InteractiveFly: GeneBrief

Inwardly rectifying potassium channel 1, 2, and 3: Biological Overview | References

BIOLOGICAL OVERVIEW

A complete understanding of the physiological pathways critical for proper function of the insect nervous system is still lacking. The recent development of potent and selective small-molecule modulators of insect inward rectifier potassium (Kir) channels, Kir1, Kir2, or Kir3 in Drosophila, has enabled the interrogation of the physiological role and toxicological potential of Kir channels within various insect tissue systems. Therefore, this study aimed to highlight the physiological and functional role of neural Kir channels the central nervous system, muscular system, and neuromuscular system through pharmacological and genetic manipulations. The data provide significant evidence that Drosophila neural systems rely on the inward conductance of K(+) ions for proper function, since pharmacological inhibition and genetic ablation of neural Kir channels yielded dramatic alterations of the CNS spike discharge frequency and broadening and reduced amplitude of the evoked EPSP at the neuromuscular junction. Based on these data, it is concluded that neural Kir channels in insects (1) are critical for proper function of the insect nervous system; (2) represents an unexplored physiological pathway that is likely to shape the understanding of neuronal signaling, maintenance of membrane potentials, and maintenance of the ionic balance of insects, and (3) are capable of inducing acute toxicity to insects through neurological poisoning (Chen, 2018).

The establishment of insecticide resistance within multiple arthropod vectors of human pathogens has been, at least in part, the driving force behind the prolific advancement of the fields of insecticide science and insect molecular physiology. The goal of mitigating the various resistance mechanisms has been a multidisciplinary and transdisciplinary approach that has resulted in a detailed understanding of molecular genetics, transcriptomics, biochemistry, cellular physiology, and neuroendocrinology of non-model insects, such as mosquitoes. In addition to these fields, the reduced efficacy of currently approved classes of insecticides has dramatically increased interest of identifying novel molecular targets for insecticide design and/or development of novel chemical scaffolds targeting previously exploited proteins. A variety of new target sites and chemical scaffolds have been identified and characterized in the past decade that include transient receptor proteins, G-protein coupled receptors, dopaminergic pathways, and K+ ion channels (Chen, 2018).

Inward rectifier potassium (Kir) channels belong to a large 'superfamily' of K+ ion channels that includes the voltage-gated, two-pore, calcium-gated, and cyclic nucleotide-gated channels. Kir channels function as biological diodes due to their unique ability to mediate the inward flow of K+ ions at hyperpolarizing membrane voltages more readily than the outward flow of K+ at depolarizing voltages. On a molecular level, Kir channel are structurally simple ion channels that consists of 4 subunits assembled around a central, water-filled pore, through which K+ ions move down their electrochemical gradient to traverse the plasma membrane. Each subunit consists of a central transmembrane domain, a re-entrant pore-forming loop, and a cytoplasmic domain comprised of amino and carboxyl termini (Chen, 2018).

Recent genetic and pharmacological evidence suggests that Kir channels could represent viable targets for new insecticides. In Drosophila melanogaster, embryonic depletion of Kir1, Kir2, or Kir3 mRNA leads to death or defects in wing development (Dahal, 2012). Reduction of Kir1 and Kir2 mRNA expression in the Malpighian (renal) tubules of Drosophila or inhibition of Kir channels in isolated mosquito Malpighian tubules with barium chloride (BaCl2) dramatically reduces the transepithelial secretion of fluid and K+ (Wu, 2015; Scott, 2004), indicating Kir channels expressed in the Malpighian tubules may be an exploitable insecticide target site. Considering this, high-throughput screens (HTS) of chemical libraries were performed to identify small-molecule modulators of mosquito Kir1 channels, which is the principal conductance pathway in mosquito Malpighian tubules. Structurally distinct small molecules were identified (i.e., VU573, VU590, or VU625) and pharmacological inhibition of Aedes aegypti Kir1 was shown to disrupt the secretion of fluid and K+ in isolated Malpighian tubules, urine production, and K+ homeostasis in intact females (Raphemot, 2013; Rouhier, 2014a; Beyenbach, 2015). Similarly, a Kir1 inhibitor, termed VU041, was identified in a subsequent HTS campaign and was shown to (1) be highly potent against the Anopheles gambiae Kir1 (ca. 500 nanomolar), (2) exhibit topical toxicity (ca. 1 μg/mosquito) to insecticide-susceptible and carbamate/pyrethroid-resistant strains of mosquitoes, (3) and display high selectivity for mosquito Kir channels over mammalian Kir channel orthologs (Chen, 2018).

Previous work indicates that VU041-mediated toxicity stems from inhibition of the Kir1 channel within the Malpighian tubules to induce tubule failure and an inability to maintain K+ homeostatsis after blood feeding (Swale, 2016). However, after exposure to lethal doses of VU041, An. gambiae and A. aegypti were found to display both hyperexcitatory and lethargic tendencies that were complexed with uncoordinated movements (Swale, 2016), which is reminiscent of neurological poisoning. Furthermore, acute toxicity (ca. 1-3 hours) was observed after exposure to VU041, similar to other insecticides that poison the nervous system. Lastly, previous studies have shown that select Kir channel inhibitors were capable of inducing a flightless behavior where mosquitoes were ambulatory, yet were not able to fly, presumably due to failure of the nervous or muscular systems (Raphemot, 2014). Although it is possible that the mortality is due to complete systems failure stemming from ubiquitous expression of Kir channels or due to accumulated waste that remains due to impaired Malpighian tubule function, it is also reasonable to predict that VU041 is directly altering the functional capacity of Kir channels expressed in the nervous system to yield toxicity. Unfortunately, there have been no studies to characterize the physiological role of Kir channels in the insect nervous systems, which limits the ability to infer the toxicological potential of these neural proteins. Studies using RT-PCR have shown that the head of A. aegypti is enriched with Kir2B' (vector base accession number: AEL013373) mRNA (Dr. Peter Piermarini, The Ohio State University, personal communication to R. Chen, 2018), suggesting that poisoning of the mosquito central nervous system (CNS) through Kir inhibition is indeed possible. Unfortunately, electrophysiological recordings of mosquito CNS activity have yet to be achieved, which limits the ability to infer the physiological role or toxicological potential of neural Kir channels of mosquitoes. However, electrophysiological recordings from an excised CNS of D. melanogaster is possible and further, the gene encoding Kir2, termed irk2, is highly concentrated in the adult head, CNS, and the thoracic-abdominal ganglia. This suggests that D. melanogaster may represent a suitable substitute for mosquitoes and will enable the characterization of the physiological role Kir channels have in the insect nervous system (Chen, 2018).

