Inwardly rectifying potassium channels: Critical insights for insect species and Apis mellifera

ABSTRACT

Kir (inwardly rectifying potassium) channels that play key roles in maintaining potassium homeostasis, neuronal excitability, and osmoregulation have been cloned and characterized in a variety of insects. In Drosophila melanogaster, three Kir channels (dKir1 dKir2, and dKir3) have been cloned and characterized, and share significant homology with mammalian Kir channels. The dKir channels are essential for various developmental processes, such as wing patterning, by modulating bone morphogenetic protein signaling pathways. Electrophysiological studies have confirmed that Drosophila Kir channels function in a way analogous to their mammalian counterparts, indicating that their roles in cellular and developmental signaling have been evolutionarily conserved. Several Kir channels have also been identified and characterized in mosquitoes (Aedes aegypti and Anopheles gambiae). Interestingly, insect Kir channel orthologs cluster into three gene “clades” or subfamilies (Kir1, Kir2, Kir3) that are distinct from mammal Kir channels based on sequence comparisons. Insect Kir channel paralogs range from two to eight Kir channel genes per species genome representing separate gene duplication events. These differences may be attributed to distinct physiological adaptations associated with their respective taxonomic groups. The honeybee Apis mellifera genome contains two Kir channel genes, AmKir1 and AmKir2, producing six Kir channel isoforms via alternative splicing, which have been cloned and expressed in heterologous systems to study their electrophysiological properties. This review provides a comprehensive overview of current knowledge about Kir channel structures, activities, and gating as well as of their roles in insects, including evolutionary genomic aspects, molecular biology, physiological roles, and pharmacological targeting.

Introduction

The superfamily of potassium channels is integral to the regulation of the membrane potential across a wide range of excitable cells and plays particularly significant roles in neurons [1–5] and muscle cells (cardiac and skeletal). These channels are essential for maintaining the electrical stability of these cells, thereby enabling crucial physiological processes such as neurotransmission in the nervous system and cardiac rhythmicity. Potassium channels also play a vital role in extracellular potassium transport and maintenance via non-excitable cells that include glial cells in the nervous system and epithelial cells in the renal and gastrointestinal systems. In humans, disruptions in the normal functioning of potassium channels due to spontaneous mutations can lead to a variety of pathologies, including cardiac arrhythmias, neurological disorders, and renal dysfunctions [6–8]. Such variants can alter channel activity, leading to either hyper or hypoexcitability of affected cells, which underscores the critical importance of potassium channels in maintaining cellular homeostasis across multiple organ systems.

The potassium channel superfamily is one of the most diverse ion channel families in humans and incorporates a diverse array of subgroups, each with distinct functional properties and physiological roles. Among these are voltage-dependent potassium channels (Kv), which are crucial for action potential repolarization, calcium-activated potassium channels (KCa), which link intracellular calcium levels to membrane potential, inward-rectifier potassium channels (Kir), which are essential in potassium homeostasis and stabilizing the resting membrane potential to regulate cellular excitability [9], and two-pore domain potassium channels (K2P), which gives rise to a leak, a background, potassium current to stabilize the negative resting potential and counterbalance depolarization [10]. The present review focuses specifically on Kir channels, with the aim of providing a comprehensive overview of their structure, functions, and significance in various physiological processes. It provides a comparative analysis of the unique structural and functional properties of insect Kir channels versus vertebrate Kir channels. The review delves into the diverse roles insect Kir channels play in various tissues, offering insights into their physiological significance and as targets for insecticide development across different species.

The mammalian Kir channel superfamily is divided into several distinct subfamilies, each with specialized functions that contribute to the diverse physiological roles of inward-rectifier potassium channels. These subfamilies include potassium transport Kir channels (ROMK-type, Kir1.x, Kir4.x, Kir5.1x, and Kir7.x), classical Kir channels (IRK, Kir2.x), G-protein-activated channels (GIRK, Kir3.x), and ATP-sensitive Kir channels (KATP, Kir6.x) [11]. All members of the Kir superfamily function as tetramers in the cell membrane formed via the assembly of four Kir protein subunits [12,13]. First observed in skeletal muscle fibers, potassium current through these channels was initially described as abnormal due to their unique inward-rectifying properties [14]. Indeed they exhibit a large potassium-selective conductance at negative membrane potentials, and are mostly non-conducting at positive membrane potentials, which was an unprecedented observation at the time [14]. This observation contrasted with those made on voltage-dependent potassium channels characterized in the squid giant axon that are found extensively in animal tissues and are opened by membrane depolarization to positive potentials.

The biophysical and pharmacological understanding of Kir channels in arthropods has garnered significant attention over the past decade [15]. They regulate numerous aspects of arthropod physiology, making Kir channels prime targets for the development of insecticides in relation to these channels. Today, an increasing number of molecules are being designed to target and disrupt the activity of these channels to control insect pest populations such as the fruit fly Drosophila melanogaster, the cotton aphid Aphis gossypii, and the mosquito Aedes aegypti (see below). However, less attention seems to have been paid to the potential effects these molecules may have on important beneficial insects such as pollinators. Honeybees play a capital role in pollination in terms of both the stability of ecosystems and for domestic honey production and consumption [16–18]. Their Kir channels however, have only recently been characterized [19].

Structural features of Kir channels

Kir channel genes and phylogenetics

Cloning of the first Kir channel genes roughly 30 years ago originated from the screening of three different mammalian cDNA libraries [20–22]. Subsequent genomic analysis has revealed the presence of 16 distinct human Kir channel genes that are assigned to the “potassium inwardly rectifying channel subfamily J” (KCNJ) classification (HUGO Gene Nomenclature Committee. Group ID 276). Multiple KCNJ genes also undergo alternative mRNA splicing to produce additional isoform variants that add to the structural diversity of encoded Kir channel subunit proteins [12,13].

The application of whole genome sequencing technologies across thousands of animal species reveals that Kir channels represent an ancient innovation found in eukaryotic organisms as well as prokaryotic microbes and plants (Figure 1). They are identified as a distinct protein family based on conserved signature sequences (Potassium channel, inwardly rectifying, Kir; IPR016449) [23]. Orthologs for each of the human Kir channel genes have been identified across vertebrate species demonstrating an abundant genomic repertoire for Kir channel expression and tissue differentiation (Figure 1) [24]. The number of Kir channel genes per genome is notably less in invertebrate species, ranging from two to eight distinct genes in insects (Piermarini, 2022). The difference in the genomic footprint for invertebrate (~4 genes) versus mammals Kir channel genes (~16 genes) is consistent with the two-round whole genome duplication (2 R-WGD) hypothesis implicated with the initiation of vertebrate evolution approximately 550 Mya [25,26]. Insect Kir channel genes have a distinct phylogenetic history from vertebrates, having split from early crustaceans where Kir channels may have provided novel osmoregulatory adaptations with the transition to a new terrestrial habitat (cf. Supplemental table S1) [27].

Figure 1.

