The Effectiveness in Activating M-Type K+ Current Produced by Solifenacin ([(3R)-1-azabicyclo[2.2.2]octan-3-yl] (1S)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate): Independent of Its Antimuscarinic Action

Solifenacin (Vesicare®, SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH3 cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K+ current (IK(M)) with effective EC50 value of 0.34 μM. The activation time constant of IK(M) was concurrently shortened in the SOL presence, hence yielding the KD value of 0.55 μM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of IK(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of IK(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 μM) failed to modify SOL-stimulated IK(M); however, subsequent addition of thyrotropin releasing hormone (TRH, 1 μM) was able to counteract SOL-induced increase in IK(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K+ (KM) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH3 cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the IK(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with KM channels and hence in stimulating IK(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH3 or mHippoE-14 cells.

Many types of anterior pituitary cells have been previously demonstrated to sec acetylcholine [33]. Earlier studies have also revealed that pituitary GH3 cells could exh the activity of muscarinic receptors and that muscarinic agonists were able to inhibit monal secretion through a reduction in intracellular cyclic AMP [33][34][35][36][37][38][39][40]. In these cells binding of acetylcholine to M2-muscarinic receptor might induce a weak stimulation the hydrolysis of phosphatidylinositol 4,5-bisphosphate [41]. The binding of acetylcho to muscarinic receptors in GH3 cells was also reported to activate the activity of G prot coupled K + channels directly [42,43] and to inhibit voltage-gated Ca 2+ currents [ Whether SOL could perturb the electrical activities directly or indirectly through its b ing of acetylcholine to muscarinic receptors in pituitary cells is uncertain.
The KCNQ2, KCNQ3, or KCNQ5 gene is viewed to encode the core subunit of KV KV7.3, or KV7.5 channel, respectively. The enhanced activity of this family of voltage-ga K + channels (KCNQx or KM channels) can generate the macroscopic M-type K + cur (IK(M)), which is biophysically characterized by current activation in response to l threshold voltage [45]. Once being activated, this type of K + currents can be sensitiv block by linopirdine and it is demonstrated to exhibit a slowly activating and deactiva property [46][47][48][49][50][51]. Alternatively, targeting IK(M) has been noticeably viewed as an adju tive regimen for the management of various neurological, smooth muscle, or endoc disorders closely linked to membrane hyperexcitability, which include cognitive dysfu tion, epilepsy, and over-active bladder [47,[52][53][54][55]. However, to our knowledge, how whether this agent can interact directly with KV channels to modify the amplitude gating of voltage-gated K + currents (e.g., M-type K + current) remain largely unknown Therefore, in terms of the considerations stated above, in the current study, we cided to explore the possible perturbations of SOL on IK(M) in pituitary GH3 cells mouse mHippoE-14 hippocampal neurons. Findings from the present observations en us to reflect that the IK(M) inherent in different cell types could be an additional and non-canonical target through which SOL can act to govern the functional activities of Earlier clinical investigations have revealed the efficacy and safety of the antimuscarinic, solifenacin (SOL), for treating patients with overactive bladder or neurogenic detrusor overactivity [1][2][3][4][5]7,8,[10][11][12][13][14][15][16][17][18][19]. However, recent evidence has been reported showing that the treatment with SOL could be closely linked to an increased risk of the impairment in cognitive functions [20][21][22][23][24][25][26][27][28][29][30][31]. Therefore, it is pertinent to reappraise the mechanism of SOL actions on electrical behaviors in varying excitable cells, given that its growing clinical use occurs [6,32].
Many types of anterior pituitary cells have been previously demonstrated to secrete acetylcholine [33]. Earlier studies have also revealed that pituitary GH 3 cells could exhibit the activity of muscarinic receptors and that muscarinic agonists were able to inhibit hormonal secretion through a reduction in intracellular cyclic AMP [33][34][35][36][37][38][39][40]. In these cells, the binding of acetylcholine to M 2 -muscarinic receptor might induce a weak stimulation on the hydrolysis of phosphatidylinositol 4,5-bisphosphate [41]. The binding of acetylcholine to muscarinic receptors in GH 3 cells was also reported to activate the activity of G protein-coupled K + channels directly [42,43] and to inhibit voltage-gated Ca 2+ currents [44]. Whether SOL could perturb the electrical activities directly or indirectly through its binding of acetylcholine to muscarinic receptors in pituitary cells is uncertain.
The KCNQ2, KCNQ3, or KCNQ5 gene is viewed to encode the core subunit of K V 7.2, K V 7.3, or K V 7.5 channel, respectively. The enhanced activity of this family of voltagegated K + channels (KCNQx or K M channels) can generate the macroscopic M-type K + current (I K(M) ), which is biophysically characterized by current activation in response to low-threshold voltage [45]. Once being activated, this type of K + currents can be sensitive to block by linopirdine and it is demonstrated to exhibit a slowly activating and deactivating property [46][47][48][49][50][51]. Alternatively, targeting I K(M) has been noticeably viewed as an adjunctive regimen for the management of various neurological, smooth muscle, or endocrine disorders closely linked to membrane hyperexcitability, which include cognitive dysfunction, epilepsy, and over-active bladder [47,[52][53][54][55]. However, to our knowledge, how and whether this agent can interact directly with K V channels to modify the amplitude and gating of voltage-gated K + currents (e.g., M-type K + current) remain largely unknown.
Therefore, in terms of the considerations stated above, in the current study, we decided to explore the possible perturbations of SOL on I K(M) in pituitary GH 3 cells and mouse mHippoE-14 hippocampal neurons. Findings from the present observations enable us to reflect that the I K(M) inherent in different cell types could be an additional and yet noncanonical target through which SOL can act to govern the functional activities of the cells involved, presuming that similar in vitro or in vivo findings occur. It thus merit attention that the stimulation of I K(M) and the antagonistic effect on the binding to muscarinic receptors may potentially converge to act on the functional activities of neurons, and neuroendocrine or endocrine cells.

