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Rhodol-based thallium sensors for cellular imaging of potassium channel activity.
Dutter BF, Ender A, Sulikowski GA, Weaver CD
(2018) Org Biomol Chem 16: 5575-5579
MeSH Terms: Fluorescent Dyes, HEK293 Cells, Humans, Methylation, Microscopy, Confocal, Optical Imaging, Potassium Channels, Spectrometry, Fluorescence, Thallium, Xanthones
Show Abstract · Added April 10, 2019
Thallium (Tl+) flux assays enable imaging of potassium (K+) channel activity in cells and tissues by exploiting the permeability of K+ channels to Tl+ coupled with a fluorescent Tl+ sensitive dye. Common Tl+ sensing dyes utilize fluorescein as the fluorophore though fluorescein exhibits certain undesirable properties in these assays including short excitation wavelengths and pH sensitivity. To overcome these drawbacks, the replacement of fluorescein with rhodols was investigated. A library of 13 rhodol-based Tl+ sensors was synthesized and their properties and performance in Tl+ flux assays evaluated. The dimethyl rhodol Tl+ sensor emerged as the best of the series and performed comparably to fluorescein-based sensors while demonstrating greater pH tolerance in the physiological range and excitation and emission spectra 30 nm red-shifted from fluorescein.
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Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.
Kharade SV, Kurata H, Bender AM, Blobaum AL, Figueroa EE, Duran A, Kramer M, Days E, Vinson P, Flores D, Satlin LM, Meiler J, Weaver CD, Lindsley CW, Hopkins CR, Denton JS
(2018) Mol Pharmacol 94: 926-937
MeSH Terms: Animals, Binding Sites, Diuretics, Electrolytes, HEK293 Cells, Humans, Male, Models, Molecular, Molecular Docking Simulation, Molecular Structure, Mutagenesis, Site-Directed, Potassium Channels, Inwardly Rectifying, Rats, Small Molecule Libraries, Substrate Specificity
Show Abstract · Added April 10, 2019
The inward rectifier potassium (Kir) channel Kir4.1 () carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)--(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC value of 0.97 M and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC = 9 M) at -120 mV. In thallium (Tl) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction () in rat plasma ( = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
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Arrhythmia genetics: Not dark and lite, but 50 shades of gray.
Roden DM, Glazer AM, Kroncke B
(2018) Heart Rhythm 15: 1231-1232
MeSH Terms: Arrhythmias, Cardiac, Humans, Long QT Syndrome, Phenotype, Potassium Channels, Voltage-Gated
Added March 26, 2019
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Neuroinflammation Alters Integrative Properties of Rat Hippocampal Pyramidal Cells.
Frigerio F, Flynn C, Han Y, Lyman K, Lugo JN, Ravizza T, Ghestem A, Pitsch J, Becker A, Anderson AE, Vezzani A, Chetkovich D, Bernard C
(2018) Mol Neurobiol 55: 7500-7511
MeSH Terms: Animals, Dendrites, Down-Regulation, Hippocampus, Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels, Inflammation, Lipopolysaccharides, Male, Membrane Proteins, Microglia, Potassium Channels, Pyramidal Cells, Rats, Sprague-Dawley, Time Factors, Toll-Like Receptor 4
Show Abstract · Added April 2, 2019
Neuroinflammation is consistently found in many neurological disorders, but whether or not the inflammatory response independently affects neuronal network properties is poorly understood. Here, we report that intracerebroventricular injection of the prototypical inflammatory molecule lipopolysaccharide (LPS) in rats triggered a strong and long-lasting inflammatory response in hippocampal microglia associated with a concomitant upregulation of Toll-like receptor (TLR4) in pyramidal and hilar neurons. This, in turn, was associated with a significant reduction of the dendritic hyperpolarization-activated cyclic AMP-gated channel type 1 (HCN1) protein level while Kv4.2 channels were unaltered as assessed by western blot. Immunohistochemistry confirmed the HCN1 decrease in CA1 pyramidal neurons and showed that these changes were associated with a reduction of TRIP8b, an auxiliary subunit for HCN channels implicated in channel subcellular localization and trafficking. At the physiological level, this effect translated into a 50% decrease in HCN1-mediated currents (I) measured in the distal dendrites of hippocampal CA1 pyramidal cells. At the functional level, the band-pass-filtering properties of dendrites in the theta frequency range (4-12 Hz) and their temporal summation properties were compromised. We conclude that neuroinflammation can independently trigger an acquired channelopathy in CA1 pyramidal cell dendrites that alters their integrative properties. By directly changing cellular function, this phenomenon may participate in the phenotypic expression of various brain diseases.
