The Molecular Physiology and Toxicology of Inward Rectifier Potassium Channels in Insects

Inward rectifier K+ (Kir) channels have been studied extensively in mammals, where they play critical roles in health and disease. In insects, Kir channels have recently been found to be key regulators of diverse physiological processes in several tissues. The importance of Kir channels in insects has positioned them to serve as emerging targets for the development of insecticides with novel modes of action. In this article, we provide the first comprehensive review of insect Kir channels, highlighting the rapid progress made in understanding their molecular biology, physiological roles, pharmacology, and toxicology. In addition, we highlight key gaps in our knowledge and suggest directions for future research to advance our understanding of Kir channels and their roles in insect physiology. Further knowledge of their functional roles will also facilitate their exploitation as targets for controlling arthropod pests and vectors of economic, medical, and/or veterinary relevance. Expected final online publication date for the Annual Review of Entomology, Volume 67 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Next-generation inward rectifier potassium channel modulators: Discovery and molecular pharmacology

Inward rectifying potassium (Kir) channels play important roles in both excitable and non-excitable cells of various organ systems and could represent valuable new drug targets for cardiovascular, metabolic, immune and neurological diseases. In non-excitable epithelial cells of the kidney tubule, for example, Kir1.1 (KCNJ1) and Kir4.1 (KCNJ10) are linked to sodium reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively, and have been explored as novel-mechanism diuretic targets for managing hypertension and edema. G-protein-coupled Kir channels (Kir3) channels expressed in the in the central nervous system and are critical effectors of numerous signal transduction pathways underlying analgesia, addiction, and respiratory-depressive effects of opioids. The historical dearth of pharmacological tool compounds for exploring the therapeutic potential of Kir channels has led to a molecular target-based approach using high-throughput screen (HTS) of small-molecule libraries and medicinal chemistry to develop "next generation" Kir channel modulators that are both potent and specific for their targets. In this article, we review recent efforts focused specifically on discovery and improvement of target-selective molecular probes. The reader is introduced to fluorescence-based thallium flux assays that has enabled much of this work and then provided with an overview of progress made toward developing modulators of Kir1.1 (VU590, VU591), Kir2.x (ML133), Kir3.X (ML297, GAT1508, GiGA1, VU059331), Kir4.1 (VU0134992), and Kir7.1 (ML418). We discuss what is known about the small molecules' molecular mechanisms of action, in vitro and in vivo pharmacology, and then close with our view of what critical work remains to be done.

Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg2+. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K+-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K+-flux is convergent with the gating of PIP2 because, at a saturating concentration, PIP2 greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K+-flux gating.

Immunoreactivity of Muscarinic Acetylcholine M2 and Serotonin 5-HT2B Receptors, Norepinephrine Transporter and Kir Channels in a Model of Epilepsy

Epilepsy is characterized by an imbalance in neurotransmitter activity; an increased excitatory to an inhibitory activity. Acetylcholine (ACh), serotonin, and norepinephrine (NE) may modulate neural activity via several mechanisms, mainly through its receptors/transporter activity and alterations in the extracellular potassium (K+) concentration via K+ ion channels. Seizures may disrupt the regulation of inwardly rectifying K+ (Kir) channels and alter the receptor/transporter activity. However, there are limited data present on the immunoreactivity pattern of these neurotransmitter receptors/transporters and K+ channels in chronic models of epilepsy, which therefore was the aim of this study. Changes in the immunoreactivity of epileptogenesis-related neurotransmitter receptors/transporters (M2, 5-HT2B, and NE transporter) as well as Kir channels (Kir3.1 and Kir6.2) were determined in the cortex, hippocampus and medulla of adult Wistar rats by utilizing a Pentylenetetrazol (PTZ)-kindling chronic epilepsy model. Increased immunoreactivity of the NE transporter, M2, and 5-HT2B receptors was witnessed in the cortex and medulla. While the immunoreactivity of the 5-HT2B receptor was found increased in the cortex and medulla, it was decreased in the hippocampus, with no changes observed in the M2 receptor in this region. Kir3.1 and Kir6.2 staining showed increase immunoreactivity in the cerebral cortex, but channel contrasting findings in the hippocampus and medulla. Our results suggest that seizure kindling may result in significant changes in the neurotransmitter system which may contribute or propagate to future epileptogenesis, brain damage and potentially towards sudden unexpected death in epilepsy (SUDEP). Further studies on the pathogenic role of these changes in neurotransmitter receptors/transporters and K+ channel immunoreactivity may identify newer possible targets to treat seizures or prevent epilepsy-related comorbidities.

