Modulation of I_Na, I_h, and I_K(erg) by Extracellular or Intracellular QX-314 (N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide) in Pituitary Tumor Cells

Abstract

QX-314 is a positively charged lidocaine derivative with the membrane-impermeant property. This compound applied at the intracellular side has been shown to suppress the voltage-gated Na⁺ current (I_Na), while lidocaine itself acts to suppress the hyperpolarization-activated cation current (I_h). To what extent this drug may exert any effects on various plasmalemmal ionic currents still remains largely unknown. This investigation focused on the impact of QX-314 on ionic currents in GH₃ cells derived from pituitary tumors. This compound applied extracellularly was noted to differentially suppress the amplitude of transient and late I_Na with an IC₅₀ value of 93 and 42 μM, respectively. In GH₃ cells dialyzed with QX-314 (10 μM), the I_Na(T) amplitude evoked by a brief depolarizing step was decreased, and its inactivation was increased. Moreover, QX-314, when applied extracellularly at 100 μM, diminished the amplitude of the I_h current with an IC₅₀ of 68 μM. Intracellular dialysis with QX-314 also suppressed I_h amplitude; moreover, the later application of oxaliplatin reversed this suppression. As cells were extracellularly and continually exposed to QX-314, the magnitude of the erg-mediated K⁺ current (I_K(erg)) was also effectively suppressed with an IC₅₀ value of 73 μM. Furthermore, upon intracellular dialysis with QX-314 (10 μM), the degree of the voltage-dependent hysteresis (Hys_(V)) of I_K(erg) during the long-lasting isosceles-triangular ramp voltage was decreased; during continued exposure to QX-314, further extracellular bath additions of PD118057 (10 μM) counteracted QX-314-induced suppression. However, the extracellular addition of QX-314 (100 μM) mildly suppressed the outward delayed rectifier K⁺ current in GH₃ cells. Collectively, QX-314 effectively suppressed I_Na, I_h, and I_K(erg) in GH₃ cells, a model of endocrine function, and these actions may contribute to their physiological functions, if similar effects are observed in vivo.

1. Introduction

QX-314, a quaternary lidocaine derivative, has been increasingly recognized as a modulator of ionic currents, including voltage-gated Na⁺ current and transient receptor potential currents [1,2,3,4,5,6,7,8]. Previous work has demonstrated the capability of lidocaine to suppress hyperpolarization-activated cation currents in various types of neurons and in heart cells [9,10,11,12]. QX-314 has been demonstrated to produce long-lasting local anesthesia with a slow onset in animal models in vivo [13,14,15]. This compound was also previously reported to suppress the acid-induced activation of esophageal nociceptive C fiber neurons [16].

Nine isoforms of voltage-gated Na⁺ (Na_V) channels—designated Na_V1.1 through 1.9 (corresponding to SCN1A-SCN5A and SCN8A-SCN11A)—are known to be distributed across various excitable tissues in mammals, including those of the endocrine system [17,18,19]. These Na_V channels mediated whole-cell voltage-gated Na⁺ currents (I_Na), which are characterized by a rapid and transient activation phase (transient I_Na or I_Na(T)), followed by swift inactivation. Despite this inactivation, a small but physiologically significant component of the current persists, referred to as late I_Na (I_Na(L)) [18,20]. Previous studies have shown that the intracellular application of QX-314 can effectively suppress I_Na in several types of excitable cells [21,22]. However, the extent to which QX-314—administered either intracellularly or extracellularly—can influence the amplitude and/or gating properties of I_Na remains largely uninvestigated.

The hyperpolarization-activated cation current (I_h), also known as the funny current (I_f), plays a critical role in regulating rhythmic electrical activity across various excitable cell types, including cardiac cells, neurons, and both neuroendocrine and endocrine cells [23,24,25,26,27,28,29,30,31]. This type of current exhibits mixed Na⁺/K⁺ permeability, reflecting an unusual ion selectivity that permits the conduction of Na⁺ and K⁺ ions alike. It is susceptible to inhibition by CsCl, ZD7288, and ivabradine, and can be activated by oxaliplatin (OXAL) [24,27,32,33,34,35,36]. When activated at resting membrane potential, these currents may generate a net inward current primarily carried by Na⁺, which subsequently depolarizes the membrane potential toward the threshold necessary for triggering an action potential [2,23,28,29,32,35,37,38,39]. These ionic currents are attributed to channels encoded by the hyperpolarization-activated cyclic nucleotide-gated (HCN) gene family, a subset of the larger superfamily comprising voltage-gated K⁺ channels and cyclic nucleotide-gated channels. Notably, lidocaine has been shown to attenuate the amplitude of I_h in both substantia gelatinosa neurons and thalamocortical neurons [10,11,12,40,41]. In contrast, prior investigations demonstrated that intracellular application of QX-314 effectively reduced I_h in hippocampal neurons [42,43], while its extracellular application failed to elicit a comparable effect [10]. These results point to an ongoing discussion surrounding the mechanism through which QX-314 engages HCN channels to alter I_h amplitude and gating dynamics.