Considering (1) the foundational role of Kir channels in mammalian and insect cellular physiology, (2) deletion of irk2 gene in Drosophila is homozygous lethal, (3) the signs of intoxication after exposure to Kir channel modulators being reminiscent of neurological poisoning, and (4) the overexpression of Kir mRNA in mosquito and Drosophila neural tissues, it was hypothesized that Kir channels regulate neuronal signaling and excitability of insect nervous systems and are a critical conductance pathway for proper functioning of the insect nervous system. Therefore, the goals of the present study were to employ electrophysiological methods combined with genetic and pharmacological techniques to determine the physiological importance of Kir channels in insect CNS, neuromuscular junction, and muscular systems that will provide insight into targeting neural Kir channels as a novel insecticide target site. Additionally, data collected in this study begin to bridge the fundamental knowledge gap regarding unexplored physiological pathways in the insect nervous system that will provide a more holistic understanding to neuronal excitability and neurotransmission of insects (Chen, 2018).

Currently, there have been no efforts to characterize the physiological role or toxicological potential of insect neural Kir channels. However, the current findings demonstrate that the recently discovered Kir-directed insecticide, VU0413, is capable of dramatically altering the neural activity of flies and, in a more general sense, that Kir channels constitute a critical K+ ion conductance pathway in the insect nervous system. Despite the nervous system being the target tissue of the extreme majority of deployed insecticides, a complete understanding of the physiological pathways critical for proper function of the insect nervous system is still lacking. This represents a critical gap in knowledge of the complex relationship between the dozens of functionally coupled ion channels, transporters, and enzyme systems that require tight regulation for proper neuronal function. This fundamental gap pertaining to the foundational neural physiology must be filled to develop a holistic understanding of insect nervous system function that will lead to the development of new insecticides (Chen, 2018).

Knowledge of the physiological role and toxicological potential of insect Kir channels is growing rapidly with studies suggesting these channels serve a critical role in Malpighian tubule function of mosquitoes and Drosophila (Wu, 2015), insect salivary gland function, honey bee dorsal vessel function, and insect antiviral immune pathways. Furthermore, these channels represent a critical K+ conductance pathway in the mammalian nervous system as Kir knockouts in glial cells leads to membrane depolarization, enhanced synaptic potentiation, and reduced spontaneous neural activity. Considering the importance of Kir channels in the function of various insect tissues and the established role of Kir channels in mammalian neuronal tissue, it is hypothesized that Kir channels also serve a critical role in insect neural tissue and aimed to highlight the general influence of Kir channel modulation to the insect nervous system through pharmacological and genetic manipulations of the Kir channel (Chen, 2018).

To begin testing the physiological role of insect neural Kir channels, neurophysiological recordings of the Drosophila CNS were performed using the voltage dependent Kir blocker, BaCl2. BaCl2 is useful pharmacological tool to test the physiological role of Kir channels since, at physiological membrane potentials, Kir channels are up to 1000-fold more sensitive to BaCl2 than other K+ ion channels. This enhanced potency to Kir channels when compared to other K+ ion channels enables selective inhibition of Kir channels at low- to mid-micromolar concentrations of BaCl2. An increase was observed in the spike discharge frequency followed by cessation of firing after exposure of the CNS to mid-micromolar concentrations of BaCl2, providing the first insight that Kir channels constitute a critical conductance pathway in insect CNS. However, the potential for BaCl2 to precipitate out of some saline solutions and the potential of BaCl2 to modulate non-target proteins limits the conclusions that can be drawn from these data. Fortunately, the recent identification of selective and potent small molecules designed to target insect Kir channels has facilitated the characterization of the physiological role of these channels in various insect tissue systems with more certainty than BaCl2 and other divalent cations (Chen, 2018).

This study used the recently discovered insect Kir channel modulator (VU041) and its inactive analog (VU937) to characterize the influence these channels have in insect nervous system function. Exposure of the Drosophila CNS to VU041 dramatically altered the spike discharge frequency in a biphasic manner with low concentrations yielding neuroexcitation and higher concentrations having a depressant effect on CNS activity. A biphasic response is oftentimes observed when multiple pathways are inhibited and it is plausible that VU041 is directly or indirectly altering the functional capacity of other ion channels or transporters, such as delayed rectifier K+ channels or calcium-activated K+ channels. Although off-target effects are possible, they are unlikely since VU937 had no influence to CNS activity, suggesting the observed phenotype is through Kir inhibition. To ensure the observed effect to CNS activity was directly due to Kir2 channel modulation, CNS-specific RNAi-mediated knockdown was performed of the Kir2 encoding gene, irk2. Results from this genetic depletion of irk2 show a dramatic increase in CNS spike discharge frequency that was also substantiated through hyperactive larval behavior. These observed responses to VU041 and irk2 genetic depletion is likely due to the physiological role of only Kir2 since no mRNA reduction was observed in other Kir-encoding genes that are expressed in the CNS or any irk gene within the whole body or carcass. Previous reports have documented compensatory functions of Kir channels that arise after genetic depletion of one Kir channel, which prevents the manifestation of an observable change in phenotype (Wu, 2015). Yet, it does not appear that a compensatory mechanism arose to account for the genetic depletion of irk2 since a direct physiological response was observed and irk1 and irk3 mRNA levels remained unchanged. The influence to expression of other K+ ion transport pathways, such as Na+-K+-2Cl- cotransporter and Na+-K+-ATPase pumps, remains unknown and should be studied prior to drawing absolute conclusions regarding the physiological basis for neural Kir channels. Furthermore, exposure of the neuromuscular junction to VU041 altered the evoked EPSP waveform and muscle excitability. These data indicate that Drosophila, and likely mosquito, central and muscular nervous systems rely on the inward conductance of K+ ions through Kir channels for proper function (Chen, 2018).