Radiation of Kir channel genes across phyla and genomic expansion in vertebrates: (a) Unrooted phylogenetic tree illustrating the distribution and clustering of different Kir channel subfamilies based on protein sequence similarities. Protein-coding Kir channel genes were identified in 252 selected reference genomes using available gene annotations together with genomic BLAST searches. 252 unique Kir channel protein sequences have been obtained (cf. Supplemental table 1), and have been modified by a multiple sequence alignment performed using the COBALT constraint-based multiple alignment tool. The phylogenetic tree was subsequently produced from the alignment using the neighbor joining algorithm with Grishin method for modeling the evolutionary distance between two sequences. The adopted Kir subfamily nomenclatures for vertebrate and insect Kir channels are shown. Dashed lines represent invertebrate chordate species (e.g. tunicates and lancelets) where their 2–3 Kir channel proteins cluster within vertebrate Kir channel subfamilies. (b) Representative species from the phylogenetic tree with their corresponding number of Kir channel genes per genome. Chordate species are shown in blue, arthropods in green, other invertebrate species in red, and bacteria in black.

The divergence of insect and vertebrate Kir channel proteins is substantial, where the closest relationship from phylogenetic analysis are Kir channels involved in potassium transport and osmoregulation (Figure 1). Even among different insect species, there are significant Kir channel sequence differences [15]. The proteins and resulting structural differences between vertebrate and insect Kir channels could indeed aid efforts to selectively target insect Kir channels while having minimal off-target effects on vertebrate Kir channels. However, the potential off-target effects on un-targeted insect Kir channel orthologs (e.g. in pollinators such as honeybees) remain an important consideration in the workflow and chemical screening process.

Protein architecture of functional Kir channels

The quaternary assembly of gene-encoded Kir channel subunit proteins expressed within a given eukaryotic cell can result in the formation of either homo-tetrameric channels (having 4 identical subunits) or hetero-tetrameric channels (having 4 subunits of more than one isoform or variant), and depends on the complement of Kir channels expressed within the cell and the determinants for tetrameric subunit assembly. Similar to other types of vertebrate potassium channels, Kir subunit assembly is biased toward members of closely related subfamily members (e.g. Kir3 isoforms assemble with Kir3 isoforms, regardless of the animal model) and is facilitated by conserved structural domains within the amino- and carboxy-termini [12,13]. In vertebrates, four major functional Kir subfamilies have emerged that generally cluster together in terms of their level of sequence similarity and heteromeric assembly potential, with the potassium transport subfamily (comprised of vertebrate Kir1.1, Kir4.1, Kir4.2, Kir5.1 and Kir7.1) being the most diverse subfamily [11].

In arthropods, three distinct Kir subfamilies emerged (Kir1, Kir2, and Kir3) based on sequence comparisons with Kir channel paralogs and orthologs [15]. The three Kir genes originally described in Drosophila melanogaster (irk1, irk2, irk3) represent each subfamily and are now designated as Kir1, Kir2, and Kir3 using the new nomenclature [15]. Interestingly, only two subfamilies (Kir1 and Kir2) are represented in Hymenoptera species including Apis mellifera [19], where the Kir3 gene has been lost (Figure 2). By comparison, species in other insect orders (e.g. Coleoptera, Diptera (mosquitos), and Lepidoptera) possess up to eight genes within all three subfamilies that likely emerged via separate gene duplication events (cf. Supplemental Table S1). Recent gene duplication events are also readily apparent in vertebrate species. For example, in humans, KCNJ12 (Kir2.2 access ID: NG_042809) and KCNJ18 (Kir2.6 access ID: NG_033093) represent two distinct genes adjacent to each other on chromosome 17p11.2 that encode Kir channels having only six amino acid differences (protein BLAST NCBI).

Figure 2.

Insect Kir channel genes across different pollinator species. (a) Cladogram of Kir channel paralogs and orthologs from selected insect and pollinator species. Reference Kir channel genes from whole genome sequencing data (NCBI genome datasets) were identified and Kir channel orthologs then confirmed (NCBI orthologs and OrthoDB, version12.0). The resulting protein reference sequences were used to generate a multiple sequence alignment using the COBALT constraint-based multiple alignment tool. The phylogenetic tree relationships were determined using the neighbor joining method and illustrates the three distinct insect Kir channel subfamilies, Kir1 (red), Kir2, (green), and Kir3 (blue) where different species possess varying numbers of genes within each Kir subfamily. Source information for each insect Kir channel subunit is provided in Supplemental Table S1. (b) the honey bee apis mellifera genome contains two genes, AmKir1 and AmKir2, which are adjacent and overlapping on chromosome LG14 (NC_037651.1). Each Kir gene (green) is predicted to produce multiple alternative mRNA splice variants (purple) yielding multiple Kir channel protein isoforms (red). The two Kir channel genes, their mRNA transcripts and corresponding protein renderings are shown and were obtained using the NCBI genome data viewer for the Amel_HAv3.1 genome assembly (GCA_003254395.2). The photo image of apis mellifera is courtesy of the global biodiversity information facility (https://www.Gbif.org/occurrence/5007606902).

Within the tetrameric ion channel assembly, each Kir subunit protein is comprised of two transmembrane domains separated by a pore-lining “H5 loop,” where both the amino- and carboxy-termini are located on the intracellular side of the membrane (Figure 3). The four subunits are arranged around a central potassium-selective pore which allows for the passage of potassium across the cell membrane. When open, potassium ions flow according to the electrochemical driving force established by the membrane potential and intracellular and extracellular potassium concentrations. The selectivity for potassium ions is determined by the highly conserved pore loop glycine-tyrosine-glycine (GYG) signature sequence [30,31]. The subunit stoichiometry for each Kir channel, either four identical subunits (homo-tetramer) or more than one type of subunit (hetero-tetramer) ultimately defines the channel’s intrinsic biophysical and pharmacological characteristics. Each Kir channel subunit possesses distinct regulatory sites for post-translational modifications (e.g. phosphorylation, glycosylation), binding to signaling molecules (e.g. PIP2, ATP), and protein-protein interactions (e.g. G proteins, GPCR’s, sulfonylurea receptor subunits) [11].

Figure 3.

Schematic representation and structural features of Kir channels. (a) Kir channels exhibit a topology consisting of two transmembrane segments, M1 and M2. (b) The region between the M1 and M2 segments (purple) forms the pore region, which constitutes the potassium ion permeation pathway. A top-down view highlights the arrangement of these segments as shown in (a). Total channel size varies between species and subtype. Transmembrane and pore vertical size ~ 40 Å, cytoplasmic domain vertical size ~ 70 Å, average total size ~ 120 Å. (c) High-resolution cryo-EM map and atomic structure of the human Kir2.1 inwardly rectifying potassium channel, providing detailed insights into channel function and regulation, particularly by the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and the GYG pore motif. For clarity, only one subunit is shown with the PIP2 binding site labelled (R80, W81, R82 and K182, K185, K187, K188, R189). The 3-dimensional structural representation was generated using the visual molecular dynamics (VMD) software [28], the model was constructed based on cryo-electron microscopy (cryo-EM) coordinates of the human inward rectifier potassium channel Kir2.1, obtained from the protein data Bank (PDB ID: 7ZDZ [29]). This high-resolution structure provided the foundation for accurate modeling of the channel’s architecture and allowed detailed visualization of its transmembrane domains and cytoplasmic regions. (d) Top-down view of the Kir2.1 structure depicted in (C), illustrating the spatial organization of the channel’s key structural elements including GYG pore motif and PIP2 binding site.

