34 results on '"Denton, Jerod S."'
Search Results
2. The Molecular Physiology and Toxicology of Inward Rectifier Potassium Channels in Insects.
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Piermarini, Peter M., Denton, Jerod S., and Swale, Daniel R.
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POTASSIUM channels , *INSECT physiology , *TOXICOLOGY , *INSECTS , *ARTHROPOD vectors , *ARTHROPOD pests - Abstract
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. [ABSTRACT FROM AUTHOR]
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- 2022
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3. Next-generation inward rectifier potassium channel modulators: discovery and molecular pharmacology.
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Weaver, C. David and Denton, Jerod S.
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MOLECULAR pharmacology , *POTASSIUM channels , *HIGH throughput screening (Drug development) , *DRUG target , *SMALL molecules , *PHARMACEUTICAL chemistry - Abstract
Inward rectifying potassium (Kir) channels play important roles in both excitable and nonexcitable cells of various organ systems and could represent valuable new drug targets for cardiovascular, metabolic, immune, and neurological diseases. In nonexcitable 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 central nervous system 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 have 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. [ABSTRACT FROM AUTHOR]
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- 2021
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4. Zinc pyrithione activates the volume-regulated anion channel through an antioxidant-sensitive mechanism.
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Figueroa, Eric E. and Denton, Jerod S.
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HIGH throughput screening (Drug development) , *COLORECTAL cancer , *REACTIVE oxygen species , *DRUG target , *BIOCIDES , *NAD (Coenzyme) - Abstract
Leucine-rich repeat-containing 8 (LRRC8) volume-regulated anion channels (VRACs) play important physiological roles in diverse cell types and may represent therapeutic targets for various diseases. To date, however, the pharmacological tools for evaluating the druggability of VRACs have been limited to inhibitors, as no activators of the channel have been reported. We therefore performed a fluorescence-based high-throughput screening (HTS) of 1,184 Food and Drug Administration-approved drugs for compounds that increase VRAC activity. The most potent VRAC potentiator identified was zinc pyrithione (ZPT), which is used commercially as an antifouling agent and for treating dandruff and other skin disorders. In intracellular Yellow Fluorescent Protein YFP(F46L/H148Q/I152L)-quenching assays, ZPT potentiates the rate and extent of swelling-induced iodide influx dose dependently with a half-maximal effective concentration (EC50) of 5.7 mM. Whole cell voltage-clamp experiments revealed that coapplication of hypotonic solution and 30mM ZPT to human embryonic kidney 293 or human colorectal carcinoma 116 cells increases the rate of swelling-induced VRAC activation by approximately 10-fold. ZPT potentiates swelling-induced VRAC currents after currents have reached a steady state and activates currents in the absence of cell swelling. Neither ZnCl2 nor free pyrithione activated VRAC; however, treating cells with a mixture of ZnCl2 and pyrithione led to robust channel activation. Finally, the effects of ZPT on VRAC were inhibited by reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and NAD(P)H oxidase inhibitor diphenyleneiodonium chloride, suggesting the mechanism of action involves ROS generation. The discovery of ZPT as a potentiator/activator of VRAC demonstrates the utility of HTS for identifying small-molecule modulators of VRAC and adds to a growing repertoire of pharmacological tool compounds for probing the molecular physiology and regulation of this important channel. [ABSTRACT FROM AUTHOR]
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- 2021
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5. Cryo-EM structures of an LRRC8 chimera with native functional properties reveal heptameric assembly.
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Hirohide Takahashi, Toshiki Yamada, Denton, Jerod S., Strange, Kevin, and Karakas, Erkan
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HOMOLOGY (Biology) , *AMINO acids , *PERMEABILITY , *ANIONS , *PHARMACOLOGY - Abstract
Volume-regulated anion channels (VRACs) mediate volume regulatory Cl- and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a small pore, hexameric structure. However, these channels are either non-functional or exhibit abnormal regulation and pharmacology, limiting their utility for structure-function analyses. We circumvented these limitations by developing novel homomeric LRRC8 chimeric channels with functional properties consistent with those of native VRAC/LRRC8 channels. We demonstrate here that the LRRC8C-LRRC8A(IL125) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A has a heptameric structure like that of homologous pannexin channels. Unlike homomeric LRRC8A and LRRC8D channels, heptameric LRRC8C-LRRC8A(IL125) channels have a large-diameter pore similar to that estimated for native VRACs, exhibit normal DCPIB pharmacology, and have higher permeability to large organic anions. Lipid-like densities are located between LRRC8C-LRRC8A(IL125) subunits and occlude the channel pore. Our findings provide new insights into VRAC/LRRC8 channel structure and suggest that lipids may play important roles in channel gating and regulation. [ABSTRACT FROM AUTHOR]
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- 2023
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6. Associations between KCNJ6 (GIRK2) gene polymorphisms and pain-related phenotypes.
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Bruehl, Stephen, Denton, Jerod S., Lonergan, Daniel, Koran, Mary Ellen, Chont, Melissa, Sobey, Christopher, Fernando, Shanik, Bush, William S., Mishra, Puneet, and Thornton-Wells, Tricia A.
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G protein coupled receptors , *GENETIC polymorphisms , *PHENOTYPES , *PAIN management , *PHYSIOLOGICAL effects of potassium channels , *OPIOID receptors , *HUMAN genetic variation - Abstract
Abstract: G-protein coupled inwardly rectifying potassium (GIRK) channels are effectors determining degree of analgesia experienced upon opioid receptor activation by endogenous and exogenous opioids. The impact of GIRK-related genetic variation on human pain responses has received little research attention. We used a tag single nucleotide polymorphism (SNP) approach to comprehensively examine pain-related effects of KCNJ3 (GIRK1) and KCNJ6 (GIRK2) gene variation. Forty-one KCNJ3 and 69 KCNJ6 tag SNPs were selected, capturing the known variability in each gene. The primary sample included 311 white patients undergoing total knee arthroplasty in whom postsurgical oral opioid analgesic medication order data were available. Primary sample findings were then replicated in an independent white sample of 63 healthy pain-free individuals and 75 individuals with chronic low back pain (CLBP) who provided data regarding laboratory acute pain responsiveness (ischemic task) and chronic pain intensity and unpleasantness (CLBP only). Univariate quantitative trait analyses in the primary sample revealed that 8 KCNJ6 SNPs were significantly associated with the medication order phenotype (P <.05); overall effects of the KCNJ6 gene (gene set-based analysis) just failed to reach significance (P =.054). No significant KCNJ3 effects were observed. A continuous GIRK Related Risk Score (GRRS) was derived in the primary sample to summarize each individual’s number of KCNJ6 “pain risk” alleles. This GRRS was applied to the replication sample, which revealed significant associations (P <.05) between higher GRRS values and lower acute pain tolerance and higher CLBP intensity and unpleasantness. Results suggest further exploration of the impact of KCNJ6 genetic variation on pain outcomes is warranted. [Copyright &y& Elsevier]
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- 2013
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7. Novel diuretic targets.
