H₂O₂ Sensitivity of K_v Channels in Hypoxic Pulmonary Vasoconstriction: Experimental Conditions Matter

Abstract

Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange but, when impaired, can result in life-threatening hypoxemia. Moreover, under conditions of generalized alveolar hypoxia, HPV can result in pulmonary hypertension. Voltage-gated K⁺ channels (K_v channels) are key to HPV: a change in the intracellular hydrogen peroxide (H₂O₂) levels during acute hypoxia is assumed to modulate these channels’ activity to trigger HPV. However, there are longstanding conflicting findings on whether H₂O₂ inhibits or activates K_v channels. Therefore, we hypothesized that H₂O₂ affects K_v channels depending on the experimental conditions, i.e., the H₂O₂ concentration, the channel’s subunit configuration or the experimental clamping potential in electrophysiological recordings. Therefore, cRNAs encoding the K_v1.5 channel and the auxiliary K_vβ subunits (K_vβ1.1, K_vβ1.4) were generated via in vitro transcription before being injected into Xenopus laevis oocytes for heterologous expression. The K⁺ currents of homomeric (Kv1.5) or heteromeric (K_v1.5/K_vβ1.1 or K_v1.5/K_vβ1.4) channels were assessed by two-electrode voltage clamp. The response of the K_v channels to H₂O₂ was markedly dependent on (a) the clamping potential, (b) the H₂O₂ concentration, and (c) the K_v channel’s subunit composition. In conclusion, our data highlight the importance of the choice of experimental conditions when assessing the H₂O₂ sensitivity of K_v channels in the context of HPV, thus providing an explanation for the long-lasting controversial findings reported in the literature.

1. Introduction

Hypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism of the mammalian lung that optimizes gas exchange. By rapidly matching perfusion to ventilation, HPV ensures optimal oxygenation of the blood during local alveolar O₂ shortage, i.e., hypoxia [1,2,3]. Impairment of this vital mechanism can result in insufficient blood oxygenation and thus O₂ undersupply to the organs. Such fatal hypoxemia due to disturbed HPV can occur in various clinical scenarios. This includes patients afflicted with pneumonia, those with acute respiratory distress syndrome (ARDS), or those requiring mechanical ventilation, e.g., COVID-19 patients during the recent pandemic [4]. Additionally, systemic administration of pulmonary vasodilatory agents, such as prostacyclin, has been associated with similar adverse outcomes due to impaired HPV [2,5]. Moreover, imbalanced HPV is considered to provoke high-altitude pulmonary edema (HAPE), a life-threatening condition [5,6,7], and also acquires pathophysiological significance in situations with prolonged hypoxia, e.g., at high altitudes or in lung diseases such as sleep apnea or chronic obstructive pulmonary disease (COPD) [8,9,10]. In these conditions, generalized and consistent alveolar hypoxia can occur, leading to the constriction of the blood vessels in the entire lung. If hypoxia persists, the subsequent onset of vascular remodeling processes leads—in addition to vasoconstriction—to the manifestation of pulmonary hypertension, which can ultimately result in right heart failure.

Although HPV was already described in 1946 by von Euler and Liljestrand, the underlying molecular mechanisms are not yet fully known [3,11]. A mandatory step in HPV is the hypoxia-induced inhibition of O₂-sensitive voltage-gated potassium (K_v) channels in pulmonary artery smooth muscle cells (PASMCs) [12,13,14,15,16]. K_v channels are composed of four α subunits forming a central pore in the membrane and four auxiliary β subunits to fine-tune the channel’s activity [13,17]. By mediating constant K⁺ leakage, K_v channels significantly contribute to maintaining the cellular membrane potential [16,18,19]. It is undisputed that hypoxia induces the closure of K_v channels, which in turn depolarizes the membrane potential of PASMCs, thereby increasing the Ca²⁺ influx via the activation of voltage-gated Ca²⁺ channels [2,20,21]. The resulting increase in intracellular Ca²⁺ induces the constriction of the individual cells and thus the pulmonary arterial vessels. Although much progress has been made in understanding the signaling pathways underlying O₂ sensing in HPV, the molecular mechanisms by which hypoxia affects the K_v channel activity are still not fully resolved [13].

