# Chlordecone reduces hyperpolarization-activated-current (Ih) conductance in honeybee

Patrick Bois, Alain Chavanieu, Christophe Magaud, Nassim Fares, Mahira Kaabeche, Aurelien Chatelier, Pierre Charnet, Thierry Cens

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Patrick Bois, Alain Chavanieu, Christophe Magaud, Nassim Fares, Mahira Kaabeche, et al., Chlordecone reduces hyperpolarization-activated-current (Ih) conductance in honeybee. Environmental Toxicology and Pharmacology, 2025, 114, pp.104659. 10.1016/j.etap.2025.104659. hal-04967787

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Chlordecone reduces hyperpolarization-activated-current (Ih) conductance in honeybee

Patrick Bois, Alain Chavanieu, Christophe Magaud, Nassim Fares, Mahira Kaabeche, Aurelien Chatelier, Pierre Charnet, Thierry Cens

<figure><img src="image_2.png" /></figure>

PII: S1382-6689(25)00034-1

DOI: https://doi.org/10.1016/j.etap.2025.104659

Reference: ENVTOX104659

To appear in: *Environmental Toxicology and Pharmacology*

Received date: 8 November 2024

Revised date: 13 February 2025

Accepted date: 17 February 2025

Please cite this article as: Patrick Bois, Alain Chavanieu, Christophe Magaud, Nassim Fares, Mahira Kaabeche, Aurelien Chatelier, Pierre Charnet and Thierry Cens, Chlordecone reduces hyperpolarization-activated-current (Ih) conductance in honeybee, *Environmental Toxicology and Pharmacology*, (2025) doi:https://doi.org/10.1016/j.etap.2025.104659

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
# Title: Chlordecone reduces hyperpolarization-activated-current (Ih) conductance in honeybee

Patrick Bois<sup>a</sup>, Alain Chavanieu<sup>b</sup>, Christophe Magaud<sup>a</sup>, Nassim Fares<sup>c</sup>, Mahira Kaabeche<sup>b</sup>, Aurelien Chatelier<sup>a</sup>, Pierre Charnet<sup>b</sup>, Thierry Cens<sup>#b</sup>

Keywords: Honeybee, HCN channel, insecticide, chlordecone, deltamethrin

## Abstract

The pacemaker channel (HCN) is responsible for electrical activity in a wide range of excitable cells, including those of invertebrates. Using Xenopus oocytes and HEK cell, we show here that HCN-channel from *Apis mellifera* is activated by hyperpolarization, modulated by cAMP, and blocked by cesium. Its PNa/PK relative permeability is 1:3, and its unitary conductance is 1.5 pS, which is similar to that of the mammalian HCN2 channel. Moreover, bee h-current is blocked by high concentrations of ZD7288, and organochlorine pesticide chlordecone reduces Ih amplitude in a dose-dependent manner (IC50 value was 9.37 µM) and diminishes HCN conductance, while preserving voltage dependence. In contrast, Deltamethrin exhibits no discernible impact. Molecular docking of Chlordecone in a homology model of bee HCN generated by AlphaFold3 suggests a binding site located at the end of the S6 helix that could explain the conductance inhibition caused by Chlordecone.