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Denton, Jerod S., Pao, Alan C., and Maduke, Merritt
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DISEASE management , *THERAPEUTICS , *ANTIHYPERTENSIVE agents , *DRUGS , *BLOOD pressure ,HEALTH of patients - Abstract
As the molecular revolution continues to inform a deeper understanding of disease mechanisms and pathways, there exist unprecedented opportunities for translating discoveries at the bench into novel therapies for improving human health. Despite the availability of several different classes of antihypertensive medications, only about half of the 67 million Americans with hypertension manage their blood pressure appropriately. A broader selection of structurally diverse antihypertensive drugs acting through different mechanisms would provide clinicians with greater flexibility in developing effective treatment regimens for an increasingly diverse and aging patient population. An emerging body of physiological, genetic, and pharmacological evidence has implicated several renal ion-transport proteins, or regulators thereof, as novel, yet clinically unexploited, diuretic targets. These include the renal outer medullary potassium channel, ROMK (Kir1.1), Kir4.1/5.1 potassium channels, ClC-Ka/b chloride channels, UTA/B urea transporters, the chloride/bicarbonate exchanger pendrin, and the STE20/SPS1-related proline/alanine- rich kinase (SPAK). The molecular pharmacology of these putative targets is poorly developed or lacking altogether; however, recent efforts by a few academic and pharmaceutical laboratories have begun to lessen this critical barrier. Here, we review the evidence in support of the aforementioned proteins as novel diuretic targets and highlight examples where progress toward developing small-molecule pharmacology has been made. [ABSTRACT FROM AUTHOR]
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- 2013
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8. CO2 chemosensitivity in Helix aspersa: three potassium currents mediate pH-sensitive neuronal spike timing.
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Denton, Jerod S., McCann, F. V., and Leiter, J. C.
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CARBON dioxide , *HYPERCAPNIA , *RESPIRATION , *SENSORY neurons , *POTASSIUM channels , *BROWN garden snail - Abstract
Elevated levels of carbon dioxide increase lung ventilation in Helix aspersa. The hypercapnic response originates from a discrete respiratory chemosensory region in the dorsal subesophageal ganglia that contains CO2-sensitive neurons. We tested the hypothesis that pH-dependent inhibition of potassium channels in neurons in this region mediated the chemosensory response to CO2. Cells isolated from the dorsal subesophageal ganglia retained CO2 chemosensitivity and exhibited membrane depolarization and/or an increase in input resistance during an acid challenge. Isolated somata expressed two voltage-dependent potassium channels, an A-type and a delayed-rectifier-type channel (IRA and IKDR). Both conductances were inhibited during hypercapnia. The pattern of voltage dependence indicated that IKA was affected by extracellular or intracellular pH, but the activity of IKDR was modulated by extracellular pH only. Application of inhibitors of either channel mimicked many of the effects of acidification in isolated cells and neurons in situ. We also detected evidence of a pH-sensitive calcium-activated potassium channel (IKCa) in neurons in situ. The results of these studies support the hypothesis that IKA initiates the chemosensory response, and IKDR and IKCa prolong the period of activation of CO2-sensitive neurons. Thus multiple potassium channels are inhibited by acidosis, and the combined effect of pH-dependent inhibition of these channels enhances neuronal excitability and mediates CO2 chemosensory responses in H. aspersa. We did not find a single ‘chemosensory channel,’ and the chemosensitive channels that we did find were not unique in any way that we could detect. The protein ‘machinery’ of CO2 chemosensitivity is probably widespread among neurons, and the selection process whereby a neuron acts or does not act as a respiratory CO2 chemosensor probably depends on the resting membrane potential and synaptic connectivity. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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9. Intracellular H... regulates the ...-subunit of ENaC, the epithelial Na... channel.
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Chalfant, Michael L. and Denton, Jerod S.
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SODIUM channels , *CHEMICAL reactions - Abstract
States that alpha, beta, gamma-epithelial sodium channel (ENaC) currents were regulated by changes in intracellular pH. Characterization of the ENaC currents; How acidification inhibits alpha, beta, gamma-ENaC currents; Effect of intracellular pH on single-channel ENaC currents.
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- 1999
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10. Intracellular H... regulates the alpha-subunit of ENaC, the epithelial Na... channel.
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Chalfant, Michael L. and Denton, Jerod S.
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SODIUM channels , *PHYSIOLOGICAL transport of sodium , *PHYSIOLOGY - Abstract
Focuses on a study which examined the effect of pH on the biophysical properties of epithelial sodium channel (ENaC) for the identification of molecular mechanisms of proton-mediated regulation of apical membrane sodium permeability epithelia. Characterization of ENaC currents; How acidification inhibits ENaC currents; Effects of voltage on ENaC currents.
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- 1999
11. LRRC8A homohexameric channels poorly recapitulate VRAC regulation and pharmacology.
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Toshiki Yamada, Figueroa, Eric E., Denton, Jerod S., and Strange, Kevin
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IONIC strength , *LEUCINE , *ARGININE , *VOLTAGE , *CELL size , *LOW voltage systems , *PHARMACOLOGY - Abstract
Swelling-activated volume-regulated anion channels (VRACs) are heteromeric channels comprising LRRC8A and at least one other LRRC8 paralog. Cryoelectron microscopy (cryo-EM) structures of nonnative LRRC8A and LRRC8D homohexamers have been described. We demonstrate here that LRRC8A homohexamers poorly recapitulate VRAC functional properties. Unlike VRACs, LRRC8A channels heterologously expressed in Lrr8c-/-HCT116 cells are poorly activated by low intracellular ionic strength (m) and insensitive to cell swelling with normal m. Combining low m with swelling modestly activates LRRC8A, allowing characterization of pore properties. VRACs are strongly inhibited by 10 mM 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB) in a voltage-independent manner. In contrast, DCPIB block of LRRC8A is weak and voltage sensitive. Cryo-EM structures indicate that DCPIB block is dependent on arginine 103. Consistent with this, LRRC8A R103F mutants are insensitive to DCPIB. However, an LRRC8 chimeric channel in which R103 is replaced by a leucine at the homologous position is inhibited ~90% by 10 mM DCPIB in a voltage-independent manner. Coexpression of LRRC8A and LRRC8C gives rise to channels with DCPIB sensitivity that is strongly m dependent. At normal intracellular m, LRRC8A þ LRRC8C heteromers exhibit strong, voltage-independent DCPIB block that is insensitive to R103F. DCPIB inhibition is greatly reduced and exhibits voltage dependence with low intracellular m. The R103F mutation has no effect on maximal DCPIB inhibition but eliminates voltage dependence under low m conditions. Our findings demonstrate that the LRRC8A cryo-EM structure and the use of heterologously expressed LRRC8 heteromeric channels pose significant limitations for VRAC mutagenesis-based structure-function analysis. Native VRAC function is most closely mimicked by chimeric LRRC8 homomeric channels. [ABSTRACT FROM AUTHOR]
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- 2021
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12. Kir7.1 knockdown and inhibition alter renal electrolyte handling but not the development of hypertension in Dahl salt-sensitive rats.