A notable structural difference between Kir channels and voltage-dependent potassium (Kv) channels is the four additional transmembrane domains (TM1-TM4) that include the voltage-sensor located in TM4 of Kv channels. The transmembrane domains TM1 and TM2 of Kir channels are analogous to the TM5 and TM6 domains of Kv channels. In Kir channels, however, the absence of the TM4 voltage-sensor prevents Kir channels from having an intrinsic voltage-dependent gating function rendering them active across the entire range of physiological membrane potentials [11]. This structural difference in transmembrane composition between Kv and Kir channels results in markedly different biophysical and biochemical properties. Due to the absence of voltage-sensing domains, Kir channels lack voltage sensitivity and are instead regulated by intracellular factors such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂ or PIP₂), ATP in the case of ATP-sensitive channels (KATP), or G protein subunits in G protein-gated channels (GIRK). The fundamental characteristic of the potassium current produced by Kir channels, directly observable when recording current-voltage curves, is its inward rectification. This inward rectification characteristic is independent of the channel structure and fundamentally caused by intracellular factors (e.g. magnesium and polyamines) that block outward potassium flow through the channel pore [32–34].

Pore and transmembrane domain of Kir channels

The initial structural characterization of Kir channels using crystallography was conducted on two channels. The first is the prokaryotic KirBac1.1 channel, and the second is a chimeric Kir channel, which is derived from the prokaryotic KirBac1.3 and the murine Kir3.1 channels [35,36]. These studies made it possible to confirm and validate the conserved structures in Kir channels [37]. This structural framework is common to many Kir potassium channels throughout the animal reign and various families [38]. The highly conserved channel pore is divided into three parts: the potassium selectivity filter, a central aqueous cavity, and the central face of the pore formed by the helices of the two transmembrane domains. As mentioned earlier, the pore region contains the signature sequence of the Kir channel selectivity filter, GYG. The pore region is very narrow, on a scale of 10 Å, and delineates the central aqueous cavity, which extends up to halfway through the transmembrane region where the walls formed by the two transmembrane domains create the end of the section connected to the cytoplasm [11]. Classical Kir channels and KATP channels are constitutively open but can be closed, exhibiting triggerable gating properties. In contrast, some other Kir channels, such as GIRK, are constitutively closed and can only be opened by their respective activation factors, displaying voltage-independent gating [11,39].

Cytoplasmic domains of Kir channels

Despite the pioneering crystallizations of BacKir3 and the KirBac1.3/Mouse Kir3.1 chimera, it was not until the crystallizations of Kir3.1 [40], Kir2.1 [41], and Kir3.2 [42], and their subsequent integration, that provided more precise information about the structure of the cytoplasmic domain. Analogous to the structure of the transmembrane regions, the cytoplasmic domain exhibits a general structure that is consistent across other Kir channels [43]. The cytoplasmic domain is formed by the physical coupling of the carboxy and amino terminal tails. The interaction interface occurs at an aliphatic helix on the amino terminal tail and is known as the slide-helix [40]. The cytoplasmic domain, as in nearly all transmembrane proteins, serves as an interaction region for the protein with endogenous factors that regulate its activity. In the case of Kir channels, this domain acts as an interface for interactions with various gating regulation factors such as certain ions, proteins, or regulatory ligands like PIP2 and ATP. In 2007 [44], it was hypothesized that the interaction between the cytoplasmic domain and the slide-helix, in addition to being fundamental for regulating the biophysics of Kir channels, is equally important for regulating the interaction of the cytoplasmic domain with endogenous ligands. The structure formed by the association of the carboxy and amino terminal tails creates a cylindrical shape that interacts with the slide helices to physically regulate gating [37]. Additionally, this architecture creates a structure that increases the ionic conduction pathway by 30 Å, effectively doubling the conduction distance [11,36].

Molecular regulation of Kir channels

Intracellular block by magnesium and Spermine

The primary characteristic of Kir channels is their ability to generate a current with a feature that is rare among ion channel families namely, inward rectification. While other channels, such as the chloride channel CLC-2 (CLCN2), also exhibit this characteristic, they are much less common [45]. Whether constitutively open, as with vertebrate Kir1 and Kir2, or opened via a cellular mechanism, as with GIRK and KATP, all Kir channels exhibit a current that diminishes as the membrane potential approaches the potassium reversal potential. This current reduction, known as inward rectification, results from several factors that influence the channel’s biophysical properties. The two main mechanisms responsible for this rectification are the intracellular actions of magnesium ions and endogenous polyamines (Figure 4). With a high external K+ gradient, Kir channels typically generate inward rather than outward currents. Intracellular magnesium and polyamines are now considered the primary agents driving the inward rectification of Kir channels, a role that has been well-established for over two decades [34,46,47]. Despite understanding their effect, a strict demonstration of the mechanism of action was only provided in 2016 [48]. It was shown that the blocking effect of magnesium is finely correlated with potassium flux, though less markedly so than with spermine. During physiological membrane depolarization, the efflux of potassium ions induces the movement of intracellular magnesium ions, which block the channel. Spermine alone cannot fully block the channel, and strong depolarization appears to allow spermine to force its way through the selectivity filter. The reduction in efflux caused by the pore blockade by magnesium ions seems to be amplified and stabilized by spermine, which retains the magnesium ions in the pore and appears to induce a conformational change in the internal cavity upstream from the selectivity filter. This narrowing induced by spermine seems to stabilize the magnesium, and the synergy between these two factors could thus be responsible for the pore blockage during depolarization [48].

Figure 4.

Schematic representation of the modus operandi of Kir (inwardly rectifying potassium channels. these channels facilitate the flow of potassium ions (K⁺) across the cell membrane, with the direction of current determined by the membrane potential relative to the potassium equilibrium potential (E_k). 1. at membrane potentials below E_k (more negative), inward currents predominate as K⁺ ions flow into the cell down their electrochemical gradient. 2. conversely, at membrane potentials above E_k (more positive), outward currents are observed as K⁺ ions exit the cell. 3. in the outward current regime, Kir channels are subject to voltage-dependent block by intracellular divalent magnesium ions (Mg^{2 +}) and polyamines such as spermine and spermidine. These molecules enter the channel in a coordinated manner from the cytoplasm up to the pore region in order to occlude ion flow, effectively inhibiting outward currents. This block contributes to the channel’s characteristic inward rectification, allowing efficient K⁺ influx at hyperpolarized potentials while limiting efflux at depolarized potentials.

