. Retigabine does not affect outward currents in freshly isolated mouse UBSM cells.

Representative recordings of outward current in the presence of 10 μM retigabine (Aa) or 10 μM XE‐991 (Ba), elicited by 500 ms depolarizing steps from −60 to +20 mV; 1 μM paxilline was also included to block BK channel currents. Average end‐pulse current density was not augmented in the presence of retigabine (Ab), but was reduced by XE‐991 (Bb). Difference currents in the presence of XE‐991 exhibited similar properties to control currents (Bc). ^* P < 0.05 vs. control, two‐way repeated measures ANOVA with Bonferroni's post hoc test (n = 6 cells from N = 6 mice).

Table 2.

Electrophysiological properties of K_V currents from isolated UBSM cells

Channel/condition	V 0.5 activation (mV)	Slope, k (mV)	τ (ms) (holding potential)	Reference
Control	3.49 ± 1.49	15.97 ± 1.69	16.9 ± 3.3 (+40 mV)
XE‐991	6.57 ± 1.48	19.06 ± 1.77	13.3 ± 1.8 (+40 mV)
Difference	1.51 ± 2.98	12.55 ± 3.04	16.7 ± 2.8 (+40 mV)
KV2.1/5.1 or KV6	1.1 ± 1.3	13.7 ± 1.0	29.9 ± 2.0 (+30 mV)	Thorneloe & Nelson (2003)

Data are values recorded from isolated UBSM cells.

UBSM membrane potential was also measured in the presence and absence of 10 μM retigabine using the current clamp configuration (Fig. 2). Under these conditions, retigabine did not affect resting membrane potential. This absence of an effect was not attributable to a poor seal or lack of cellular viability, as the addition of the K_ATP channel opener pinacidil (10 μM) caused a significant hyperpolarization, and exposure to equimolar K⁺ (140 mM) caused a reversible depolarization to E _K (0 mV). In a further effort to uncover any K_V7 current present in these cells, we measured K⁺ currents in the presence of 95 mM K⁺ at a physiological membrane potential (−40 mV) (Fig. 3). This approach permits the measurement of K⁺ currents with an elevated inward driving force. As was the case in the previous experiments, retigabine alone did not change membrane current, but the addition of pinacidil caused a significant and sustained inward K⁺ current.

Figure 2. Retigabine does not affect membrane potential in freshly isolated mouse UBSM cells.

A, representative trace of membrane potential of freshly isolated, current‐clamped UBSM cells in the presence of retigabine alone, retigabine with the K_ATP channel opener pinacidil, and 140 mM extracellular K⁺. B, retigabine had no effect on membrane potential, whereas pinacidil caused hyperpolarization and 140 mM K⁺ caused a reversible depolarization. Circles represent individual paired experiments. Bars represent mean ± SEM of each condition (n = 3–7 cells from N = 3–5 mice).

Figure 3. Retigabine‐sensitive inward currents are absent in the presence of 95 mM extracellular K⁺ .

A, representative recordings of inward currents in response to the addition of 95 mM extracellular K⁺. B, summary graph showing that retigabine did not alter inward currents, whereas the K_ATP channel opener pinacidil caused a significant inward K⁺ current. Circles represent individual paired experiments. Bars represent mean ± SEM of each condition. ^* P < 0.05 vs. control (n = 5 cells from N = 3–5 mice).

Retigabine inhibits voltage‐dependent Ca²⁺ channels

Inhibition of L‐type voltage‐dependent Ca²⁺ channels (VDCCs), which are responsible for the upstroke of the action potential in UBSM, significantly impairs urinary bladder contractility (Klöckner & Isenberg, 1985; Heppner et al. 1997; Sidaway & Teramoto, 2014; Heppner et al. 2016). In addition to activating K_V7 channels, retigabine has been reported to inhibit peak inward current through VDCCs by greater than 50% in vascular smooth muscle (Mani et al. 2013). Consistent with this, we found that VDCC‐mediated Ca²⁺ currents in isolated UBSM cells were significantly inhibited by 10 μM retigabine (19.7 ± 3.1% inhibition) (Fig. 4 A and B). To test the possibility that retigabine can relax UBSM through inhibition of VDCCs, isolated bladder strips were contracted with 60 mM K⁺, such that the K⁺ equilibrium potential and the membrane potential are equal (roughly −22 mV). Under these conditions, activation or inhibition of K⁺ channels would not affect membrane potential. Addition of retigabine (10 nM to 10 μM) caused a small but significant concentration‐dependent relaxation (Fig. 4 C–E) that is likely due to inhibition of VDCCs. Together, these data suggest that retigabine inhibits VDCCs, and this effect is sufficient to decrease UBSM contractility.