Effect of SOL on the M-Type K + Current (I K(M) ) Measured from GH 3 Cells
For the first stage of experiments, we intended to determine the possible effect of SOL on the amplitude and kinetics of I K(M) identified in these cells. In attempts to measure the magnitude of I K(M) , we kept cells bathed in high-K + , Ca 2+ -free solution which contained 1 µM tetrodotoxin (TTX), and the recording pipette was backfilled with a K + -containing (145 mM) solution. When the whole-cell configuration was established, we held the examined cell in voltage-clamp mode at the level of −50 mV and a 1-sec depolarizing command voltage to −10 mV was thereafter applied to it. Under these experimental conditions, a specific population of K + currents with a slowly activating and deactivating property was robustly evoked and it has been thus viewed as an I K(M) [48,49,51,56]. This type of I K(M) found in pituitary lactotrophs including GH 3 cells has been demonstrated to be sensitive to be blocked by thyrotropin releasing hormone (TRH) [48,57]. As demonstrated in Figure 2A,B, the I K(M) in response to step depolarization from −50 to −10 mV was sensitive to inhibition by 10 µM linopirdine (Lino) or 1 µM TRH, while the presence of 10 µM naringenin (NGEN) or 10 µM ML213 increased current amplitude. NGEN or ML213 was previously reported to be an activator of I K(M) [58,59]. Of particular interest, one minute after GH 3 -cell exposure to SOL, the amplitude of I K(M) upon 1-sec membrane depolarization from −50 to −10 mV progressively became increased together with a concurrent decrease in the activation time constant (τ act ) of the current ( Figure 3A). For example, the addition of 0.3 or 1 µM SOL increased I K(M) amplitude to 56 ± 7 pA (n = 8, p < 0.05) or 77 ± 9 pA (n = 8, p < 0.05), respectively, from control value of 36 ± 6 pA (n = 8). Concomitantly, the presence of 0.3 or 1 µM SOL also reduced the τ act value to 89.1 ± 12.5 msec or 56.7 ± 10.1 msec, respectively, from control value of 123.5 ± 16.8 msec (n = 8). After SOL was removed, current amplitude was returned to 39 ± 7 pA (n = 7). cells involved, presuming that similar in vitro or in vivo findings occur. It thus merit attention that the stimulation of IK(M) and the antagonistic effect on the binding to muscarinic receptors may potentially converge to act on the functional activities of neurons, and neuroendocrine or endocrine cells.

Effect of SOL on the M-Type K + Current (IK(M)) Measured from GH3 Cells
For the first stage of experiments, we intended to determine the possible effect of SOL on the amplitude and kinetics of IK(M) identified in these cells. In attempts to measure the magnitude of IK(M), we kept cells bathed in high-K + , Ca 2+ -free solution which contained 1 μM tetrodotoxin (TTX), and the recording pipette was backfilled with a K + -containing (145 mM) solution. When the whole-cell configuration was established, we held the examined cell in voltage-clamp mode at the level of −50 mV and a 1-sec depolarizing command voltage to −10 mV was thereafter applied to it. Under these experimental conditions, a specific population of K + currents with a slowly activating and deactivating property was robustly evoked and it has been thus viewed as an IK(M) [48,49,51,56]. This type of IK(M) found in pituitary lactotrophs including GH3 cells has been demonstrated to be sensitive to be blocked by thyrotropin releasing hormone (TRH) [48,57]. As demonstrated in Figure 2A,B, the IK(M) in response to step depolarization from −50 to −10 mV was sensitive to inhibition by 10 μM linopirdine (Lino) or 1 μM TRH, while the presence of 10 μM naringenin (NGEN) or 10 μM ML213 increased current amplitude. NGEN or ML213 was previously reported to be an activator of IK(M) [58,59]. Of particular interest, one minute after GH3-cell exposure to SOL, the amplitude of IK(M) upon 1-sec membrane depolarization from −50 to −10 mV progressively became increased together with a concurrent decrease in the activation time constant (τact) of the current ( Figure 3A). For example, the addition of 0.3 or 1 μM SOL increased IK(M) amplitude to 56 ± 7 pA (n = 8, P < 0.05) or 77 ± 9 pA (n = 8, P < 0.05), respectively, from control value of 36 ± 6 pA (n = 8). Concomitantly, the presence of 0.3 or 1 μM SOL also reduced the τact value to 89.1 ± 12.5 msec or 56.7 ± 10.1 msec, respectively, from control value of 123.5 ± 16.8 msec (n = 8). After SOL was removed, current amplitude was returned to 39 ± 7 pA (n = 7).