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Zietara, Adrian, Palygin, Oleg, Levchenko, Vladislav, Dissanayake, Lashodya V., Klemens, Christine A., Geurts, Aron, Denton, Jerod S., and Staruschenko, Alexander
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HIGH-salt diet , *RATS , *BLOOD pressure , *ELECTROLYTES , *HYPERTENSION , *SODIUM salts - Abstract
High K+ supplementation is correlated with a lower risk of the composite of death, major cardiovascular events, and ameliorated blood pressure, but the exact mechanisms have not been established. Inwardly rectifying K+ (Kir) channels expressed in the basolateral membrane of the distal nephron play an essential role in maintaining electrolyte homeostasis. Mutations in this channel family have been shown to result in strong disturbances in electrolyte homeostasis, among other symptoms. Kir7.1 is a member of the ATP-regulated subfamily of Kir channels. However, its role in renal ion transport and its effect on blood pressure have yet to be established. Our results indicate the localization of Kir7.1 to the basolateral membrane of aldosterone-sensitive distal nephron cells. To examine the physiological implications of Kir7.1, we generated a knockout of Kir7.1 (Kcnj13) in Dahl salt-sensitive (SS) rats and deployed chronic infusion of a specific Kir7.1 inhibitor, ML418, in the wild-type Dahl SS strain. Knockout of Kcnj13 (Kcnj13-/-) resulted in embryonic lethality. Heterozygous Kcnj13þ/- rats revealed an increase in K+ excretion on a normal-salt diet but did not exhibit a difference in blood pressure development or plasma electrolytes after 3 wk of a high-salt diet. Wild-type Dahl SS rats exhibited increased renal Kir7.1 expression when dietary K+ was increased. K+ supplementation also demonstrated that Kcnj13þ/- rats excreted more K+ on normal salt. The development of hypertension was not different when rats were challenged with high salt for 3 wk, although Kcnj13þ/- rats excrete less Na+. Interestingly, chronic infusion of ML418 significantly increased Na+ and Cl- excretion after 14 days of high salt but did not alter salt-induced hypertension development. Here, we found that reduction of Kir7.1 function, either through genetic ablation or pharmacological inhibition, can influence renal electrolyte excretion but not to a sufficient degree to impact the development of SS hypertension. [ABSTRACT FROM AUTHOR]
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- 2023
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13. A 30-year journey from volume-regulated anion currents to molecular structure of the LRRC8 channel.
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Strange, Kevin, Toshiki Yamada, and Denton, Jerod S.
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GENE families , *ANIONS , *MOLECULAR structure , *PHYSIOLOGY , *CELL size - Abstract
The swelling-activated anion channel VRAC has fascinated and frustrated physiologists since it was first described in 1988. Multiple laboratories have defined VRAC's biophysical properties and have shown that it plays a central role in cell volume regulation and possibly other fundamental physiological processes. However, confusion and intense controversy surrounding the channel's molecular identity greatly hindered progress in the field for >15 yr. A major breakthrough came in 2014 with the demonstration that VRAC is a heteromeric channel encoded by five members of the Lrrc8 gene family, Lrrc8A--E. A mere 4 yr later, four laboratories described cryo-EM structures of LRRC8A homomeric channels. As the melee of structure/function and physiology studies begins, it is critical that this work be framed by a clear understanding of VRAC biophysics, regulation, and cellular physiology as well as by the field's past confusion and controversies. That understanding is essential for the design and interpretation of structure/function studies, studies of VRAC physiology, and studies aimed at addressing the vexing problem of how the channel detects cell volume changes. In this review we discuss key aspects of VRAC biophysics, regulation, and function and integrate these into our emerging understanding of LRRC8 protein structure/function. [ABSTRACT FROM AUTHOR]
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- 2019
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14. Crosstalk between epithelial sodium channels (ENaC) and basolateral potassium channels (Kir4.1/Kir5.1) in the cortical collecting duct.
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Isaeva, Elena, Bohovyk, Ruslan, Fedoriuk, Mykhailo, Shalygin, Alexey, Klemens, Christine A., Zietara, Adrian, Levchenko, Vladislav, Denton, Jerod S., Staruschenko, Alexander, and Palygin, Oleg
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SODIUM channels , *POTASSIUM channels , *CHO cell , *AMITRIPTYLINE , *EPITHELIAL cells , *KIDNEY tubules , *FLUOXETINE - Abstract
Background and Purpose: Inwardly rectifying K+ (Kir) channels located on the basolateral membrane of epithelial cells of the distal nephron play a crucial role in K+ handling and BP control, making these channels an attractive target for the treatment of hypertension. The purpose of the present study was to determine how the inhibition of basolateral Kir4.1/Kir5.1 heteromeric K+ channel affects epithelial sodium channel (ENaC)‐mediated Na+ transport in the principal cells of cortical collecting duct (CCD). Experimental Approach The effect of fluoxetine, amitriptyline and recently developed Kir inhibitor, VU0134992, on the activity of Kir4.1, Kir4.1/Kir5.1 and ENaC were tested using electrophysiological approaches in CHO cells transfected with respective channel subunits, cultured polarized epithelial mCCDcl1 cells and freshly isolated rat and human CCD tubules. To test the effect of pharmacological Kir4.1/Kir5.1 inhibition on electrolyte homeostasis in vivo and corresponding changes in distal tubule transport, Dahl salt‐sensitive rats were injected with amitriptyline (15 mg·kg−1·day−1) for 3 days. Key Results: We found that inhibition of Kir4.1/Kir5.1, but not the Kir4.1 channel, depolarizes the cell membrane, induces the elevation of intracellular Ca2+ concentration and suppresses ENaC activity. Furthermore, we demonstrate that amitriptyline administration leads to a significant drop in plasma K+ level, triggering sodium excretion and diuresis. Conclusion and Implications: The present data uncover a specific role of the Kir4.1/Kir5.1 channel in the modulation of ENaC activity and emphasize the potential for using Kir4.1/Kir5.1 inhibitors to regulate electrolyte homeostasis and BP. [ABSTRACT FROM AUTHOR]
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- 2022
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15. Pharmacological Validation of an Inward-Rectifier Potassium (Kir) Channel as an Insecticide Target in the Yellow Fever Mosquito Aedes aegypti.
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Rouhier, Matthew F., Raphemot, Rene, Denton, Jerod S., and Piermarini, Peter M.
