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Photopharmacology relies on ligands that change their pharmacodynamics upon photoisomerization. Many of these ligands are azobenzenes that are thermodynamically more stable in their elongated trans-configuration. Often, they are biologically active in this form and lose activity upon irradiation and photoisomerization to their cis-isomer. Recently, cyclic azobenzenes, so-called diazocines, have emerged, which are thermodynamically more stable in their bent cis-form. Incorporation of these switches into a variety of photopharmaceuticals could convert dark-active ligands into dark-inactive ligands, which is preferred in most biological applications. This "pharmacological sign-inversion" is demonstrated for a photochromic blocker of voltage-gated potassium channels, termed CAL, and a photochromic opener of G protein-coupled inwardly rectifying potassium (GIRK) channels, termed CLOGO.
© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Pancreatic α-cells exhibit oscillations in cytosolic Ca (Ca), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca oscillations have not been elucidated. As β-cell Ca oscillations are regulated in part by Ca-activated K (K) currents, this work investigated the role of K in α-cell Ca handling and GCG secretion. α-Cells displayed K currents that were dependent on Ca influx through L- and P/Q-type voltage-dependent Ca channels (VDCCs) as well as Ca released from endoplasmic reticulum stores. α-Cell K was decreased by small-conductance Ca-activated K (SK) channel inhibitors apamin and UCL 1684, large-conductance Ca-activated K (BK) channel inhibitor iberiotoxin (IbTx), and intermediate-conductance Ca-activated K (IK) channel inhibitor TRAM 34. Moreover, partial inhibition of α-cell K with apamin depolarized membrane potential ( V) (3.8 ± 0.7 mV) and reduced action potential (AP) amplitude (10.4 ± 1.9 mV). Although apamin transiently increased Ca influx into α-cells at low glucose (42.9 ± 10.6%), sustained SK (38.5 ± 10.4%) or BK channel inhibition (31.0 ± 11.7%) decreased α-cell Ca influx. Total α-cell Ca was similarly reduced (28.3 ± 11.1%) following prolonged treatment with high glucose, but it was not decreased further by SK or BK channel inhibition. Consistent with reduced α-cell Ca following prolonged K inhibition, apamin decreased GCG secretion from mouse (20.4 ± 4.2%) and human (27.7 ± 13.1%) islets at low glucose. These data demonstrate that K activation provides a hyperpolarizing influence on α-cell V that sustains Ca entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when α-cell Ca is elevated during secretagogue stimulation, K activation helps to preserve GCG secretion.
Stress contributes to numerous psychiatric disorders. Corticotropin releasing factor (CRF) signaling and CRF neurons in the bed nucleus of the stria terminalis (BNST) drive negative affective behaviors, thus agents that decrease activity of these cells may be of therapeutic interest. Here, we show that acute restraint stress increases cFos expression in CRF neurons in the mouse dorsal BNST, consistent with a role for these neurons in stress-related behaviors. We find that activation of α-adrenergic receptors (ARs) by the agonist guanfacine reduced cFos expression in these neurons both in stressed and unstressed conditions. Further, we find that α- and β-ARs differentially regulate excitatory drive onto these neurons. Pharmacological and channelrhodopsin-assisted mapping experiments suggest that α-ARs specifically reduce excitatory drive from parabrachial nucleus (PBN) afferents onto CRF neurons. Given that the α-AR is a G-linked GPCR, we assessed the impact of activating the G-coupled DREADD hM4Di in the PBN on restraint stress regulation of BNST CRF neurons. CNO activation of PBN hM4Di reduced stress-induced in BNST neurons. Further, using as an additional marker of BNST neuronal identity, we uncovered a female-specific upregulation of the coexpression of in BNST neurons following stress, which was prevented by ovariectomy. These findings show that stress activates BNST CRF neurons, and that α-AR activation suppresses the activity of these cells, at least in part by suppressing excitatory drive from PBN inputs onto CRF neurons. Stress is a major variable contributing to mood disorders. Here, we show that stress increases activation of BNST CRF neurons that drive negative affective behavior. We find that the clinically well tolerated α-AR agonist guanfacine reduces activity of these cells , and reduces excitatory PBN inputs onto these cells Additionally, we uncover a novel sex-dependent coexpression of with in female BNST neurons after stress, an effect abolished by ovariectomy. These results demonstrate input-specific interactions between norepinephrine and CRF, and point to an action by which guanfacine may reduce negative affective responses.