## 1. Introduction

h-current (Ih) is distributed in many excitable cells. h-current is best known for its proposed important role in generating spontaneous pacemaker activity in the heart and brain. (Wahl-Schott and M. Biel, 2009). In addition to controlling pacemaker activity, Ih has several non-peacemaking functions such as determining and limiting the resting potential, altering membrane resistance, synaptic transmission and perception. (Biel et al 2009). In mammals, there are four different genes encoding four isoforms (HCN1-4) which contain six transmembrane helices (S1–S6) (including a positively charged S4 segment) and a cyclic nucleotide-binding domain (CNBD) located in C-terminal region. From a phylogenic tree invertebrates, including Drosophila melanogaster, sea urchin, Heliothis virescens, lobster (Marx et al., 1999; Gauss et al., 1998; Krieger et al., 1999; Zhang et al., 2003) and Apis mellifera (Gisselmann et al., 2003; Gisselmann et al., 2004) appear to have only a single gene encoding HCN channel. The expression and functional studies conducted on HCN channels indicate that the channel is expressed in olfactory receptor neurons of Apis mellifera, Drosophila melanogaster (Marx et al., 1999) and Heliothis virescens (Krieger et al., 1999). This indicates the potential involvement of HCN channels in the transduction or modulation of sensory signals. A number of studies have demonstrated that ion channels remain the primary target of the majority of small molecule insecticides (Ffrench-Constant et al., 2016). These are chemicals employed to kill insects, and it is therefore unsurprising that a significant proportion of insecticides have the potential to harm honey bees (Cens et al., 2022). Contaminated soil particles may adhere to bees, or contaminants may be absorbed by plants, water and incorporated into pollen and nectar.

<footnote><sup>a</sup>Laboratoire PRÉTI UR 24184 - Université de Poitiers - POITIERS, France</footnote>

<footnote><sup>b</sup>Institut des Biomolécules Max Mousseron, Université de Montpellier, CNRS, ENSCM, Montpellier, France</footnote>

<footnote><sup>c</sup>Université Saint-Joseph de Beyrouth, Faculté de médecine. Laboratoire de recherche de Physiologie et Physiopathologie.</footnote>

<footnote><sup>#</sup>Contributed equally</footnote>
In the present study, using stably transfected HEK293 cells or transiently expressing Xenopus
laevis oocytes, we provide a comprehensive analysis of the electrophysiological and pharmacological
properties of the bee h-current, confirming and expanding in previous studies. Moreover, certain
insecticides were tested, including the organochlorine pesticide chlordecone (CDC), which is known to
be a highly contaminating molecule (Guzelian, 1982) and the pyrethroid insecticide Deltamethrin, a
popular insecticide class that targets voltage-gated sodium (Na<sub>v</sub>) and GABA-activated channels, was
also tested (Narahashi et al., 1992).

**2. Materials and methods**

2.1. cDNA, cRNA and oocyte preparation

Honeybee total RNA was isolated and first-strand cDNA was obtained as previously described (Cens et al., 2015). cDNAs were amplified by PCR using the Herculase II Polymerase (Agilent Technologies, Inc). Primers were designed based on the sequences previously published to generate the channel transcript AMIH (Gisselmann et al., 2003). Full-length cDNA covering the entire ORF was cloned in the pcDNA3.1(+) vector, with the Alfalfa Mosaic Virus (AMV) sequence immediately before the start codon and the 3'-UTR sequence of the X. laevis β-globin gene immediately after the stop codon. The amino acid sequence deduced from the obtained cDNA was similar to the Genbank entry AAQ16312. For X. laevis oocyte injection, cRNAs were obtained from linearized plasmids using the Mmessage Mmachine Transcription Kit (Thermo Fisher), following the manufacturer's instructions. The cRNA was diluted at a final concentration of 500 ng/µL. Preparation and injection of X. laevis oocytes were previously described (Cens et al., 1996), in strict accordance with the recommendations and relevant guidelines of our institution. Surgery was performed under anesthesia, and efforts were made to minimize suffering. The experimental protocols were approved by the Direction Départementale des Services Vétérinaires (authorization N° C34.16). Each oocyte was injected with 30 nL of cRNA solution and cells were then maintained at 19°C in NDS (96mM NaCl, 2mM KCl, 1.8mM CaCl<sub>2</sub>, 1mM MgCl<sub>2</sub>, 5mM Hepes, 2.5 mM Na-Pyruvate, 0.05mM gentamycin, pH 7.2 with NaOH) renewed daily until recordings.