The ether-à-go-go-related gene, (erg)-mediated K⁺ current (I_K(erg)) is present in a variety excitable cell types, including heart cells, endocrine cells, and cells of the neuroendocrine system [44,45,46,47,48,49]. This current is carried by the EAG (ether-à-go-go) family voltage-gated K⁺ (K_V) channels, characterized by distinctive gating behavior—marked by swift inactivation and notably delayed deactivation. These currents play a vital role in maintaining the baseline potential across the cell membrane and in fine-tuning subthreshold responsiveness and rhythmic firing behavior [48,49]. Consequently, it is crucial to investigate how QX-314 influences the amplitude of I_K(erg), its channel-opening and closing dynamics, and the voltage-driven memory effect known as voltage-dependent hysteresis (Hys_(V)).

With this background, the research aims to investigate how QX-314, a compound known to block ion channels, impacts the electrical properties of GH₃ cells. Specifically, the study aims to delineate its impact on several critical transmembrane ionic currents, including the transient I_Na (I_Na(T)), the late I_Na (I_Na(L)), the hyperpolarization-activated cation current (I_h), the K⁺ current mediated by erg (I_k(erg)), and the voltage-dependent delayed-rectifier K⁺ current (I_K(DR)). The use of GH₃ cells, a well-established and stable model derived from a pituitary tumor, provides a reliable system for these experiments.

2. Results

2.1. Inhibitory Effect of QX-314 on the Amplitude of Voltage-Gated Na⁺ Currents (I_Na) Measured from GH₃ Cells

The initial phase of the experiment aimed to assess the impact of extracellular QX-314 on the amplitude of transient I_Na (I_Na(T)) and late I_Na (I_Na(L)) in these cells. To isolate I_Na, we placed cells in a Ca²⁺-free Tyrode’s solution, supplemented with 10 mM tetraethylammonium chloride (TEA) and 0.5 mM CdCl₂. TEA or CdCl₂ was included to inhibit nonspecific K⁺ and Ca²⁺ currents, respectively. Additionally, the recording pipettes were filled with a Cs⁺-enriched internal solution. Once the whole-cell configuration was successfully established, the membrane potential of a recorded cell was held at −80 mV. A 40 ms depolarizing pulse to −10 mV was then applied to evoke I_Na. As anticipated, this voltage step induced a robust inward current characterized by rapid activation and inactivation kinetics. The current was effectively blocked by tetrodotoxin (TTX, 1 μM), confirming its identity as I_Na, whereas nimodipine (1 μM) had no inhibitory effect, indicating minimal contribution from L-type Ca²⁺ channels [50,51,52]. Continued exposure of the cells to QX-314 resulted in a progressive reduction in the amplitude of I_Na, as illustrated in Figure 1A. For example, a bath addition of QX-314 at a concentration of either 100 or 300 μM markedly decreased the I_Na(T) amplitude from 594 ± 23 pA to either 452 ± 21 pA (n = 8, p < 0.05) or 312 ± 16 pA (n = 8, p < 0.05), respectively, while such maneuver, respectively, decreased the I_Na(L) amplitude to 76 ± 7 pA (n = 8, p < 0.05) or 21 ± 5 pA (n = 8, p < 0.05) from a control value of 92 ± 8 pA (n = 8). After washout of QX-314, the amplitudes of I_Na(T) and I_Na(L) recovered to 588 ± 21 pA (n = 8) and 90 ± 7 pA (n = 8), respectively. In addition, exposure to 300 μM QX-314 significantly accelerated the inactivated time course of I_Na(T), as reflected by a reduction in the slow component of the I_Na(T)-inactivation time constant (τ_inact(S)) from 7.2 ± 1.3 to 3.9 ± 0.9 ms (n = 8, p < 0.05). Figure 1B showed that cell exposure to QX-314 could result in a concentration-dependent decrease in the amplitude of I_Na(T) or I_Na(L) activated by 40 ms step depolarization. The experimental data indicated that the IC₅₀ values for extracellular QX-314-induced inhibition were 93 μM for I_Na(T) and 42 μM for I_Na(L), respectively. Our initial measurements, therefore, demonstrated that the extracellular application of QX-314 exerted a suppressive effect on both I_Na(T) and I_Na(L) natively expressed in GH₃ cells. Moreover, the drug showed a greater selectivity toward I_Na(L) compared with I_Na(T) activated by short rectangular depolarizing steps.