The Drosophila genome encodes three Kir channel proteins, termed ir, irk2 and irk3, and all three contain the structural features and biophysical properties that are found in mammalian Kir channel subunits. Although ir and irk3 mRNA has been found to be expressed at low levels in the fly head, the irk2 gene is highly expressed in the adult fly head where it is concentrated in the brain and eye, suggesting that, of the Kir channels, irk2 is the principal inward conductance pathway for K+ ions. The sequence of irk2 is similar to that of ir, and both are highly related to human Kir 2, 3, and 6 proteins, which are constitutively active, GIRK, and ATP-gated Kir channels, respectively. Interestingly, irk2 channels have been shown to be constitutively active in S2 cells, associate with sulphonylurea receptors (SUR) as is seen with KATP channels, and the presence of an Asn223 residue suggests similarity to the GPCR-gated Kirs (Kir3.x; mammalian nomenclature). The variable functional associations have led to the speculation that irk2 may have different mechanisms of gating and regulation based on the cell type the gene is expressed in. Due to this, pharmacological modulators of mammalian GIRK and KATP channels were used to determine the mechanisms of irk2 gating in the Drosophila CNS. The GIRK activator, ML297, is highly selective for mammalian GIRK1/2 subunit combination over other Kir channels and was found to induce neuroexcitation to the Drosophila CNS. The sustained increase in Drosophila CNS activity after ML297 exposure was unexpected since GIRK2 knockouts in mice revealed an epileptic phenotype, suggesting GIRK is responsible for depressing neuronal excitability and thus, an activator of GIRK should reduce CNS spike discharge frequency. It is important to note that ML297 was shown to have moderate activity on the mammalian serotonin (5-Ht2b) receptor, which is expressed in the Drosophila CNS and may be the cause for observed neuroexcitation to the Drosophila CNS. Unfortunately, the severely underdeveloped pharmacological library of GIRK inhibitors prevents further interrogation at this time. To determine if Irk2 is gated by ATP, four structurally distinct activators and inhibitors of mammalian KATP channels were used. No change in CNS spike discharge frequency was observed after exposure to these molecules at concentrations ranging into the upper micromolar range. Since other studies have shown clear effects to various insect systems with mammalian KATP modulators, the lack of response to the Drosophila CNS is likely to be due to the absence of ATP-gated Kir channels and not due to incompatibility of the structural scaffolds with the Drosophila KATP channel. These findings have led to the speculation that (1) irk2 is not likely to be expressed as a KATP channel in the CNS, (2) constitutively active Kir channels are present in the Drosophila CNS, and (3) GIRK-like channels may be present in the CNS yet further studies are required to interrogate this claim (Chen, 2018).

The data presented in this study raise the question as to what the physiological role Kir channels have in nervous system function of insects at the cellular level. In mammals, astrocyte function has received significant interest for their roles in the regulation of synaptic levels of neurotransmitters, in particular glutamate, buffering of extracellular K+, and release of neurotransmitters, all of which have been shown to directly modulate neuronal excitability and transmission. In particular, Kir4.1 channels expressed in astrocytes have been directly linked to K+ influx across neural membranes where cells take up excess extracellular potassium ions, distribute them via gap junctions, and extrude the ions at sites in which extracellular K+ concentrations ([K+]out) is low, which is termed K+ spatial buffering. It is reasonable to predict that the insect nervous system employs this method of K+ transport during neuronal activity since [K+]out is dramatically increased and must be rapidly reversed to prevent membrane depolarization of neurons. Therefore, inhibition of this process through pharmacological blockage of neural Kir channels will lead to depolarization of the nervous system and induce CNS excitation, which was observed in this study at low concentrations of VU041 and after genetic knockdown of Kirs. In mammals, a complete knockout of Kir4.1 yielded a reduction of spontaneous EPSC in pyramidal neurons, similar to what was observed after CNS exposure to concentrations of VU041 greater than 10 µM (Chen, 2018).

It is hypothesized that Kir channels provide a pathway for K+ spatial buffering during neuronal activity of Drosophila and this pathway is critical for proper CNS activity. Excitability and synaptic transmission of insect and mammalian nervous systems are dependent upon [K+]out and alteration of the K+ ion gradient directly affects excitatory neurotransmission. In accordance to this, changes were observed in the CNS spike frequency and complete cessation of evoked EPSP's at the NMJ, which is classically attributed to changes in presynaptic function that may be resultant of altered neurotransmitter release. Similarly, reduced amplitude and broadening of the evoked EPSP waveform at the neuromuscular junction were observed after pharmacological inhibition of Kirs, which may be a result of modification of postsynaptic terminal responsiveness to neurotransmitters. The influence of Kir channel inhibition to pre- and post-synaptic function can be due to changes in either extracellular ion or transmitter levels. This is evidenced by the response of the Drosophila CNS after exposure to BaCl2 and 25 μM VU041. Exposure to these pharmacological agents yielded near maximal spike discharge frequency that culminated in a relatively abrupt termination of this activity. This reduction of CNS spike frequency may be due to depolarization-induced inactivation of Na+ channels due to prolonged exposure to elevated [K+]out, thereby lowering the probability of transmitter release that will reduce neuronal firing. Therefore, it appears as though Kir channels are responsible for regulating the K+ ion gradient that ultimately controls synaptic activity and neurotransmitter release, which is essential for proper neural signaling and activity (Chen, 2018).