Comparison in IK(M) Amplitudes Caused by the Presence of SOL, SOL Plus Acetylcholine (ACh), SOL Plus Iberiotoxin (Iber), SOL Plus Apamin (Apa), SOL Plus Tolbutamide (TLB), SOL Plus Chlorotoxin (ChTx), SOL Plus Linopirdine (Lino), or SOL Plus Thyrotropin Releasing Hormone (TRH)
We continued to examine whether SOL-stimulated IK(M) in GH3 cells could be modified by further application of acetylcholine, iberiotoxin, apamin, tolbutamide, chlorotoxin, linopirdine, or thyrotropin releasing hormone. The muscarinic receptor in GH3 cells can be activated by acetylcholine [42,43], while iberiotoxin or apamin is an inhibitor of largeor small-conductance Ca 2+ -activated K + channels, respectively, whereas tolbutamide is reported to suppress ATP-sensitive K + channel. Chlorotoxin is known to suppress the activity of Clchannels, while Lino or TRH is an inhibitor of IK(M) in GH3 cells [48][49][50][51]. In the Because the I K(M) activation in response to long-last step depolarization tends to be shortened, our next goal was to determine the kinetics of SOL-stimulated currents seen in GH 3 cells. As demonstrated in Figure 3B, as cells were rapidly depolarized from −50 to −10 mV with a duration of 1 sec, it was noticed that exposure to SOL resulted in a reduction in the τ act value in a concentration-dependent manner. This finding can thus be interpreted to reflect that the stimulatory effect of SOL on I K(M) seen in GH 3 cells is explained by the state-dependent activation in situations where the molecule can preferentially bind to the open state (conformation) of the M-type K + (K M ) channel, on the assumption of the first-order reaction scheme: (1) where α or β represents kinetic constant for the opening or closing of K M channel, respectively; and k * +1 or k −1 is that for forward (on-) or reverse (off-) rate constant of the SOL binding, respectively. "C", "O", or "O [SOL]" denotes the closed, open, or open [SOL] state of the channel, respectively. Forward or reverse rate constant (k * +1 or k −1 ) in this reaction was evaluated from the τ act values for SOL-stimulated modification in the trajectory of I K(M) activation, as described under the Materials and Methods ( Figure 3B). The value of k * +1 or k −1 obtained from eight different cells was consequently determined to be 13.962 sec −1 µM −1 or 7.672 sec −1 , respectively; thereafter, the value of dissociation constant (K D = k −1 /k * +1 ) was calculated to be 0.55 µM. The relationship between the SOL concentration and the percentage increase of I K(M) was determined and thereafter constructed. In these experiments, each examined cell was held at −50 mV and the depolarizing step from −50 to −10 mV with a duration of 1 sec was delivered to it, and the I K(M) amplitudes during exposure to different concentrations (0.3-10 µM) of SOL were measured at the end of depolarizing step. As illustrated in Figure 3C, SOL increased I K(M) amplitude in a concentration-dependent fashion. By use of a non-linear least-squares fit to the experimental data, the EC 50 value required for the stimulatory effect of SOL on I K(M) in GH 3 cells was calculated to be 0.34 µM, a value that was noticeably similar to the K D value estimated above. As such, these emerging data reflect that SOL alone is able to render I K(M) to be sensitive to stimulation attainable in these cells, which appears to be unlinked to its binding to muscarinic receptors.

Comparison in I K(M) Amplitudes
Caused by the Presence of SOL, SOL plus Acetylcholine (ACh), SOL plus Iberiotoxin (Iber), SOL plus Apamin (Apa), SOL plus Tolbutamide (TLB), SOL plus Chlorotoxin (ChTx), SOL plus Linopirdine (Lino), or SOL plus Thyrotropin Releasing Hormone (TRH) We continued to examine whether SOL-stimulated I K(M) in GH 3 cells could be modified by further application of acetylcholine, iberiotoxin, apamin, tolbutamide, chlorotoxin, linopirdine, or thyrotropin releasing hormone. The muscarinic receptor in GH 3 cells can be activated by acetylcholine [42,43], while iberiotoxin or apamin is an inhibitor of largeor small-conductance Ca 2+ -activated K + channels, respectively, whereas tolbutamide is reported to suppress ATP-sensitive K + channel. Chlorotoxin is known to suppress the activity of Clchannels, while Lino or TRH is an inhibitor of I K(M) in GH 3 cells [48][49][50][51]. In the examined cells bathed in high-K + , Ca 2+ -free solution, the potential was held at −50 mV and the depolarizing step from −50 to −10 with a duration of 1 sec was applied to the cell. Summary bar graph demonstrated in Figure 4 revealed that cell exposure to 1 µM SOL increased I K(M) amplitude and that neither further addition of acetylcholine (10 µM), iberiotoxin (200 nM), apamin (200 nM), tolbutamide (10 µM), nor chlorotoxin (1 µM) resulted in any adjustments in SOL-stimulated I K(M) , while that of Lino or TRH was able to reverse the stimulation of I K(M) caused by SOL. The results indicate that the I K(M) amplitude stimulated by SOL seen in GH 3 cells is unlinked to its effects on the activity of large-or small-conductance Ca 2+ -activated K + channels or on that of ATP-sensitive K + channels, and that its stimulatory effect on I K(M) is unable to be adjusted by further application of acetylcholine.
in any adjustments in SOL-stimulated IK(M), while that of Lino or TRH was able to reverse the stimulation of IK(M) caused by SOL. The results indicate that the IK(M) amplitude stimulated by SOL seen in GH3 cells is unlinked to its effects on the activity of large-or smallconductance Ca 2+ -activated K + channels or on that of ATP-sensitive K + channels, and that its stimulatory effect on IK(M) is unable to be adjusted by further application of acetylcholine. In these experiments, we bathed GH3 cells in high-K + , Ca 2+ -free solution and the recording electrode was filled with K + -enriched (145 mM) solution. Current amplitude was measured at the end of the depolarizing step from −50 to −10 mV. Each bar represents the mean ± SEM (n = 7). * Significantly different from controls (P < 0.05) and ** significantly different from SOL (1 μM) alone group (P < 0.05).

Current-Voltage (I-V) Relationship and Steady-State Activation Curve of IK(M) in the Absence and Presence of SOL
We next studied whether the presence of SOL can modify the amplitude of IK(M) measured at different level of membrane potentials. The averaged I-V relationship of IK(M) with or without the addition of SOL (1 μM) is illustrated in Figure 5A. The current amplitude was significantly increased as the membrane potential was less negative to −30 mV, and the magnitude of SOL-stimulated current at the level of −10 mV was noted to be greater than that at −20 mV. The relationship of IK(M) conductance versus membrane potential gained in the control period (i.e., SOL was not present) and during cell exposure to SOL (1 μM) was collated ( Figure 5B). The smooth sigmoidal curve derived from data sets was reliably fitted with a modified Boltzmann function (described under Materials and Methods). That is, the value of V1/2 or q taken in the control period was −17.4 ± 2.1 mV (n = 8) or 6.4 ± 0.9 e (n = 8), respectively, while that in the presence of 1 μM SOL was −28.2 ± 2.2 mV (n = 8) or 6.1 ± 0.9 e (n = 8), respectively. The data enable us to reflect that, in addition to increasing IK(M) conductance, the addition of SOL was capable of producing a leftward In these experiments, we bathed GH 3 cells in high-K + , Ca 2+ -free solution and the recording electrode was filled with K + -enriched (145 mM) solution. Current amplitude was measured at the end of the depolarizing step from −50 to −10 mV. Each bar represents the mean ± SEM (n = 7). * Significantly different from controls (p < 0.05) and ** significantly different from SOL (1 µM) alone group (p < 0.05).