Phosphatidylinositol PIP2 binding

Vertebrate Kir6.X KATP channels were the first Kir channels in which PIP2-dependent activity was discovered [49]. Although molecules from the phosphoinositide (PIP) family are known to regulate the activity of Kir channels, the Pi(4,5)P2 (PIP2) isoform is the most active in this process [50]. PIP2 interacts with specific interaction sites in the cytoplasmic domain known as “PIP2 binding pockets,” which are formed by the movement of the cytoplasmic domain toward the membrane by attachment to the TM2 transmembrane domain [51–53]. The crystallization of Kir channels revealed that each channel possesses four PIP2 binding sites [51,52]. However, this identical number of binding sites does not explain the observed differences in PIP2 sensitivity between channels. In human Kir2.1, the PIP2 binding site is located at the interface between the transmembrane domain (TMD) and the cytoplasmic domain (CTD) of the channel. Nine key amino acids form this interaction site: within the TMD, the RWR triad (Arg78, Trp79, Arg80) interacts with the 1′ phosphate, glycerol backbone, and acyl chains of PIP2; this sequence is highly conserved among Kir channels, reflecting its functional importance [51] (Figure 5). In the CTD, the inositol ring and its 4′ and 5′ phosphates interact with Lys183, Arg186, Lys188, Lys189, and Arg190, which are located on the tether helix, a secondary structure induced by PIP2 binding. PIP2 binding results in a 6 Å translation of the CTD toward the TMD, triggering large conformational changes necessary for channel opening [51]. These amino acids, particularly those in the CTD, provide the specificity for inositol-phosphate recognition, while interactions with the TMD facilitate lipid anchoring. This set of residues is highly conserved across the Kir family, highlighting a common regulatory mechanism mediated by PIP2 activation. Salt-bridges at the interface of the cytoplasmic domain, were identified by Wang, S. et al. [54], who referred to these regions as Cytoplasmic Domain Subunit-Interface (CD-I) that directly impact PIP2-accessible conformation of Kir channels. They also demonstrated that the interaction sequences responsible for the structure of these salt bridges are conserved across different organisms, both prokaryotic and eukaryotic (Figure 5). Additionally, mutations in the amino acids responsible for these interactions at the CD-I sites negatively and stoichiometrically impact the PIP2 sensitivity of the Kir channel [53,55]. The amino acids that are most active in the interactions at the CD-1 sites are arginine, serine, and methionine (in the case of human and bacterial Kir channels) downstream, which interact with a glutamate upstream. Disruption of the CD-I sites increases the open probability (P_o) of the KirBac1.1 channel, which normally is constitutively open and is closed by PIP2 [53]. For eukaryotic Kir channels which are constitutively closed and opened by PIP2 binding [54], CD-I site disruptions result in a decrease in P_o. In human Kir2.1, loss-of-function variants result in Andersen-Tawil Syndrome by causing impaired channel regulation by PIP2 or other molecular defects, including reduced membrane expression [56].

Figure 5.

Sequence alignment of pore and salt-bridge regions in Kir channels across species. sequence alignment of Kir channels from various species including Homo sapiens, Mus musculus, Burkholderia (bacteria), apis mellifera (honeybee), Drosophila melanogaster (fruit fly) and Aedes Aegypti (mosquito), performed using nucleotide BLAST from the National center for biotechnology information (NCBI). (a) Kir1.1 ROMK channels from each species, with the corresponding NCBI GenBank accession IDs. (b) sequence alignment highlighting the Kir channel-specific and amino acid described as responsible for the PIP2 binding-pocket structure (Hansen et al., 2012). For clarity, only one subunit is shown with the PIP₂ binding site labelled. (c-d) sequence alignment highlighting the Kir channel-specific selectivity filter motif (G-Y-G) and the amino acid R and E described as main candidate for the PIP2 CD-I salt bridge formation (Wang et al., 2017). Amino acid differences compared to hKir1.1 channel are highlighted in blue. Amino acids of interest are in red.

Extracellular block by barium and cesium ions

Historically, the obstruction of the selectivity filter by exogenous cations such as barium and cesium was considered the primary blockade mechanism [57]. This type of blockade is dependent on the concentration of potassium and the direction of the current and primarily affects the inward component of the Kir current. However, it has been suggested that this obstruction alone cannot explain the effect observed on the outward component of the Kir current. In 2022, Gilles Ouanounou [58] provided an in-depth analysis of the intrinsic mechanisms involved in the regulation of endogenous Kir channels in vertebrate Xenopus laevis embryological myotomal cells caused by cesium and barium ions, which addressed gaps in the literature regarding the action of these ions in the context of outward potassium currents. Indeed, similar to the inward current, the outward component is inhibited by cesium and barium in a manner that is independent of extracellular potassium concentration and current polarity, suggesting the involvement of a mechanism beyond simple pore occlusion. The author suggested that this additional potassium-independent blockade mechanism results from the binding of cesium and barium cations to specific sites located outside the selectivity filter, likely on the external side of the channel, due to their foreign nature. Unlike the obstruction of the selectivity filter, this new mechanism does not require the foreign cations to enter the channel pore to exert their effect. Experiments have shown that the blockade of the outward component of the Kir current by cesium and barium is not dependent on potassium concentrations and that these cations seem to bind to common sites upstream from the selectivity filter [58]. Under physiological conditions, where the membrane potential is typically equal to or less negative than the potassium equilibrium potential, this new blockade mechanism, which is specific to Kir channels, would explain for the first time why cesium and barium can be used to selectively block Kir currents without affecting Kv channels. This is particularly relevant in contexts where it is crucial to specifically target Kir channels. Structurally, it has been suggested that this potassium-independent blockade might involve a conformational change in the Kir channel. Although Kir channels lack the S1-S4 helices present in Kv channels that provide voltage sensitivity, X-ray crystallography studies [36,59,60] have revealed that there is an intrinsic gating mechanism in Kir channels that could be related to this new mode of blockade. The author proposes that the binding of cesium or barium to an extracellular loop connecting the transmembrane helices TM1 and TM2 might induce the closure of the channel below the selectivity filter, thereby explaining the independence of this blockade mechanism from the filter. This study [58] provides strong evidence for a mechanism of Kir channel blockade by cesium and barium that is distinct from the sole concept of selectivity filter obstruction that is independent of potassium. This mechanism appears to involve interactions with specific sites located upstream from the selectivity filter that are possibly related to conformational changes in the channel [58].

Kir channel blockade by venom peptides

Attributed to convergent evolution, venom-derived peptides are deployed by diverse venomous species for both defensive and predatory purposes, with many targeting the ion channels in predators and prey to mediate the beneficial physiological responses for the host [61]. From venom screening assays, a 21 amino acid bee venom peptide called tertiapin (TPN) was discovered, which reversibly blocks certain mammalian Kir channels with high affinity [62]. TPN blocks Kir channels by binding to the extracellular mouth of the channel and effectively plugging the selectivity pore like a “cork in a bottle” [63–65]. Bee venom, which contains numerous substances, is typically deployed for defensive purposes where female worker bees protect the hive from invading predators via their venom and stinger apparatus. The role of TPN in this process is not known but may involve the targeting and block of Kir channels in the natural pests and predators of bees, thereby contributing to the noxious sting and an effective defensive response [66,67]. Insect venom peptides that target the Kir channels of insect predators are plausible and would provide the mechanistic underpinnings for the adaptive evolutionary process that leads to the emergence of venom peptides like TPN that block Kir channels with high affinity and specificity.

Kir channels in Drosophila melanogaster: Functions and insights

Kir cloning and tissue-specific expression

In insects, the cDNAs encoding inwardly rectifying potassium channels were first isolated from Drosophila melanogaster [68]. Five distinct Kir channel transcripts from the three different genes were identified. These transcripts are differentially expressed in various tissues, indicating that different Kir channels are utilized at various stages and in various tissues during development. In situ hybridization has been used to show that dKir1 transcripts are absent in embryos, suggesting that this channel is not required and is thus not expressed at early developmental stages. However, dKir2 and dKir3 are present in specific regions during embryonic development. dKir2 is expressed in the embryonic hindgut while dKir3 is expressed in the Malpighian tubules, the key organ responsible for osmoregulation and excretion, collectively including the osmoregulatory system of the developing Drosophila fly [68]. In adult Drosophila, dKir2 transcripts are predominantly detected in the head, indicating that dKir2 plays a specific role in neural and/or sensory functions in adult flies.