## 2.2. Electrophysiological recording on Xenopus oocytes in double electrodes and in macropatch configurations

For two-electrode voltage-clamp recording of Xenopus oocytes, pipettes (Harward, GC150T10) of 0.2-1 MΩ were filled with 3 M KCl, and the recording solution contained (in mM): 110 NaCl; 35 KCl; 1.8 CaCl₂; 5 BaCl₂; 10 HEPES; pH 7.4. BaCl₂ was used to block the intrinsic potassium currents. In macro patch inside/out configuration (where pipette resistance is < 1MΩ) this external recording solution is used as intrapipette solution. In this configuration the bath solution contained (in mM): 130 KCl; 2 NaCl; 10 HEPES; 5 EGTA; pH 7.2. The amplifier (Geneclamp 500B, Axon Inst.) was connected to the bath using the virtual ground headstage and agar-KCl (3M) bridges. The holding potential was set to -30 mV, and hyperpolarizing pulses of different amplitudes and durations were applied every 20 sec, unless otherwise mentioned. To obtain the isochronal activation curve of Ih, currents were elicited by hyperpolarizing pulses applied from holding potential of -30 mV. Potentials pulses ranging from -40 to -150 mv were applied in 10 mV increments followed by step at -150 mV. We performed protocols with isochronal pre-pulses of an average duration of 3 seconds to limit the run-down process. The limit is that the values of V1/2 are slightly less negative than those reported. This is also true for HEK cells.
## 2.3. Generation of stably-transfected cell lines HEK293/Am Kv HCN -188820L1.1.

After plasmid amplification by transformation SIG10 Chemically Competent Cells (sigma-aldrich), HEK293 cells (ECACC-cell-line: 85120602) were Calcium Phosphate transfected with 4 µg of pCMV-PL10/Am Kv HCN -188820L1.1 plasmid. The next day, Medium was changed. After 48 hours post-transfection, cells suspension was diluted into culture plates and selection agent G418 was added to medium at 400µg/ml. Cells were grown in fresh medium EMEM supplemented with 10% FBS, 2mM glutamine, 1% antibiotics (100 IU.mL-1 penicillin-G-Na; 50 IU.mL-1 streptomycin sulphate) and in presence of the selection agent during 2 weeks. G418 Concentration was 400µg/ml for cell selection or 200 µg/ml for cell culture maintenance.

## 2.4. Electrophysiological recording on HEK293 by patch clamp technique

The cells were seeded at a density of 2.10³ cells/cm² in 35 mm diameter culture dishes 24 hours before the patch clamp experiments. The ionic currents were recorded on HEK293 in the whole-cell configuration using an Axopatch 200A amplifier (Molecular Devices, CA, USA). The voltage clamps were generated using an analogue to digital converter (Digidata 1322a, Molecular Devices) and the pClamp software v10 (Molecular Devices). Cells were superfused with an extracellular solution containing (in mM): 110 NaCl; 35 KCl; 1.8 CaCl₂: 5 BaCl₂; 10 HEPES; pH 7.4. BaCl₂ was used to block the intrinsic potassium currents. Pipette resistance was between 1.5 and 3 MΩ when filled with the intrapipette solution containing (in mM): 130 KCl; 2 NaCl; 10 HEPES; 2 CaCl₂; 5 EGTA; 0.1 GTP; 5 Na2-phosphocreatine; 5 Mg-ATP; pH 7.2 ($P_{ca}$ = 6.3). The currents were recorded in control (with 0.01% DMSO (Sigma-Aldrich) as vehicle) or in deltamethrin (10 µM) (Sigma-Aldrich) or chlordecone (10 µM) (Sigma-Aldrich) conditions. ZD7288 (Sigma-Aldrich) was dissolved in water.

## 2.5. Ionic currents, variance and statistical analysis

Current traces analysis, fitting of activation curve and current variance analysis was performed with Clampfit (ver 11, Molec Dev.). The variance ($\sigma^2$) is estimated by subtracting the average current (Ih) of 75 consecutive individual recordings made in macropatch configuration at a stimulation of -150 mV. The variance which is the squared difference (point by point) between the average current and each individual record. The variance is then plotted against the mean courant and fitted by parabolic equation $\sigma^2 = Ih i - 1/N Ih^2$ where i is the unitary current and N the number of channels, (see Hille 1984).