Current-Voltage (I-V) Relationship and Steady-State Activation Curve of I K(M) in the Absence and Presence of SOL
We next studied whether the presence of SOL can modify the amplitude of I K(M) measured at different level of membrane potentials. The averaged I-V relationship of I K(M) with or without the addition of SOL (1 µM) is illustrated in Figure 5A. The current amplitude was significantly increased as the membrane potential was less negative to −30 mV, and the magnitude of SOL-stimulated current at the level of −10 mV was noted to be greater than that at −20 mV. The relationship of I K(M) conductance versus membrane potential gained in the control period (i.e., SOL was not present) and during cell exposure to SOL (1 µM) was collated ( Figure 5B). The smooth sigmoidal curve derived from data sets was reliably fitted with a modified Boltzmann function (described under Materials and Methods). That is, the value of V 1/2 or q taken in the control period was −17.4 ± 2.1 mV (n = 8) or 6.4 ± 0.9 e (n = 8), respectively, while that in the presence of 1 µM SOL was −28.2 ± 2.2 mV (n = 8) or 6.1 ± 0.9 e (n = 8), respectively. The data enable us to reflect that, in addition to increasing I K(M) conductance, the addition of SOL was capable of producing a leftward shift along the voltage axis, albeit with no marked change in the gating charge of the current. shift along the voltage axis, albeit with no marked change in the gating charge of the current. The statistical analyses in (A) and (B) were undertaken by ANOVA-2 for repeated measures, P (factor 1, groups among data taken at different level of membrane potentials) < 0.05, P (factor 2, groups between the absence and presence of SOL) < 0.05, P (interaction) < 0.05, followed by post-hoc Fisher's least-significance difference test (P < 0.05).

Effect of SOL on Voltage-Dependent Hysteresis (Vhys) of IK(M) Activated by Long Isosceles-Triangular Ramp Pulse
The Vhys of membrane ionic currents (i.e., a lag in the current amplitude as the linear voltage ramp is changed in the opposite direction) has been recently noticed with an impact on the electrical signal events of varying excitable cells [51,[60][61][62][63][64][65][66]. In other words, Vhys behavior is thought to dynamically adjust the voltage sensitivity and kinetics to optimize channel function for appropriately matching its physiological or pathophysiological role in regulation of AP firing [62,63,65]. Toward this goal, we continued to determine how the presence of SOL might adjust the Vhys strength of IK(M). In this separate set of experiments, as soon as the whole-cell configuration was achieved, we maintained the examined cell in voltage clamp at −50 mV, and a long-lasting upright isosceles-triangular ramp pulse with a duration of 2 sec at voltages between −45 and +5 mV (i.e., a ramp slope of ±50 mV/sec) was digitally created and, through DA conversion via Digidata 1440A device, thereafter, delivered to the examined cell at a rate of 0.025 Hz. Of notice, as demonstrated in Figure 6, the IK(M) trajectories elicited in response to the forward upsloping (i.e., voltage change from −45 to +5 mV) ramp pulse and by the backward downsloping (i.e., the change from +5 to −45 mV) as a function of time (as indicated by the dashed arrows in Figure 6A) were markedly distinguishable between these two limbs. In other words, the IK(M) amplitude activated by the upsloping (forward or ascending) limb of the triangular voltage ramp was demonstrated to be smaller than that by the downsloping (backward or descending) end of the ramp. These observations led us to indicate that there was a Vhys phenomenon ranging between −45 and −5 mV for this type of recorded currents in GH3 cells.

Effect of SOL on Voltage-Dependent Hysteresis (V hys ) of I K(M) Activated by Long Isosceles-Triangular Ramp Pulse
The V hys of membrane ionic currents (i.e., a lag in the current amplitude as the linear voltage ramp is changed in the opposite direction) has been recently noticed with an impact on the electrical signal events of varying excitable cells [51,[60][61][62][63][64][65][66]. In other words, V hys behavior is thought to dynamically adjust the voltage sensitivity and kinetics to optimize channel function for appropriately matching its physiological or pathophysiological role in regulation of AP firing [62,63,65]. Toward this goal, we continued to determine how the presence of SOL might adjust the V hys strength of I K(M) . In this separate set of experiments, as soon as the whole-cell configuration was achieved, we maintained the examined cell in voltage clamp at −50 mV, and a long-lasting upright isosceles-triangular ramp pulse with a duration of 2 sec at voltages between −45 and +5 mV (i.e., a ramp slope of ±50 mV/sec) was digitally created and, through DA conversion via Digidata 1440A device, thereafter, delivered to the examined cell at a rate of 0.025 Hz. Of notice, as demonstrated in Figure 6, the I K(M) trajectories elicited in response to the forward upsloping (i.e., voltage change from −45 to +5 mV) ramp pulse and by the backward downsloping (i.e., the change from +5 to −45 mV) as a function of time (as indicated by the dashed arrows in Figure 6A) were markedly distinguishable between these two limbs. In other words, the I K(M) amplitude activated by the upsloping (forward or ascending) limb of the triangular voltage ramp was demonstrated to be smaller than that by the downsloping (backward or descending) end of the ramp. These observations led us to indicate that there was a V hys phenomenon ranging between −45 and −5 mV for this type of recorded currents in GH 3 cells.
(i.e., Δarea) between the forward and backward currents activated in response to a 2-sec isosceles-triangular ramp pulse. Of notice, when the whole-cell IK(M) was identified, the addition of 0.3 or 1 μM SOL actually increased the area up to 1.2-or 1.5-fold, respectively, while the subsequent application of 10 μM linopirdine, an inhibitor of KM channels, markedly attenuated SOL-induced increase in the area by around 30%. It is conceivable, therefore, that the Vhys of IK(M) in these cells can be augmented by the presence of SOL. , SOL was not present) or during exposure to SOL and SOL plus linopirdine (Lino). The area encircled by current amplitudes activated in the ascending and descending limb at the voltages between −45 and 0 mV was calculated. Each bar indicates the mean ± SEM (n = 7 for each bar). Data analysis was performed by ANOVA−1 (P < 0.05). * Significantly different from control (P < 0.05) and ** significantly different from SOL (1 μM) alone group (P < 0.05).