When dKir channels are heterologously expressed in Xenopus oocytes (a common model for studying ion channel function), channel activity is only observed after specific amino acid substitutions in their cytosolic tails. For example, substituting a unique valine in the NH2 terminus (dKirIV34Q) is required to observe functional inwardly rectifying potassium channel activity [68]. When wildtype dKir1 and dKir2 are expressed individually in Drosophila S2 cells (a cell line derived from Drosophila embryos), they readily exhibit typical inwardly rectifying potassium currents, suggesting that the native cellular environment in Drosophila cells provides additional factors necessary for the proper functioning of these channels. The potassium currents generated by dKir channels in Drosophila S2 cells are weakly sensitive to barium blockade compared to mammals Kir channels [68]. This provides insights into the ion selectivity and inhibitory characteristics of these channels, which are important for understanding their physiological role. No functional activity of dKir3 has been observed in Drosophila S2 cells or Xenopus oocytes, although high expression levels have been detected. The lack of functional activity of dKir3 channels in Drosophila S2 cells or in Xenopus oocytes, despite high tissue expression levels, is not understood. However, it could be due to several factors, including the absence of essential accessory proteins such as a G-protein βγ subunit [68], improper post-translational modifications, incorrect cell surface trafficking, or assembly with other Kir channel subunits forming heteromeric channels. Further studies are warranted to elucidate this lack of functional expression.

Function of Kir channels in Malpighian tubule physiology

In 2005, building on the work of Döring 2002 [68], Evans, J., et al. [69] determined the distribution of each of the three Kir channels in Drosophila in its Malpighian tubules, which is analogous to the renal system in many insects (with some exceptions such as aphids, see below) [69]. A microarray analysis revealed that Kir channels are involved in the functioning of renal Malpighian tubules. The three dKir channels in Drosophila are localized and abundant in principal cells and show both distinct and overlapping expression patterns. For instance, dKir3 is expressed in the principal cells of the main segment of the tubules and in the lower parts, indicating that it may be involved in fluid secretion and reabsorption. The secretion mechanism in Malpighian tubules is inhibited by several agents, including sulfonylureas, which are specific inhibitors that target ATP-sensitive Kir channels. However, the sulfonylurea sensitivity alone is insufficient to affirm that there is an association between Kir channels and the SUR protein of Drosophila melanogaster. Complete inhibition was observed with sulfonylureas, while inhibitors of the Na⁺/K⁺-ATPase pump and Na⁺/K⁺/2Cl^– cotransporter only partially reduced secretion in the tubules, further supporting the idea that Kir channels play a major role in this secretion function. The significant amounts of mRNA coding for dKir1, dKir2, and dKir3 channels in the tubules suggest that they play an important role in potassium entry at the basolateral level of the cells. Kir channels seem to play a limited role in basal secretion but contribute to potassium entry during tubule stimulation, which is consistent with the high secretion rates observed in insects [70]. The Kir channels of Drosophila melanogaster, like those of other species including Anopheles gambiae and Tenebrio molitor, share strong similarities, despite being less sensitive to barium [71,72]. These results indicate that the Kir channels in Drosophila melanogaster are located in the same cells as the apical V-ATPase, which allows the passage of potassium ions through the apical membrane, and that they play a key role in fluid secretion. These mechanisms appear to be conserved in other insects [73–75].

In Drosophila melanogaster, the physiological role of Kir channels has been compared to the activity of Kir channels in other insects, including Aedes aegypti. Kir1, Kir2, and Kir3 channels are essential for regulating potassium flux in the Malpighian tubules of insects such Aedes aegypti, Formica polyctena [76], Rhodnius prolixus [77], Locusta migratoria [74], and Tenebrio molitor [72,78]. It has been shown that barium decreases fluid secretion and reduces potassium flux in the Malpighian tubules of Drosophila melanogaster [78]. However, half the reduction in potassium flux by barium might be attributed to other unidentified channels. The simultaneous inhibition of Kir1 and Kir2 is required to reduce potassium flux by approximately 50%, indicating that they play a central role in renal function. The exact function of Kir3 and its homologs (such as AeKir3 in Aedes aegypti) remains unknown [78] despite the high levels in the tubules in some insects.

Functional significance of Kir channels in salivary gland physiology

The salivary glands of arthropods are essential for feeding and pathogen transmission. Despite growing interest over the past decade in their physiological roles and the impact of Kir channels in regulating their activity, further investigations are needed to obtain a better understanding of all the underlying mechanisms of salivary glands. Pharmacological studies have revealed that pathways such as dopaminergic, Na⁺-K⁺-ATPase, GABA, and muscarinic receptors are involved in regulating salivary secretion [79–81]. However, Kir channels, particularly dKir1, have proven to be critical, being highly expressed in the salivary glands of Drosophila melanogaster, especially in the larval and adult stages. The inhibitor VU041, which specifically targets certain dKir channels [82], significantly reduces sucrose intake by flies. In contrast, the inactive analogue VU937 has no effect, proving that the reduction in ingestion is due to Kir channel inhibition. These findings highlight the importance of Kir channels in the proper functioning of the salivary glands and how they directly affect feeding efficiency. Genetic suppression of the dKir1 gene via RNAi results in a significant decrease in salivary secretion after as soon as day 2 [82]. Genetically modified flies exhibit a marked reduction in salivary secretion and feeding efficiency, which validates the fundamental role of dKir1 in regulating potassium flux and saliva production. Kir channels play a critical role in rapidly buffering potassium ions, facilitating their entry following depolarization. This process establishes a gradient that drives potassium efflux through the activation of calcium-activated potassium channels and is comparable to mammalian salivary glands, where Kir channels help maintain the membrane potential polarization necessary for chloride ion secretion. When dKir1 is suppressed, compensation is observed through the increased expression of dKir2, which explains the lack of noticeable effects on feeding on day 1. This phenomenon is also observed in the Malpighian tubules, where the Na⁺/K⁺/2Cl⁻ transporters and Na⁺/K⁺/ATPase pump help maintain high ionic gradients. As such, VU041 could potentially serve as a basis for insecticides targeting Kir channels. By disrupting the feeding of disease-carrying pests like mosquitoes, this could limit pathogen transmission by affecting the salivary secretion of these insects [82].