Resulting values are given as mean ± S.E.M. Student's t-test or ANOVA was used to assess differences between means. The significance level was set at $P < 0.05$.

## 2.6 Protein model prediction and molecular docking

The homotetrameric structure of the honeybee hyperpolarization-activated ion channel was predicted using AlphaFold 3 (Abramson et al., 2024) via the online server (https://alphafold.com) based on the sequence of Q6WL04_APIME (isoform X11). The highest-ranked model was subsequently prepared using the Prepare Protein module of Discovery Studio (Dassault Systèmes, BIOVIA). Molecular docking of the ligand chlordecone was carried out using DiffDock-L via the platform on Hugging Face (https://huggingface.co/spaces/simonduerr/diffdock). The ligand structure, in .sdf format, was obtained from PubChem.
## 3. Results

### 3.1. Electrophysiological properties of bee HCN current

To confirm and complete the functional properties of bee HCN channel, we either transiently expressed this channel in Xenopus oocytes or used a stably-transfected cell lines HEK293/Am Kv HCN (see materials and methods 2.3). The hyperpolarizing current is recorded in the presence of a hyper-potassium solution to increase its conductance and of 5 mM BaCl₂ to suppress the intrinsic potassium currents (DiFrancesco et al., 1986). Fig. 1A1 shows representative traces of h-current recorded from holding potential of -30 mV to various hyperpolarizing potentials with increments of 10 mV (-40 to -150 mV during 2.5 s) followed by step to -150 mV. To analyze voltage dependence of pacemaker channel activation, HCN tail currents on hyperpolarization to -150 mV (Fig. 1A1) were normalized by the maximum tail-current value and plotted as a function of preceding step potential. The curve was fitted by Boltzmann function to obtain the isochronal voltage dependence of bee HCN channel activation. The V₁/₂ and slope-constant values obtained from Boltzmann fits of data in each experiments averaged -101.8 ± 0.9 mV and k = -9.91 ± 1.30 mV (n = 7), respectively (Fig. 1A2). Next to evaluate the reversal potential we determined the current-voltage (I/V) relationship by measuring the tail current at different potentials (-10 to -50 mV in 10 mV intervals) after a pre-pulse (2 s) to -150 mV (see protocol in Fig. 1B insert). As can be seen in Fig. 1B the I-V relation is linear with a reversal potential of -34.6 ± 0.7 mV (n=7). The permeability ratio PNa/PK calculated from Goldman-Hodgkin-Katz equation was 0.31. Macro-patch inside/out configuration on oocyte expressing h-current has been used to estimate the unitary conductance of the hyperbolizing activated current (Fig 1C). From the variance method (see materials and methods) the parabolic fitting of variance/mean current relationship yielded a conductance close to 1.4 pS.

### 3.2. Direct activation of Ih by cyclic nucleotide

Direct activation by cAMP is a well-studied property of HCN channels (DiFrancesco and Tortora, 1991; Bois et al., 1997). To verify this direct regulation on the bee HCN channel, we measured the effect produced by cAMP application (have investigated the effect of a cAMP) on the intracellular side of HCN channels in inside-out macro-patch recordings in HEK cells. In these conditions Ih does not arise from single channels but reflects the activation of a large number of channels per patch (Bois et al., 1997). In Fig. 2A, Ih was activated by 1s hyperpolarizing steps to -130 mV in one macro-patch. Perfusion of 3µM or 10 µM cAMP on the intracellular side of the patch accelerated the current activation time course and increased current amplitude. When plotted as a function of time, this increase has a fast onset and is reversible (Fig. 2B). The same effect was observed in two other macro-patch experiments.

