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. 2024 Apr 3;14(1):7834.
doi: 10.1038/s41598-024-58234-0.

Cloning, functional expression, and pharmacological characterization of inwardly rectifying potassium channels (Kir) from Apis mellifera

Fabien Sourisseau  1 , Chaimaa Chahine  1 , Valérie Pouliot  1 , Thierry Cens  2 , Pierre Charnet  2 , Mohamed Chahine  3   4
Affiliations

Cloning, functional expression, and pharmacological characterization of inwardly rectifying potassium channels (Kir) from Apis mellifera

Fabien Sourisseau et al. Sci Rep. .

Abstract

Potassium channels belong to the super family of ion channels and play a fundamental role in cell excitability. Kir channels are potassium channels with an inwardly rectifying property. They play a role in setting the resting membrane potential of many excitable cells including neurons. Although putative Kir channel family genes can be found in the Apis mellifera genome, their functional expression, biophysical properties, and sensitivity to small molecules with insecticidal activity remain to be investigated. We cloned six Kir channel isoforms from Apis mellifera that derive from two Kir genes, AmKir1 and AmKir2, which are present in the Apis mellifera genome. We studied the tissue distribution, the electrophysiological and pharmacological characteristics of three isoforms that expressed functional currents (AmKir1.1, AmKir2.2, and AmKir2.3). AmKir1.1, AmKir2.2, and AmKir2.3 isoforms exhibited distinct characteristics when expressed in Xenopus oocytes. AmKir1.1 exhibited the largest potassium currents and was impermeable to cesium whereas AmKir2.2 and AmKir2.3 exhibited smaller currents but allowed cesium to permeate. AmKir1 exhibited faster opening kinetics than AmKir2. Pharmacological experiments revealed that both AmKir1.1 and AmKir2.2 are blocked by the divalent ion barium, with IC50 values of 10-5 and 10-6 M, respectively. The concentrations of VU041, a small molecule with insecticidal properties required to achieve a 50% current blockade for all three channels were higher than those needed to block Kir channels in other arthropods, such as the aphid Aphis gossypii and the mosquito Aedes aegypti. From this, we conclude that Apis mellifera AmKir channels exhibit lower sensitivity to VU041.