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DENGUE , *THERAPEUTICS , *PHARMACOLOGY , *POTASSIUM channels , *INSECTICIDES , *AEDES aegypti , *MOSQUITO vectors , *MALARIA treatment - Abstract
Mosquitoes are important disease vectors that transmit a wide variety of pathogens to humans, including those that cause malaria and dengue fever. Insecticides have traditionally been deployed to control populations of disease-causing mosquitoes, but the emergence of insecticide resistance has severely limited the number of active compounds that are used against mosquitoes. Thus, to improve the control of resistant mosquitoes there is a need to identify new insecticide targets and active compounds for insecticide development. Recently we demonstrated that inward rectifier potassium (Kir) channels and small molecule inhibitors of Kir channels offer promising new molecular targets and active compounds, respectively, for insecticide development. Here we provide pharmacological validation of a specific mosquito Kir channel (AeKir1) in the yellow fever mosquito Aedes aegypti. We show that VU590, a small-molecule inhibitor of mammalian Kir1.1 and Kir7.1 channels, potently inhibits AeKir1 but not another mosquito Kir channel (AeKir2B) in vitro. Moreover, we show that a previously identified inhibitor of AeKir1 (VU573) elicits an unexpected agonistic effect on AeKir2B in vitro. Injection of VU590 into the hemolymph of adult female mosquitoes significantly inhibits their capacity to excrete urine and kills them within 24 h, suggesting a mechanism of action on the excretory system. Importantly, a structurally-related VU590 analog (VU608), which weakly blocks AeKir1 in vitro, has no significant effects on their excretory capacity and does not kill mosquitoes. These observations suggest that the toxic effects of VU590 are associated with its inhibition of AeKir1. [ABSTRACT FROM AUTHOR]
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- 2014
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16. Cardiac and renal inward rectifier potassium channel pharmacology: emerging tools for integrative physiology and therapeutics.
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Swale, Daniel R, Kharade, Sujay V, and Denton, Jerod S
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POTASSIUM channels , *PHARMACOLOGY , *TARGETED drug delivery , *HEART physiology , *KIDNEY physiology , *BIOCHEMICAL mechanism of action - Abstract
Highlights: [•] Some Kir channels are putative drug targets, but their pharmacology is limited. [•] Newly discovered inhibitors or activators of Kir2.x, 4.1, and 7.1 are discussed. [•] Mechanisms of action involve pore-lining residues that control rectification. [•] This work is a critical first step toward developing more specific modulators. [ABSTRACT FROM AUTHOR]
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- 2014
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17. A computational analysis of central CO2 chemosensitivity in Helix aspersa.
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Chernov, Mykyta M., Daubenspeck, Andrew, Denton, Jerod S., Pfeiffer, Jason R., Putnam, Robert W., and Leiter, J. C.
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CARBON dioxide , *SENSORY neurons , *BROWN garden snail , *COMPUTATIONAL biology , *DIFFERENTIAL equations , *MATHEMATICAL analysis - Abstract
We created a single-compartment computer model of a CO2 chemosensory neuron using differential equations adapted from the Hodgkin-Huxley model and measurements of currents in CO2 chemosensory neurons from Helix aspersa. We incorporated into the model two inward currents, a sodium current and a calcium current, three outward potassium currents, an A-type current (IKA), a delayed rectifier current (IKDR), a calcium-activated potassium current (IKCa), and a proton conductance found in invertebrate cells. All of the potassium channels were inhibited by reduced pH. We also included the pH regulatory process to mimic the effect of the sodium-hydrogen exchanger (NHE) described in these cells during hypercapnic stimulation. The model displayed chemosensory behavior (increased spike frequency during acid stimulation), and all three potassium channels participated in the chemosensory response and shaped the temporal characteristics of the response to acid stimulation, pH-dependent inhibition of IKA initiated the response to CO2, but hypercapnic inhibition of IKDR and IKCa affected the duration of the excitatory response to hypercapnia. The presence or absence of NHE activity altered the chemosensory response over time and demonstrated the inadvisability of effective intracellular pH (pHi) regulation in cells designed to act as chemostats for acid-base regulation. The results of the model indicate that multiple channels contribute to CO2 chemosensitivity, but the primary sensor is probably IKA. pHi may be a sufficient chemosensory stimulus, but it may not be a necessary stimulus: either pHi or extracellular pH can be an effective stimuli if chemosensory neurons express appropriate pH-sensitive channels. The lack of pHi regulation is a key feature determining the neuronal activity of chemosensory cells over time, and the balanced lack of pHi regulation during hypercapnia probably depends on intracellular activation of pHi regulation but extracellular inhibition of pHi regulation. These general principles are applicable to all CO2 chemosensory cells in vertebrate and invertebrate neurons. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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18. CysLT1 receptor antagonists pranlukast and zafirlukast inhibit LRRC8-mediated volume regulated anion channels independently of the receptor.
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Figueroa, Eric E., Kramer, Meghan, Strange, Kevin, and Denton, Jerod S.
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Volume-regulated anion channels (VRACs) encoded by the leucine-rich repeat containing 8 (LRRC8) gene family play critical roles in myriad cellular processes and might represent druggable targets. The dearth of pharmacological compounds available for studying VRAC physiology led us to perform a high-throughput screen of 1,184 of US Food and Drug Administration-approved drugs for novel VRAC modulators. We discovered the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, pranlukast, as a novel inhibitor of endogenous VRAC expressed in human embryonic kidney 293 (HEK293) cells. Pranlukast inhibits VRAC voltage-independently, reversibly, and dose-dependently with a maximal efficacy of only ~50%. The CysLT1R pathway has been implicated in activation of VRAC in other cell types, prompting us to test whether pranlukast requires the CysLT1R for inhibition of VRAC. Quantitative PCR analysis demonstrated that CYSLTR1 mRNA is virtually undetectable in HEK293 cells. Furthermore, the CysLT1R agonist leukotriene D4 had no effect on VRAC activity and failed to stimulate Gq-coupled receptor signaling. Heterologous expression of the CysLT1R reconstituted LTD4-CysLT1R- Gq-calcium signaling in HEK293 cells but had no effect on VRAC inhibition by pranlukast. Finally, we show the CysLT1R antagonist zafirlukast inhibits VRAC with an IC50 of ~17µM and does so with full efficacy. Our data suggest that both pranlukast and zafirlukast are likely direct channel inhibitors that work independently of the CysLT1R. This study provides clarifying insights into the putative role of leukotriene signaling in modulation of VRAC and identifies two new chemical scaffolds that can be used for development of more potent and specific VRAC inhibitors. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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19. Pharmacological Inhibition of Inward Rectifier Potassium Channels Induces Lethality in Larval Aedes aegypti.
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Rusconi Trigueros, Renata, Hopkins, Corey R., Denton, Jerod S., and Piermarini, Peter M.