### 3.3. Inhibition of h-current by ZD7288 and cesium

We next tested the effect produced by known blockers of this conductance such as the bradycardic agent ZD7288 (BoSmith et al., 1993) and cesium (DiFrancesco and Ojeda, 1980) in stably-
transfected cell lines. Currents were elicited by hyperpolarizing pulses applied from holding potential of -30 mV. Potentials pulses ranging from -40 to -150 mv were applied in 10 mV increments followed by step at -150 mV. Fig. 3A shows an example of current traces in control solution (upper panel) and after steady state block by 30 µM ZD 7288 (lower panel). Perfusion of 30 µM ZD 7288 significantly reduces Ih amplitude. The amplitude of tail currents measured at -150 mV were normalized to the maximum value, plotted as a function to test voltage, and fitted with a Boltzmann function to obtain the isochronal voltage dependence of bee HCN channel activation. As shown in Fig. 3B, 30 µM ZD7288 significantly reduces the maximum of activation curve by 53 ± 9.3% without changing Ih half-maximal activation potential (V₁/₂ = -97.9 ± 2.4 mV in control and -89.4 ± 3.2 mV in the presence of 30 µM ZD 7288, n = 5, p > 0.05). Likewise, we have verified the cesium blockage in the same experimental conditions. As expected, 4 mM cesium drastically blocks bee Ih current (by 94 ± 0.3% when compared to control at -150 mV, n = 5).

### 3.4. Effect of chlordecone and deltamethrin on honeybee h-current

We next evaluate the potential effect produced by two insecticides, chlordecone and deltamethrin, on the bee pacemaker current stably expressed in HEK cells. In Fig. 4, Ih was activated by hyperpolarizing pulses to -120 mV from holding potential of -30 mV. In this experiment the voltage-clamp pulse train was applied at a frequency of 1/15 HZ. Fig 4Aa shows an example of the application of chlordecone. In the presence of 1µM chlordecone, h-current amplitude decreased by 29%. Note that this effect was only partially reversible after washing out chlordecone Fig. 4 Aa. A concentration-response curve of Ih inhibition is shown in Fig. 4 Ab. The percentage of Ih reduction was measured at steady-state of blockage. The estimated IC₅₀ value was 9.37 µM. To test whether chlordecone affects the I/V curve, we evaluate its effect on the isochronal voltage dependence curve, as we have previously realized (see above 3.3). As illustrated in Fig. 4B, 1 µM chlordecone reduces the maximum of the activation curve by 28 ± 2.4% without changing Ih half-maximal activation potential (V₁/₂ = -96 +/- 3.2 mV in control vs -95 +/- 3.8 mV in the presence of 1 µM chlordecone, n = 6, p < 0.001). When tested in the same way, the pyrethroid insecticide deltamethrin at 10 µM did not produce any effect on Ih activation curve (n = 6; p > 0.05). We also tested the effects produced by abamectin, which belongs to the ivermectin insecticide group. At a concentration of 10 µM, this active molecule has no effect on Ih, as does deltamethrin (not shown).

### 3.5. Bee HCN channel model prediction and chlordecone docking

Functional HCN channel is assembled as a homotetramer. Fig. 5A shows a structural model of the honeybee HCN channel generated using AlphaFold 3 (See Materials and Methods). The subunit comprises six critical transmembrane domains (S1-S6), as well as intracellular C- and N-terminals. As with other ion channels of the voltage-gated family as documented by Zhou et al. (2001), the S4 transmembrane domain contains the voltage sensor and comprises several charged amino acids (arginine and lysine residues). The S1 to S4 helices of each tetramer are shown in green, the pore domain including helices S5 and S6 and the C-linker disk are colored red. A Cyclic nucleotide-binding domains found at the intracellular C-terminus of HCN channels are displayed in orange.

The Fig. 5A (focused by the blue circle) and 5B illustrate the chlordecone docking, with the binding site located at the end of the S6 helix. The confidence score for the best binding pose was -0.45. The residues that appear to be critical for ligand binding are Q347, R354 and E358 (Fig. 5B).
4. Conclusion

The results obtained in this study on the electrophysiological properties of the bee h-current confirm those already published. Indeed, several basic features of this current closely resemble those describe on recombinant and native Ih-channels from Apis mellifera (Gisselmann et al., 2003) and similar to those of vertebrate HCN channels with respect to activation by hyperpolarizing voltage steps, modulation by cAMP, cesium block and ion selectivity (Biel et al., 2009).