A proposed signaling mechanism linking hypoxia to the K_v channel function in PASMCs involves reactive oxygen species (ROS) and hydrogen peroxide (H₂O₂) in particular [3,13,22,23,24]. Although there is a consensus on the involvement of ROS in hypoxia-induced K_v channel inhibition, there is a long-lasting disagreement as to whether hypoxia induces an increase or decrease in ROS [2,13]. The original redox hypothesis of Archer, Weir, and colleagues proposes that under normoxic conditions, a certain level of intracellular ROS, i.e., H₂O₂, maintains the K_v channels in an oxidized open state. Conversely, acute hypoxia causes an increase in the NADH/NAD⁺ ratio and a concomitant decrease in the H₂O₂ level that shifts the cellular redox state to a more reduced state, resulting in K_v channel inhibition and subsequent vasoconstriction [12,17,25,26,27]. In contrast, Paul Schumacker’s lab presented an opposing concept, often referred to as the mitochondrial ROS theory, which describes a hypoxia-induced increase in the intracellular ROS levels being causative for the inhibition of K_v channels [13,28]. This theory was not only confirmed by previous studies by our research group, but we could also identify the signaling molecule responsible as H₂O₂ [3] and further decipher the role of mitochondria in this regard [3,22]. Although there is a large body of evidence for increased ROS levels during acute hypoxia underlying HPV, both theories emphasize that the K_v channels are inhibited by the indisputable hypoxia-induced change in the intracellular H₂O₂ levels [3,13].

Consequently, several studies investigated the influence of H₂O₂ on K_v channel activity. In line with the discrepancies between the two distinct theories, contrary results were also reported by distinct research groups in this context. While some studies showed an activation of the K_v channels in response to H₂O₂ [29,30,31], other groups observed an H₂O₂-induced inhibition of these channels [3,32,33,34]. Upon thorough examination of these reports, discrepancies in the experimental conditions became apparent, specifically the widely varying and occasionally nonphysiologically high concentrations of H₂O₂ utilized. Furthermore, the studies were conducted at distinct experimental clamping potentials and on very different cell types, including various primary cells as well as overexpression systems that may be featured by different subunit compositions of K_v channels. In particular, the auxiliary K_vβ subunits are differentially expressed in the pulmonary circulation [35]. The reported correlation between their expression level and the strength of HPV suggests a role for these subunits in O₂ sensitivity [35]. Thus, the presence or absence of K_vβ subunits could be responsible for the different responses of K_v channels to H₂O₂, thereby delivering a potential explanation for the contrary results described in the literature.

Considering the controversy as to whether H₂O₂ inhibits or activates K_v channels, we hypothesized that H₂O₂ regulates K_v channel activity (a) in a concentration-dependent manner and/or (b) in a manner dependent on the molecular composition of the channel, i.e., the presence or absence of auxiliary K_vβ subunits.

2. Results

2.1. Homomeric Kvβ Subunits Do Not Form Functional Kv Channels

To address these questions, we selected the K_v1.5 channel as a suitable candidate. K_v1.5 has long been recognized as a channel involved in HPV [36,37]. In line with this, mice lacking K_v1.5 exhibited impaired HPV [38], which could be fully restored by its subsequent introduction via gene transfer in vivo [39]. To address the H₂O₂ sensitivity of K_v1.5, this subunit was heterologously expressed either as a homomeric or heteromeric channel, i.e., in combination with either K_vβ1.1 or K_vβ1.4 subunits in Xenopus laevis oocytes, which are supposed to not express endogenous K_v channels [40,41]. The potassium (K⁺) currents were assessed via the two-electrode voltage clamp technique (TEVC) by applying depolarizing 200 ms voltage pulses from a holding potential of −60 mV to +50 mV in 10 mV steps. Neither water-injected control oocytes (Figure 1a) nor oocytes injected with the auxiliary subunits K_vβ1.1 (Figure 1b) or K_vβ1.4 (Figure 1c) alone exhibited voltage-dependent currents when depolarized. In contrast, oocytes injected with cRNA-encoding K_v1.5 subunits showed typical rapid outward K⁺ currents upon depolarization to potentials more positive than −20 mV (Figure 1d). In agreement with previous studies, the homomeric K_v1.5 currents exhibited no detectable inactivation in response to such short pressure pulses [42]. Co-expression with K_vβ1.1 altered the K_v1.5 activity by inducing a rapid voltage-dependent inactivation (Figure 1e), while co-expression with K_vβ1.4 lowered the currents’ amplitude (Figure 1f). The final application of 4-aminopyridine (4-AP) largely inhibited the K⁺ currents (Figure 1d–f), which confirmed the successful expression of the three K_v channel combinations.