Kir channels represent a critical K+ ion conductance pathway within the Drosophila, and likely mosquito, central and neuromuscular nervous systems. Considering this, it is reasonable to suggest that the recently identified Kir-directed mosquitocide, VU041, is capable of inducing toxicity through neurological poisoning in addition to inducing Malpighian tubule failure that leads to toxicity by an inability to perform osmoregulatory actions. These data provides a proof-of-concept that novel chemical scaffolds targeting neural Kir channels in insects represent a novel mechanism of action with insecticide resistance mitigating potential. Based on the data collected in this study, it is hypothesized that the function of Kir channels in the insect nervous system is responsible for reducing [K+]out during neuronal activity by the process known as K+ spatial buffering, similar to that described in mammals. It is important to note that this hypothesis cannot be fully validated until whole-cell electrophysiological recordings are performed to determine the role of Kirs in (1) glutamate and K+ uptake during neural activity, (2) maintenance of neural membrane properties (e.g., Vm, Rm, etc.), and 3) synaptic transmission and plasticity (Chen, 2018).

Role of inward rectifier potassium channels in salivary gland function and sugar feeding of the fruit fly, Drosophila melanogaster

The arthropod salivary gland is of critical importance for horizontal transmission of pathogens, yet a detailed understanding of the ion conductance pathways responsible for saliva production and excretion is lacking. A superfamily of potassium ion channels, known as inward rectifying potassium (Kir) channels, is overexpressed in the Drosophila salivary gland by 32-fold when compared to the whole body mRNA transcripts. Therefore, this study aimed to test the hypothesis that pharmacological and genetic depletion of salivary gland specific Kir channels alters the efficiency of the gland and reduced feeding capabilities using the fruit fly Drosophila melanogaster as a model organism that could predict similar effects in arthropod disease vectors. Exposure to VU041, a selective Kir channel blocker, reduced the volume of sucrose consumption by up to 3.2-fold and was found to be concentration-dependent with an EC50 of 68mµM. Importantly, the inactive analog, VU937, was shown to not influence feeding, suggesting the reduction in feeding observed with VU041 is due to Kir channel inhibition. Next, a salivary gland specific knockdown of Kir1 was performed to assess the role of these channels specifically in the salivary gland. The genetically depleted fruit flies had a reduction in total volume ingested and an increase in the time spent feeding, both suggestive of a reduction in salivary gland function. Furthermore, a compensatory mechanism appears to be present at day 1 of RNAi-treated fruit flies, and is likely to be the Na(+)-K(+)-2Cl(-) cotransporter and/or Na(+)-K(+)-ATPase pumps that serve to supplement the inward flow of K(+) ions, which highlights the functional redundancy in control of ion flux in the salivary glands. These findings suggest that Kir channels likely provide, at least in part, a principal potassium conductance pathway in the Drosophila salivary gland that is required for sucrose feeding (Swale, 2017).

Despite the critical role the arthropod salivary gland serves in horizontal transmission of pathogens, an understanding of the machinery required for proper gland function is limited. Although rather limited in scope, pharmacological studies against the isolated tick salivary gland have implicated several components involved in the process of salivary secretion: dopaminergic pathway, Na+-K+-ATPase, GABA, and the muscarinic acetylcholine receptor. Although these pathways are clearly important for saliva production and excretion, the complex nature of the gland suggests other pathways are likely critical for proper function of the salivary gland. Indeed, the results of the present study provide compelling data that a superfamily of potassium ion channels, known as inward rectifier potassium channels, is an essential conductance pathway in the salivary gland that mediates proper feeding in the model organism Drosophila melanogaster (Swale, 2017).

Recent work on insect Kir channels have yielded insightful data suggesting these channels serve a critical role in Malpighian tubule function and fluid secretion. The Malpighian tubules and salivary glands are physiologically related tissues as both are a polarized epithelial tissue, require water and ion transport for function, and are considered, at least in part, to be an exocrine tissue. Furthermore, the Kir1 channel has been shown to constitute the primary inward K+ conductance in the mosquito Malpighian tubule and the analogous gene that encodes Kir1 in Drosophila is highly upregulated in the salivary glands of larval and adult flies. Therefore, it was hypothesized Kir channels also serve a critical role in salivary gland function, and this study aimed to elucidate the role of these channels through pharmacological and genetic manipulations of the Kir1 channel measured through feeding efficiency (Swale, 2017).

The recent identification of selective and potent small-molecules designed to target insect Kir channels have enabled researchers to begin to characterize the physiological role of these channels in various tissue systems. This study used the recently discovered insect Kir channel modulator (VU041) and its inactive analog (VU937) to characterize the influence these molecules have in the feeding cascade. Exposure to VU041 during feeding significantly reduced the volume of sucrose ingested, whereas VU937 had no influence to feeding efficiency, suggesting the observed phenotype is through Kir inhibition. However, due to the capability that small-molecules can inhibit unintended target sites and the fact Kir channels are highly expressed in the Malpighian tubules, it was impossible to ensure the observed effect to feeding was directly due to salivary gland failure. Therefore, salivary gland specific RNAi-mediated knockdown of the Kir1 encoding gene was performed. Results from this genetic depletion of Kir1 show a significantly less efficient salivary gland and, when combined with the VU041-mediated reduction in sucrose consumption, strongly suggests the Drosophila salivary gland relies on the inward conductance of K+ ions through Kir channels (Swale, 2017).

