H2O2 Sensitivity of Kv 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.

Keywords: Kv channels; Kvβ subunits; Xenopus laevis oocytes; hydrogen peroxide (H2O2); hypoxic pulmonary vasoconstriction (HPV); two-electrode voltage clamp (TEVC).

Figure 1

Expression and electrophysiological characterization of distinct K_v channel combinations in Xenopus laevis oocytes. (a–c) Representative current traces of control oocytes exposed to depolarizing voltage steps. Neither (a) water (black) nor (b) homomeric K_vβ1.1 (light turquois) or (c) K_vβ1.4 subunits (light purple) built functional channels. (d–f) Electrophysiological characterization of (d) homomeric K_v1.5 channels (gray), (e) K_v1.5 co-expressed with the auxiliary subunits K_vβ1.1 (K_v1.5/K_vβ1.1, dark turquoise) or (f) together with K_vβ1.4 (K_v1.5/K_vβ1.4, dark purple). Current traces were elicited by depolarizing voltage pulses (200 ms) between −80 mV and +50 mV in 10 mV steps from a holding potential of −60 mV before (Control) and after application of the K_v channel inhibitor 4-aminopyridine (4-AP) to validate the successful K_v channel expression. The current–voltage relationship (I–V curve) was determined by plotting the averaged current amplitude (plateau) against the corresponding voltage step (K_v1.5: n = 13; K_v1.5/K_vβ1.1: n = 12; K_v1.5/K_vβ1.4: n = 13). The mean K_v current amplitudes were each normalized relatively to the current value at + 50 mV under control conditions (no 4-AP). Data were statistically analyzed by 2-way ANOVA and the uncorrected Fisher’s least significant difference test for multiple comparisons (*: p ≤ 0.05; **: p ≤ 0.01, ***: p ≤ 0.001; ****: p ≤ 0.0001).

Figure 2

Determination of the H₂O₂ concentration present at the time point of electrophysiological recording. (a) Schematical illustration of the experimental setup. Oocyte Ringer’s solution (ORi) was supplemented with H₂O₂ within the perfusion reservoir (input). One sample was taken directly after H₂O₂ addition (input), while the second sample was extracted from the recording chamber 90 s post starting the perfusion—which represents the time point at which the electrophysiological recording was performed. Both samples were subjected to an Amplex Red Hydrogen Peroxide/Peroxidase Assay to determine the H₂O₂ decomposition for distinct concentrations within the experimental setup, i.e., the H₂O₂ concentration at the time of recording (b). The dotted line represents the input concentration (100%).

Figure 3

Experimental approach to evaluate the effect of H₂O₂ on K_v channel activity. (a) Representative current trace of a K_v-channel-expressing oocyte that was exposed to depolarizing voltage steps in the (a) absence (Control, gray) and (b) presence of H₂O₂ (10 mM, blue), as well as (c) the corresponding statistical analysis. The current–voltage relationship (I–V curve) was determined by plotting the averaged current amplitude (plateau) against the corresponding voltage step (control: n = 12; H₂O₂: n = 11). The I–V curve was fitted using the Boltzmann equation. No differences in the V₅₀ (Control: 7.1 ± 1.6 mV, H₂O₂; 6.1 ± 1.4 mV; p = 0.6523) and gating charge (q) (control: 1.4 ± 0.038 e₀, H₂O₂: 1.4 ± 0.028 e₀; p = 0.4252) were observed in response to H₂O₂. The mean K_v current amplitudes were each normalized relatively to the current amplitude in the control condition at +50 mV. H₂O₂ increased the channel’s activity at clamping potentials more positive than −30 mV, with a prominent effect at −20 mV, a value within the physiological range of the membrane potential of PASMCs. Therefore, clamping potentials of −20 mV and +50 mV (characterized by the strongest H₂O₂-induced activation) were chosen as the clamping potentials for analysis in all the following experiments (respective current traces and data points are highlighted in darker colors). Data were statistically analyzed by 2-way ANOVA and uncorrected Fisher’s least significant difference test for multiple comparisons (**: p ≤ 0.01, ***: p ≤ 0.001).

Figure 4

The effect of H₂O₂ on K_v channels is dependent on the concentration, step potential and ion channel subunit composition. (a) The effect of increasing H₂O₂ concentrations on homomeric K_v1.5 channels at step potentials of −20 mV and (b) +50mV (light gray background). (c,d) Assessment of the H₂O₂ response of heteromeric K_v1.5/K_vβ1.1 as well as (e,f) K_v1.5/K_vβ1.4 channels. The H₂O₂ response was assessed by normalizing the current amplitude upon H₂O₂ application to the control conditions (represented by the dotted line). A new oocyte and freshly prepared H₂O₂ were used for each experiment. Number of experiments per H₂O₂ concentration: n = 8–13 (K_v1.5); n = 8–13 (K_v1.5/K_vβ1.1); n = 6–13 (K_v1.5/K_vβ1.4). For statistical analysis, one-sample t-tests were performed, with p ≤ 0.05 being considered indicative of significance. Group data are expressed as the mean ± SEM.