2.2. Effect of Intracellular Dialysis with QX-314 on the Amplitude of I_Na

Next, we examined whether I_Na(T) or I_Na(L) in GH₃ cells could be affected by intracellular dialysis with QX-314. For these experiments, the recording pipette was filled with a Cs⁺-enriched solution containing 10 μM QX-314. Following establishment of the whole-cell configuration, cells were voltage clamped at −80 mV. A 30 ms hyperpolarizing prepulse to −100 mV was applied every 20 s, followed by a depolarizing step to −10 mV to elicit I_Na. As shown in Figure 2, the intracellular application of 10 μM QX-314 led to a progressive decline in I_Na amplitude in response to the repetitive depolarizing pulse delivered at a frequency of 0.05 Hz. For instance, immediately following the establishment of the whole-cell configuration, the amplitudes of I_Na(T) and I_Na(L) recorded at the first voltage-clamp pulse were 491 ± 31 and 17.3 ± 2.2 pA (n = 8), respectively. Notably, these values were significantly reduced by the 20th pulse—approximately 7 min after membrane rupture—with I_Na(T) and I_Na(L) declining to 297 ± 19 pA and 3.2 ± 0.3 pA, respectively (n = 8, p < 0.05 for both). Additionally, the slow inactivation time constant (τ_inact(S)) of I_Na(T) decreased markedly from 5.6 ± 0.3 ms at the first pulse to 1.9 ± 0.2 ms at the 20th pulse (n = 8, p < 0.05). Furthermore, this reduction in I_Na(T) and I_Na(L) amplitude observed at the 20th pulse was reversed upon the subsequent application of 10 μM tefluthrin (Tef), a type-I pyrethroid insecticide recently identified as an activator of I_Na [50,53].

The extracellular and intracellular application of QX-314 resulted in differential inhibition, which is consistent with previous reports demonstrating distinct pharmacokinetics and access routes for the compound [6,7]. The higher concentrations used in bath applications were necessary to overcome the limited membrane permeability of QX-314 and achieve effective inhibition. In contrast, intracellular dialysis shows direct access to the intracellular compartment, enabling robust effects at significantly lower concentrations. We have clarified this rationale in the manuscript to improve transparency.

2.3. Inhibitory Effect of QX-314 on the Current Versus Voltage (I-V) Relationship of Hyperpolarization-Activated Cation Currents (I_h) in GH₃ Cells

Past research has demonstrated that lidocaine can alter HCN currents in substantia gelatinosa neurons and thalamocortical neurons [11,40]. Building on this, we next examined how QX-314 affects I_h in GH₃ cells at varying membrane potentials. In these experiments, cells were bathed in a Ca²⁺-free Tyrode’s solution and the recording pipette was filled with K⁺-enriched solution. As demonstrated in Figure 3A,B, in the control period (i.e., neither extracellular nor intracellular QX-314 was present), the currents became progressively increased at a voltage more negative than −80 mV; furthermore, the time course of current activation was noted to become faster with a greater step hyperpolarization, as reported previously [30,33,34,40]. The addition of QX-314 (100 μM) to the bath medium resulted in a reduction in the amplitude of I_h by responding to 2 s membrane hyperpolarization (Figure 3A). Figure 3B displays the averaged I-V relationship of I, which was measured at the conclusion of each hyperpolarizing pulse. The I_h amplitudes in Figure 3B between the absence and presence of 100 μM QX-314 were significantly distinguishable at the voltages between −80 and −120 mV. Furthermore, the activation time constant (τ_act) of I_h at the level of −120 mV was conceivably increased (1.28 ± 0.18 s [control] versus 1.62 ± 0.17 s [in the presence of 100 μM QX-314]; n = 8, p < 0.05). It is thus important to mention, from these results, that in addition to a reduction in the initial component of I_h, the I-V relationship of sustained I_h (Figure 3B) became more pronounced. Figure 3C showed that the addition of QX-314 to the bath medium could concentration-dependently decrease the amplitude of I_h in response to the 2 s membrane hyperpolarization from −40 to −120 mV. According to the modified Hill equation described in the Section 4. the IC₅₀ value for QX-314-mediated suppression of the I_h amplitude in GH₃ cells was determined to be 68 μM.