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TALK-1 reduces delta-cell endoplasmic reticulum and cytoplasmic calcium levels limiting somatostatin secretion.
Vierra NC, Dickerson MT, Jordan KL, Dadi PK, Katdare KA, Altman MK, Milian SC, Jacobson DA
(2018) Mol Metab 9: 84-97
MeSH Terms: Animals, Calcium Signaling, Cells, Cultured, Cytoplasm, Endoplasmic Reticulum, Glucagon, Humans, Male, Mice, Mice, Inbred C57BL, Potassium Channels, Tandem Pore Domain, Somatostatin, Somatostatin-Secreting Cells
Show Abstract · Added February 7, 2018
OBJECTIVE - Single-cell RNA sequencing studies have revealed that the type-2 diabetes associated two-pore domain K (K2P) channel TALK-1 is abundantly expressed in somatostatin-secreting δ-cells. However, a physiological role for TALK-1 in δ-cells remains unknown. We previously determined that in β-cells, K flux through endoplasmic reticulum (ER)-localized TALK-1 channels enhances ER Ca leak, modulating Ca handling and insulin secretion. As glucose amplification of islet somatostatin release relies on Ca-induced Ca release (CICR) from the δ-cell ER, we investigated whether TALK-1 modulates δ-cell Ca handling and somatostatin secretion.
METHODS - To define the functions of islet δ-cell TALK-1 channels, we generated control and TALK-1 channel-deficient (TALK-1 KO) mice expressing fluorescent reporters specifically in δ- and α-cells to facilitate cell type identification. Using immunofluorescence, patch clamp electrophysiology, Ca imaging, and hormone secretion assays, we assessed how TALK-1 channel activity impacts δ- and α-cell function.
RESULTS - TALK-1 channels are expressed in both mouse and human δ-cells, where they modulate glucose-stimulated changes in cytosolic Ca and somatostatin secretion. Measurement of cytosolic Ca levels in response to membrane potential depolarization revealed enhanced CICR in TALK-1 KO δ-cells that could be abolished by depleting ER Ca with sarco/endoplasmic reticulum Ca ATPase (SERCA) inhibitors. Consistent with elevated somatostatin inhibitory tone, we observed significantly reduced glucagon secretion and α-cell Ca oscillations in TALK-1 KO islets, and found that blockade of α-cell somatostatin signaling with a somatostatin receptor 2 (SSTR2) antagonist restored glucagon secretion in TALK-1 KO islets.
CONCLUSIONS - These data indicate that TALK-1 reduces δ-cell cytosolic Ca elevations and somatostatin release by limiting δ-cell CICR, modulating the intraislet paracrine signaling mechanisms that control glucagon secretion.
Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.
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13 MeSH Terms
Cytokine-mediated changes in K channel activity promotes an adaptive Ca response that sustains β-cell insulin secretion during inflammation.