The data presented in this study raises the question as to what the physiological role Kir channels have in salivary gland function at the cellular level. Consideration of knowledge and hypotheses based on the role of Kir channels in mammalian salivary gland function and saliva production can be applied to expand understanding of salivary gland physiology in arthropods. First, it has been shown that electrolyte secretion in the mammalian salivary glands is based on the secondary active transport of anions, principally Cl- (and/or HCO3-) ions. In this model, K+ channels in the basolateral membrane of acinar cells maintain the membrane potential of the apical cell membrane to be more negative than the Nernst potential for anions, thereby providing a driving force for the sustained electrogenic anion efflux across the apical membrane. The second model for a role of Kir channels in the mammalian salivary gland was described through cell-attached patch and whole-cell patch-clamp studies. Here, researchers demonstrated the presence of four primary K+ channels, two of which are the outward mediated Ca2 +-activated K+ channel and a Kir channel. The inwardly rectifying property of the Kir channel was hypothesized to perform fast uptake of accumulated K+ ions, in concert with Na+-K+-ATPase, into acinar cells with the K+ influx depending on the relation between the membrane potential and the concentration gradient of K+ across the basolateral membrane. This buffering action likely provides an ion gradient enabling the outward flow of K+ ions through Ca2 +-activated K+ channels. Such K+ buffering action of Kir channels has been proposed in brain astrocytes, in retinal Müller cells and also in retinal pigmented epithelial cells (Swale, 2017).

To begin elucidating the role of Kir channels in the insect salivary gland based on the mammalian hypotheses, the potassium ion concentration in the sucrose solution was augmented to increase the potassium equilibrium constant (Ek; based on the Nernst equation), which ultimately reduces the efficacy of intracellular K+ channel inhibitors, such as Kir channel blockers. The loss of VU041 potency supports the notion that Kir channels function as a means to provide a pathway for rapid influx of K+ ions after depolarization events, a phenomenon oftentimes referred to as K+-spatial buffering. Therefore, it was hypothesize that Kir channels in Drosophila salivary glands are responsible, at least in part, for maintaining the high intracellular K+ concentration through a buffering-like action, which provides the K+ ion gradient to enable the outward flow of potassium ions, presumably through Ca2 +-activated K+-channels as is seen in mammals. However, further studies rooted in cellular electrophysiology and membrane physiology are needed to provide additional support for this hypothesis (Swale, 2017).

Although the data of this study suggest Kir channels are likely the primary mechanism for K+ spatial buffering within insect salivary gland cells, it is also evident that it is not the only transport pathway facilitating inward flow of K+ ions. Genetic depletion of Kir1 channels yielded a reduction of feeding at days 2, 3, and 4, but not on day 1 and similarly, the time spent feeding was not statistically different to controls at day 1 when compared to subsequent days. These data suggest the presence of a compensatory mechanism that accounts for the reduced expression of Kir1 in the genetically depleted animals, but one that is lost after day 1. Compensatory mechanisms are commonly observed in animals with genetic depletions of Kir channels and most oftentimes arise through upregulation of a different Kir gene. For instance, Wu and colleagues (2015) showed individual knockdown of any of the three Kir channel genes in Drosophila Malpighian tubules had no effect on the organ function, yet simultaneous knockdown of irk1 and irk2 had significant effects on transpeithelial K+ transport, suggesting Kir1 and Kir2 play redundant roles in Malpighian tubule function. Due to the expression of Kir1 and Kir2 mRNA in the Drosophila salivary gland, albeit at dramatic differences in mRNA expression level, it is plausible that the Ir2 gene is upregulated after genetic depletion of Kir1, which may account for the absence of an effect to feeding at day 1. Furthermore, the Malpighian tubules partially rely on the Na+-K+-2Cl- cotransporter and Na+-K+-ATPase pump to establish a high intracellular K+ ion gradient. The compensatory systems of the Malpighian tubules and the expression of the same conductance pathways in the salivary glands highlights the possibility that the Drosophila salivary gland is capable of utilizing these same pathways for establishing the intracellular K+ ion concentration as well as providing redundancy into the system for salivary gland K+ excretion. However, additional studies are required to validate this notion (Swale, 2017).

This study provided the first insight into the role of K+ ion channels in arthropod salivary gland physiology. Such knowledge helped understand the machinery arthropods evolved in the salivary gland to facilitate food acquisition. A significant amount of research remains to be performed to elucidate all the mechanisms of K+ ion transport that is required for proper gland function, but this study provides clear evidence that inward rectifying potassium channels expressed in the insect salivary gland are critical for its proper function as evidenced by alterations in feeding ability after chemical- or genetic- depletion. This study serves as a proof-of-concept that VU041 could serve as a lead compound for the development of new/novel vector control agents aimed at disrupting blood feeding and pathogen transport. Therefore, future studies will aim to expound on these data to characterize the functional relationship between Kir channels, Na+-K+-2Cl- cotransporter and Na+-K+-ATPase pumps as well as identify the role of Kir channels in the salivary glands of arthropod disease vectors as a means for the development of new/novel vector control agents aimed at disrupting blood feeding and pathogen transport (Swale, 2017).

Two inwardly rectifying potassium channels, Irk1 and Irk2, play redundant roles in Drosophila renal tubule function

Inwardly rectifying potassium channels play essential roles in renal physiology across phyla. Barium-sensitive K(+) conductances are found on the basolateral membrane of a variety of insect Malpighian (renal) tubules, including Drosophila melanogaster. This study found that barium decreases the lumen-positive transepithelial potential difference in isolated perfused Drosophila tubules and decreases fluid secretion and transepithelial K(+) flux. In those insect species in which it has been studied, transcripts from multiple genes encoding inwardly rectifying K(+) channels are expressed in the renal (Malpighian) tubule. In Drosophila melanogaster, this includes transcripts of the Irk1, Irk2, and Irk3 genes. The role of each of these gene products in renal tubule function is unknown. This study found that simultaneous knockdown of Irk1 and Irk2 in the principal cell of the fly tubule decreases transepithelial K(+) flux, with no additive effect of Irk3 knockdown, and decreases barium sensitivity of transepithelial K(+) flux by approximately 50%. Knockdown of any of the three inwardly rectifying K(+) channels individually has no effect, nor does knocking down Irk3 simultaneously with Irk1 or Irk2. Irk1/Irk2 principal cell double-knockdown tubules remain sensitive to the kaliuretic effect of cAMP. Inhibition of the Na(+)/K(+)-ATPase with ouabain and Irk1/Irk2 double knockdown have additive effects on K(+) flux, and 75% of transepithelial K(+) transport is due to Irk1/Irk2 or ouabain-sensitive pathways. In conclusion, Irk1 and Irk2 play redundant roles in transepithelial ion transport in the Drosophila melanogaster renal tubule and are additive to Na(+)/K(+)-ATPase-dependent pathways (Wu, 2015).