2.2. Evaluation of H₂O₂ Decomposition in the Experimental Setup

Beside the temperature and the presence of decomposing impurities, the stability of H₂O₂ in aqueous solution is affected by the pH value [43]. For optimum stability, the pH of pure H₂O₂ is below pH 4.5, while increasing it to above pH 5 sharply increases the decomposition of H₂O₂ into water and oxygen. Since the pH value of the oocyte Ringer’s solution (ORi) used in our experiments was at a physiological value of pH 7.4, we first tested the decomposition of H₂O₂ in our experimental setup via an Amplex Red Hydrogen Peroxide/Peroxidase Assay. As schematically shown in Figure 2a, the ORi in the reservoir was analyzed directly after adding H₂O₂ to the perfusate (input). Afterwards, the perfusion was started to deliver the H₂O₂-containing ORi to the recording chamber. The second sample was then taken from the recording chamber 90 s post starting the perfusion—which corresponds to the time point at which the K⁺ currents were recorded in the electrophysiological experiments. We observed that at the time point of current recording, approximately 50% of the H₂O₂ of the distinct input concentrations was decomposed (Figure 2b).

2.3. Experimental Approach to Evaluate the Effect of H₂O₂ on K_v Channel Activity

For evaluation of the effect of H₂O₂ on K_v channel activity, K_v currents were elicited by depolarizing voltage pulses between −80 mV and +50 mV in 10 mV steps from a holding potential of −60 mV in the absence (Control, Figure 3a) and after the addition of H₂O₂ (Figure 3b). As shown in Figure 3c, H₂O₂ (10 mM) significantly activated K⁺ currents at all the clamping potentials of −30 mV and more positive—with a prominent effect at −20 mV, a value within the physiological range of the membrane potential of PASMCs under hypoxia [3,23]. Therefore, two distinct voltage steps were chosen for statistical analysis in all the following experiments: the one that lies within the physiological range of the membrane potential of PASMCs (−20 mV) and one at a positive membrane potential, which was characterized by the strongest H₂O₂-induced activation (+50 mV).

2.4. The Effect of H₂O₂ on Homomeric K_v1.5 Channels Is Voltage- and Concentration-Dependent

To assess the effect of distinct H₂O₂ concentrations on the activity of homomeric K_v1.5 channels, H₂O₂ was applied in concentrations between 0.01 µM and 100 mM (Figure 4a,b), using a new oocyte and freshly prepared H₂O₂ for each individual experiment. As previously described, the K⁺ current amplitudes were analyzed at step potentials of −20 mV (Figure 4a) and +50 mV (Figure 4b) to determine the potential voltage-dependency of the H₂O₂ effect. In our investigation, we observed dependencies on both voltage and concentration: while low H₂O₂ concentrations (ranging from 0.1 µM to 1 mM) did not affect the activity of K_v1.5 channels at a physiological step potential of −20 mV (Figure 4a), these same concentrations were found to inhibit the channels at a positive step potential (+50 mV, Figure 4b). Interestingly, higher H₂O₂ concentrations (10 mM and above) were observed to enhance the K⁺ currents at −20 mV (Figure 4a), yet no such effect was noted for the same concentrations at +50 mV (Figure 4b). These findings underscore a dual relationship involving both the voltage- and concentration-dependency of H₂O₂ modulation on K_v1.5 channel activity.

2.5. Co-Expression of K_v1.5 with K_vβ Subunits Modulates the Channel’s Response to H₂O₂

To determine whether co-expression with accessory β subunits alters the channel’s response to H₂O₂, K_v1.5 was co-expressed with the β-isoform K_vβ1.1 and the channels’ response to H₂O₂ was assessed by TEVC (Figure 4c,d). While low H₂O₂ concentrations (0.01 µM–10 µM) did not affect the activity of the K_v1.5/K_vβ1.1 channels at a step potential of −20 mV (Figure 4c), the same concentrations significantly inhibited the K_v1.5/K_vβ1.1 currents at +50 mV (see enlargement in Figure 4d). Importantly, this inhibition was induced even with the lowest H₂O₂ concentration, i.e., 0.01 µM. In contrast, application of higher H₂O₂ concentrations (1 mM to 100 mM) significantly and concentration-dependently increased the K⁺ currents at both step potentials, i.e., −20 mV and +50 mV (Figure 4c,d).