2.4. Impact of Intracellular Dialysis with QX-314 on the I_h Amplitude

Next, we investigated whether intracellular dialysis with QX-314 could alter the I_h amplitude in response to prolonged membrane hyperpolarization. To isolate I_h, cells were perfused with a Ca²⁺-free Tyrode’s solution supplemented with 1 μM tetrodotoxin (TTX) and 0.5 mM CdCl₂. The recording electrode was filled with a K⁺-rich internal solution containing 10 μM QX-314. As shown in Figure 4, the intracellular application of 10 μM QX-314 led to a substantial attenuation of the I_h amplitude. The I_h amplitude obtained between the 1st pulse and 20th pulse was found to differ vastly. Furthermore, when oxaliplatin (10 μM, OXAL) was subsequently added following the 20th pulse, the I_h amplitude suppressed by intracellular dialysis with QX-314 was markedly reversed. OXAL, a platinum-based chemotherapeutic drug, was demonstrated to activate I_h [33].

2.5. Suppressive Effect of QX-314 on the Amplitude of erg-Mediated K⁺ Current (I_K(erg)) in GH₃ Cells

We also investigated whether extracellular QX-314 modified I_K(erg) in these cells. To identify I_K(erg), cells were bathed in a high-K⁺, Ca²⁺-free solution, while the recording pipette was filled with a K⁺-enriched solution. With the cell held at a holding potential of −10 mV, we then applied a series of voltage steps from −110 to −10 mV in 20 mV increments. As depicted in Figure 5A, one minute after cell exposure to 100 μM QX-314, the peak I_K(erg) was significantly suppressed measured across membrane potentials of −90 and −110 mV. The averaged I-V relationships for peak and sustained I_K(erg) are shown in Figure 5B, comparing data from the control condition (without QX-314) to those obtained with 100 μM QX-314 acquired in the control period and during cell exposure to 100 μM QX-314.

2.6. Impact of Intracellular Dialysis with QX-314 on Hys_(V) of I_K(erg) Activated by the Upright Isosceles-Triangular Ramp Voltage (V_ramp)

The Hys_(V) behavior of I_K(erg), which has been studied in multiple cell types and has been previously characterized in various cell types, is known to significantly influence the electrical properties of diverse, electrically active cells [44,50]. Therefore, we next explored how intracellular dialysis with QX-314 alters the Hys_(V) behavior in GH₃ cells, using a long-lasting isosceles-triangular V_ramp. In this set of whole-cell current recordings, the cell was held at a membrane potential of −10 mV and subjected to an upright isosceles-triangular V_ramp ranging from −110 to 0 mV over 2.4 s (corresponding to a ramp speed of ±0.092 mV/s) delivered via digital-to-analog conversion (Figure 6A). Notably, the current amplitudes elicited by the ascending and descending phases of the triangular V_ramp were markedly different. The clear difference between the instantaneous I_K(erg) and the membrane potential indicates the presence of Hys_(V) in the channel’s elicitation, as shown in Figure 6A. Furthermore, when the cell was intracellularly dialyzed with 10 μM QX-314, the I_K(erg) elicited during the ascending phase of the triangular V_ramp was reduced, albeit to a lesser extent than the current observed during the descending phase. For example, in the current trace taken at the first pulse (i.e., immediately after membrane rupture occurred), I_K(erg)s at the level of −60 mV activated by the ascending and descending phases of triangular V_ramps were noted to be 197 ± 18 and 87 ± 11 pA (n = 8), respectively, the values of which were found to differ significantly (p < 0.05). Furthermore, during intracellular dialysis with 10 μM QX-314, subsequent bath application of PD118057 (10 μM) led to a significant reduction in the amplitude of both ascending and descending I_K(erg)s recorded at −60 mV during the 20th pulse, decreasing to 101 ± 17 and 49 ± 7 pA (n = 8, p < 0.05). PD118057 was previously reported to enhance the I_K(erg) [51].

2.7. Mild Inhibitory Effect of QX-314 on Averaged I-V Relationship of Delayed Rectifier K⁺ Current (I_K(DR)) Recorded from GH₃ Cells

Lidocaine at higher concentrations was previously reported to suppress voltage-gated K⁺ currents [52,53]. In another set of measurements, we thus wanted to explore if cell exposure to QX-314 could modify various K⁺ currents, specifically the delayed rectifier K⁺ current (I_K(DR)), in GH₃ cells. To isolate these currents, GH₃ cells were perfused with a Ca²⁺-free Tyrode’s solution. The cells were held at a membrane potential of −50 mV under voltage-clamp conditions and subsequently subjected to 1 s depolarizing voltage steps ranging from −50 to +50 mV in 10 mV increments. Under this voltage-clamp protocol, a distinct family of I_K(DR) was reliably evoked in response to sustained membrane depolarization. These currents exhibited outward rectification and a modest relaxation in their inactivation kinetics. Of note, following a 1 min application of 100 μM QX-314, the I_K(DR) amplitude exhibited a modest reduction, especially at membrane potentials between +30 and +50 mV (Figure 7).