Copyright © 2019 the authors 0270-6474/19/390472-13$15.00/0.
Positive allosteric modulators (PAMs) of the M subtype of muscarinic acetylcholine receptor have attracted intense interest as an exciting new approach for improving the cognitive deficits in schizophrenia and Alzheimer's disease. Recent evidence suggests that the presence of intrinsic agonist activity of some M PAMs may reduce efficacy and contribute to adverse effect liability. However, the M PAM PF-06827443 was reported to have only weak agonist activity at human M receptors but produced M-dependent adverse effects. We now report that PF-06827443 is an allosteric agonist in cell lines expressing rat, dog, and human M and use of inducible cell lines shows that agonist activity of PF-06827443 is dependent on receptor reserve. Furthermore, PF-06827443 is an agonist in native tissue preparations and induces behavioral convulsions in mice similar to other ago-PAMs. These findings suggest that PF-06827443 is a robust ago-PAM, independent of species, in cell lines and native systems.
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.
OBJECTIVE - The mutant γ-aminobutyric acid type A (GABA ) receptor γ2(Q390X) subunit (Q351X in the mature peptide) has been associated with the epileptic encephalopathy, Dravet syndrome, and the epilepsy syndrome genetic epilepsy with febrile seizures plus (GEFS+). The mutation generates a premature stop codon that results in translation of a stable truncated and misfolded γ2 subunit that accumulates in neurons, forms intracellular aggregates, disrupts incorporation of γ2 subunits into GABA receptors, and affects trafficking of partnering α and β subunits. Heterozygous Gabrg2 knock-in (KI) mice had reduced cortical inhibition, spike wave discharges on electroencephalography (EEG), a lower seizure threshold to the convulsant drug pentylenetetrazol (PTZ), and spontaneous generalized tonic-clonic seizures. In this proof-of-principal study, we attempted to rescue these deficits in KI mice using a γ2 subunit gene (GABRG2) replacement therapy.
METHODS - We introduced the GABRG2 allele by crossing Gabrg2 KI mice with bacterial artificial chromosome (BAC) transgenic mice overexpressing HA (hemagglutinin)-tagged human γ2 subunits, and compared GABA receptor subunit expression by Western blot and immunohistochemical staining, seizure threshold by monitoring mouse behavior after PTZ-injection, and thalamocortical inhibition and network oscillation by slice recording.
RESULTS - Compared to KI mice, adult mice carrying both mutant allele and transgene had increased wild-type γ2 and partnering α1 and β2/3 subunits, increased miniature inhibitory postsynaptic current (mIPSC) amplitudes recorded from layer VI cortical neurons, reduced thalamocortical network oscillations, and higher PTZ seizure threshold.
SIGNIFICANCE - Based on these results we suggest that seizures in a genetic epilepsy syndrome caused by epilepsy mutant γ2(Q390X) subunits with dominant negative effects could be rescued potentially by overexpression of wild-type γ2 subunits.
Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.
G-protein coupled inwardly rectifying potassium (GIRK) channels are an integral part of inhibitory signal transduction pathways, reducing the activity of excitable cells via hyperpolarization. They play crucial roles in processes such as cardiac output, cognition and the coordination of movement. Therefore, the precision control of GIRK channels is of critical importance. Here, we describe the development of the azobenzene containing molecule VLOGO (Visible Light Operated GIRK channel Opener), which activates GIRK channels in the dark and is promptly deactivated when illuminated with green light. VLOGO is a valuable addition to the existing tools for the optical control of GIRK channels as it circumvents the need to use potentially harmful UV irradiation. We therefore believe that VLOGO will be a useful research tool for studying GIRK channels in biological systems.
BACKGROUND - Genome-wide association studies have implicated variants in SCN10A, which encodes Nav1.8, as modulators of cardiac conduction. Follow-up work has indicated the SCN10A sequence includes an intronic enhancer for SCN5A. Yet the role of the Nav1.8 protein in the myocardium itself is still unclear. To investigate this, we use homozygous knockout mice (Scn10a) generated by disruption of exons 4 and 5, leaving the Scn5a enhancer intact.