To assess whether distinct isoforms differently modulate the response of K_v1.5 to H₂O₂, an additional K_vβ isoform (K_vβ1.4) was co-expressed with K_v1.5 (Figure 4e,f). In contrast to the homomeric K_v1.5 and heteromeric K_v1.5/K_vβ1.1 channels, this channel combination was significantly inhibited by low H₂O₂ concentrations at −20 mV (Figure 4e). Importantly, this inhibition occurred under physiologically relevant conditions in terms of both the clamping potential and the H₂O₂ concentration. Notably, K_v1.5/K_vβ1.4 was the only channel combination that remained unaffected by low H₂O₂ concentrations at a depolarized clamping potential (+50 mV) while being inhibited when exposed to high concentrations of 500 µM and above (Figure 4f).

3. Discussion

It is well accepted that a shift in the intracellular ROS levels in response to acute hypoxia inhibits K_v channels in PASMCs, thereby triggering HPV. However, although the signaling molecule responsible could be identified as H₂O₂ [3], long-lasting divergences and contradictory reports exist in the literature on whether H₂O₂ inhibits or activates K_v channels. We hypothesized that these contrary observations depend on (a) the experimental clamping potential, (b) the H₂O₂ concentration utilized and/or (c) the K_v channel subunit composition, i.e., the presence of auxiliary K_vβ subunits. Therefore, the aim of the present study was to determine whether H₂O₂ modulates K_v channel activity in a concentration-dependent manner, and at the same time, to address the potential role of the accessory β subunits K_vβ1.1 and K_vβ1.4 as well as the experimental clamping potential in H₂O₂ sensitivity. For our studies, we heterologously expressed K_v1.5 as a homomeric channel and in combination with either K_vβ1.1 or K_vβ1.4, respectively, before exposing them to H₂O₂ at various concentrations between 0.01 µM and 100 mM. We then analyzed the K_v currents at two distinct clamping potentials, i.e., (a) at −20 mV, which falls within the physiological resting membrane potential of PASMCs and (b) at +50 mV, which was characterized by the highest voltage-induced current.

As an expression system, we used Xenopus laevis oocytes that are supposed to not express endogenous 4-AP-sensitive K_v channels [40,41]. This assumption was confirmed in our study as neither the water-injected control oocytes, nor the oocytes injected with K_vβ subunits alone exhibited voltage-induced currents upon depolarization, thereby confirming the choice of the expression system as ideally suited for investigating the effect of H₂O₂ on defined K_v channel combinations.

3.1. The Reaction of K_v Channels to H₂O₂ Depends on the Experimental Clamping Potential

K_v currents are typically measured at positive clamping potentials of up to +70 mV [29,34], which not only ensures channel activation but also maximizes the driving force for K⁺ efflux, thereby facilitating current detection and analysis. However, such positive clamping potentials do not reflect physiological conditions, as the membrane potentials in PASMCs typically range from approximately −50 mV to −10 mV [3,44]. As a result, the K_v channel behavior observed at such strongly positive (depolarized) voltages may not fully reflect their natural activation, inactivation, or response to pharmacological modulation under physiological conditions. In line with this, we observed a clear voltage-dependency in the response to H₂O₂ for all three channel combinations, i.e., K_v1.5, K_v1.5/K_vβ1.1 and K_v1.5/K_vβ1.4 (see Figure 4). For example, at a depolarizing voltage step of +50 mV, the homomeric K_v1.5 channels were inhibited by low concentrations of H₂O₂ (0.8 µM–1 mM), while higher H₂O₂ concentrations (5 mM–100 mM) had no effect on the channels’ activity. In contrast, at a physiological clamping potential of −20 mV, the channels exhibited a completely different response pattern, being unaffected by low H₂O₂ concentrations but strongly activated by H₂O₂ concentrations of 10 mM and above. Similar observations of voltage-dependent effects were also made for the heteromeric channels, i.e., K_v1.5/K_vβ1.1 and K_v1.5/K_vβ1.4. These data clearly indicate that the response of the K_v channels to H₂O₂ is markedly dependent on the experimental clamping potential.