Stimulatory Effect of SOL on the Activity of M-Type K + (KM) Channels in GH3 Cells
The SOL-induced raise in whole-cell IK(M) stated above could be due to either changes in channel open probability, single-channel amplitude, gating kinetics of the KM channels, or in any combinations. Such reasons thus urged us to assess the single-channel activities of the channels residing in GH3 cells. In this stage of cell-attached current recordings, we bathed cells in high-K + , Ca 2+ -free solution and the recording electrode used was filled up with low-K + (5.4 mM) solution. As demonstrated in Figure 7, as the examined cell was held at +20 mV relative to the bath, the activity of single-KM channels was robustly detected [51,56,66]. One minute after bath application of SOL, the channel open probability was markedly raised. For example, at the level of +20 mV relative to the bath, the presence of 1 μM SOL significantly increased the probability of channel openings from 0.023 ± 0.006 to 0.082 ± 0.012 (n = 7, P < 0.01); conversely, no appreciable modification in the singlechannel amplitude was shown in its presence (28 ± 2 pS [control] versus 29 ± 2 pS [in the presence of SOL]; n = 7, P > 0.05). Meanwhile, the mean open time of KM channels in its presence was appreciably increased to 5.2 ± 1.1 msec (n = 7, P < 0.05) from a control value of 2.8 ± 0.9 msec (n = 7). Furthermore, as cells were continually exposed to SOL, subsequent addition of Lino (10 μM) or TRH (1 μM) could attenuate SOL-stimulated channel activity, while that of acetylcholine (10 μM) failed to influence it. However, no detectable change In this study, we continued to quantify the V hys strength of I K(M) on the basis of the area encircled by the curvilinear trajectory in response to the upsloping and downsloping direction in ramp voltage. Figure 6B illustrates a summary of the area under the curve (i.e., ∆area) between the forward and backward currents activated in response to a 2-sec isosceles-triangular ramp pulse. Of notice, when the whole-cell I K(M) was identified, the addition of 0.3 or 1 µM SOL actually increased the area up to 1.2-or 1.5-fold, respectively, while the subsequent application of 10 µM linopirdine, an inhibitor of K M channels, markedly attenuated SOL-induced increase in the area by around 30%. It is conceivable, therefore, that the V hys of I K(M) in these cells can be augmented by the presence of SOL.

Stimulatory Effect of SOL on the Activity of M-Type K + (K M ) Channels in GH 3 Cells
The SOL-induced raise in whole-cell I K(M) stated above could be due to either changes in channel open probability, single-channel amplitude, gating kinetics of the K M channels, or in any combinations. Such reasons thus urged us to assess the single-channel activities of the channels residing in GH 3 cells. In this stage of cell-attached current recordings, we bathed cells in high-K + , Ca 2+ -free solution and the recording electrode used was filled up with low-K + (5.4 mM) solution. As demonstrated in Figure 7, as the examined cell was held at +20 mV relative to the bath, the activity of single-K M channels was robustly detected [51,56,66]. One minute after bath application of SOL, the channel open probability was markedly raised. For example, at the level of +20 mV relative to the bath, the presence of 1 µM SOL significantly increased the probability of channel openings from 0.023 ± 0.006 to 0.082 ± 0.012 (n = 7, p < 0.01); conversely, no appreciable modification in the singlechannel amplitude was shown in its presence (28 ± 2 pS [control] versus 29 ± 2 pS [in the presence of SOL]; n = 7, p > 0.05). Meanwhile, the mean open time of K M channels in its presence was appreciably increased to 5.2 ± 1.1 msec (n = 7, p < 0.05) from a control value of 2.8 ± 0.9 msec (n = 7). Furthermore, as cells were continually exposed to SOL, subsequent addition of Lino (10 µM) or TRH (1 µM) could attenuate SOL-stimulated channel activity, while that of acetylcholine (

Effect of SOL on Spontaneous Action Potentials (APs) Recorded from GH 3 Cells
For another stage of the experiments, the measurements were repurposed to wholecell potential recordings, in attempts to assess the possible perturbations of SOL on the firing frequency of APs found in these cells. For this stage of measurements, we suspended cells to be bathed in normal Tyrode's solution containing 1.8 mM CaCl 2 , the recording pipet was filled with K + -enriched solution, and whole-cell current-clamp configuration was carried out. As demonstrated in Figure 8, one minute after cell exposure to 0.3 or 1 µM SOL, the firing rate (i.e., spikes/sec) of spontaneous APs was noticeably diminished in combination with concurrent membrane hyperpolarization. For example, the presence of SOL at a concentration of 1 µM overly decreased the firing frequency of spontaneous APs to 0.47 ± 0.03 Hz (n= 8, p < 0.05) from a control value of 1.10 ± 0.05 Hz (n = 8). Moreover, during continued exposure to SOL, subsequent addition of TRH (1 µM) or Lino (10 µM) was able to reverse SOL-mediated inhibition of spontaneous APs effectively. It is likely, therefore, that SOL-mediated decrease in firing frequency of spontaneous APs is mostly mediated through its stimulation of I K(M) identified in GH 3 cells.