Dickerson MT, Bogart AM, Altman MK, Milian SC, Jordan KL, Dadi PK, Jacobson DA
(2018) Sci Rep 8: 1158
MeSH Terms: Adult, Animals, Calcium, Female, Gene Expression Regulation, Glucose, Humans, Insulin, Insulin Secretion, Insulin-Secreting Cells, Interferon-gamma, Interleukin-1beta, Ion Transport, Islets of Langerhans, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Potassium, Potassium Channels, Tandem Pore Domain, Primary Cell Culture, RNA, Messenger, Sarcoplasmic Reticulum Calcium-Transporting ATPases, Tissue Culture Techniques, Tumor Necrosis Factor-alpha
Show Abstract · Added February 7, 2018
Cytokines present during low-grade inflammation contribute to β-cell dysfunction and diabetes. Cytokine signaling disrupts β-cell glucose-stimulated Ca influx (GSCI) and endoplasmic reticulum (ER) Ca ([Ca]) handling, leading to diminished glucose-stimulated insulin secretion (GSIS). However, cytokine-mediated changes in ion channel activity that alter β-cell Ca handling remain unknown. Here we investigated the role of K currents in cytokine-mediated β-cell dysfunction. K currents, which control the termination of intracellular Ca ([Ca]) oscillations, were reduced following cytokine exposure. As a consequence, [Ca] and electrical oscillations were accelerated. Cytokine exposure also increased basal islet [Ca] and decreased GSCI. The effect of cytokines on TALK-1 K currents were also examined as TALK-1 mediates K by facilitating [Ca] release. Cytokine exposure decreased KCNK16 transcript abundance and associated TALK-1 protein expression, increasing [Ca] storage while maintaining 2 phase GSCI and GSIS. This adaptive Ca response was absent in TALK-1 KO islets, which exhibited decreased 2 phase GSCI and diminished GSIS. These findings suggest that K and TALK-1 currents play important roles in altered β-cell Ca handling and electrical activity during low-grade inflammation. These results also reveal that a cytokine-mediated reduction in TALK-1 serves an acute protective role in β-cells by facilitating increased Ca content to maintain GSIS.
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25 MeSH Terms
Simultaneous Real-Time Measurement of the β-Cell Membrane Potential and Ca Influx to Assess the Role of Potassium Channels on β-Cell Function.
Vierra NC, Dickerson MT, Philipson LH, Jacobson DA
(2018) Methods Mol Biol 1684: 73-84
MeSH Terms: Animals, Calcium, Cells, Cultured, Humans, Insulin-Secreting Cells, Membrane Potentials, Mice, Patch-Clamp Techniques, Potassium Channels
Show Abstract · Added November 13, 2017
Stimulus-secretion coupling in pancreatic β-cells requires Ca influx through voltage-dependent Ca channels, whose activity is controlled by the plasma membrane potential (V ). Here, we present a method of measuring fluctuations in the β-cell V and Ca influx simultaneously, which provides valuable information about the ionic signaling mechanisms that underlie insulin secretion. This chapter describes the use of perforated patch clamp electrophysiology on cells loaded with a fluorescent intracellular Ca indicator, which permits the stable recording conditions needed to monitor the V and Ca influx in β-cells. Moreover, this chapter describes the protocols necessary for the preparation of mouse and human islet cells for the simultaneous recording of V and Ca as well as determining the specific islet cell type assessed in each experiment.
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TALK-1 channels control β cell endoplasmic reticulum Ca homeostasis.
Vierra NC, Dadi PK, Milian SC, Dickerson MT, Jordan KL, Gilon P, Jacobson DA
(2017) Sci Signal 10:
MeSH Terms: Animals, Calcium, Diabetes Mellitus, Endoplasmic Reticulum, HEK293 Cells, Homeostasis, Humans, Insulin-Secreting Cells, Mice, Mice, Knockout, Potassium Channels, Tandem Pore Domain
Show Abstract · Added November 13, 2017
Ca handling by the endoplasmic reticulum (ER) serves critical roles in controlling pancreatic β cell function and becomes perturbed during the pathogenesis of diabetes. ER Ca homeostasis is determined by ion movements across the ER membrane, including K flux through K channels. We demonstrated that K flux through ER-localized TALK-1 channels facilitated Ca release from the ER in mouse and human β cells. We found that β cells from mice lacking TALK-1 exhibited reduced basal cytosolic Ca and increased ER Ca concentrations, suggesting reduced ER Ca leak. These changes in Ca homeostasis were presumably due to TALK-1-mediated ER K flux, because we recorded K currents mediated by functional TALK-1 channels on the nuclear membrane, which is continuous with the ER. Moreover, overexpression of K-impermeable TALK-1 channels in HEK293 cells did not reduce ER Ca stores. Reduced ER Ca content in β cells is associated with ER stress and islet dysfunction in diabetes, and islets from TALK-1-deficient mice fed a high-fat diet showed reduced signs of ER stress, suggesting that TALK-1 activity exacerbated ER stress. Our data establish TALK-1 channels as key regulators of β cell ER Ca and suggest that TALK-1 may be a therapeutic target to reduce ER Ca handling defects in β cells during the pathogenesis of diabetes.