Inwardly rectifying K+ channels play important roles in invertebrate and vertebrate renal physiology. In insects, basolateral membrane barium-sensitive K+ conductances have been demonstrated in a variety of insect renal tubules. Examples include the yellow fever mosquito Aedes aegypti, the forest ant Formica polyctena, the Chagas vector Rhodnius prolixus, the agricultural pest Locusta migratoria, and the mealworm Tenebrio molitor. In addition, transcripts for inwardly rectifying K+ channels are expressed in the Malpighian tubules of Aedes aegypti, the vector for yellow fever, dengue, and chikungunya; Anopheles gambiae, the malaria vector; and the bed bug Cimex lectularius. Drugs targeting renal tubule inwardly rectifying K+ channels are currently being developed as mosquitocidal insecticides in Aedes aegypti. These channels may represent targets for the control of other insect pests as well, although killing of benign or beneficial insects will need to be avoided. This study extends the understanding of the role of these channels in insect renal tubule function (Wu, 2015).

Barium has been extensively used to probe the function of inwardly rectifying K+ channels in insects. This study replicated previous findings that barium decreases fluid secretion in the fly renal tubule and showed that it decreases transepithelial K+ flux. A decrease (lumen less positive) was observed in the transepithelial potential difference of 21 mV. This is consistent with prior reports of hyperpolarization of the basolateral membrane potential of similar magnitude, likely explaining the effect on the transepithelial potential difference. Both Irk1 and Irk2 are sensitive to barium when expressed in heterologous cells, and barium blocks the basolateral K+ conductance of the Drosophila tubule. Barium may also have additional effects on other, undefined ion channels on the basolateral membrane of the fly tubule. Indeed, Irk1 and Irk2 appear to account for only half of the barium sensitivity observed for transepithelial K+ flux. Genetic studies, in which specific channels can be manipulated, therefore provide complementary information to pharmacological studies. This is particularly important given the number of genes encoding inwardly rectifying K+ channels. The genomes of the mosquito species Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus encode Irk1 and Irk3 homologs, whereas the Irk2/Kir2 gene has undergone a duplication event, resulting in Kir2A and Kir2B genes. Additional duplication events have resulted in Kir2A and Kir2A' genes in Anopheles and Culex, and Kir2B and Kir2B' genes in Aedes. In Culex and Anopheles, the Kir3 gene has also been duplicated to result in Kir3A and Kir3B genes (Wu, 2015).

This study observed that Irk1 and Irk2, but not Irk3, are important for transepithelial fluid secretion and K+ flux. Interestingly, no functional channel activity was observed with attempts at expression of Irk3 in Xenopus oocytes or Drosophila S2 cultured cells, whereas both Irk1 and Irk2 possessed inwardly rectifying K+ channel activity. Similarly, no channel activity was observed in Xenopus oocytes expressing the Aedes aegypti Irk3 homolog, AeKir3, although it is highly expressed in the mosquito tubule. In addition, recent immunolocalization data indicates that AeKir3 is expressed in intracellular punctae in the mosquito tubule. In bed bugs, the Irk3 homolog ClKir3 transcript is expressed at very high levels in the Malpighian tubules, yet whole-organism silencing using RNA interference had no effect on bed bug viability. The functional role of Irk3 and its homologs in other insects in the renal tubule is therefore unclear (Wu, 2015).

In contrast, compounds that inhibit AeKir1 and AeKir2B have inhibitory effects on whole-mosquito urine excretion and on transepithelial fluid secretion and K+ flux in the Ramsay assay. This is broadly consistent with the current results that both Irk1 and Irk2 play roles in the fly tubule although there are interesting differences; in Aedes, AeKir1 is located in the stellate cell, whereas AeKir2B is in the principal cell, and inhibition of both AeKir1 and AeKir2 has additive effects on K+ flux. However, this may also reflect the effects of acutely inhibiting the channels pharmacologically, as opposed to the longer-term genetic knockdown used in this study. Additional open questions remain. For example, why is expression of AeKir2B enriched in the mosquito tubule, rather than the fly Irk2 homolog, AeKir2A? Does AeKir2B play a specific role in fluid secretion and ion flux after a blood meal, a situation not faced by Drosophila? Do specific splice isoforms of Irk2/Kir2A, demonstrated in Drosophila, Aedes aegypti, and Anopheles gambiae, have specific functional roles in the tubule? Given the ease of genetic manipulation and transgenesis in Drosophila, the fly renal tubule could serve as a platform to explore, not only the role of the Drosophila channels, but potentially also the physiological roles and/or the drug sensitivities of various inwardly rectifying K+ channels of other insects, aiding in the development of pharmacological agents to control insect disease vectors or insect pests (Wu, 2015).

Irk1 and Irk2 both have K+ channel activity as homomeric channels when heterologously expressed. It is possible that they could also function as heteromeric channels, as is the case for some other inwardly rectifying K+ channels. However, the fact that Irk1 and Irk2 must simultaneously be knocked down to see a change in transepithelial flux suggests that, even if the two channels do have heteromeric interactions, these are not absolutely required for their function in the tubule (Wu, 2015).