Figure 4. Retigabine inhibits Ca²⁺ currents in freshly isolated mouse UBSM cells.

A, representative trace of inward Ca²⁺ currents in response to a 20 ms voltage step from −60 to +10 mV. B, retigabine (10 μM) significantly reduced inward Ca²⁺ currents in isolated UBSM cells. C–E, isometric contractility of urinary bladder strips in response to 60 mM K⁺ in the absence (C) or presence (D) of increasing concentrations of retigabine. Upward, square deflections in D are an artefact of tissue oxygenation and are not muscle contractions. Retigabine (1–10 μM) reduced steady‐state contractile responses to 60 mM K⁺ as compared to vehicle (DMSO) (E). ^* P < 0.05 vs. control (n = 6 cells from N = 6 mice (A–C); N = 4 mice (C–E)).

K_V7 activation reduces afferent activity associated with simulated TCs

Transient contractions (TCs) of discrete portions of the bladder wall drive bursts of afferent nerve activity, which comprise the major part of the sensory outflow from the urinary bladder. These naturally occurring TCs were mimicked by gently compressing the bladder wall with plastic‐covered forceps. This manoeuvre produces an increase in intravesical pressure with consistent and reproducible TC integrals that are similar to naturally occurring TCs and elicited similar bursts of afferent activity. This technique allowed for the measurement of TC‐dependent afferent activity without engaging VDCCs in UBSM (Heppner et al. 2016). To investigate how afferent bursting activity during a TC is affected by K_V7 modulators, we simultaneously measured intravesical pressure and afferent nerve activity in an ex vivo bladder preparation during simulated TCs (sTCs) (Fig. 5). Afferent activity generated by sTCs was nearly abolished by retigabine, but unchanged by XE‐991 alone (Fig. 5 E). Retigabine, but not XE‐991, significantly reduced the relationship between peak afferent nerve activity and sTC integral, such that less afferent activity is generated per change in sTC integral (Fig. 5 F). Pre‐incubation with XE‐991 prevented the effects of retigabine, restoring afferent activity to that of controls (Fig. 5 E and F).

Figure 5. Retigabine inhibits bursts of afferent activity during simulated TCs.

A–D, representative traces of intravesical pressure (top) and afferent nerve activity (bottom) during simulated TCs in the absence of drug (A) or in the presence of 10 μM retigabine (B), 10 μM XE‐991 (C), or both drugs (D). TCs were simulated by gentle compression of the bladder wall with plastic‐covered forceps. E, comparison of simulated TC peak afferent activity in the presence of retigabine, XE‐991, or a combination of both. Peak afferent activity per change in simulated TC integral was reduced by retigabine (F), an effect that was reversed by pre‐incubation of tissue with XE‐991. ^* P < 0.05 vs. control, one‐way ANOVA with Bonferroni's post hoc test (N = 5–6 animals; n = 50–60 events). [Color figure can be viewed at wileyonlinelibrary.com]

Effects of K_V7 modulators on afferent nerve activity and TCs during bladder filling

To extend this analysis to a more physiological setting, we measured naturally occurring TCs and afferent outflow during urinary bladder filling using the same ex vivo bladder preparation (Fig. 6 A–H). In the absence of K_V7 modulators, continuous filling of the urinary bladder caused an increase in afferent nerve activity that was composed of two parts: activity associated with increases in intravesical pressure produced by TCs, and a graded increase in baseline activity. Retigabine inhibited TCs and associated afferent activity in a concentration‐dependent manner (Fig. 7 A–C). XE‐991 alone had no effect on TC integral (Fig. 6 E) or bursts of afferent activity (Fig. 6 G), but did significantly increase TC frequency (Figs 6 F and 7 D–F). Pre‐incubation with XE‐991 countered the actions of retigabine, suggesting that retigabine effects on TCs and afferent activity are attributable to activation of K_V7 channels. Retigabine also significantly reduced baseline afferent activity, an effect that was prevented by pre‐incubation with XE‐991 (Fig. 6 H).

Figure 6. K_V7 channel modulators alter TCs and afferent activity.