Functional role of Kir channels in the nervous system

Kir channels play a significant role in potassium ion conduction in the nervous system of insects. Although their exact role is not fully understood, VU041 significantly disrupts neuronal activity in Drosophila melanogaster, demonstrating that Kir channels are essential for regulating neuronal excitability, similar to mammals [83]. Recordings from the central nervous system (CNS) of Drosophila melanogaster in the presence of barium have shown that there is an increase in neural discharges followed by a complete cessation, indicating that Kir channels form a critical pathway for potassium ions in the CNS. However, the use of barium has limitations as it can bind to proteins other than Kir channels or precipitate in certain saline solutions, making the data less reliable. VU041 induces a biphasic response in the CNS of Drosophila melanogaster. At low concentrations, it stimulates neural excitation, while at higher concentrations, it reduces activity. RNAi experiments targeting dKir2 have confirmed this interaction, showing an increase in neural discharge frequency similar to that observed with VU041, suggesting that dKir2 plays a crucial role in regulating CNS excitability. The action of glial Kir channels in buffering potassium ions during neuronal activity, helps to prevent excessive depolarizations. The inhibition by VU041 or the suppression of dKir2 disrupts this mechanism, altering synaptic transmission and reducing neural excitability. Pharmacological tests using known mammalian KATP channel modulators have shown no significant modification of neuronal activity in the CNS of Drosophila melanogaster, even at high concentrations [83]. These channels may play a similar role as their mammalian counterparts in regulating potassium levels and moderating synaptic transmission, ensuring precise neuronal control. Given that Kir channels play an important role in regulating potassium ion actions and that inhibiting them leads to prolonged depolarization and synaptic dysfunction, the development of insecticides that specifically target Kir channels offers a promising strategy for pest control. By precisely inhibiting these channels in pest insects, while avoiding any impact on non-target species such as pollinators, this approach will provide a more selective and environmentally friendly solution [83].

The insulin-producing cells (IPCs) of Drosophila melanogaster function in a similar way as mammalian pancreatic β-cells, particularly in how KATP channels regulate insulin secretion. Electrophysiological studies have shown that increasing mitochondrial uncoupling protein activity in IPCs causes membrane depolarization and calcium influx in response to glucose, mimicking the mechanism of insulin secretion [84]. Molecular analyses have confirmed the presence of KATP channel SUR subunits in IPCs, highlighting their role in glucose regulation. These channels also protect against metabolic and infectious stress, underscoring their importance in maintaining glucose homeostasis. The results of pharmacologically modulating KATP activity in CNS neurons [83] and depolarizing cells expressing SUR regulatory subunits give credence to the hypothesis that there is a close connection between Kir2 and KATP channels, as proposed recently [15]. It is well-established that KATP currents are essential for many physiological processes in arthropods. However, unlike mammals, where KATP channels are formed by distinct Kir6.X subtypes, the exact formation of KATP channels in arthropods remains unclear. It is also not yet understood what activates the SUR subunit in conjunction with Kir1 and/or Kir2 in these organisms [15].

Role of Kir channels in immune system function and regulation

Cardiac KATP channels play a crucial role in resistance to Flock House Virus (FHV) infections in Drosophila melanogaster by modulating RNA interference (RNAi), a key antiviral defense mechanism [85]. RNAi must be regulated to control infections, with components like TRBP, AGO, and PIWI undergoing post-translational modifications (phosphorylation, hydroxylation, etc.) [85]. An unexpected finding is that FHV is a cardiotropic virus in Drosophila, which provides a useful model for studying virus-cardiomyocyte interactions. In humans, cardiotropic viruses cause myocarditis, but only 10% of those infected develop the disease, suggesting that genetic background and environmental factors play an important role in whether a person becomes infected. KATP channels play an ancestral role in antiviral cardiac immunity in both insects and mammals. They regulate both innate immunity via the inflammasome and RNAi in insects, while also contributing to cardiac protection [86]. KATP channels are also known to be fundamental for the antiviral protection of Apis mellifera [87].

Contribution of Kir channels to morphogenesis and development

The vertebrate Kir2.1 channel has proven to be of crucial importance in development and morphogenesis. Morphological anomalies associated with Andersen-Tawil syndrome (ATS) suggest that Kir2.1 function is essential for human development [88,89]. These anomalies have been reproduced in murine models where the Kir2.1 gene is inactivated [90]. Similarly, a reduction in dKir2 function in Drosophila leads to developmental defects, likely by disrupting Dpp signaling, the homolog of TGFβ/BMP signaling, which regulates development [5]. Phenotypes related to dKir2 modification, using dKir2-deficient flies, dKir2 mutant alleles, dKir2 siRNA, or dominant-negative dKir2DN expression, resemble dpp mutants in wing formation. These anomalies include loss of the wing blade, as observed in dpp mutants. Reducing dKir2 expression via siRNA decreases Mad factor phosphorylation in wing discs, confirming a reduction in Dpp signaling [5,91]. Blocking apoptosis in cells expressing dKir2DN does not restore wing phenotypes, as Dpp signaling is key for compensating for apoptotic cells, a mechanism that appears dysfunctional in the presence of dKir2DN. The alteration in Dpp signaling seems to be due to the influence of dKir2 on the membrane potential, which is crucial for Dpp signal production and distribution. Changes in potassium concentrations may interfere with HSPGs (heparan sulfate proteoglycans), which facilitate BMP/TGFβ signaling. In mammals, developmental alterations caused by Kir2.1 loss of function, such as dental and limb malformations, are similar to those observed in BMP/TGFβ signaling dysfunctions. Potassium channels like dKir2 are essential for BMP signaling in Drosophila due to their influence on various aspects of morphogenesis [5].

Aedes aegypti Kir channel: Function and interest in pest control

Cloning and functional expression of Aedes aegypti Kir channels

The first cloning and functional characterization of the Kir channel subunits (AeKir1, AeKir2B, AeKir3) in the Malpighian tubules of Aedes aegypti mosquitoes was reported by Piermarini et al. [92]. The expression patterns of these subunits show a strong similarity to those observed in Anopheles gambiae and Drosophila melanogaster [68,93]. A notable distinction is the specific expression of AeKir2B in mosquitoes. However, its functional impact appears to be minimal. The AeKir1 and AeKir2B channels are both constitutively active, potassium-selective, and sensitive to barium blockade, and exhibit properties comparable to mammalian and Drosophila melanogaster Kir channels. However, AeKir1 demonstrates higher activity than AeKir2B, likely due to differences in their PIP2 binding motifs (R²⁶⁵PKK for AeKir1 vs. R²³²PKS for AeKir2B). This suggests that AeKir2B may have a reduced capacity to bind PIP2 or may require an additional agonist for full activation.

Furthermore, a difference in cesium conductance has also been observed between AeKir1 and AeKir2B. AeKir2B generates inward cesium currents, whereas cesium acts as a blocker for AeKir1. Although this discrepancy could potentially be attributed to variations near the selectivity filter, the corresponding residues are identical in both subunits, leaving the underlying cause of this difference uncertain. Similar to dKir3 of Drosophilia melanogaster, AeKir3 subunits from mosquitoes do not exhibit functional activity when expressed in Xenopus oocytes [68], suggesting that their functionality may require an accessory protein, co-expression with other Kir subunits, or an intracellular modulator. AeKir1 channels in Malpighian tubules are responsible for potassium conductance on the basolateral membrane of principal cells with high permeability to Tl⁺ -like mammalian Kir channels. The AeKir1 channel conductance is sensitive to barium, which causes approximately 60% inhibition. AeKir1 is considered the main mediator of potassium transport from the hemolymph to the cytoplasm of principal cells in parallel with the Na⁺-K⁺-2Cl⁻ co-transporter (NKCC). Upon stimulation by kinins (leucokinins or aedeskinins), which increase intracellular Ca^{2 +} levels and hyperpolarize the basolateral membrane, potassium uptake by AeKir1 is enhanced, which supports diuresis. In contrast, stimulation by the calcitonin-like peptide (CNP) via cAMP induces depolarization of the basolateral membrane, reducing potassium entry, and promoting its efflux, which contributes to natriuresis. This potassium recycling mechanism by AeKir1 is similar to that observed in mammalian kidneys via Kir1.1 channels [11,94].