The biophysical properties of this bee pacemaker current are analogous to those of the mammalian HCN1 and HCN2 isoforms cloned by Ludwig et al., 1998. The V₀.₅ values are situated at approximately -101 or -96 mV for the bee HCN in our experiments, in comparison to -94 and -103 mV for HCN1 and HCN2, respectively (Ludwig et al., 1999). For example, patch-clamp analysis of single HCN2 channels yielded conductance values of about 1.5 pS in HEK293 cells (Dekker et al., 2006) and 1.67 pS in oocytes (Thon et al., 2013) which corresponds to the conductance obtained in the present data. The relative permeability ratio for Na⁺ versus K⁺ (PNa/K) as determined by Goldmann-Hodgkin-Katz is 1:3 for the present bee pacemaker current, which is consistent with the value observed for native h-current, including Heliothis virescens HCN (Krieger et al., 1999) or for each mammalian isoform ranging from 1:3 to 1:5, yielding values for reversal potential between -25 and -40 mV, as reported by Biel et al., 2009.

As expected, the bee HCN channel is modulated directly by cAMP. This effect, which is fully reversible, is analogous to the direct regulation of cAMP observed with human HCN2 and HCN4 isoforms (Biel et al., 2009). Moreover, the bee pacemaker channel is blocked by external application of cesium (at 4 mM) or ZD7288 (at 30 µM), which are known inhibitors of mammalian HCN channels. In rat hippocampal CA1 neurons (Gasparini and DiFrancesco, 1997), blocking h-current requires higher doses of ZD7288 than those required to block If in the sino-atrial node (30 µM vs 0.3 µM) (BoSmith et al., 1993). This may be due to the different HCN isoform composition of native channels in these two tissues (HCN1 and HCN4 in hippocampal neurons and sino-atrial nodes, respectively). The overall effect of ZD7288 is comparable to that of zatebradine and cilobradine (other HCN blockers), as all of these drugs affect the h-current by inhibiting its maximal conductance without significantly altering the voltage dependence of current activation (Bois et al., 2007, Chen at al. 2023). Sensitivity of the bee pacemaker current to ZD7288 suggests that the current is carried by an isoform similar to that found in neurons, rather than that found in the Sino Atrial Node. It is probable that the bee isoform belongs to the HCN1 or HCN2 type. Using computational docking experiments, Tanguay and colleagues have shown that ZD7288 binding in the human HCN1 isoform is facilitated when the channel pore is open (Tanguay et al., 2019). The residues that appear to be critical for ZD7288 binding are C358, A383, Y386, A387, V390. The bee HCN channel retains four of the five residues (C358, A383, Y386 and A387).

The second part of the study aimed to assess the impact of chlordecone on bee HCN, a neurotoxic, endocrine-disrupting and carcinogenic insecticide employed to control banana weevil populations in the French West Indies between 1972 and 1993. This pesticide is a highly persistent pollutant that will remain present in soil and water for centuries. It contaminates plants and animals, and subsequently humans who consume them. It is an endocrine disruptor that also increases the risk of prostate cancer and premature birth. Several types of studies have been undertaken to elucidate the mechanism underlying Chlordecone toxicity, including studies on enzymes such as Na-K ATPase and Mg ATPase (Desaiah et al., 1980; Mishra et al., 1980; Desaiah, 1981), mitochondrial energy production (End et al., 1979), intracellular calcium mobilization (Komulainen and Bondy., 1987) and neurotransmitter binding (Aronstam and Hong., 1986).