Effect of SOL on Spontaneous Action Potentials (APs) Recorded from GH3 Cells
For another stage of the experiments, the measurements were repurposed to wholecell potential recordings, in attempts to assess the possible perturbations of SOL on the firing frequency of APs found in these cells. For this stage of measurements, we suspended cells to be bathed in normal Tyrode's solution containing 1.8 mM CaCl2, the recording pipet was filled with K + -enriched solution, and whole-cell current-clamp configuration was carried out. As demonstrated in Figure 8, one minute after cell exposure to 0.3 or 1 μM SOL, the firing rate (i.e., spikes/sec) of spontaneous APs was noticeably diminished in combination with concurrent membrane hyperpolarization. For example, the presence of SOL at a concentration of 1 μM overly decreased the firing frequency of spontaneous APs to 0.47 ± 0.03 Hz (n= 8, P < 0.05) from a control value of 1.10 ± 0.05 Hz (n = 8). Moreover, during continued exposure to SOL, subsequent addition of TRH (1 μM) or Lino (10 μM) was able to reverse SOL-mediated inhibition of spontaneous APs effectively. It is likely, therefore, that SOL-mediated decrease in firing frequency of spontaneous APs is mostly mediated through its stimulation of IK(M) identified in GH3 cells.

Stimulatory Effect of SOL on IK(M) Present in mHippoE-14 Neurons
Evidence has recently accumulated that the treatment with SOL could be linked to an increased risk of the impairment in cognitive functions [20][21][22][23][24][25][26][27][28][29][30][31]. Earlier reports have also reported the ability of this drug to influence the muscarinic activity in cerebral cortex and hippocampus [67,68]. For these reasons, we further assessed the possible adjustments of SOL on IK(M) in hippocampal mHippoE-14 neurons. This cell line tends to be a homogenous population and it is known to possess the characteristics of embryonic hippocampal neurons valuable for the investigations on different types of neurological disorders [56,[69][70][71]. In this series of experiments, we bathed mHippoE-14 cells in high-K + , Ca 2+ -free

Stimulatory Effect of SOL on I K(M) Present in mHippoE-14 Neurons
Evidence has recently accumulated that the treatment with SOL could be linked to an increased risk of the impairment in cognitive functions [20][21][22][23][24][25][26][27][28][29][30][31]. Earlier reports have also reported the ability of this drug to influence the muscarinic activity in cerebral cortex and hippocampus [67,68]. For these reasons, we further assessed the possible adjustments of SOL on I K(M) in hippocampal mHippoE-14 neurons. This cell line tends to be a homogenous population and it is known to possess the characteristics of embryonic hippocampal neurons valuable for the investigations on different types of neurological disorders [56,[69][70][71]. In this series of experiments, we bathed mHippoE-14 cells in high-K + , Ca 2+ -free solution which contained 1 µM TTX, and we filled up the recording electrodes by using K + -enriched (145 mM) solution. As whole-cell configuration was established, the examined cell was held at −50 mV in voltage-clamp mode and the depolarizing pulse to −10 mV with a duration of 1 sec was delivered to it. As shown in Figure 9 as cells were acutely exposed to different concentrations of SOL, the amplitude of I K(M) activated by such voltage-clamp protocol progressively rose. For example, the presence of 1 µM SOL augmented I K(M) amplitude from 28 ± 4 to 65 ± 8 pA (n = 7, p < 0.05); and, after removal of SOL, current amplitude was returned to 30 ± 5 pA (n = 7). In the continued presence of 1 µM SOL, further application of Lino (µM) attenuated SOL-stimulated I K(M) , as demonstrated by an appreciable reduction of I K(M) amplitude to 36 ± 5 pA (n = 7, p < 0.05). Therefore, it is plausible to assume that indistinguishable from those identified above in GH 3 cells, I K(M) present in mHippoE-14 neurons, to which I K(M) confers excitability, is subject to stimulation by SOL. solution which contained 1 μM TTX, and we filled up the recording electrodes by using K + -enriched (145 mM) solution. As whole-cell configuration was established, the examined cell was held at −50 mV in voltage-clamp mode and the depolarizing pulse to −10 mV with a duration of 1 sec was delivered to it. As shown in Figure 9 as cells were acutely exposed to different concentrations of SOL, the amplitude of IK(M) activated by such voltage-clamp protocol progressively rose. For example, the presence of 1 μM SOL augmented IK(M) amplitude from 28 ± 4 to 65 ± 8 pA (n = 7, P < 0.05); and, after removal of SOL, current amplitude was returned to 30 ± 5 pA (n = 7). In the continued presence of 1 μM SOL, further application of Lino (μM) attenuated SOL-stimulated IK(M), as demonstrated by an appreciable reduction of IK(M) amplitude to 36 ± 5 pA (n = 7, P < 0.05). Therefore, it is plausible to assume that indistinguishable from those identified above in GH3 cells, IK(M) present in mHippoE-14 neurons, to which IK(M) confers excitability, is subject to stimulation by SOL.

Discussion
The salient findings noticed in the current investigations are as follows: (a) In pituitary GH3 cells, during exposure to SOL, the IK(M) amplitude upon long membrane depolarization was concentration-dependently increased and the activation time course of the current concurrently became shortened; (b) the EC50 or KD value of SOL-stimulated IK(M) was calculated to be 0.34 or 0.55 μM, respectively; (c) there is a leftward shift of the steadystate activation curve of IK(M) in its presence; (d) the Vhys area of IK(M) activated by isoscelestriangular ramp pulse increased during cell exposure to SOL; (e) the KM-channel activity was elevated by adding SOL; however, no change in single-channel conductance of the channel was detected; (f) under current-clamp conditions, the firing frequency of spontaneous APs was measured to be appreciably decreased in the presence of this drug; and (g) the IK(M) inherently in hippocampal mHippoE-14 neurons was also subject to stimulation by SOL. Altogether, regardless of the unresolved detailed ionic mechanism of its actions on KM (or KCNQx) channels, the present results provide an unanticipated and yet