Physiological significance of Kir channels in the salivary glands of Aedes aegypti

Aedes aegypti Kir channels, particularly the Kir2A isoform, are expressed in the salivary glands, similar to what has been observed in the Malpighian tubules [95]. The salivary glands of Aedes aegypti however, do not express Kir1 [82]. The secretory activity of mosquito salivary glands has also been reported to be sensitive to KATP channel activators such as pinacidil and VU0071063 that target blood feeding. In contrast, inhibitors of classical Kir channels (such as VU041 and VU625) or KATP channel antagonists (such as tolbutamide and glibenclamide) do not affect fluid secretion or blood feeding. These results suggest that KATP channels are more involved in regulating salivary secretion than classical Kir channels. Kir channels play a critical role in regulating ionic conductance and fluid transport in the salivary glands, which is essential for blood feeding and pathogen transmission [95]. KATP channels may be more involved in adapting the salivary secretion required for mosquito blood feeding. The pharmacological activation of KATP channels reduces the ability of mosquitoes to feed and transmit pathogens, highlighting the importance of these channels in the feeding process and in the horizontal transmission of viruses, such as dengue virus 2 (DENV2).

Physiological role of the Kir channel of Anopheles gambiae in egg production

Mosquitoes ingest large quantities of blood, which poses a significant physiological challenge due to the need to rapidly excrete excess water, weight, and solutes [96]. Kir channels, particularly AgKir1 in Anopheles gambiae, play a key role in this regulation. The inhibition of AgKir1 disrupts urine production and potassium homeostasis in the hemolymph, making these channels promising targets for mosquito population control. In Anopheles gambiae, the genome contains between five and six Kir genes (AgKir1, AgKir2A, AgKir2A,’ AgKir2B, AgKir3A, and AgKir3B) [97], a greater number than that of Drosophila melanogaster, suggesting that these channels are more functionally complex. This diversity likely allows for finer regulation of physiological processes, particularly in modulating membrane excitability and ion transport signaling. AgKir1 behaves similarly to AeKir1 in Aedes aegypti. This channel mediates inward potassium currents that are blocked by barium. The electrophysiological properties of AgKir1 have been investigated in Xenopus oocytes, where resting potentials and current-voltage relationship curves can be compared to those of oocytes expressing AeKir1, where both produce canonical Kir currents. AgKir1 expression is maximal at the pupal stage, indicating that it is involved in metamorphosis, a process associated with changes in membrane excitability [98]. Expression is also enriched in the ovaries, suggesting that AgKir1 plays a potential role in oocyte maturation. Indeed, the reduction in fecundity following AgKir1 gene silencing (RNAi) supports this hypothesis. Although inhibiting AgKir1 reduces fecundity, it does not lead to a lethal phenotype or flight incapacity after a blood meal, possibly due to compensation by other Kir channels or the relative inefficiency of RNAi compared to the hypothetical effects of pharmacological inhibition. In conclusion, AgKir1 and Kir channels in general are essential for renal regulation and mosquito fecundity, making these channels potential targets for new insecticides [97].

Dynamic expression of Kir channels in mosquitoes during development

In Aedes aegypti, the general expression of AeKir1 mRNA remains constant throughout development, whereas in Anopheles gambiae, expression increases during the pupal stage [97] and in Drosophila melanogaster, it increases progressively [68,99]. In the Malpighian tubules, AeKir1 expression rises in pupae and adult females, and plays a critical role in transepithelial potassium excretion via the basolateral membrane of stellate cells, which display an enhanced capacity following a blood meal. In the midgut, AeKir1 is more highly expressed in pupae than in larvae or adult females, although less so than in the Malpighian tubules, and its functional role in this tissue remains unknown. AeKir2A-c exhibits high mRNA levels in the anal papillae of larvae and the ovaries of adult females, suggesting that it may be involved in ionic absorption and ovarian maturation, respectively. Its reduced expression in pupae reflects the loss of anal papillae during metamorphosis. However, its expression remains stable in the Malpighian tubules and midgut throughout development [99]. AeKir2B, which is primarily expressed in the principal cells of the Malpighian tubules, plays a complementary but quantitatively smaller role than AeKir1 in renal potassium excretion [100]. Its expression is low in larvae but progressively increases in pupae and adult females, potentially contributing to active fluid absorption following blood meals [99,101]. AeKir2B′, a gene duplication specific to Aedes aegypti, is weakly expressed across life stages, except for the elevated levels observed in the midgut of pupae where its function remains unclear [92]. AeKir3 mRNA is highly expressed in the larvae and pupae of Aedes aegypti but is reduced in adult females [99]. A similar profile has been observed in Cimex lectularius (high expression during nymphal stages) and contrasts with Drosophila melanogaster, where dKir3 is predominantly expressed in adults compared to larval and pupae stages [68]. In Aedes aegypti, AeKir3 expression is higher in the Malpighian tubules than in the midgut across all stages, with the tubules accounting for global variations. Unlike Aedes aegypti (single Kir3 gene), Anopheles gambiae possesses AgKir3A and AgKir3B, with AgKir3A being expressed at higher levels in adults. Similarly, in Drosophila melanogaster, dKir3 is more highly expressed in adults, with no significant impact on potassium excretion following RNA silencing [68]. AeKir3 is localized in the intracellular vesicles of principal and stellate cells within the Malpighian tubules of adult females and does not form functional channels when expressed in Xenopus oocytes. The elevated expression in larvae and pupae suggests that it plays an enhanced role in mitigating ionic losses during aquatic stages, while the reduced expression in terrestrial, hematophagous adults indicates that it plays a diminished role in this phase

Targeting Kir channels for mosquito population control

Classified as pests, mosquitoes are significant disease vectors capable of transmitting substantial quantities of pathogens to humans. As alluded to earlier, the critical roles of Kir channels in regulating key physiological processes such as salivary secretion, renal function, and reproduction, and their influence in major developmental stages and in reproduction have made them targets of interest in the past decade for the development of molecules designed to disrupt their functions. As reviewed earlier, chemical blockers of insect Kir channels developed over the years and commonly used in toxicological studies include molecules from the Institute of Chemical Biology at Vanderbilt University, which are extensively documented and characterized in the literature [102–105]. VU590 was identified as a selective inhibitor of the AeKir1 channel of Aedes aegypti [102]. This compound also affects the mammalian Kir1.1 and Kir7.1 channels but not the Kir2.1 and Kir4.1 channels [106]. On the other hand, VU573 has a lower affinity for AeKir1 and, intriguingly, acts as both a blocker of AeKir1 and an agonist of AeKir2B, which highlights the significant structural and functional differences between these channels. VU590 has been shown to be lethal to mosquitoes by disrupting secretory functions [102]. Similar observations have been made for VU573 and VU625, with particular interest given to VU625, which exhibits 20- to 80-fold higher selectivity for AeKir1 than for human Kir channels [103]. In 2016, the discovery of VU041 and its use on Anopheles gambiae was reported and compared to the effects of VU937, which was used as an inactive analog due to its 60-fold lower efficacy in reducing AgKir1 currents [104]. VU041 caused a decrease in renal function, which was confirmed in 2017 [105], as well as a reduction in fertility. Interestingly, topical application of VU041 on the honeybee did not induce lethality within bee colonies [104]. A depth analysis of the electrophysiological effects of VU041 on the Apis mellifera AmKir channels will be explored later (see Apis mellifera section below).