3. Discussion

The results of this study show that the structural features of QX-314 are key to its ability to block I_Na, I_h, and I_K(erg) when applied extracellularly at high concentrations. As QX-314 was applied on the extracellular side, the IC₅₀ value needed for its block of I_Na(T), I_Na(L), I_h, or I_K(erg) observed in GH₃ cells was optimally estimated to be 93, 42, 68, or 73 μM, respectively, although the I_K(DR) amplitude was mildly suppressed. Since QX-314 is a membrane-impermeant molecule that does not diffuse through the lipid bilayer [7], it is plausible to assume it acts on these ionic currents via extracellular binding sites. However, its intracellular binding site may have a higher affinity for the specified ionic channels.

In accordance with previous studies, QX-314 could be an intracellular blocker of I_Na [1,21,22]. However, in our study, intracellular dialysis with QX-314 suppressed the amplitude of I_Na(T) less than the amplitude of I_Na(L). Furthermore, adding Tef, a known activator of I_Na [48,49], attenuated the decrease in the I_Na magnitude caused by QX-314. QX-314 applied intracellularly was also noted to shorten the τ_inact(S) value of I_Na(T). As a result, the QX-314 molecule might have a greater affinity for the open state of Na_V channels than for their closed or resting state in GH₃ cells [1]. This might explain the slow onset and long-lasting anesthesia observed in animal models [13,14,53].

Research has previously established the suppressive effect of lidocaine on I_h in various types of neurons and in heart cells [9,10,11,12,39]. In this study, we demonstrated that intracellular QX-314 suppressed the amplitude of I_h and decreased its activation time constant. The blocking of I_h by QX-314, applied extracellularly or intracellularly, is likely not instantaneous. Instead, the block develops over time after the HCN channel opens, causing a subsequent slowing of the current during QX-314 exposure. With continued dialysis with QX-314, additional application of OXAL, an activator of I_h [32], could effectively reverse the QX-314-mediated suppression of the I_h amplitude. In previous studies, HCN2, HCN3, or mixed HCN2+HCN3 channels have been expressed in GH₃ cells or other endocrine or neuroendocrine cells [24,31]. Based on the lack of isoform-specific effects of QX-314 on the magnitude and gating of these HCN-encoded currents, it is likely that QX-314 inhibits at least three of the four mammalian HCN isoforms: HCN1, HCN2, and HCN4. These three isoforms are all found in both neurons and cardiomyocytes [19,23]. Moreover, the QX-314-mediated suppression of I_h observed in this study would likely affect the spontaneous electrical activity in diverse excitable cell types, such as GH₃ cells [23,25,36]. It is also reasonable to reflect that the inhibition of HCN-encoded currents by QX-314 may be intimately linked to changes in either stimulus–secretion coupling (e.g., exocytosis) in endocrine/neuroendocrine cells, peripheral sensation, or analgesic actions [2,10,15,16,22,24,30].

This study provides evidence that QX-314 applied intracellularly or extracellularly was able to suppress the magnitude of I_K(erg) evoked by long-lasting membrane hyperpolarization. This work also demonstrated that GH₃ cells exhibited the dynamically changing Hys_(V) behavior of I_K(erg) when activated by an upright isosceles-triangular V_ramp, which strongly reflects the remarkable voltage dependence of double V_ramp-induced I_K(erg) in GH₃ cells [50]. It is further important to mention that intracellular dialysis with QX-314 (10 μM) was found to decrease the Δarea of I_K(erg)’s Hys_(V). With continued dialysis with QX-314, the further addition of PD118057, an activator of I_K(erg) [51], was found to counteract QX-314 mediated decrease inf Hys_(V)’s strength of the current. Consequently, these results strongly suggest that the Hys_(V)’s degree of I_K(erg) observed in these cells can be evidently perturbed. It is therefore reasonable to assume that QX-314 applied intracellularly possesses positive charges, which may become the protonated or quaternized forms and in turn exert multiple interactions with negative charge amino acid residues in the pore region and/or voltage-sensing domains of K_erg channels, thereby blocking the pore and restricting the movement of the S4 voltage-sensing segments. The QX-314 molecule can enter K_erg-channel pores from the intracellular side and block K⁺ ion movements through the channel at a depolarized potential. To what extent an QX-314-mediated block of I_K(erg) (i.e., rapidly activating delayed rectifier K⁺ current) affects cardiac function [43] remains to be further resolved.