What roles are Irk1 and Irk2 playing in transepithelial ion flux? One possibility is that Irk1 and Irk2 constitute all or part of the basolateral K+ conductance and are important for maintaining the basolateral membrane potential. Could they also serve as a conductive pathway for K+ entry from the hemolymph into the principal cell? EK (-52 mV) is close to the basolateral membrane potential (-43 mV), with a net outward driving force for K+ movement from cell to bath. Because EK and the basolateral membrane potential are close to one another, relatively modest changes in conditions could affect the direction of the driving force. Indeed, the bathing solution for Ramsay assay studies of genetically modified tubules differed from those used in another study. In the Formica tubule, EK was also close to the basolateral membrane potential, and, depending on the measurement technique as well as the bath K+ concentration, driving forces were observed that were either inward, outward, or zero. Another subsequent study proposed that, at high hemolymph K+ concentrations, K+ uptake occurs through basolateral K+ channels. Similarly, studies in the deoxycorticosterone-treated rabbit cortical collecting duct demonstrated an inward (bath to cell) driving force for potassium across the basolateral membrane. In this preparation, transepithelial K+ flux from bath to lumen increased when bath K+ concentration increased, and this increase was attenuated by the basolateral application of barium, indicating that basolateral K+ channels allow K+ uptake into the cortical collecting duct principal cell. Similarly, application of an AeKir1/AeKir2B inhibitor to the basolateral membrane of the Aedes aegypti tubule depolarized the basolateral membrane potential and decreased input conductance under high bath K+ (34 mM) conditions (Wu, 2015).

It was found in a previous study that the Na+/K+-ATPase generates the driving force for NKCC activity in the fly renal tubule. Another potential role for K+ channels could be to recycle the K+ entering through the Na+/K+-ATPase. However, this study observed additive effects of the Na+/K+-ATPase inhibitor ouabain and knockdown of Irk1 and Irk2, indicating that Irk1 and Irk2 have functions beyond recycling the K+ entering through the Na+/K+-ATPase. Indeed, about 75% of transepithelial K+ flux was mediated by Irk1, Irk2, and ouabain-sensitive pathways, which could include the Na+/K+-ATPase itself as well as secondary active K+ uptake by the NKCC (Wu, 2015).

These genetic and pharmacological results are most consistent with a role for the inwardly rectifying K+ channels, Irk1 and Irk2, on the basolateral membrane of the Drosophila melanogaster main segment principal cell. Possible roles of Irk1 and Irk2 are the maintenance of the basolateral membrane potential or to allow the movement of K+ from the hemolymph into the principal cell during transepithelial K+ flux. Because flies eat a K+-rich diet, the existence of multiple mechanisms to allow principal cell K+ uptake, Irk1 and Irk2 as well as the Na+/K+-ATPase and NKCC, builds redundancy into the system for renal K+ excretion (Wu, 2015).

An inwardly rectifying K+ channel is required for patterning

Mutations that disrupt function of the human inwardly rectifying potassium channel KIR2.1 are associated with the craniofacial and digital defects of Andersen-Tawil Syndrome, but the contribution of Kir channels to development is undefined. Deletion of mouse Kir2.1 also causes cleft palate and digital defects. These defects are strikingly similar to phenotypes that result from disrupted TGFbeta/BMP signaling. This study used Drosophila melanogaster to show that a Kir2.1 homolog, Irk2, affects development by disrupting BMP signaling. Phenotypes of irk2 deficient lines, a mutant irk2 allele, irk2 siRNA and expression of a dominant-negative Irk2 subunit (Irk2DN) all demonstrate that Irk2 function is necessary for development of the adult wing. Compromised Irk2 function causes wing-patterning defects similar to those found when signaling through a Drosophila BMP homolog, Decapentaplegic (Dpp), is disrupted. To determine whether Irk2 plays a role in the Dpp pathway, flies were generated in which both Irk2 and Dpp functions are reduced. Irk2DN phenotypes are enhanced by decreased Dpp signaling. In wild-type flies, Dpp signaling can be detected in stripes along the anterior/posterior boundary of the larval imaginal wing disc. Reducing function of Irk2 with siRNA, an irk2 deletion, or expression of Irk2DN reduces the Dpp signal in the wing disc. As Irk channels contribute to Dpp signaling in flies, a similar role for Kir2.1 in BMP signaling may explain the morphological defects of Andersen-Tawil Syndrome and the Kir2.1 knockout mouse (Dahal, 2012).

Insulin signaling, lifespan and stress resistance are modulated by metabotropic GABA receptors on insulin producing cells in the brain of Drosophila

Insulin-like peptides (ILPs) regulate growth, reproduction, metabolic homeostasis, life span and stress resistance in worms, flies and mammals. A set of insulin producing cells (IPCs) in the Drosophila brain that express three ILPs (DILP2, 3 and 5) have been the main focus of interest in hormonal DILP signaling. Little is, however, known about factors that regulate DILP production and release by these IPCs. This study shows that the IPCs express the metabotropic GABA(B) receptor (GBR), but not the ionotropic GABA(A) receptor subunit RDL. Diminishing the GBR expression on these cells by targeted RNA interference abbreviates life span, decreases metabolic stress resistance and alters carbohydrate and lipid metabolism at stress, but not growth in Drosophila. A direct effect of diminishing GBR on IPCs is an increase in DILP immunofluorescence in these cells, an effect that is accentuated at starvation. Knockdown of irk3, possibly part of a G protein-activated inwardly rectifying K(+) channel that may link to GBRs, phenocopies GBR knockdown in starvation experiments. These experiments suggest that the GBR is involved in inhibitory control of DILP production and release in adult flies at metabolic stress and that this receptor mediates a GABA signal from brain interneurons that may convey nutritional signals. This is the first demonstration of a neurotransmitter that inhibits insulin signaling in its regulation of metabolism, stress and life span in an invertebrate brain (Enell, 2010).