However, it is important to consider that—in contrast to Xenopus laevis oocytes—in both primary cells and overexpression systems (particularly mammalian cell lines), endogenous ion channels might be active at physiological clamping potentials and contribute to the recorded currents, thus potentially confounding the detection and analysis of K_v-channel-specific responses. Furthermore, at physiological clamping potentials, the electrochemical driving force for K⁺ is diminished, resulting in reduced current amplitudes and less pronounced differences, thereby potentially masking subtle differences that are more apparent under depolarized conditions. Thus, the choice of clamping protocol constitutes a critical experimental parameter that can profoundly affect the biophysical properties and pharmacological response of K_v channels to H₂O₂.

3.2. The Effect of H₂O₂ on K_v Channel Activity Is Concentration-Dependent

The estimation of the intracellular concentration of H₂O₂ is based on the kinetics of production and elimination, while its determination is still technically difficult [45,46]. The physiological estimate of the cytosolic H₂O₂ concentration has been reported to be in the pico- to nanomolar range, e.g., 2.2 nM [47], 1–700 nM [48], 1–10 nM [46] or even in the picomolar range [47,49]. Consequently, H₂O₂ concentrations above the nanomolar range are regarded as pathological levels that are related to oxidative stress [46].

In previous studies investigating the effect of H₂O₂ on K_v channel activity, various concentrations of H₂O₂ were applied, resulting in contradictory outcomes. Higher concentrations, ranging from 0.1 to 10 mM, were reported to activate K_v channels [29,30,31]. In contrast, other studies employing lower concentrations, i.e., between 5 µM and 400 µM, observed an inhibitory effect on K_v channel activity [32,33,34]. These divergent observations prompted us to hypothesize that the concentration of H₂O₂ used under experimental conditions underlies the reported inconsistencies in H₂O₂-mediated K_v channel modulation, i.e., whether H₂O₂ inhibits or activates K_v channels. Based on this rationale, we applied a broad range of H₂O₂ concentrations, spanning from physiological to pathophysiological levels (10 nM to 100 mM).

At a physiological clamping potential of −20 mV, we observed the clear concentration-dependency of H₂O₂ on the homomeric K_v1.5 channel activity: whereas H₂O₂ did not affect the K_v1.5 activity at physiological concentrations, an activation of up to 200% was observed in response to pathophysiological concentrations of 10 mM and above. As discussed in the previous paragraph, a different response pattern was observed at a depolarized clamping potential (+50 mV), although there was still a clear difference between the physiological and pathophysiological H₂O₂ concentrations. Interestingly, the channel’s response to high doses of H₂O₂ appeared to be an “all or nothing” effect as no difference was observed between the concentrations applied. This indicates that oxidation by H₂O₂ above a certain level might induce a conformational change within the channel structure that increases the channel’s activity. However, although K_v1.5 has been implicated in HPV and is therefore presumed to be inhibited by hypoxia-induced H₂O₂ release, we did not observe an inhibition of homomeric K_v1.5 in response to H₂O₂ at physiological clamping potentials—regardless of the H₂O₂ concentration applied. Given that the accessory β subunits are reported to exert regulatory functions on K_v channels, including modulation of their redox sensitivity [50], we next investigated their influence on the H₂O₂ sensitivity of K_v1.5 channels.

3.3. K_vβ Subunits Modulate the Response of K_v1.5 to H₂O₂

Interestingly, the H₂O₂-induced activation in response to pathophysiological concentrations above 10 mM could be further potentiated by the co-expression of K_v1.5 with K_vβ1.1. In these heteromeric channels (K_v1.5/K_vβ1.1), H₂O₂ of 1 mM and above induced a channel activation of up to 900%, which in contrast to the homomeric K_v1.5 channels, was clearly concentration-dependent. This observation suggests that the accessory β subunits not only alter the channel’s activity per se, as described in several studies [51,52,53], but also equip them with the ability to fine-tune the kinetics of K_v1.5 channels in response to H₂O₂, most likely as a result of their oxidoreductase properties [50]. However, an H₂O₂-induced inhibition of the channel, as would be necessary for HPV initiation, was only observed in response to an H₂O₂ concentration of 100 µM—which, however, is not in the assumed physiological range of intracellular H₂O₂ concentrations [46].