The limited research that has been conducted on the effects of chlordecone on ion channels shows that this drug blocks at least three types of ionic currents (ATP-activated inward current, inward calcium current and outward potassium current) recorded in neural cells line (rat pheochromocytoma PC12 cells, Inoue et al., 1990). In the present study, and similarly to ZD7288, chlordecone markedly diminishes the HCN conductance without affecting channel voltage dependence. Molecular docking experiments suggest that chlordecone binding involves three residues of the bee HCN channel pore (Q347, R354, E358). Binding of chlordecone in the channel pore may explain full conductance decrease.
Sequential mutation of these three residues coupled with a functional investigation should confirm
the chlordecone binding site. It is worth noting that these three residues are conserved in the four
human HCN1-4 isoforms, which suggests that Chlordecone may also affect human pacemaker currents.

By in-situ hybridization and immunohistochemistry, expression of HCN was seen in olfactory
receptor neurons of the bee antennae (ORNs) (Gisselmann et al. 2003). Like vertebrate heart and brain
cells, ORNs from insect antennae show spontaneous activity (Boeckh et al., 1965; Kaissling, 1986) and
an increase as well as decrease of the action potential frequency has been observed upon application
of odorants (Dubin and Harris, 1997). One potential mediator of this effect may be the antennal HCN
channel, whose activity is regulated by cyclic adenosine monophosphate (cAMP), a key second
messenger in insect olfactory signal transduction.

In this context, it is essential to carry out a behavioral and neurophysiological study of olfactory
perception and learning in bees in the presence of chlordecone, focusing particularly the
octopaminergic and dopaminergic pathways. Indeed, appetitive reinforcement and aversive
reinforcement depends in bees as in other insects on octopamine and dopamine, respectively (Sandoz,
2011).

As part of our study, we propose to carry out an exhaustive study of channel expression in bees
in all tissues showing spontaneous or oscillatory activity. In addition, we will assess the toxicity of
chlordecone in particular, including on human HCN. It would be beneficial to test other organochlorine
pesticides (Zhou et al., 2011) on the electrophysiological properties of pacemaker channels.

Declaration of Competing Interest

The authors report no declarations of interest

CRediT author statement

**Patrick Bois**: Conceptualization, Methodology, Software Writing - Original draft. **Alain Chavanieu**: Software, Writing - Original Draft. **Christophe Magaud**: Methodology. **Nassim Fares**: Writing - Review & Editing. **Mahira Kaabeche**: Methodology. **Aurélien Chatelier**: Investigation, Writing - Review & Editing. **Thierry Cens**: Conceptualization, Methodology, Software, Supervision. **Pierre Charnet**: Investigation, Conceptualization, Methodology, Software, Supervision.

Acknowledgments

This work was financed by the Agence National pour la Recherche (ANR Synaptic-Bee: ANR-20-CE34-0017-01). Special thanks to Isabelle Fixe for invaluable help with cell cultures.

Legends
A

1

<figure><img src="image_3.png" /></figure>

2

<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>

B

<figure><img src="image_5.png" /></figure>

C

<figure><img src="image_6.png" /></figure>

**Fig. 1. Characterization of bee HCN current.** Bee HCN channel was expressed in xenopus oocytes. A. A1 Ih current with slow activation kinetics in response to 2s membrane hyperpolarizations from -40 mV to -150 mV in steps of -10 mV from holding potential (HP) of -30 mV followed by a voltage step to -150 mV to record Ih tail current (see insert). A2 The normalized Ih tail current at -150 mV was plotted versus step potential. The activation curve was fitted using a Boltzmann distribution (n = 7). B I/V relationship obtained by applying 2s lasting prepulses to -150 mV (HP = -30 mV) and subsequent 1s test pulses ranging from -10 to -50 mV. The I/V relationship was determined by plotting Ih as a function of the test pulses. The linear regression of the data point yielded a reversal potential of -34.6 ± 0.7 mV (n = 7). C Variance estimation after Ih recorded in macro patch inside/out configuration after 75 consecutive individual recordings (see materials and methods) and plotted versus mean Ih amplitude. The data points were adjusted by the parabolic equation σ2= Ih i -1/N Ih2 where σ2 is the variance, Ih mean h-current, i unitary h-current and N the number of functional channels.