A–D, representative traces of bladder pressure (top) and afferent nerve activity (mean frequency, bottom) during ex vivo bladder filling, before drug exposure (A) and after addition of the K_V7 channel activator retigabine (10 μM, B), the K_V7 channel blocker XE‐991 (10 μM, C), or XE‐991 and retigabine combined (D). While nearly abolished, discernable TCs still occurred in the presence of retigabine (B, inset). E–G, summary bar graphs showing TC integral (E), TC frequency (F), and peak afferent nerve activity (G) in the absence or presence of retigabine, XE‐991, or a combination of both drugs. Retigabine (10 μM) significantly reduced TC amplitude and peak afferent activity without significantly reducing leading slope, effects that were blocked by XE‐991. XE‐991 alone had no effect on TC amplitude, TC slope or afferent activity, but did cause a significant increase in TC frequency that was reversed by retigabine. H, summary graph showing that baseline afferent nerve activity (black circles) is nearly abolished by retigabine at all pressures, but is significantly augmented by XE‐991 only at intravesical pressures ≥ 18 mmHg (open circles). Both effects were blocked by incubation with the combination of the two drugs. E–G, ^* P < 0.05 vs. control; one‐way ANOVA (N = 4–5). H, ^* P < 0.05 vs. control; two‐way ANOVA (N = 4). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7. Concentration dependent effects of retigabine and XE‐991 on TCs and associated afferent activity.

A–C, summary bar graphs showing TC integral (A), peak afferent nerve activity (B), and TC frequency (C) in the absence or presence of retigabine (10 nM to 10 μM). Retigabine reduced TC amplitude and associated afferent activity in a concentration‐dependent manner. D–F, summary bar graphs showing TC integral (D), peak afferent nerve activity (E), and TC frequency (F) in the absence or presence of XE‐991 (10 nM to 10 μM). XE‐991 did not significantly change reduced TC amplitude, TC leading slope, or afferent activity; TC frequency was only increased with 10 μM XE‐991. ^* P < 0.05 vs. control, repeated measures one‐way ANOVA with Bonferroni's post hoc test (N = 4–5). [Color figure can be viewed at wileyonlinelibrary.com]

To determine if the decrease in afferent activity caused by retigabine was solely due to inhibition of TCs, we measured intravesical pressure and afferent nerve activity simultaneously in the presence of retigabine (10 μM) or the VDCC blocker diltiazem (50 μM). Diltiazem eliminated TCs and associated afferent activity, but had no effect on baseline afferent activity (Fig. 8; Heppner et al. 2016). Retigabine (10 μM) nearly abolished TCs (Fig. 8 B), and unlike diltiazem, retigabine nearly abolished baseline afferent nerve activity at physiological pressures (Fig. 8 C). This suggests that, in addition to blocking VDCCs in UBSM, retigabine also directly inhibits urinary bladder afferent nerve outflow during filling.

Figure 8. Retigabine inhibits all afferent nerve activity.

A–B, representative traces of intravesical pressure (top) and afferent nerve activity (bottom) in the presence of the L‐type Ca²⁺ channel blocker diltiazem (A) or the K_V7 agonist retigabine (B). C, diltiazem (grey circles) blocked all transient contractions and associated bursts of nerve activity, but did not affect baseline afferent activity. Retigabine (black circles) not only abolished TCs and associated afferent activity, but also eliminated the remaining baseline afferent nerve activity. ^* P < 0.05 vs. control, repeated measures one‐way ANOVA with Bonferroni's post hoc test (N = 4).

Discussion

Using electrophysiology, isometric contractility and ex vivo bladder pressure/afferent nerve recordings, we comprehensively assessed the role of K_V7 channels as regulators of urinary bladder function in mice. Our results support the concept that K_V7 channels can be activated in sensory nerves to decrease activity. The Kv7 agonist retigabine inhibited transient contractions without directly affecting the detrusor smooth muscle cell excitability or membrane potential. Rather, these changes reflect impaired bladder sensation and reduced TC generation, possibly by non‐specific effects on VDCCs and K_V2.1 channels, or by activating K_V7 channels in other cell types within the urinary bladder (e.g. interstitial cells).

As noted in Table 1, K_V7 channels activate slowly (activation time constant, >100 ms), exhibit half‐activation voltages of about −40 mV, and have little to no inactivation (Robbins, 2001). Combined with our previous extensive investigation of the biophysical properties of mouse UBSM K_V currents (Thorneloe & Nelson, 2003), our present study found that UBSM K_V currents bear the characteristic signature of K_V2.1/5.1 heteromultimers with relatively rapid activation (<30 ms), half‐activation around 0 mV, and insensitivity to retigabine. These properties are very different from all members of the Kv7 family (see Table 1), and suggest that K_V7 channels are not functional in mouse UBSM. While the K_V7 antagonist XE‐991 did reduce K_V currents, this effect more likely reflects previously reported non‐specific inhibition of K_V2.1 channels (Wladyka & Kunze, 2006). Given the high input impedance (∼100 MΩ) of UBSM cells (Bramich & Brading, 1996)), it is conceivable that K_V7 currents could be present and necessary for maintaining UBSM membrane potential, but were undetectable using our patch‐clamp parameters. However, even in the presence of 95 mM extracellular K⁺ – conditions that would uncover and amplify any retigabine‐sensitive current – patch‐clamp experiments revealed no retigabine‐sensitive currents whatsoever (Fig. 3). This conclusion is reinforced by the absence of retigabine‐sensitive hyperpolarization in UBSM cells recorded under current‐clamp conditions (Fig. 2). This differs markedly from responses to the K_ATP channel opener pinacidil, which caused significant hyperpolarization.