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POTASSIUM channels , *PHARMACOLOGY , *AEDES aegypti , *INSECT larvae , *MOSQUITO physiology , *INSECTICIDE analysis , *INSECTS - Abstract
The inward rectifier potassium (Kir) channels play key roles in the physiology of mosquitoes and other insects. Our group, among others, previously demonstrated that small molecule inhibitors of Kir channels are promising lead molecules for developing new insecticides to control adult female mosquitoes. However, the potential use of Kir channel inhibitors as larvicidal agents is unknown. Here we tested the hypothesis that pharmacological inhibition of Kir channels in the larvae of Aedes aegypti, the vector of several medically important arboviruses, induces lethality. We demonstrated that adding barium, a non-specific blocker of Kir channels, or VU041, a specific small-molecule inhibitor of mosquito Kir1 channels, to the rearing water (deionized H2O) of first instar larvae killed them within 48 h. We further showed that the toxic efficacy of VU041 within 24 h was significantly enhanced by increasing the osmolality of the rearing water to 100 mOsm/kg H2O with NaCl, KCl or mannitol; KCl provided the strongest enhancement compared to NaCl and mannitol. These data suggest: (1) the important role of Kir channels in the acclimation of larvae to elevated ambient osmolality and KCl concentrations; and (2) the disruption of osmoregulation as a potential mechanism of the toxic action of VU041. The present study provides the first evidence that inhibition of Kir channels is lethal to larval mosquitoes and broadens the potential applications of our existing arsenal of small molecule inhibitors of Kir channels, which have previously only been considered for developing adulticides. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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20. Towards a TREK-1/2 (TWIK-Related K+ Channel 1 and 2) dual activator tool compound: Multi-dimensional optimization of BL-1249.
- Author
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Iwaki, Yuzo, Yashiro, Kentaro, Kokubo, Masaya, Mori, Takahiro, Wieting, Joshua M., McGowan, Kevin M., Bridges, Thomas M., Engers, Darren W., Denton, Jerod S., Kurata, Haruto, and Lindsley, Craig W.
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POTASSIUM channels , *ROBUST optimization , *STRUCTURE-activity relationships , *ION channels , *POTASSIUM ions - Abstract
• First detailed SAR account around TREK1/2 activators. • First optimization campaign directed at BL-1249. • Discovery of new in vitro and in vivo tool compound, ONO-2960632/VU6011992. This letter describes a focused, multi-dimensional optimization campaign around BL-1249, a fenamate class non-steroidal anti-inflammatory and a known activator of the K 2P potassium channels TREK-1 (K 2P 2.1) and TREK-2 (K 2P 10.1). While BL-1249 has been widely profiled in vitro as a dual TREK-1/2 activator, poor physicochemical and DMPK properties have precluded a deeper understanding of the therapeutic potential of these key K 2P channels across a broad spectrum of peripheral and central human disease. Here, we report multi-dimensional SAR that led to a novel TREK-1/2 dual activator chemotype, exemplified by ONO-2960632/VU6011992, with improved DMPK properties, representing a new lead for further optimization towards robust in vivo tool compounds. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
21. G protein-coupled receptors differentially regulate glycosylation and activity of the inwardly rectifying potassium channel Kir7.1.
- Author
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Carrington, Sheridan J., Hernandez, Ciria C., Swale, Daniel R., Aluko, Oluwatosin A., Denton, Jerod S., and Cone, Roger D.
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G protein coupled receptors , *GLYCOSYLATION , *POTASSIUM channels , *WESTERN immunoblotting , *MELANOCORTIN receptors - Abstract
Kir7.1 is an inwardly rectifying potassium channel with important roles in the regulation of the membrane potential in retinal pigment epithelium, uterine smooth muscle, and hypothalamic neurons. Regulation of G protein-coupled inwardly rectifying potassium (GIRK) channels by G protein-coupled receptors (GPCRs) via the G protein βγ subunits has been well characterized. However, how Kir channels are regulated is incompletely understood. We report here that Kir7.1 is also regulated by GPCRs, but through a different mechanism. Using Western blotting analysis, we observed that multiple GPCRs tested caused a striking reduction in the complex glycosylation of Kir7.1. Further, GPCR-mediated reduction of Kir7.1 glycosylation in HEK293T cells did not alter its expression at the cell surface but decreased channel activity. Of note, mutagenesis of the sole Kir7.1 glycosylation site reduced conductance and open probability, as indicated by single-channel recording. Additionally, we report that the L241P mutation of Kir7.1 associated with Lebers congenital amaurosis (LCA), an inherited retinal degenerative disease, has significantly reduced complex glycosylation. Collectively, these results suggest that Kir7.1 channel glycosylation is essential for function, and this activity within cells is suppressed by most GPCRs. The melanocortin-4 receptor (MC4R), a GPCR previously reported to induce ligand-regulated activity of this channel, is the only GPCR tested that does not have this effect on Kir7.1. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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- View/download PDF
22. Inward rectifier potassium (Kir) channels in the soybean aphid Aphis glycines: Functional characterization, pharmacology, and toxicology.
- Author
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Piermarini, Peter M., Inocente, Edna Alfaro, Acosta, Nuris, Hopkins, Corey R., Denton, Jerod S., and Michel, Andrew P.
- Subjects
- *
SOYBEAN diseases & pests , *APHIDS , *INSECT genomes , *APHIS glycines , *POTASSIUM channels , *PHARMACOLOGY , *TOXICOLOGY - Abstract
Graphical abstract Highlights • The genome of Aphis glycines possessed 2 Kir channel genes: ApKir1 and ApKir2. • ApKir1 and ApKir2 formed barium-inhibitable Kir channels in Xenopus oocytes. • ApKir1 produced larger K+-currents with stronger rectification compared to ApKir2. • ApKir1 was more sensitive to barium compared to ApKir2. • Small molecule inhibition of Kir channels in vivo was lethal to aphids within 24 h. Abstract Inward rectifier K+ (Kir) channels contribute to a variety of physiological processes in insects and are emerging targets for insecticide development. Previous studies on insect Kir channels have primarily focused on dipteran species (e.g., mosquitoes, fruit flies). Here we identify and functionally characterize Kir channel subunits in a hemipteran insect, the soybean aphid Aphis glycines , which is an economically important insect pest and vector of soybeans. From the transcriptome and genome of Ap. glycines we identified two cDNAs, ApKir1 and ApKir2, encoding Kir subunits that were orthologs of insect Kir1 and Kir2, respectively. Notably, a gene encoding a Kir3 subunit was absent from the transcriptome and genome of Ap. glycines , similar to the pea aphid Acyrthosiphon pisum. Heterologous expression of ApKir1 and ApKir2 in Xenopus laevis oocytes enhanced K+-currents in the plasma membrane; these currents were inhibited by barium and the small molecule VU041. Compared to ApKir2, ApKir1 mediated currents that were larger in magnitude, more sensitive to barium, and less inhibited by small molecule VU041. Moreover, ApKir1 exhibited stronger inward rectification compared to ApKir2. Topical application of VU041 in adult aphids resulted in dose-dependent mortality within 24 h that was more efficacious than flonicamid, an established insecticide also known to inhibit Kir channels. We conclude that despite the apparent loss of Kir3 genes in aphid evolution, Kir channels are important to aphid survival and represent a promising target for the development of new insecticides. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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23. Synthesis and SAR of a novel Kir6.2/SUR1 channel opener scaffold identified by HTS.