The co-expression of K_v1.5 with the auxiliary isoform K_vβ1.4 rendered the channel sensitive to inhibition by physiological H₂O₂ concentrations—even as low as 10 nM. Most interestingly, this inhibition was observed at a physiological clamping potential—thereby closely reflecting the physiological conditions present in PASMCs under hypoxia. Given that H₂O₂ was decomposed by approximately 50% under our experimental conditions, this suggests that K_v1.5/K_vβ1.4 channels can be inhibited by H₂O₂ concentrations of less than 10 nM. Sommer et al. reported in a study from our laboratory that 124 nM H₂O₂ inhibits the K_v currents in primary mouse PASMCs [3], thereby contributing to the initiation of HPV. This finding is consistent with the present data, which demonstrate that even lower concentrations of H₂O₂ can significantly inhibit K_v currents, an effect that is—at least for Kv1.5—critically dependent on the clamping potential and the ion channel composition, particularly the presence of the auxiliary subunit K_vβ1.4.

It should be noted, however, that in addition to K_v1.5, a variety of other K_v channel subunits contribute to HPV initiation and further studies would be required to investigate the effect of H₂O₂ on these channels as well. However, our study clearly highlights the importance of carefully selecting experimental parameters—particularly the necessity of utilizing physiological conditions—when attempting to elucidate a physiological process.

3.4. Conclusions

In conclusion, our findings indicate that the response of K_v channels to H₂O₂ is markedly dependent on (a) the experimental clamping potential, (b) the H₂O₂ concentration and (c) the K_v channel subunit composition. Thus, our data clearly highlight the importance of the choice of experimental conditions when assessing the H₂O₂ sensitivity of K_v channels in the context of HPV, thus providing an explanation for the long-lasting controversial findings reported in the literature.

4. Materials and Methods

4.1. Generation of cRNA for Heterologous Expression of K_v Channels

Plasmids encoding murine K_v1.5 (NM_145983.2), K_vβ1.1 (NM_010597.4) or K_vβ1.4 (NM_001403872.1) in a pTNT expression vector (Promega, Madison, WI, USA) were purchased from the Eurofins gene synthesis service (Eurofins, Ebersberg, Germany) and transformed into competent JM109 E. coli cells (Promega, Madison, WI, USA) for amplification. Upon subsequent plasmid purification using the GenElute Plasmid Miniprep Kit (Sigma Aldrich, St Louis, MO, USA) according to the manufacturer’s instructions, the purified plasmids were used for cRNA synthesis via in vitro transcription using the mMESSAGE mMACHINE T7 kit (Invitrogen, Waltham, MA, USA). The resulting full-length capped cRNAs were then purified using the MEGAclear transcription cleaning kit (Invitrogen, Waltham, MA, USA). The cRNAs were stored at –80 °C and diluted in nuclease-free water immediately prior to injection.

4.2. Preparation, Storage and Injection of Xenopus Laevis Oocytes

Xenopus laevis oocytes at stages V–VI were purchased from Ecocyte Bioscience (Dr. Lohmann Diaclean GmbH, Dortmund, Germany) and plated individually in a 96 Conical Bottom MicroWell Plate (Fischer Scientific, Schwerte, Germany) filled with modified Barth’s solution (MBS, in mM: NaCl 88, KCl 1, CaCl₂ 0.4, Ca(NO₃)₂ 0.33, MgSO₄ 0.8, TRIS-HCl 5, NaHCO₃ 2.4, pH 7.4, Dr. Lohmann Diaclean GmbH, Dortmund, Germany) that was supplemented with sodium pyruvate (2.5 mM, Sigma Aldrich, St Louis, MO, USA), penicillin (20 µg/mL, Sigma Aldrich, St Louis, MO, USA) and streptomycin (25 µg/mL, Sigma Aldrich, St Louis, MO, USA). The oocytes were stored in a digital mini-incubator (Gilson, Middleton, WI, USA) at 5–8 °C until use.

At 24–48 h prior to the electrophysiological recordings, the oocytes were injected with K_v1.5: 0.1 ng/oocyte; K_vβ1.1: 1.75 ng/oocyte; K_vβ1.4: 0.1 ng/oocyte in a total injection volume of 10 nL/oocyte (for K_v1.5 and K_v1.5/K_vβ1.4, resp.) and 17.5 nL/oocyte (for K_v1.5/K_vβ1.1) using the Roboinject fully automated injection system (Multichannel Systems, Reutlingen, Germany). Therefore, the injection needle was filled with mineral oil (Sigma Aldrich, St Louis, MO, USA) before being mounted onto the device. Afterwards, the cRNAs that were diluted in nuclease-free water were drawn into the injection needle and injected into the oocytes. The impalement and injection depth for the cRNAs were defined for all the samples as 550 µm and 450 µm, respectively. The injected oocytes were incubated for 24 h at 18 °C for heterologous expression.