Membrane currents were measured in freshly isolated smooth muscle cells at 25°C to increase the success rate of high‐quality seals and minimize cell contraction. Temperature is unlikely to be a significant factor, in that expressed K_V7 channels are routinely measured at room temperature (see references in Table 1) and also have been characterized in vascular smooth muscle at these temperatures (Mackie et al. 2008). Overall, we found no electrophysiological evidence for K_V7 channel currents in mouse UBSM cells under any conditions, suggesting that these channels do not contribute to resting membrane potential in these cells.

Effects of K_V7 channel modulation on sensory nerve activity

To examine other roles for K_V7 channels in the urinary bladder, we tested the effects of K_V7 modulators on urinary bladder afferent nerve activity. Retigabine has been reported to inhibit mechanosensitive primary bladder afferent nerves in the rat (Aizawa et al. 2017), a possibility we sought to explore in the mouse using an ex vivo bladder preparation. We previously showed that the major part of the afferent nerve activity generated by the bladder occurs as bursts during TCs, and that these bursts could be simulated by gently compressing the bladder wall (Heppner et al. 2016). Retigabine nearly abolished these bursts of afferent activity (Fig. 5), an effect that was prevented by the K_V7 antagonist XE‐991. Retigabine also nearly abolished afferent nerve activity in the absence of naturally occurring TCs (Fig. 7). Collectively, these observations suggest that activation of K_V7 channels in sensory nerves decreases sensory outflow in the mouse urinary bladder and could account for the urological side effects of retigabine.

Effects of K_V7 channel modulators on urinary bladder smooth muscle

Several studies have suggested the presence of K_V7 channels in UBSM from rat and guinea pig, as well as direct effects of K_V7 modulators on UBSM function (Rode et al. 2010; Afeli et al. 2013; Anderson et al. 2013; Provence et al. 2015). However, to date, the site of action of these K_V7 modulators has been incompletely evaluated, leaving the door open to alternative interpretations of their effects on urinary bladder function. In addition, given the high concentrations of retigabine used in these studies (>10 μM), the observed responses to retigabine may be more consistent with VDCC inhibition than with K_V7 activation. This conclusion is supported by our findings (Fig. 4) and previous reports that K_V7 agonists significantly reduce VDCC currents (Mani et al. 2013) and inhibit 60 mM KCl‐induced contractions (Provence et al. 2018). This off‐target inhibition of VDCCs confounds the interpretation of all studies on the effects of retigabine on contractile activity of urinary bladder strips, whether spontaneous (Afeli et al. 2013) or induced by electrical field stimulation (Rode et al. 2010). Nonetheless, several studies have reported K_V7 agonist‐dependent hyperpolarization and K⁺ currents in bladder cells from species other than mouse (Anderson et al. 2013; Provence et al. 2015), supporting the possibility of species‐dependent differences. However, no study has described Kv7 agonist‐ or antagonist‐sensitive currents in smooth muscle which have the properties (e.g. voltage dependence of activation and inactivation) consistent with known properties of Kv7 channels (Table 1). Furthermore, XE991 reduced voltage‐dependent K⁺ currents (presumably K_V1.5) in smooth muscle cells from KCNQ3, 4 and 5 knockout mice (Schleifenbaum et al. 2014), consistent with off target effect of this antagonist.

One possibility is that the absence of retigabine‐sensitive K⁺ current in UBSM may be pharmacological in nature. Due to an absence of a key amino acid in the retigabine binding site, K_V7.1 channels are insensitive to retigabine (Schenzer et al. 2005). If mouse bladder predominantly expressed K_V7.1 channels, retigabine would thus have no effect. The dominant K_V7 subtypes expressed in UBSM from rat and guinea pig appears to be K_V7.2, K_V7.3 and possibly K_V7.5, so this scenario seems unlikely (Ohya et al. 2002; Anderson et al. 2013). Furthermore, the inability of XE‐991 to block any appreciable current at membrane potentials negative of −30 mV suggests that the lack of retigabine‐sensitive current is not due to differences in K_V7 channel subtype expression, as XE‐991 blocks all K_V7 subtypes (Robbins, 2001).