- Author
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Dodd, Cayden J., Chronister, Keagan S., Rathnayake, Upendra, Parr, Lauren C., Li, Kangjun, Chang, Sichen, Mi, Dehui, Days, Emily L., Bauer, Joshua A., Cho, Hyekyung P., Boutaud, Olivier, Denton, Jerod S., Lindsley, Craig W., and Han, Changho
- Subjects
- *
POTASSIUM channels , *NEUROPEPTIDES , *STRUCTURE-activity relationships , *ION channels , *SECRETION , *ADENOSINE triphosphate - Abstract
[Display omitted] Kir6.2/SUR1 is an ATP-regulated potassium channel that acts as an intracellular metabolic sensor, controlling insulin and appetite-stimulatory neuropeptides secretion. In this Letter, we present the SAR around a novel Kir6.2/SUR1 channel opener scaffold derived from an HTS screening campaign. New series of compounds with tractable SAR trends and favorable potencies are reported. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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24. Dynamic expression of genes encoding subunits of inward rectifier potassium (Kir) channels in the yellow fever mosquito Aedes aegypti.
- Author
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Yang, Zhongxia, Statler, Bethanie-Michelle, Calkins, Travis L., Alfaro, Edna, Esquivel, Carlos J., Rouhier, Matthew F., Denton, Jerod S., and Piermarini, Peter M.
- Subjects
- *
YELLOW fever , *AEDES aegypti , *GENES , *MESSENGER RNA , *LARVAE - Abstract
Inward rectifier potassium (Kir) channels play fundamental roles in neuromuscular, epithelial, and endocrine function in mammals. Recent research in insects suggests that Kir channels play critical roles in the development, immune function, and excretory physiology of fruit flies and/or mosquitoes. Moreover, our group has demonstrated that mosquito Kir channels may serve as valuable targets for the development of novel insecticides. Here we characterize the molecular expression of 5 mRNAs encoding Kir channel subunits in the yellow fever mosquito, Aedes aegypti : Kir1 , Kir2A-c , Kir2B , Kir2B’ , and Kir3 . We demonstrate that 1) Kir mRNA expression is dynamic in whole mosquitoes, Malpighian tubules, and the midgut during development from 4th instar larvae to adult females, 2) Kir2B and Kir3 mRNA levels are reduced in 4th instar larvae when reared in water containing an elevated concentration (50 mM) of KCl, but not NaCl, and 3) Kir mRNAs are differentially expressed in the Malpighian tubules, midgut, and ovaries within 24 h after blood feeding. Furthermore, we provide the first characterization of Kir mRNA expression in the anal papillae of 4th instar larval mosquitoes, which indicates that Kir2A-c is the most abundant. Altogether, the data provide the first comprehensive characterization of Kir mRNA expression in Ae. aegypti and offer insights into the putative physiological roles of Kir subunits in this important disease vector. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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- View/download PDF
25. Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations.
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Martin, Gregory M., Rex, Emily A., Devaraneni, Prasanna, Denton, Jerod S., Boodhansingh, Kara E., DeLeon, Diva D., Stanley, Charles A., and Shyng, Show-Ling
- Subjects
- *
POTASSIUM channels , *ADENOSINE triphosphate , *SULFONYLUREAS , *GENETIC mutation , *MEMBRANE potential - Abstract
ATP-sensitive potassium (KATP) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to β-cell membrane potential. Loss of KATP channel function due to mutations in ABCC8 or KCNJ11, genes encoding the sulfonylurea receptor 1 (SUR1) or the inwardly rectifying potassium channel Kir6.2, respectively, results in congenital hyperinsulinism. Many SUR1 mutations prevent trafficking of channel proteins from the endoplasmic reticulum to the cell surface. Channel inhibitors, including sulfonylureas and carbamazepine, have been shown to correct channel trafficking defects. In the present study, we identified 13 novel SUR1 mutations that cause channel trafficking defects, the majority of which are amenable to pharmacological rescue by glibenclamide and carbamazepine. By contrast, none of the mutant channels were rescued by KATP channel openers. Cross-linking experiments showed that KATP channel inhibitors promoted interactions between the N terminus of Kir6.2 and SUR1, whereas channel openers did not, suggesting the inhibitors enhance intersubunit interactions to overcome channel biogenesis and trafficking defects. Functional studies of rescued mutant channels indicate that most mutants rescued to the cell surface exhibited WT-like sensitivity to ATP, MgADP, and diazoxide. In intact cells, recovery of channel function upon trafficking rescue by reversible sulfonylureas or carbamazepine was facilitated by theKATP channel opener diazoxide. Our study expands the list of KATP channel trafficking mutations whose function can be recovered by pharmacological ligands and provides further insight into the structural mechanism by which channel inhibitors correct channel biogenesis and trafficking defects. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
26. ROMK inhibitor actions in the nephron probed with diuretics.
- Author
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Kharade, Sujay V., Flores, Daniel, Lindsley, Craig W., Satlin, Lisa M., and Denton, Jerod S.
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DIURETICS , *KIDNEY tubules - Abstract
Diuretics acting on specific nephron segments to inhibit Na+ reabsorption have been used clinically for decades; however, drug interactions, tolerance, and derangements in serum K+ complicate their use to achieve target blood pressure. ROMK is an attractive diuretic target, in part, because its inhibition is postulated to indirectly inhibit the bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and the amiloride- and benzamil-sensitive epithelial Na+ channel (ENaC). The development of small-molecule ROMK inhibitors has created opportunities for exploring the physiological responses to ROMK inhibition. The present study evaluated how inhibition of ROMK alone or in combination with NKCC2, ENaC, or the hydrochlorothiazide (HCTZ) target NCC alter fluid and electrolyte transport in the nephron. The ROMK inhibitor VU591 failed to induce diuresis when administered orally to rats. However, another ROMK inhibitor, termed compound A, induced a robust natriuretic diuresis without kaliuresis. Compound A produced additive effects on urine output and Na+ excretion when combined with HCTZ, amiloride, or benzamil, but not when coadministered with bumetanide, suggesting that the major diuretic target site is the thick ascending limb (TAL). Interestingly, compound A inhibited the kaliuretic response induced by bumetanide and HCTZ, an effect we attribute to inhibition of ROMK-mediated K+ secretion in the TAL and CD. Compound A had no effect on heterologously expressed flow-sensitive large-conductance Ca2+-activated K+ channels (Slo1/β1). In conclusion, compound A represents an important new pharmacological tool for investigating the renal consequences of ROMK inhibition and therapeutic potential of ROMK as a diuretic target. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
27. Localization and role of inward rectifier K+ channels in Malpighian tubules of the yellow fever mosquito Aedes aegypti.