Discussion
The salient findings noticed in the current investigations are as follows: (a) In pituitary GH 3 cells, during exposure to SOL, the I K(M) amplitude upon long membrane depolarization was concentration-dependently increased and the activation time course of the current concurrently became shortened; (b) the EC 50 or K D value of SOL-stimulated I K(M) was calculated to be 0.34 or 0.55 µM, respectively; (c) there is a leftward shift of the steady-state activation curve of I K(M) in its presence; (d) the V hys area of I K(M) activated by isosceles-triangular ramp pulse increased during cell exposure to SOL; (e) the K M -channel activity was elevated by adding SOL; however, no change in single-channel conductance of the channel was detected; (f) under current-clamp conditions, the firing frequency of spontaneous APs was measured to be appreciably decreased in the presence of this drug; and (g) the I K(M) inherently in hippocampal mHippoE-14 neurons was also subject to stimulation by SOL. Altogether, regardless of the unresolved detailed ionic mechanism of its actions on K M (or KCNQx) channels, the present results provide an unanticipated and yet non-canonical ionic mechanisms through which the SOL molecule can interact with K M channels to increase whole-cell I K(M) and, consequently, to diminish the firing rate of spontaneous of APs.
One element that is pertinent to notable findings in this study is that the presence of SOL has the propensity to interact with K M channels to increase the magnitude of I K(M) as well as to fasten the activation rate of the current during long depolarizing steps. In other words, although the SOL addition was effective at stimulating I K(M) , the activation time course of I K(M) evoked by long-step membrane depolarization became raised. The interaction of SOL with K M channels could also be enhanced by repetitive opening of the channel pore to provide drug access. According to minimal binding scheme, the K D value was yielded to be 0.55 µM, a value which noticeably bears a similarity to effective EC 50 value needed for SOL-stimulated I K(M) . The steady-state activation curve of I K(M) attained in the SOL presence was also found to be shifted along the voltage axis in a leftward direction (i.e., a more negative potential), with no modifications in the gating charge of the current. The mean open time of K M channels was also found to become lengthened in its presence. In this regard, it is plausible to assume that the SOL molecule can preferentially bind to the open state of the K M channel; consequently, the magnitude of I K(M) activated upon long membrane depolarization became elevated during its exposure.
In the present study, the inability of iberiotoxin, apamin, tolbutamide, or chlorotoxin to modify the stimulatory effect on I K(M) caused by the presence of SOL was demonstrated. Iberiotoxin or apamin is viewed to inhibit the activity of large-or small-conductance Ca 2+activated K + channels, respectively. Tolbutamide can suppress the activity of ATP-sensitive K + channels, and chlorotoxin is a blocker of Clchannels. Therefore, it seems unlikely that SOL-mediated stimulation of I K(M) in GH 3 cells is associated with its perturbations on the activities of large-or small-conductance Ca 2+ -activated K + channels, ATP-sensitive K + channels, or Clchannels, which were reportedly present in GH 3 cells. Additionally, in continued presence of SOL, the subsequent addition of acetylcholine failed to reverse SOL-mediated increase in I K(M) amplitude, reflecting that the stimulatory action on I K(M) would not solely be explained by its competitive binding of acetylcholine to muscarinic receptors in these cells, although GH 3 cells have been previously demonstrated to exhibit the activity of muscarinic receptors [33][34][35][36][37][38][39][40]. Of note, the SOL molecule is structurally similar to tetrahydropyrrolopyrazines demonstrated to activate I K(M) [72], suggesting that 1-phenyl-3,4-dihydro-1H-isoquinoline moiety residing in the molecule is an active site for the binding to the channel.
In accordance with the preceding reports, the V hys phenomenon of I K(M) evoked by the long isosceles-triangular ramp pulse (i.e., the upsloping and downsloping ramp) was revealed in GH 3 cells [73]. The adjustments of such V hys have been recently noticed to serve a role in fine-tuning the activity of ionic channels (e.g., K M channels) to respond when they are virtually needed [62,63,66,73]. We further determined the possible perturbations of SOL on such dynamic and non-equilibrium properties of I K(M) present in GH 3 cells. The emerging results allowed us to bespeak that the presence of SOL was able to increase the hysteretic strength of the current efficiently (i.e., ∆area in Figure 6) associated with the voltage-dependent activation of instantaneous I K(M) . Under such scenario, it is possible that intrinsic changes in the voltage dependence of the voltage-sensing machinery in K M (KCNQx) channels, namely voltage-sensing domain relaxation would be dynamically modulated during exposure to SOL.
According to previous pharmacokinetic studies, the peak plasma concentrations of SOL with 24.0 ng/mL (0.066 µM) or 40.6 ng/mL (0.11 µM) were reported to reach 3-8 h after long-term oral administration of a 5 or 10 mg SOL dose, respectively [7,[74][75][76]. The SOL plasma level was also found to be even higher (i.e., around 52 ng/mL or 0.14 µM) in patients with renal insufficiency [77]; and, it could have a long duration of action as it is usually taken once daily. As such, it is possible that, apart from interfering with the binding to muscarinic receptors, SOL-mediated stimulation of I K(M) is of clinical or therapeutic relevance.
Considering all of the experimental results together, the effects of SOL on I K(M) demonstrated herein appears to be acute and robust in onset; moreover, meanwhile, such stimulatory actions tend to be non-canonical and they are presumably mediated via a mechanism independent of its blockade of muscarinic receptors. These actions probably result in its perturbations on the functional activities of electrically excitable cells (e.g., GH 3 or mHippoE-14 cells), in the case that in vivo findings occur. Whether the impairment of cognitive function after long-term administration of SOL [20][21][22][23][24][25][26][27][28][29][30][31] could be intimately connected with its stimulation of I K(M) in central neurons remains to be further investigated.
It is worth noting that different types of smooth muscle cells, including smooth myocytes of the urinary bladder, have been demonstrated to be functionally expressed in the activity of K M (KCNQx) channels [52,53,[78][79][80][81][82][83][84][85][86][87][88][89]. The SOL-induced interaction with K M channels to modify the magnitude and gating of I K(M) has the propensity to change muscarinic cholinergic activation involved in the micturition reflex, presuming that the in vivo results happen. It turns out that whether the actions of SOL or other structurally similar compounds (e.g., darifenacin) on overactive bladder or neurogenic detrusor overactivity [90] are related to its enhanced actions on K M -channel activity [82][83][84][85], warrants further investigations, despite its high-affinity binding to muscarinic receptors.