METHODS AND RESULTS - We previously reported that pharmacologic blockade of Nav1.8 in wild-type animals blunts action potential prolongation by ATX-II at slow drive rates (≤1 Hz). Here we present evidence of the same blunting in Scn10a compared to wild-type ventricular myocytes, supporting the conclusion that Nav1.8 contributes to late sodium current at slow rates. In contrast to earlier studies, we found no differences in electrocardiographic parameters between genotypes. Low-dose ATX-II exposure in lightly anesthetized animals and Langendorff-perfused hearts prolonged QTc and generated arrhythmias to the same extent in wild-type and Scn10a. RNA sequencing failed to identify full-length Scn10a transcripts in wild-type or knockout isolated ventricular myocytes. However, loss of late current in Scn10a myocytes was replicated independently in a blinded set of experiments.
CONCLUSIONS - While Scn10a transcripts are not detectible in ventricular cardiomyocytes, gene deletion results in reproducible loss of late sodium current under extreme experimental conditions. However, there are no identifiable consequences of this Scn10a deletion in the intact mouse heart at usual rates. These findings argue that common variants in SCN10A that affect ventricular conduction do so by modulating SCN5A.
© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.
Purpose - Many proteins in the lens undergo extensive posttranslational modifications (PTMs) with age, leading to alterations in their function. The extent to which lens gap junction proteins, Cx46 and Cx50, accumulate PTMs with aging is not known. In this study, we identified truncations in Cx46 and Cx50 in the human lens using mass spectrometry. We also examined the effect of truncations on channel function using electrophysiological measurements.
Methods - Human lenses were dissected into cortex, outer nucleus, and nucleus regions, and fiber cell membranes were subjected to trypsin digestion. Tryptic peptides were analyzed by liquid chromatography (LC)-electrospray tandem mass spectrometry (ESI/MS/MS). Effects of truncations on channel conductance, permeability, and gating were assessed in transfected cells.
Results - Cleavage sites were identified in the C-terminus, the cytoplasmic loop, and the N-terminus of Cx46 and Cx50. Levels of C-terminal truncations, which were found at residues 238 to 251 in Cx46 and at residues 238 to 253 and 274 to 284 in Cx50, were similar in different lens regions. In contrast, levels of truncations in cytoplasmic loop and N-terminal domains of Cx46 and Cx50 increased dramatically from outer cortex to nucleus. Most of the C-terminally truncated proteins were functional, whereas truncations in the cytoplasmic loop did not result in the formation of functional channels.
Conclusions - Accumulation of cytoplasmic loop and N-terminal truncations in the core might lead to decreases in coupling with age. This reduction is expected to lead to an increase in intracellular calcium and a decrease in levels of glutathione in the nucleus. These changes may ultimately lead to age-related nuclear cataracts.
The inward rectifier potassium (Kir) channel Kir7.1 (KCNJ13) has recently emerged as a key regulator of melanocortin signaling in the brain, electrolyte homeostasis in the eye, and uterine muscle contractility during pregnancy. The pharmacological tools available for exploring the physiology and therapeutic potential of Kir7.1 have been limited to relatively weak and nonselective small-molecule inhibitors. Here, we report the discovery in a fluorescence-based high-throughput screen of a novel Kir7.1 channel inhibitor, VU714. Site-directed mutagenesis of pore-lining amino acid residues identified glutamate 149 and alanine 150 as essential determinants of VU714 activity. Lead optimization with medicinal chemistry generated ML418, which exhibits sub-micromolar activity (IC50 = 310 nM) and superior selectivity over other Kir channels (at least 17-fold selective over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and Kir4.1) except for Kir6.2/SUR1 (equally potent). Evaluation in the EuroFins Lead Profiling panel of 64 GPCRs, ion-channels, and transporters for off-target activity of ML418 revealed a relatively clean ancillary pharmacology. While ML418 exhibited low CLHEP in human microsomes which could be modulated with lipophilicity adjustments, it showed high CLHEP in rat microsomes regardless of lipophilicity. A subsequent in vivo PK study of ML418 by intraperitoneal (IP) administration (30 mg/kg dosage) revealed a suitable PK profile (Cmax = 0.20 μM and Tmax = 3 h) and favorable CNS distribution (mouse brain/plasma Kp of 10.9 to support in vivo studies. ML418, which represents the current state-of-the-art in Kir7.1 inhibitors, should be useful for exploring the physiology of Kir7.1 in vitro and in vivo.