- Author
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Piermarini, Peter M., Dunemann, Sonja M., Rouhier, Matthew F., Calkins, Travis L., Raphemot, Rene, Denton, Jerod S., Hine, Rebecca M., and Beyenbach, Klaus W.
- Subjects
- *
YELLOW fever , *AEDES , *EXANTHEMA , *AEDES aegypti , *EPITHELIAL cells - Abstract
Malpighian tubules of adult female yellow fever mosquitoes Aedes aegypti express three inward rectifier K + (Kir) channel subunits: Ae Kir1, Ae Kir2B and Ae Kir3. Here we 1) elucidate the cellular and membrane localization of these three channels in the Malpighian tubules, and 2) characterize the effects of small molecule inhibitors of Ae Kir1 and Ae Kir2B channels (VU compounds) on the transepithelial secretion of fluid and electrolytes and the electrophysiology of isolated Malpighian tubules. Using subunit-specific antibodies, we found that Ae Kir1 and Ae Kir2B localize exclusively to the basolateral membranes of stellate cells and principal cells, respectively; Ae Kir3 localizes within intracellular compartments of both principal and stellate cells. In isolated tubules bathed in a Ringer solution containing 34 mM K + , the peritubular application of VU590 (10 μM), a selective inhibitor of Ae Kir1, inhibited transepithelial fluid secretion 120 min later. The inhibition brings rates of transepithelial KCl and fluid secretion to 54% of the control without a change in transepithelial NaCl secretion. VU590 had no effect on the basolateral membrane voltage (V bl ) of principal cells, but it significantly reduced the cell input conductance (g in ) to values 63% of the control within ∼90 min. In contrast, the peritubular application of VU625 (10 μM), an inhibitor of both Ae Kir1 and Ae Kir2B, started to inhibit transepithelial fluid secretion as early as 60 min later. At 120 min after treatment, VU625 was more efficacious than VU590, inhibiting transepithelial KCl and fluid secretion to ∼35% of the control without a change in transepithelial NaCl secretion. Moreover, VU625 caused the V bl and g in of principal cells to respectively drop to values 62% and 56% of the control values within only ∼30 min. Comparing the effects of VU590 with those of VU625 allowed us to estimate that Ae Kir1 and Ae Kir2B respectively contribute to 46% and 20% of the transepithelial K + secretion when the tubules are bathed in a Ringer solution containing 34 mM K + . Thus, we uncover an important role of Ae Kir1 and stellate cells in transepithelial K + transport under conditions of peritubular K + challenge. The physiological role of Ae Kir3 in intracellular membranes of both stellate and principal cells remains to be determined. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
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28. High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility.
- Author
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Herington, Jennifer L., Swale, Daniel R., Brown, Naoko, Shelton, Elaine L., Choi, Hyehun, Williams, Charles H., Hong, Charles C., Paria, Bibhash C., Denton, Jerod S., and Reese, Jeff
- Subjects
- *
CALCIUM in the body , *UTERINE contraction , *PREMATURE labor , *HEMORRHAGE , *OXYTOCIN - Abstract
The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
29. G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons.
- Author
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Ghamari-Langroudi, Masoud, Digby, Gregory J., Sebag, Julien A., Millhauser, Glenn L., Palomino, Rafael, Matthews, Robert, Gillyard, Taneisha, Panaro, Brandon L., Tough, Iain R., Cox, Helen M., Denton, Jerod S., and Cone, Roger D.
- Subjects
- *
G proteins , *APPETITE depressants , *MELANOCYTES , *APPETITE stimulants , *HOMEOSTASIS - Abstract
The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
30. Discovery and Characterization of a Potent and Selective Inhibitor of Aedes aegypti Inward Rectifier Potassium Channels.
- Author
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Raphemot, Rene, Rouhier, Matthew F., Swale, Daniel R., Days, Emily, Weaver, C. David, Lovell, Kimberly M., Konkel, Leah C., Engers, Darren W., Bollinger, Sean F., Hopkins, Corey, Piermarini, Peter M., and Denton, Jerod S.
- Subjects
- *
AEDES aegypti , *VIRUS disease transmission , *POTASSIUM channels , *DISEASE vectors , *INSECTICIDE resistance , *ELECTROPHYSIOLOGY - Abstract
Vector-borne diseases such as dengue fever and malaria, which are transmitted by infected female mosquitoes, affect nearly half of the world's population. The emergence of insecticide-resistant mosquito populations is reducing the effectiveness of conventional insecticides and threatening current vector control strategies, which has created an urgent need to identify new molecular targets against which novel classes of insecticides can be developed. We previously demonstrated that small molecule inhibitors of mammalian Kir channels represent promising chemicals for new mosquitocide development. In this study, high-throughput screening of approximately 30,000 chemically diverse small-molecules was employed to discover potent and selective inhibitors of Aedes aegypti Kir1 (AeKir1) channels heterologously expressed in HEK293 cells. Of 283 confirmed screening ‘hits’, the small-molecule inhibitor VU625 was selected for lead optimization and in vivo studies based on its potency and selectivity toward AeKir1, and tractability for medicinal chemistry. In patch clamp electrophysiology experiments of HEK293 cells, VU625 inhibits AeKir1 with an IC50 value of 96.8 nM, making VU625 the most potent inhibitor of AeKir1 described to date. Furthermore, electrophysiology experiments in Xenopus oocytes revealed that VU625 is a weak inhibitor of AeKir2B. Surprisingly, injection of VU625 failed to elicit significant effects on mosquito behavior, urine excretion, or survival. However, when co-injected with probenecid, VU625 inhibited the excretory capacity of mosquitoes and was toxic, suggesting that the compound is a substrate of organic anion and/or ATP-binding cassette (ABC) transporters. The dose-toxicity relationship of VU625 (when co-injected with probenecid) is biphasic, which is consistent with the molecule inhibiting both AeKir1 and AeKir2B with different potencies. This study demonstrates proof-of-concept that potent and highly selective inhibitors of mosquito Kir channels can be developed using conventional drug discovery approaches. Furthermore, it reinforces the notion that the physical and chemical properties that determine a compound's bioavailability in vivo will be critical in determining the efficacy of Kir channel inhibitors as insecticides. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
31. Molecular and functional characterization of Anopheles gambiae inward rectifier potassium (Kir1) channels: A novel role in egg production.
- Author
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Raphemot, Rene, Estévez-Lao, Tania Y., Rouhier, Matthew F., Piermarini, Peter M., Denton, Jerod S., and Hillyer, Julián F.