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
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Discovery and Characterization of 1H-Pyrazol-5-yl-2-phenylacetamides as Novel, Non-Urea-Containing GIRK1/2 Potassium Channel Activators.
Wieting JM, Vadukoot AK, Sharma S, Abney KK, Bridges TM, Daniels JS, Morrison RD, Wickman K, Weaver CD, Hopkins CR
(2017) ACS Chem Neurosci 8: 1873-1879
MeSH Terms: Acetamides, Animals, Brain, G Protein-Coupled Inwardly-Rectifying Potassium Channels, HEK293 Cells, Humans, Liver, Membrane Transport Modulators, Mice, Microsomes, Liver, Molecular Structure, Pyrazoles, Structure-Activity Relationship
Show Abstract · Added April 3, 2018
The G protein-gated inwardly-rectifying potassium channels (GIRK, K3) are a family of inward-rectifying potassium channels, and there is significant evidence supporting the roles of GIRKs in a number of physiological processes and as potential targets for numerous indications. Previously reported urea containing molecules as GIRK1/2 preferring activators have had significant pharmacokinetic (PK) liabilities. Here we report a novel series of 1H-pyrazolo-5-yl-2-phenylacetamides in an effort to improve upon the PK properties. This series of compounds display nanomolar potency as GIRK1/2 activators with improved brain distribution (rodent K > 0.6).
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Pore Polarity and Charge Determine Differential Block of Kir1.1 and Kir7.1 Potassium Channels by Small-Molecule Inhibitor VU590.
Kharade SV, Sheehan JH, Figueroa EE, Meiler J, Denton JS
(2017) Mol Pharmacol 92: 338-346
MeSH Terms: HEK293 Cells, Heterocyclic Compounds, 1-Ring, Humans, Mutation, Potassium Channel Blockers, Potassium Channels, Inwardly Rectifying, Structure-Activity Relationship
Show Abstract · Added September 15, 2017
VU590 was the first publicly disclosed, submicromolar-affinity (IC = 0.2 M), small-molecule inhibitor of the inward rectifier potassium (Kir) channel and diuretic target, Kir1.1. VU590 also inhibits Kir7.1 (IC ∼ 8 M), and has been used to reveal new roles for Kir7.1 in regulation of myometrial contractility and melanocortin signaling. Here, we employed molecular modeling, mutagenesis, and patch clamp electrophysiology to elucidate the molecular mechanisms underlying VU590 inhibition of Kir1.1 and Kir7.1. Block of both channels is voltage- and K-dependent, suggesting the VU590 binding site is located within the pore. Mutagenesis analysis in Kir1.1 revealed that asparagine 171 (N171) is the only pore-lining residue required for high-affinity block, and that substituting negatively charged residues (N171D, N171E) at this position dramatically weakens block. In contrast, substituting a negatively charged residue at the equivalent position in Kir7.1 enhances block by VU590, suggesting the VU590 binding mode is different. Interestingly, mutations of threonine 153 (T153) in Kir7.1 that reduce constrained polarity at this site (T153C, T153V, T153S) make wild-type and binding-site mutants (E149Q, A150S) more sensitive to block by VU590. The Kir7.1-T153C mutation enhances block by the structurally unrelated inhibitor VU714 but not by a higher-affinity analog ML418, suggesting that the polar side chain of T153 creates a barrier to low-affinity ligands that interact with E149 and A150. Reverse mutations in Kir1.1 suggest that this mechanism is conserved in other Kir channels. This study reveals a previously unappreciated role of membrane pore polarity in determination of Kir channel inhibitor pharmacology.
Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.
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7 MeSH Terms