QX-314 is known to preferentially block channels in their open state of the Na_V channel, and our protocol involved a series of pulses to promote channel opening. However, when QX-314 is applied intracellularly, its access to the channel pore is direct and not dependent on extracellular activation. In this context, the presence of external agonists (e.g., Tef) may alter channel gating kinetics or induce conformational states that reduce QX-314’s binding affinity or accessibility from the intracellular side [48,49]. One possible explanation for the cancelation of the blocking effect is that agonist-induced conformational changes stabilize a channel state that is less susceptible to intracellular QX-314 binding, despite increased open-state probability. Alternatively, agonist binding may promote the rapid inactivation or desensitization of the channel, thereby limiting the time window during which QX-314 can exert its blocking action.

Previous reports have indicated that QX-314 is a membrane-impermeable derivative of lidocaine [7]. However, our findings and those of other studies [39,40,41] have suggested that its site of action is intracellular. How this compound gains access to the cytosol remains unclear. One possible explanation is that it enters the cell via other permeabilizing mechanisms, thereby exerting its inhibitory effects on ionic currents. This unique property has made QX-314 a valuable tool in neuroscience and pain research, especially in efforts to achieve targeted, long-lasting local anesthesia without affecting surrounding tissues.

QX-314 is a quaternary lidocaine derivative that carries a permanent positive charge, which prevents it from passively crossing the lipid bilayer of the neuronal membrane [7]. However, recent studies have demonstrated that QX-314 can selectively enter nociceptive neurons via the transient receptor potential vanilloid 1 (TRPV1) channel, which forms large, cation-permeable pores upon activation by agents such as capsaicin [3]. Once inside the cell, QX-314 binds to the intracellular vestibule of voltage-gated ion channels, thereby modulating the initiation and propagation of action potentials. This blockade results in long-lasting analgesia with minimal motor impairment due to the selective expression of TRPV1 on pain-sensing neurons [3,5].

Lidocaine is a well-established local anesthetic known for its non-selective blockade of Na_V channels [2,52]. It inhibits neuronal excitability by stabilizing the neuronal membrane and preventing the initiation and conduction of action potential. In addition to Na_V channels, lidocaine has been shown to modulate other ion channels, including transient outward potassium currents. These broader effects contribute to its widespread use but also limit its specificity in mechanistic studies. Therefore, in this study, we focused on QX-314, which—when delivered intracellularly, possibly via TRPV1 channels—offers a more targeted approach to ion channel blockade [2,3,5,6,7,9].

4. Materials and Methods

4.1. Chemicals, Drugs, Reagents, and Solutions Used in This Study

QX-314 (CAS No.: 21306-56-9; also known as lidocaine N-ethyl bromide, N-ethyllidocaine bromide, and N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide purchased from Sigma-Aldrich (Genechain, Kaohsiung, Taiwan); and chemical formula C₁₆H₂₇BrN₂O), along with nimodipine, oxaliplatin (OXAL), tefluthrin (Tef), tetraethylammonium chloride (TEA), and tetrodotoxin (TTX) were procured from Sigma-Aldrich (Genechain, Kaohsiung, Taiwan). PD118057 was obtained from Tocris (Union Biomed, Taipei, Taiwan). Unless stated otherwise, horse and fetal calf sera, along with L-glutamine, trypsin/EDTA, and culture media (e.g., Ham’s F-12 medium) were sourced from HyClone^TM (Thermo Fisher; Level Biotech, Tainan, Taiwan). Other chemicals and reagents were primarily supplied by Sigma-Aldrich or Merck (Genechain) and were of laboratory grade, acquired from standard, reliable sources.

In this study, the standard extracellular medium consisted of HEPES-buffered normal Tyrode’s solution, formulated with the following ionic compositions (in mM): 136.5 NaCl, 1.8 CaCl₂, 5.4 KCl, 0.53 MgCl₂, 5.5 glucose, and 5.5 HEPES, and the pH was brought to 7.4 using NaOH. To examine I_h or I_Na, a Ca²⁺-free variant of Tyrode’s solution—matching the standard composition but lacking CaCl₂—was employed.