Sulphonylurea sensitivity and enriched expression implicate inward rectifier K+ channels in Drosophila melanogaster renal function

Insect Malpighian (renal) tubules are capable of transporting fluid at remarkable rates. Secondary active transport of potassium at the apical surface of the principal cell must be matched by a high-capacity basolateral potassium entry route. A recent microarray analysis of Drosophila tubule identified three extremely abundant and enriched K(+) channel genes encoding the three inward rectifier channels of Drosophila: ir, irk2 and irk3. Enriched expression of inward rectifier channels in tubule was verified by quantitative RT-PCR, and all three IRKs localised to principal cells of the main segment (and ir and irk3 to the lower tubule) by in situ hybridisation, suggesting roles both in primary secretion and reabsorption. A new splice form of irk2 was also identified. The role of inward rectifiers in fluid secretion was assessed with a panel of selective inhibitors of inward rectifier channels, the antidiabetic sulphonylureas. All completely inhibited fluid secretion, with IC(50)s of 0.78 mmol l(-1) for glibenclamide and approximately 5 mmol l(-1) for tolbutamide, 0.01 mmol l(-1) for minoxidil and 0.1 mmol l(-1) for diazoxide. This pharmacology is consistent with a lower-affinity class of inward rectifier channel that does not form an obligate multimer with the sulphonylurea receptor (SUR), although effects on non-IRK targets cannot be excluded. Glibenclamide inhibited fluid secretion similarly to basolateral K(+)-free saline. Radiolabelled glibenclamide is both potently transported and metabolised by tubule. Furthermore, glibenclamide is capable of blocking transport of the organic dye amaranth (azorubin S), at concentrations of glibenclamide much lower than required to impact on fluid secretion. Glibenclamide thus interacts with tubule in three separate ways; as a potent inhibitor of fluid secretion, as an inhibitor (possibly competitive) of an organic solute transporter and as a substrate for excretion and metabolism (Evans, 2005).

Inwardly rectifying K+ (Kir) channels in Drosophila. A crucial role of cellular milieu factors Kir channel function

Three cDNAs encoding inwardly rectifying potassium (Kir) channels were isolated from Drosophila melanogaster. The protein sequences of Drosophila KirI (dKirI) and dKirII are moderately (<44%) and dKirIII sequence is weakly (<27%) identical to human Kir channel subunits. During fly development, five dKir channel transcripts derived from three genes are differentially expressed. Whole mount in situ hybridizations revealed dKirI transcripts absent from embryos, but dKirII and dKirIII are expressed in the embryonic hind gut and in Malpighian tubules, respectively, thus covering the entire osmoregulatory system of the developing fly. In the head of adult flies, predominantly dKirII transcripts were detected. When expressed in Xenopus oocytes, dKir channel activity was only observed after amino acid substitutions in their cytosolic tails (e.g. exchange of a unique valine in the NH(2) terminus). In contrast, heterologous expression of wild type dKirI and dKirII in Drosophila S2 cells readily evoked typical inwardly rectifying K(+) currents, which were weakly sensitive to Ba(2+). Thus, the specific milieu of insect cells provides a crucial cellular environment for proper function of dKir channels (Doring, 2002).

Characterization of Dir: a putative potassium inward rectifying channel in Drosophila

Potassium channels vary in their function and regulation, yet they maintain a number of important features -- they are involved in the control of potassium flow, cell volume, cell membrane resting potential, cell excitability and hormone release. The potassium (K(+)) inward rectifier (Kir) superfamily of channels are potassium selective channels, that are sensitive to the concentration of K(+) ions. They are termed inward rectifiers since they allow a much greater K(+) influx than efflux. There are at least seven subfamilies of Kir channels, grouped according to sequence and functional similarities. While numerous Kir channels have been discovered in a variety of organisms, Drosophila inward rectifier (Dir) is the first putative inward rectifier to be studied in Drosophila. In fact, there are only three genes (including Dir) encoding putative inward rectifiers in the Drosophila genome. Though there are other known potassium channels in Drosophila such as Ether-a-go-go and Shaker, most are voltage-gated channels. As an important first step in characterizing Kir channels in Drosophila, studies on Dir have been initiated (MacLean, 2002).

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Raphemot, R., Rouhier, M. F., Swale, D. R., Days, E., Weaver, C. D., Lovell, K. M., Konkel, L. C., Engers, D. W., Bollinger, S. R., Hopkins, C., Piermarini, P. M. and Denton, J. S. (2014). Discovery and characterization of a potent and selective inhibitor of Aedes aegypti inward rectifier potassium channels. PLoS One 9(11): e110772. PubMed ID: 25375326

Rouhier, M. F. and Piermarini, P. M. (2014a). Identification of life-stage and tissue-specific splice variants of an inward rectifying potassium (Kir) channel in the yellow fever mosquito Aedes aegypti. Insect Biochem Mol Biol 48: 91-99. PubMed ID: 24657620

Rouhier, M. F., Raphemot, R., Denton, J. S. and Piermarini, P. M. (2014b). Pharmacological validation of an inward-rectifier potassium (Kir) channel as an insecticide target in the yellow fever mosquito Aedes aegypti. PLoS One 9(6): e100700. PubMed ID: 24959745

Scott, B. N., Yu, M. J., Lee, L. W. and Beyenbach, K. W. (2004). Mechanisms of K+ transport across basolateral membranes of principal cells in Malpighian tubules of the yellow fever mosquito, Aedes aegypti. J Exp Biol 207(Pt 10): 1655-1663. PubMed ID: 15073198

Swale, D. R., Engers, D. W., Bollinger, S. R., Gross, A., Inocente, E. A., Days, E., Kanga, F., Johnson, R. M., Yang, L., Bloomquist, J. R., Hopkins, C. R., Piermarini, P. M. and Denton, J. S. (2016). An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria. Sci Rep 6: 36954. PubMed ID: 27849039

Swale, D. R., Li, Z., Guerrero, F., Perez De Leon, A. A. and Foil, L. D. (2017). Role of inward rectifier potassium channels in salivary gland function and sugar feeding of the fruit fly, Drosophila melanogaster. Pestic Biochem Physiol 141: 41-49. PubMed ID: 28911739

Wu, Y., Baum, M., Huang, C. L. and Rodan, A. R. (2015). Two inwardly rectifying potassium channels, Irk1 and Irk2, play redundant roles in Drosophila renal tubule function. Am J Physiol Regul Integr Comp Physiol 309(7): R747-756. PubMed ID: 26224687

date revised: 25 April 2024

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