Phylogenomics of Tick Inward Rectifier Potassium Channels and Their Potential as Targets to Innovate Control Technologies

This study was conducted to enhance the identification of novel targets to develop acaricides that can be used to advance integrated tick-borne disease management. Drivers for the emergence and re-emergence of tick-borne diseases affecting humans, livestock, and other domestic animals in many parts of the world include the increased abundance and expanded geographic distribution of tick species that vector pathogens. The evolution of resistance to acaricides among some of the most important tick vector species highlights the vulnerability of relying on chemical treatments for tick control to mitigate the health burden of tick-borne diseases. The involvement of inward rectifier potassium (Kir) channels in homeostasis, diuresis, and salivary gland secretion in ticks and other pests identified them as attractive targets to develop novel acaricides. However, few studies exist on the molecular characteristics of Kir channels in ticks. This bioinformatic analysis described Kir channels in 20 species of hard and soft ticks. Summarizing relevant investigations on Kir channel function in invertebrate pests allowed the phylogenomic study of this class of ion channels in ticks. How this information can be adapted to innovate tick control technologies is discussed.

Neuromodulation of Astrocytic K+ Clearance

Potassium homeostasis is fundamental for brain function. Therefore, effective removal of excessive K+ from the synaptic cleft during neuronal activity is paramount. Astrocytes play a key role in K+ clearance from the extracellular milieu using various mechanisms, including uptake via Kir channels and the Na+-K+ ATPase, and spatial buffering through the astrocytic gap-junction coupled network. Recently we showed that alterations in the concentrations of extracellular potassium ([K+]o) or impairments of the astrocytic clearance mechanism affect the resonance and oscillatory behavior of both the individual and networks of neurons. These results indicate that astrocytes have the potential to modulate neuronal network activity, however, the cellular effectors that may affect the astrocytic K+ clearance process are still unknown. In this study, we have investigated the impact of neuromodulators, which are known to mediate changes in network oscillatory behavior, on the astrocytic clearance process. Our results suggest that while some neuromodulators (5-HT; NA) might affect astrocytic spatial buffering via gap-junctions, others (DA; Histamine) primarily affect the uptake mechanism via Kir channels. These results suggest that neuromodulators can affect network oscillatory activity through parallel activation of both neurons and astrocytes, establishing a synergistic mechanism to maximize the synchronous network activity.

Correlation between structure and function in phosphatidylinositol lipid-dependent Kir2.2 gating

Inward rectifier K+(Kir) channels regulate cell membrane potential. Different Kir channels respond to unique ligands, but all are regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Using planar lipid bilayers we show that Kir2.2 exhibits bursts of openings separated by long quiescent inter-burst periods. Increasing PI(4,5)P2 concentration shortens the Kir2.2 inter-burst duration and lengthens the burst duration without affecting dwell times within a burst. From this, we propose that burst and inter-burst durations correspond to the CTD-docked and CTD-undocked conformations observed in the presence and absence of PI(4,5)P2 in atomic structures. We also studied the effect of different phosphatidylinositol lipids on Kir2.2 activation and conclude that the 5’ phosphate is essential to Kir2.2 pore opening. Other phosphatidylinositol lipids can compete with PI(4,5)P2 but cannot activate Kir2.2 without the 5’ phosphate. PI(4)P, which is directly interconvertible to and from PI(4,5)P2, might thus be a regulator of Kir channels in the plasma membrane.