To examine I_K(erg), the extracellular medium was replaced with a high-K⁺, Ca²⁺-free bath composition containing (in mM): 130 KCl, 10 NaCl, 3 MgCl₂, and 5 HEPES, with the pH adjusted to 7.4 using KOH. Patch pipettes used for recording I_h and I_K(erg) currents were loaded with an internal solution comprising (in mM): 130 K-aspartate, 20 KCl, 1 KH₂PO₄, 1 MgCl₂, 3 Na₂ATP, 0.1 Na₂GTP, 0.1 EGTA, and 5 HEPES. The pH of this solution was brought to 7.2 using KOH. For I_Na, the K⁺ ions in the internal solution were substituted with Cs⁺ ions, and the pH was adjusted to 7.2 using CsOH. In specific experimental protocol, the intracellular recording medium was loaded with 10 μM QX-314 to measure changes in ionic currents. All reagents were formulated using deionized water that had been further refined via the Milli-Q^® ultrapure purification system (Merck, Tainan, Taiwan).

4.2. Cell Preparation

GH₃ pituitary tumor cells (BCRC-60015), obtained from the Bioresources Collection and Research Center in Hsinchu, Taiwan, were cultured in Ham’s F-12 medium supplemented with 15% (v/v) horse serum, 2.5% (v/v) fetal calf serum, and 2 mM L-glutamine. Cells were maintained at 37 °C in plastic culture flasks with a 50 mL capacity under monolayer conditions within a humidified incubator containing a CO₂/air mixture at a ratio of 1:19) [46,47]. Cell proliferation was routinely assessed using a colorimetric method based on a tetrazolium salt (WST), conducted in microtiter plates. Absorbance was measured with an ELISA microplate reader (Dynatech, Chantily, VA, USA). Measurements were performed once cultures attained a 50–70% confluence, typically between 5 and 7 days after seeding.

4.3. Electrophysiological Assessment via Patch-Clamp Current Recording

On the day of experimentation, GH₃ cells were gently dissociated using a 1% trypsin-EDTA solution. A small volume of the resulting cell suspension was transferred into a custom-built chamber mounted on the stage of an inverted DM-II phase-contrast microscope (Leica; Major Instruments, Kaohsiung, Taiwan). Cells were maintained at room temperature (20–25 °C) in normal Tyrode’s solution, with ionic compositions as previously described. Before measurements, cells were given time to settle onto the bottom surface of the chamber.

Patch electrodes were crafted from Kimax-51 borosilicate capillaries (#DWK34500-99; Kimble^®, Merck, Tainan, Taiwan) using a vertically oriented microelectrode puller (PP-83; Narishige, Major Instruments, Taipei, Taiwan). The resulting electrodes featured tip diameters of approximately 1 μm and exhibited resistances between 2 and 4 MΩ. During electrophysiological recordings, each electrode was securely positioned in an airtight holder equipped with a lateral suction port. Electrical continuity with the internal pipette solution was maintained via a silver-chloride wire.

Transmembrane ionic fluxes were monitored using an adapted variant of conventional whole-cell configuration of the patch-clamp technique, which incorporated dynamic adaptive suction—where negative pressure was progressively reduced in accordance with rising seal resistance. This approach, implemented with either an Axoclamp-2B amplifier (Molecular Devices, Sunnyvale, CA, USA) or an RK-400 amplifier (Bio-Logic, Claix, France), has been shown to enhance gigaseal formation and stability during prolonged recordings [33,47].

Once a high-resistance seal (>1 GΩ) was achieved, the cell membrane was disrupted by controlled suction to establish the whole-cell configuration. To eliminate potential offset artifacts arising from differences between pipette and bath solutions, the potential was zeroed immediately prior to seal formation, and all recordings obtained in whole-cell configuration were subsequently adjusted.

4.4. Whole-Cell Current Recordings

Electrophysiological signals, including potential and current tracings, were recorded using an Asus ExpertBook laptop (Tainan, Taiwan) and a high-resolution Digidata 1440A interface (Molecular Devices). The Digidata 1440A performed both analog-to-digital (A/D) and digital-to-analog (D/A) conversions, operating at a sampling rate of 10 kHz or more. Data acquisition was controlled by pCLAMP 10.6 software (Molecular Devices) running on a Microsoft Window 7 operating system. Using D/A conversion, the voltage-clamp protocols employing rectangular or ramp voltage (V_ramp) waveforms were specifically tailored to measure the steady-state current versus voltage (I-V) relationship and voltage-dependent hysteresis (Hys_(V)) of the target ionic currents specified, such as I_K(erg).