Electrophysiological Measurements
GH 3 cells or mHippoE-14 neurons were gingerly harvested and cell suspension was rapidly placed in a customized chamber immediately before the electrical recordings. The recording chamber was positioned on the stage of an inverted DM-IL fluorescence microscope (Leica, Uranus Technology, Taipei, Taiwan) coupled to a digital video system (DCR-TR30; Sony, Tokyo, Japan) with a magnification of up to 1500×. Cells were kept immersed at room temperature (20-25 • C) in normal Tyrode's solutions containing 1.8 mM CaCl 2 , and the composition of this solution is stated above. The patch-clamp procedure in either whole-cell (voltage-and current-clamp mode) or cell-attached configuration was implemented by using an RK-400 patch amplifier (Biologic, Echirolles, France) [51,91]. When filled with internal solution, patch-clamp glass pipettes had tip resistances ranging between 3 and 5 MΩ and they were made from Kimax-51 capillaries (#34500 [1.5-1.8 mm in outer diameter]; Dogger, Tainan, Taiwan), by using either a PP-830 vertical puller (Narishige, Tokyo, Japan) or a P-97 horizontal puller (Sutter, Novato, CA, USA), and their tips were fire-polished with an MF-83 microforge (Narishige). The potentials were corrected for the liquid-liquid junction potential which emerged when the composition of the pipette solution was different from that in the bath. An anti-vibration air table was used to ensure mechanical stability during the measurements.

Data Recordings
The signals comprising voltage and current tracings were monitored on an HM-507 oscilloscope (Hameg, East Meadow, NY, USA) and stored online in an ASUS ExpertBook laptop computer (P2451F; ASUS, Tainan, Taiwan) at 10 kHz interfaced with a Digidata 1440A converter (Molecular Devices; Bestogen Biotech, New Taipei City, Taiwan), which proceeded for efficient analog-to-digital/digital-to-analog (AD/DA) conversion. During the measurements, the process in data acquisition equipped with this device was controlled by pCLAMP 10.6 program suite (Molecular Devices) run under Windows 7 (Redmond, WA, USA), and the signals were simultaneously displayed on an LCD monitor through USB type-C connection. Current signals were low-pass filtered at 2 kHz with an FL-4 four-pole Bessel Filter (Dagan, Minneapolis, MN, USA) to minimize possible electrical interference. After the recorded data were digitally collected, we off-line collated them using various analytical tools that include LabChart 7.0 program (ADInstruments; KYS Technology, Taipei, Taiwan), OriginPro ® 2021 (OriginLab; Scientific Formosa, Kaohsiung, Taiwan) and varying custom-made macros built in Excel ® 2021 under Microsoft 365 (Redmond, WA, USA).

Whole-Cell Current Analyses
To evaluate the effect of concentration-dependent stimulation of SOL on I K(M) , GH 3 cells were allowed to be immersed in high-K + (145 mM K + ), Ca 2+ -free solution. As the whole-cell mode was established, each cell was voltage-clamped at a holding potential of −50 mV, and a 1-sec depolarizing voltage command to −10 mV was delivered to it. The amplitude of I K(M) at the end-pulse of 1-sec depolarizing pulse measured during cell exposure to 10 µM SOL was taken as 100%, and current amplitudes were thereafter compared to those in the presence of different SOL concentrations. The concentration required to increase the I K(M) amplitude by 50% was determined by use of the Hill function: The time-dependent rate constant of forward (k * +1 ) or backward (k −1 ) was broadly evaluated from the activation time constant (τ act ) of I K(M) activated by the long depolarizing pulse from −50 to −10 mV. The τ act values in the presence of different SOL concentrations were approximated by fitting single exponential function to the trajectory of each current trace. Since a Hill coefficient of about 1 was found according to the concentration-dependent curve, the forward or backward rate constant was extended to be determined using the following equation: 1 where [SOL] is the SOL concentration applied, and k * +1 or k −1 was gained from the slope and the y-axis intercept at [SOL] = 0 of the interpolated regression line, where the relation of the reciprocal time constant of I K(M) activation (1.e., 1/τ act ) versus different SOL concentration was constructed.
The relationship of the membrane potential versus the I K(M) conductance gained in the absence or presence of SOL was well approximated by a modified Boltzmann function (or the Fermi-Dirac distribution) of the following form: where G is the I K(M) conductance, G max the maximal conductance of I K(M) , V 1/2 the voltage at which half-maximal activation of the current is achieved, q the apparent gating charge, F Faraday's constant, R the universal gas constant, and T the absolute temperature.

Analyses of Single M-Type K + (K M ) Channels
Single K M -channel currents experimentally measured from GH 3 cells were collated using pCLAMP 10.7 suite (Clampfit 10.7 subroutine). We determined single-channel amplitude taken with or without the addition of SOL by reliably fitting Gaussian distributions to the amplitude histograms of the closed (resting) or open state. The channel open probabilities were defined as N·PO, which was determined by using the following expression: where N is a number of active K M channels residing in the patch examined, A 0 is an area under the curve of an all-points histogram corresponding to the closed (resting) state, and A 1 . . . A n represents a histogram area that corresponds to the level of distinct open state for 1 to n channels in the patch. The single-channel conductance of K M channels with or without the SOL addition was calculated using a linear I-V approximation with mean values of single-channel amplitudes measured at the different membrane potentials relative to the bath, while open lifetime distribution of K M channels was fitted with single exponential function.

Curve-Fitting Procedures and Statistical Analyses
Linear (e.g., single-channel conductance) or nonlinear (e.g., Hill or Boltzmann equation and single exponential) curves fitting to experimental data sets demonstrated here was performed from the goodness-of-fit test using either the Solver add-in bundled with