<caption>Figure 2</caption>

A

<figure><img src="image_7.png" /></figure>

B

<figure><img src="image_8.png" /></figure>

**Fig. 2. Action of cAMP on Ih in inside-out configuration macropatch.** A representative trace of Ih current activated by a voltage step of -130 mV applied every 3s from holding potential of -30 mV in
control (1) the presence of 3µM (2) or 10 µM (3) cAMP. B Time course of Ih amplitude (start-current subtracted from end-current) before and during the bath perfusion of cAMP at 3 µM and 10 µM. Note that for both concentrations the effect is fully reversible.

A

<figure><img src="image_9.png" /></figure>

B

<figure><img src="image_10.png" /><figcaption>Figure 3</figcaption></figure>

<caption>Fig. 3. Action of ZD7288 and cesium on the bee Ih current stably expressed in the HEK293 cell line.</caption>

A Ih current activation traces in response to 2s membrane hyperpolarizations from -40 mV to -150 mV in steps of -10 mV from HP of -30 mV followed by a voltage step to -150 mV to record Ih tail current in absence (upper panel) or in the presence (lower panel) of 30 µM ZD7288. B The normalized Ih tail current at -150 mV was plotted versus step potential, in control and in the presence of 30 µM ZD 7288 or 4 mM cesium. Notes that at 30 µM ZD 7288 significantly reduces the overall conductance of Ih by 54% (n=5).

A

<figure><img src="image_11.png" /></figure>

B

<figure><img src="image_12.png" /><figcaption>Figure 4</figcaption></figure>

Fig. 4. Effet of Chlordecone and de deltamethrin on the bee Ih current stably expressed in the HEK293 cell line. Aa Ih was activated by hyperpolarizing pulses to -120 mV from holding potential of -30 mV. Time course of the absolute value of Ih amplitude (start-current subtracted from end-current at -120
mV) before and during the perfusion of 1 µM of Chlordecone. The arrow indicates the 0 current level. **Ab** Dose-response relationship of Ih normalized amplitude as a function of Chlordecone concentration. The data were fitted with the Hill equation $f(x) = 100[1+(IC₅₀/x)nth]$ where $IC₅₀ = 9.37$ µM and Hill coefficient estimated at 9.98. The number of cells (n) is indicated above each point of the curve. **B** The normalized Ih tail current at -150 mV (see protocol in the text) was plotted versus step potential in control and in the presence of 10 µM Chlordecone or 10 µM Deltamethrin. Note that at 1 µM Chlordecone significantly reduces the overall conductance of Ih by 31% (n=6) whereas at 10 µM Deltamethrin has no effect (n =6).

<figure><img src="image_13.png" /><figcaption>**Fig. 5.** Structural model of the honeybee HCN channel docked with Chlordecone, generated via AlphaFold 3 and DiffDock-L. **A** Structural representation of the HCN channel tetramer in ribbon form, aligned parallel to the membrane. The S1 to S4 helices of each tetramer are shown in green (residues 40-238), while the pore domain, including helices S5 and S6, and the C-linker disk containing the A'- and B'-helices, are colored red (residues 239-392). The C-linker disk with the C'-, D'-, E'-, and F'-helices and the Cyclic Nucleotide-Binding Domain (CNBD) are displayed in orange (residues 393-554). Approximate membrane bilayer boundaries are marked by blue bars. The Chlordecone molecule is docked near the pore exit on the cytoplasmic side, close to the membrane (highlighted by the cyan circle). **B** Close-up view of Chlordecone docking interactions within the channel. Two opposite subunits are shown in light gray. Key interacting residues within 4 Å of chlordecone include Q347 at the end of the S6 helix, and R354 and E358 located in the A'-helix of the C-linker disk.</figcaption></figure>

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**Declaration of Interest Statement**

[x] The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐ The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for this journal and was not involved in the editorial review or the decision to publish this article.

☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

## Highlights

* The biophysical properties of bee pacemaker current are similar to those of the human

* The neurotoxic insecticide chlordecone reduces the pacemaker current in bees

* The current data set can be expanded to encompass human toxicology