4.3. Electrophysiological Recordings via Two-Electrode Voltage Clamp (TEVC)

The K_v currents of the cRNA injected oocytes were recorded via the two-electrode voltage clamp (TEVC) technique using an automated TEVC system (Roboocyte2, Multichannel Systems, Reutlingen, Germany). Therefore, the glass capillaries of the measure head were filled with 1 M KCl. Only electrodes with tip resistances between 300 and 800 kΩ were used for the experiments. All the electrophysiological recordings were carried out at room temperature (20–22 °C) under continuous perfusion with oocyte Ringer’s solution (ORi; Normal Frog Ringer, Ecocyte Biosciences, composition in mM: NaCl 64, KCl 2, CaCl₂ 2, MgCl₂ 1, NaHCO₃ 26). The ORi inside the perfusion reservoir was continuously gassed with 21% O₂, 5.3% CO₂, 73.7 N₂ for pH stabilization at pH 7.4. Due to the rapid decomposition of H₂O₂ at physiological pH values, the H₂O₂ (Sigma Aldrich, St Louis, MO, USA) was freshly prepared directly before the start of each individual experiment. Being a strong oxidizing agent, H₂O₂ has the potential to corrode Ag/AgCl electrodes upon direct contact, which would manifest in a shift of the baseline current. Anticipating this potential issue, a rigorous maintenance protocol involving regular and frequent re-chlorination and replacement of the electrodes was implemented. Additionally, the baseline current was continuously monitored to ensure stability throughout the experiments.

For the electrophysiological recordings, the oocytes were voltage-clamped at a holding potential of −60 mV. The current amplitudes were elicited by depolarizing voltage pulses in 10 mV increment steps starting from −80 mV to +50 mV and 200 ms duration.

4.4. Experimental Design and Analysis

As a current rundown was already observed in the control recordings (two recordings in succession with an interval of 90 s between the two recordings), a different procedure was used. Two sets of experiments were performed for each experiment. As a control, two recordings were performed in the absence of H₂O₂ (control), separated by 90 s. In the second set of experiments, only one control recording was performed, before H₂O₂ was delivered to the recording chamber for 90 s. The recording taken after the 90 s (+H₂O₂) was then compared with the second current trace from the control measurements (−H₂O₂). The recordings +/−H₂O₂ were always performed on oocytes obtained from the same individual. The mean K_v current amplitudes were normalized relatively to the current at +50 mV in the control experiment. All the current traces were leak-subtracted prior to statistical evaluation. The current–voltage relationship (I–V curve) was obtained by plotting the averaged plateau current against the respective clamping potential. The specific blocker 4-aminopyrimidine (4-AP, 10 mM) was added at the end of experiment to validate the K_v channel expression.

4.5. Amplex Red Hydrogen Peroxide/Peroxidase Assay

To determine the decomposition of H₂O₂ inside the experimental system, H₂O₂ was added to the perfusate and delivered to the recording chamber via the perfusion system. At 90 s post starting the perfusion, a sample was taken from the recording chamber, representing the time point where the electrophysiological recording (+H₂O₂) was performed. The H₂O₂ concentration (input, i.e., [H₂O₂] present at time of recording) was determined by Amplex^® Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The assay was performed according to the supplier’s instructions. The absorbance was measured at 560/590 nm using a microplate reader TECAN Spark 10M (Tecan Trading AG, Männedorf, Switzerland). The manuscript consistently reports the input H₂O₂ concentration. However, it should be considered that—due to the decomposition of H₂O₂—the effective concentration at the oocyte during the electrophysiological recordings is lower (see Figure 2).

4.6. Statistical Analysis

The statistical differences were assessed via two-way ANOVA and the uncorrected Fisher’s least significant difference test for multiple comparisons or one-sample t-tests. All the analyses were considered statistically significant at p ≤ 0.05. Data are expressed as the mean ± SEM. The statistical analysis was performed using Prism 10 (GraphPad Software Inc., San Diego, CA, USA).