Keywords: Apis mellifera; Cloning; Expression; Insecticides; Insects; Kir channels; Potassium channels; VU041.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(A) Neighbor-joining phylogenic tree of amino acid sequences encoding Kir channel subunits in insects and humans. Geneious Prime 2023.2.2 (www.geneious.com) was used to construct the tree. The Genbank® accession numbers of the Kir sequences are shown in parentheses. Underlines denote Apis mellifera Kir channel subunits. The abbreviations of the species are as follows: Aag: Aedes aegypti, Agam: Anopheles gambiae, Agly: Aphis glycines, Amel: Apis mellifera, Apisu: Acyrthosiphon pisum, Clec: Cimex lectularius, Dmel: Drosophila melanogaster, Hsap: Homo sapiens, Nlug: Nilaparvata lugens. (B) Evaluation of expression of AmKir channel isotypes in honeybee tissues and life stage. Tissue-specific expression of the AmKir channel isotype was determined using RT-PCR. All honeybee tissue samples underwent identical preparation steps, from dissection to gel electrophoresis. The sizes of the ladder marker next to the fist blot is the same for all the other blots, and are indicated in base pairs (bp). Abbreviations: LE: legs, LA: larvae, HE: heads, BR: brains, GAG: ganglia, GU: guts, MU: muscles, ANT: antennae (C) Sequence alignments of the three current-generating AmKir channel isoforms were performed using MegAlign from Lasergen. The blue squares indicate the hydropathy estimation of the transmembrane regions. The red square represents the estimated region of the selectivity filter. The black squares indicate arginine-glutamate salt bridge interactions forming the PIP2-binding site in the cytoplasmic domain.
Figure 2
Figure 2
Comparison of the current amplitudes of Apis mellifera AmKir channels. (A–C) Representative raw current traces of the three Apis mellifera AmKir channel isoforms at a holding potential of − 80 mV under different conditions: Control (2 mM KCl), 25 mM KCl, 96 mM KCl, and washout (2 mM KCl). (D) Comparison bar plots of current amplitudes observed for the three channels tested in (A), (B), and (C) (AmKir1.1 n = 9, AmKir2.2 n = 12, AmKir2.3 n = 6, mean ± SEM). In the AmKir1.1 section, *p < 0.05 and **p < 0.005 vs. AmKir1.1 control. One-way ANOVA multiple comparison test. In the AmKir2.2 section, *p < 0.05 vs. AmKir2.2 control. One-way ANOVA multiple comparison test. In the AmKir2.3 section, *p < 0.05 vs. AmKir2.3 control. One-way ANOVA multiple comparison test.
Figure 3
Figure 3
Visual comparison of raw current traces of Apis mellifera AmKir channels and H2O-injected Xenopus oocytes in 2 mM KCl, 25 mM KCl, and 96 mM KCl. (A–C) Raw current traces of AmKir1.1, (D–F) AmKir2.2, (G–I) AmKir2.3 and (J–L) oocytes H2O-injected.
Figure 4
Figure 4
Comparison of I-V curves of Apis mellifera AmKir channels and H2O-injected s oocytes in 2 mM KCl, 25 mM KCl, and 96 mM KCl. (A) Curves of I/V curves from AmKir1.1 (n = 9, mean + SEM), (B) AmKir2.2 (n = 6, mean + SEM), (C) AmKir2.3 (n = 24, mean + SEM) and (D) H2O-injected Xenopus oocytes (n = 19, mean + SEM). (E) Current amplitude comparison between AmKir2.3 and H2O-injected Xenopus oocytes at − 150 mV and − 80 mV for each KCl external concentration condition, *p < 0.05, **p < 0.005, ****p < 0.0001 vs H2O-injected. Two-Way ANOVA multiple comparison test (mean + SEM). (F) Half-time to peak (t1/2) of the three AmKir channels while perfused with 96 mM KCl solution, *p < 0.05 vs. AmKir1.1. Two-way ANOVA multiple comparison test.
Figure 5
Figure 5
Ionic permeability rankings of the Apis mellifera AmKir channel isoforms. (A–D) Representative raw current traces recorded at a holding potential of − 80 mV with 10 mM of specific monovalent cations including, thallium, potassium, rubidium, sodium, cesium, and lithium (A–C) and 96 mM of potassium, rubidium, sodium, cesium and lithium (D). The grey segment represents the perfusion time of a Ringer’s analog solution without any monovalent cation. (E–H) Average peak current intensity as a function of the cation used. Data are normalized to the mean potassium peak current. Mean ± SEM, *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005. One-way ANOVA multiple comparison test.
Figure 6
Figure 6
Blockage of Apis mellifera AmKir1.1 and AmKir2.2 isoforms by barium. (A–B) Representative raw current traces of the pharmacological inhibition of AmKir1.1 and AmKir2.2 currents in the presence of increasing concentrations of barium (10−7–10−2 M). (C) Dose-responses curves of the inhibition of AmKir1.1 and AmKir2.2 currents in the presence of increasing barium concentrations (mean ± SEM). (D) I/V curves of the AmKir1.1 channel in the presence of high potassium (96 mM KCl), high potassium + barium (96 mM KCl + 1 mM barium), and washout (2 mM KCl) conditions (mean ± SEM). (E, F) Current traces of the AmKir1.1 channel in the presence of high potassium (96 mM KCl) and high potassium + barium (96 mM KCl + 1 mM barium) conditions, respectively. (G) I/V curves of the AmKir2.2 channel in the presence of high potassium (96 mM KCl), high potassium + barium (96 mM KCl + 1 mM barium), and washout (2 mM KCl) conditions (mean ± SEM). (H, I) Current traces of the AmKir2.2 channel in the presence of high potassium (96 mM KCl) and high potassium + barium (96 mM KCl + 1 mM barium) conditions, respectively.
Figure 7
Figure 7
Blockade of Apis mellifera AmKir channel isoforms by the insecticide VU041. (A–C) Representative raw current traces of the pharmacological inhibition of AmKir1.1, AmKir2.2, and AmKir2.3 currents by increasing concentrations of VU041 (10−8–10−4 M for AmKir1.1 and AmKir2.2 and 10−8–10−5 M for AmKir2.3). (D) Dose–response curves of the inhibition of AmKir1.1, AmKir2.2, and AmKir2.3 currents in the presence of increasing concentrations of VU041 (mean ± SEM, AmKir1.1: n = 12, AmKir2.2: n = 7, AmKir2.3: n = 5).
Figure 8
Figure 8
Blockade of Apis mellifera AmKir channel isoforms expressed in HEK293T by the insecticide VU041. (A–C) Comparison of current potential traces of Apis mellifera AmKir1.1 in the presence of the bath, 0.2% DMSO, and 10 µM VU041 conditions, respectively. (D) I/V curves of the AmKir1.1 channel isoform in the presence of the bath, 0.2% DMSO, and 10 µM VU041 conditions (mean + SEM, Bath n = 9, 0.2% DMSO n = 5, 10 µM VU041).
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