- Subjects
- *
MOSQUITO physiology , *ANOPHELES gambiae , *PHYSIOLOGICAL effects of potassium channels , *INSECT eggs , *INSECT genomes , *REVERSE transcriptase polymerase chain reaction , *BLOOD meal as feed , *RNA interference - Abstract
Inward rectifier potassium (Kir) channels play essential roles in regulating diverse physiological processes. Although Kir channels are encoded in mosquito genomes, their functions remain largely unknown. In this study, we identified the members of the Anopheles gambiae Kir gene family and began to investigate their function. Notably, we sequenced the A. gambiae Kir1 (AgKir1) gene and showed that it encodes all the canonical features of a Kir channel: an ion pore that is composed of a pore helix and a selectivity filter, two transmembrane domains that flank the ion pore, and the so-called G-loop. Heterologous expression of AgKir1 in Xenopus oocytes revealed that this gene encodes a functional, barium-sensitive Kir channel. Quantitative RT-PCR experiments then showed that relative AgKir1 mRNA levels are highest in the pupal stage, and that AgKir1 mRNA is enriched in the adult ovaries. Gene silencing of AgKir1 by RNA interference did not affect the survival of female mosquitoes following a blood meal, but decreased their egg output. These data provide evidence for a new role of Kir channels in mosquito fecundity, and further validates them as promising molecular targets for the development of a new class of mosquitocides to be used in vector control. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
32. Eliciting Renal Failure in Mosquitoes with a Small-Molecule Inhibitor of Inward-Rectifying Potassium Channels
- Author
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Raphemot, Rene, Rouhier, Matthew F., Hopkins, Corey R., Gogliotti, Rocco D., Lovell, Kimberly M., Hine, Rebecca M., Ghosalkar, Dhairyasheel, Longo, Anthony, Beyenbach, Klaus W., Denton, Jerod S., and Piermarini, Peter M.
- Subjects
- *
KIDNEY failure , *MOSQUITOES , *POTASSIUM channels , *MALARIA , *DENGUE , *NERVOUS system - Abstract
Mosquito-borne diseases such as malaria and dengue fever take a large toll on global health. The primary chemical agents used for controlling mosquitoes are insecticides that target the nervous system. However, the emergence of resistance in mosquito populations is reducing the efficacy of available insecticides. The development of new insecticides is therefore urgent. Here we show that VU573, a small-molecule inhibitor of mammalian inward-rectifying potassium (Kir) channels, inhibits a Kir channel cloned from the renal (Malpighian) tubules of Aedes aegypti (AeKir1). Injection of VU573 into the hemolymph of adult female mosquitoes (Ae. aegypti) disrupts the production and excretion of urine in a manner consistent with channel block of AeKir1 and renders the mosquitoes incapacitated (flightless or dead) within 24 hours. Moreover, the toxicity of VU573 in mosquitoes (Ae. aegypti) is exacerbated when hemolymph potassium levels are elevated, suggesting that Kir channels are essential for maintenance of whole-animal potassium homeostasis. Our study demonstrates that renal failure is a promising mechanism of action for killing mosquitoes, and motivates the discovery of selective small-molecule inhibitors of mosquito Kir channels for use as insecticides. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
33. The LRRC8 volume‐regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel.
- Author
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Afzal, Aqeela, Figueroa, Eric E., Kharade, Sujay V., Bittman, Kevin, Matlock, Brittany K., Flaherty, David K., and Denton, Jerod S.
- Subjects
- *
RESPIRATION , *MITOCHONDRIAL membranes , *MEMBRANE potential , *POTASSIUM channels , *CELL morphology , *POTASSIUM antagonists - Abstract
There has been a resurgence of interest in the volume‐regulated anion channel (VRAC) since the recent cloning of the LRRC8A‐E gene family that encodes VRAC. The channel is a heteromer comprised of LRRC8A and at least one other family member; disruption of LRRC8A expression abolishes VRAC activity. The best‐in‐class VRAC inhibitor, DCPIB, suffers from off‐target activity toward several different channels and transporters. Considering that some anion channel inhibitors also suppress mitochondrial respiration, we systematically explored whether DCPIB inhibits respiration in wild type (WT) and LRRC8A‐knockout HAP‐1 and HEK‐293 cells. Knockout of LRRC8A had no apparent effects on cell morphology, proliferation rate, mitochondrial content, or expression of several mitochondrial genes in HAP‐1 cells. Addition of 10 µM DCPIB, a concentration typically used to inhibit VRAC, suppressed basal and ATP‐linked respiration in part through uncoupling the inner mitochondrial membrane (IMM) proton gradient and membrane potential. Additionally, DCPIB inhibits the activity of complex I, II, and III of the electron transport chain (ETC). Surprisingly, the effects of DCPIB on mitochondrial function are also observed in HAP‐1 and HEK‐293 cells which lack LRRC8A expression. Finally, we demonstrate that DCPIB activates ATP‐inhibitable potassium channels comprised of heterologously expressed Kir6.2 and SUR1 subunits. These data indicate that DCPIB suppresses mitochondrial respiration and ATP production by dissipating the mitochondrial membrane potential and inhibiting complexes I‐III of the ETC. They further justify the need for the development of sharper pharmacological tools for evaluating the integrative physiology and therapeutic potential of VRAC in human diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
34. An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria.
- Author
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Swale, Daniel R., Engers, Darren W., Bollinger, Sean R., Gross, Aaron, Inocente, Edna Alfaro, Days, Emily, Kanga, Fariba, Johnson, Reed M., Yang, Liu, Bloomquist, Jeffrey R., Hopkins, Corey R., Piermarini, Peter M., and Denton, Jerod S.
- Abstract
Insecticide resistance is a growing threat to mosquito control programs around the world, thus creating the need to discover novel target sites and target-specific compounds for insecticide development. Emerging evidence suggests that mosquito inward rectifier potassium (Kir) channels represent viable molecular targets for developing insecticides with new mechanisms of action. Here we describe the discovery and characterization of VU041, a submicromolar-affinity inhibitor of Anopheles (An.) gambiae and Aedes (Ae.) aegypti Kir1 channels that incapacitates adult female mosquitoes from representative insecticide-susceptible and -resistant strains of An. gambiae (G3 and Akron, respectively) and Ae. aegypti (Liverpool and Puerto Rico, respectively) following topical application. VU041 is selective for mosquito Kir channels over several mammalian orthologs, with the exception of Kir2.1, and is not lethal to honey bees. Medicinal chemistry was used to develop an analog, termed VU730, which retains activity toward mosquito Kir1 but is not active against Kir2.1 or other mammalian Kir channels. Thus, VU041 and VU730 are promising chemical scaffolds for developing new classes of insecticides to combat insecticide-resistant mosquitoes and the transmission of mosquito-borne diseases, such as Zika virus, without harmful effects on humans and beneficial insects. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
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