4.5. Data Analyses

To assess the percentage inhibition of the I_Na amplitude (specifically I_Na(T) and I_Na(L)) by the extracellular application of QX-314, cells were voltage-clamped at −80 mV. A 40 msec depolarizing pulse to −10 mV was then applied to elicit I_Na. The amplitudes of I_Na(T) and I_Na(L) were measured at the onset and end of each depolarizing pulse, respectively, under varying concentrations of QX-314. For I_h measurements, the cell was held at −40 mV. A 2 s hyperpolarizing step to −120 mV was applied, and the I_h amplitude was measured at the end of this step. To record I_K(erg), cells were bathed in a high-K⁺, Ca²⁺-free solution. The membrane potential was set at −10 mV, and a 1 s step hyperpolarization to −110 mV was applied to evoke deactivating I_K(erg)s. The peak current amplitude was measured at the onset of the hyperpolarization pulse.

To evaluate the concentration-dependent inhibition of I_Na(T), I_Na(L), I_h, and I_K(erg) by extracellular QX-314, the data were fitted using a modified Hill equation, specifically the three-parameter logistic equation, expressed as follows:

$$\mathrm{Percentage} \mathrm{inhibition}\left(\%\right)=\left(E_{max}\times {\left[QX\right]}^{n_H}\right)/\left({IC}_{50}^H+{\left[QX\right]}^{n_H}\right)$$

In this equation, [QX] is the extracellular concentration of QX-314. IC₅₀ is the concentration needed for 50% inhibition. n_H is the Hill coefficient, which indicates the degree of cooperativity in the drug’s effect. Lastly, E_max represents the maximal inhibition of the ionic current that can be induced by QX-314.

4.6. Curve-Fitting Procedures and Statistical Analyses

For linear or nonlinear curve-fitting analyses, we employed a range of computational approaches, including the Microsoft^® “Solver” add-in embedded in Excel^TM 2022 (Microsoft^® 365), as well as the 64-bit OriginPro^® 2021 software (OriginLab; Scientific Formosa, Kaohsiung, Taiwan) [48]. Ionic currents were expressed as mean ± standard error of the mean (SEM), with sample sizes (n) representing the number of cells from which independent measurements were obtained. All graphical results were presented as mean ± SEM.

To assess statistical differences between two groups, we initially applied either paired or unpaired Student’s t-tests. For comparisons involving more than two groups, we conducted a one-way or two-way analysis of variance (ANOVA), followed by Duncan’s multiple range post hoc tests. Formal assessments of data normality were conducted using the Shapiro–Wilk test across all relevant datasets. The results demonstrated no significant deviation from a normal distribution, with p-values exceeding 0.05 for all groups. Statistical significance was defined as p < 0.05, and significant differences are indicated in the figures by asterisks (* or **).

5. Conclusions

The major findings in this study demonstrate seven key effects of the compound QX-314 on ionic currents. First, when applied to GH₃ cells from the outside, QX-314 was more effective at suppressing I_Na(L) than I_Na(T) amplitudes. Second, the intracellular application of QX-314 (10 μM in the patch pipette) caused a progressive decrease in the I_Na amplitude and accelerated its inactivation; moreover, this suppressive effect on I_Na was partially reversed by the presence of Tef. Third, as QX-314 was applied extracellularly, the amplitude of I_h in response to long-lasting membrane hyperpolarization was suppressed with considerable slowing in the activation time course of the current (i.e., an increase in τ_act value). Fourth, in cells internally dialyzed with QX-314, the I_h amplitude responding to the long-lasting hyperpolarizing step was progressively suppressed. This inhibition was subsequently reversed by the addition of oxaliplatin (OXAL). Fifth, with the presence of extracellular QX-314, the I_K(erg) amplitude activated by membrane hyperpolarization was overly depressed. Sixth, upon intracellular dialysis with QX-314, the I_K(erg) measured at the entire voltage-clamp level was decreased, and, with continued dialysis with QX-314, the additional presence of PD118057, a stimulator of I_K(erg), reversed the QX-314-induced decrease in I_K(erg) amplitude. Seventh, the amplitude of I_K(DR), in response to membrane depolarization, was slightly decreased with the extracellular presence of QX-314.

The differential effects of QX-314 via extracellular and intracellular routes suggest potential strategies for the targeted modulation of neuronal excitability, which may be relevant for developing localized analgesic or anti-epileptic interventions. Furthermore, the presence of QX-314, whether inside or outside the cell, may alter the magnitude or gating of I_Na, I_h, and I_K(erg). This may lead to the disruption of electrophysiological activity in diverse excitable cells, a phenomenon observable in both in vitro culture models and in vivo biological systems [5,7,41,53].