The role of Kir channel in the feeding process of aphids

Insect Kir ion channels demonstrate unique evolutionary and functional features, with ApKir channels in aphids like Aphis glycines having been cloned and characterized [107]. ApKir2 channels have undergone faster and less constrained evolution than Kir1 and Kir3 channels. Aphid ApKir2 orthologs possess a “non-canonical” selectivity filter sequence (GFG instead of GYG) [11,68,107] and cluster separately from other hemipteran orthologs in phylogenetic analyses. Unlike other insects [68,92], but similar to the honeybee Apis mellifera [19], aphids lack Kir3 orthologs, perhaps because they lack the Malpighian tubules where Kir3 is typically expressed. Functional studies of ApKir1 and ApKir2 channels in Xenopus oocytes have revealed that ApKir1 is more active and sensitive to barium than ApKir2, while ApKir2 exhibits weaker rectification and greater sensitivity to the inhibitor VU041 than to VU937. VU041 disrupts feeding and excretion and is more toxic to Aphis glycines than the insecticide flonicamid. The cotton aphid Aphis gossypii has been reported to display high resistance to many insecticides such as carbamates [108], neonicotinoids [109], and pyrethroids [110,111].

With annual losses reaching $1 billion USD [112], interest in VU small molecules targeting aphid Kir channels has grown significantly over the past decade. Although the Aphis gossypii Kir1 and Kir2 channels have yet to be cloned and tested, they are hypothesized to be key targets. The treatment of leaves with VU041 [111] and VU730 [113] effectively reduces salivary secretion and alters probing behavior by Aphis gossypii, ultimately disrupting feeding and resulting in death.

AmKir channels of Apis mellifera: The interesting case of VU041

As discussed previously, two Kir channel genes have been identified in the honeybee genome [19], AmKir1 and AmKir2, which have two and four isoforms, respectively (Figure 2). These isoform mRNA transcripts have been detected in various honeybee organs, including the brain, where they likely contribute to essential physiological processes such as neuronal signaling and ion homeostasis. PCR analyses have confirmed that AmKir channels are differentially expressed in various organs. For instance, certain isoforms may be enriched in neural tissues and may support synaptic activity while others may be enriched in organs involved in osmoregulation (Figure 6). Isoforms AmKir1.1, AmKir2.2, and AmKir2.3 generate functional, inwardly rectifying potassium currents when expressed alone in Xenopus oocytes [19]. In contrast, AmKir1.2, AmKir2.1, and AmKir2.4 do not generate functional currents when similarly expressed. While AmKir2.2 and AmKir2.3 share similar sequence similarity, they exhibit distinct biophysical properties. AmKir2.3, due to a 31-amino-acid shorter N-terminal tail, displays a smaller current amplitude and distinct activation kinetics potentially related to an altered stability or interaction with its cytoplasmic domain.

Figure 6.

Expression of Kir channels within different arthropods in the Malpighian tubules, salivary glands, brain, immunity function, heart function and wing patterning. d: Drosophilia melanogaster (fruit fly), Ae: Aedes aegypti (mosquito), Ag: Anopheles gambiae (mosquito), Ap: Aphis glycines (Aphid), ago: Aphis gossypii (Aphid), Am: Apis mellifera (honeybee), n.A: not available, n.O: not observed. Channels in parentheses are those whose presence is suggested but not demonstrated.

Ion permeability experiments have revealed that, unlike AmKir1.1, both AmKir2.2 and AmKir2.3 are permeable to cesium, a rare characteristic among Kir channels [92]. All three isoforms are highly permeability to potassium and thallium but display low permeability to sodium and lithium. In terms of pharmacology, barium, a known nonselective Kir channel blocker, inhibits both AmKir1.1 and AmKir2.2, although a ten-fold higher concentration is required to block AmKir1.1. Amino acid sequence differences in the pore region, which is seven amino acids upstream from the selectivity filter, may explain the varying sensitivity to barium observed between honeybee (isoleucine) [19], mosquito (leucine) [92], and aphid (valine) [107] Kir channels. Lastly, unlike previous findings in other insects, VU041 has no significant effect on AmKir1.1, AmKir2.2, or AmKir2.3 channels, even at high concentrations (up to 10 µM). Although VU041 is effective in blocking Kir1 channels in mosquitoes [104], its lack of efficacy in Apis mellifera may be due to structural differences in binding sites or non-conserved regions between species. Although AeKir1 and AmKir1 channels share approximately 55% sequence similarity, critical variations in amino acid sequences could alter the binding affinity or orientation of VU041 [15]. These differences may lead to distinct channel conformations, impacting the inhibitor’s interaction with the binding pocket or even its access to the site. Additionally, non-conserved regions may contribute to variations in channel gating, sensitivity, or pharmacological profile, potentially rendering VU041 ineffective in honeybees. VU041 has previously been topically tested on live bees and has displayed no lethality in bee colonies [104]. These findings confirm that VU041 does not inhibit Kir channels in honeybees, underscoring the importance of conducting further investigations into the molecular interactions specific to AmKir channels. This selective insensitivity suggests that there may be unique structural or functional features within the AmKir channel that prevent VU041 from binding effectively. Understanding these differences is crucial not only for developing targeted insecticides that minimize their impact on beneficial species like honeybees, but also for advancing our knowledge of Kir channel pharmacology across species. Future research should focus on characterizing the binding sites and gating mechanisms of AmKir channels, examining possible conformational differences, and identifying alternative small molecules or modifications to VU041 that would enable selective inhibition in pest species while safeguarding pollinators.

Conclusion

In conclusion, Kir channels play pivotal roles in maintaining potassium homeostasis, neuronal excitability, and osmoregulation across various physiological functions and insect species. The cloning and characterization of these channels in model organisms such as Drosophila melanogaster, Aedes aegypti, and Apis mellifera have highlighted their evolutionary conservation and functional significance. These channels exhibit structural and functional homology with mammalian Kir channels, underscoring their conserved roles in cellular signaling and developmental processes such as wing patterning and bone morphogenetic protein signaling. The identification of species-specific variations in the number and types of Kir channels reflects adaptive evolutionary mechanisms tailored to unique physiological needs. Electrophysiological studies have deepened our understanding of Kir channel properties, including ion selectivity, gating mechanisms, and pharmacological profiles, paving the way for their consideration as potential insecticide targets. By disrupting ion homeostasis, compounds that modulate Kir channel activity offer a promising strategy for pest control. Despite the significant progress made to date, much remains to be explored with respect to the ecological and physiological roles of Kir channels in insects. This review provides a comprehensive foundation for future research, emphasizing the need to investigate unexplored insect Kir channel types and their broader involvement in insect biology and pest management strategies.

Funding Statement

The present study was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant [RGPIN-2020–06359] to MC, an NSERC Alliance International Catalyst Grants [572132 – 2022 and 590085 – 23] to MC.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Supplementary Table S1 is available using the following link: https://usf.box.com/shared/static/ema8x2vdc8cdobwlpymz9zcxasxni7uj.xlsx