Regulation of transient contractions by K_V7 modulators

The most puzzling effect we observed was the near elimination of TCs by retigabine. TCs are driven by local excitation of smooth muscle cells in bladder (Hashitani et al. 2001). During excitation, smooth muscle cells exhibit action potentials, ranging from −40 to nearly +50 mV (Heppner et al. 1997). Under these conditions, K_V7 channels would be maximally activated and thus insensitive to retigabine. So, how can retigabine block TCs, and why does XE‐991 increase TC frequency? Our current findings suggest that the inhibition of TCs is most likely due to the inhibitory effects of retigabine on VDCCs, and not due to K_V7 channel activation in UBSM. Likewise, the potentiating effects of XE‐991 on TC frequency is most likely caused by non‐specific inhibition of K_V2.1 channels, as has been previously noted (Zhong et al. 2010). Our data support this conclusion as well, since the biophysical properties of the XE‐991‐sensitive currents we recorded from UBSM cells is similar in nature to K_V2.1/5.1 dimers, and not K_V7 channels. Thus, the responses to both retigabine and XE‐991 can be explained by their non‐specific effects on other ion channel types.

Another possibility is that activation of K_V7 channels in another cell type is responsible for the inhibition of TCs in UBSM. One potential cellular target for this regulation is a population of interstitial cells of Cajal‐like cells of unknown function that are present in the bladder wall (McCloskey, 2010; Koh et al. 2012). While it has been suggested that these interstitial cells inhibit UBSM excitability through interactions between small‐conductance K⁺ channels and TRPV4 channels (Lee et al. 2012; 2017), it is possible that they may act as an excitatory influence on the bladder smooth muscle. K_V7 currents have been recorded from guinea pig interstitial cells, and evidence suggests K_V7 channels are integral to the maintenance of interstitial cell resting membrane potential and excitability (Anderson et al. 2009). This suggests that at least a subpopulation of interstitial cells is excitable in nature and may be responsible for generating urinary bladder TCs.

Summary

Taken together, our findings indicate that retigabine primarily affects mouse urinary bladder function by inhibiting sensory nerve outflow and TC generation, and not through direct inhibition of UBSM excitability. These data also imply that K_V7 channels, perhaps in interstitial cells, may be important in the generation of TCs through a mechanism independent of direct effects on UBSM. This suggests that afferent nerves and interstitial cells are potential targets for therapeutic modulation of urinary bladder function. Since most treatments for bladder overactivity inhibit efferent input to the bladder or UBSM contractility – resulting in urinary retention without adequate relief of symptoms of overactivity – targeting sensory transduction pathways and not UBSM directly represents a more attractive means of delaying micturition without impairing the ability of the bladder to efficiently void urine when needed.

Additional information

Competing interests

The authors declare no competing financial interests.

Author contributions

Experiments were performed in the laboratory of M.T.N. at the University of Vermont (Burlington, VT, USA). N.R.T. and M.T.N. contributed to the design and conception of this work. N.R.T., T.J.H., T.D., A.D.B. and M.T.N. contributed to the acquisition, analysis or interpretation of data for this work. N.R.T., T.J.H., T.D., A.D.B. and M.T.N. contributed to drafting this work or revising it critically for important intellectual content. All authors approved the final version of the manuscript; all authors agree to be accountable for all aspects of this work in ensuring that questions related to the accuracy or integrity of any part of it are appropriately investigated and resolved; and all persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was supported by grants to M.T.N. (National Institute of Diabetes and Digestive and Kidney Diseases (R37‐DK053832), National Heart, Lung, and Blood Institute (R01‐HL121706, R01‐HL131181), Fondation Leducq, and the Totman Medical Research Trust) and N.R.T. (National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK103840)).

Biography

Nathan Tykocki earned his PhD in Pharmacology and Toxicology from Michigan State University and is currently a Research Assistant Professor at the University of Vermont Larner College of Medicine. His foci are pharmacology and physiology of smooth muscle, from the single‐channel level to the whole animal. His research explores the roles of ion channels as signalling regulators in the urinary bladder urothelium, detrusor muscle, vasculature and sensory nerves. Ultimately, these studies aim to uncover the mechanisms responsible for bladder dysfunction that develops with stress and ageing, as well as in metabolic and vascular disease.