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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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9 MeSH Terms
Overexpressing wild-type γ2 subunits rescued the seizure phenotype in Gabrg2 Dravet syndrome mice.
Huang X, Zhou C, Tian M, Kang JQ, Shen W, Verdier K, Pimenta A, MacDonald RL
(2017) Epilepsia 58: 1451-1461
MeSH Terms: Action Potentials, Animals, Convulsants, Electric Stimulation, Epilepsies, Myoclonic, Humans, In Vitro Techniques, Inhibitory Postsynaptic Potentials, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mutation, Neural Pathways, Patch-Clamp Techniques, Pentylenetetrazole, Protein Subunits, Pyramidal Cells, Receptors, GABA-A, Somatosensory Cortex, Thalamus
Show Abstract · Added June 21, 2017
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.
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21 MeSH Terms
Optical control of GIRK channels using visible light.
Trads JB, Burgstaller J, Laprell L, Konrad DB, de la Osa de la Rosa L, Weaver CD, Baier H, Trauner D, Barber DM
(2016) Org Biomol Chem 15: 76-81
MeSH Terms: Azo Compounds, G Protein-Coupled Inwardly-Rectifying Potassium Channels, HEK293 Cells, Humans, Light, Patch-Clamp Techniques, Photochemical Processes
Show Abstract · Added April 3, 2018
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.
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Contrasting Nav1.8 Activity in Scn10a-/- Ventricular Myocytes and the Intact Heart.
Stroud DM, Yang T, Bersell K, Kryshtal DO, Nagao S, Shaffer C, Short L, Hall L, Atack TC, Zhang W, Knollmann BC, Baudenbacher F, Roden DM
(2016) J Am Heart Assoc 5:
MeSH Terms: Action Potentials, Animals, Electrocardiography, Heart, Heart Ventricles, Isolated Heart Preparation, Mice, Mice, Knockout, Myocardium, Myocytes, Cardiac, NAV1.8 Voltage-Gated Sodium Channel, Patch-Clamp Techniques, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction
Show Abstract · Added February 3, 2017
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.
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14 MeSH Terms
Identification and Functional Assessment of Age-Dependent Truncations to Cx46 and Cx50 in the Human Lens.
Slavi N, Wang Z, Harvey L, Schey KL, Srinivas M
(2016) Invest Ophthalmol Vis Sci 57: 5714-5722
MeSH Terms: Aging, Cataract, Cell Membrane Permeability, Cells, Cultured, Connexins, Humans, Lens, Crystalline, Membrane Glycoproteins, Middle Aged, Patch-Clamp Techniques, Tandem Mass Spectrometry
Show Abstract · Added May 6, 2017
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.
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11 MeSH Terms
ML418: The First Selective, Sub-Micromolar Pore Blocker of Kir7.1 Potassium Channels.
Swale DR, Kurata H, Kharade SV, Sheehan J, Raphemot R, Voigtritter KR, Figueroa EE, Meiler J, Blobaum AL, Lindsley CW, Hopkins CR, Denton JS
(2016) ACS Chem Neurosci 7: 1013-23
MeSH Terms: Animals, Dose-Response Relationship, Drug, HEK293 Cells, Humans, Membrane Potentials, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Patch-Clamp Techniques, Potassium, Potassium Channel Blockers, Potassium Channels, Inwardly Rectifying, Structure-Activity Relationship, Time Factors, Transfection
Show Abstract · Added April 8, 2017
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.
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15 MeSH Terms
Electrophysiological Measurement of Cannabinoid-Mediated Synaptic Modulation in Acute Mouse Brain Slices.
Báldi R, Ghosh D, Grueter BA, Patel S
(2016) Curr Protoc Neurosci 75: 6.29.1-6.29.19
MeSH Terms: Animals, Brain, Cannabinoid Receptor Modulators, Endocannabinoids, In Vitro Techniques, Mice, Neuronal Plasticity, Patch-Clamp Techniques, Synapses, Synaptic Transmission
Show Abstract · Added March 14, 2018
Endocannabinoids (eCBs) are a class of bioactive lipids that mediate retrograde synaptic modulation at central and peripheral synapses. The highly lipophilic nature of eCBs and the pharmacological tools available to interrogate this system require unique methodological consideration, especially when applied to ex vivo systems such as electrophysiological analysis in acute brain slices. This unit provides protocols for measuring cannabinoid and eCB-mediated synaptic signaling in mouse brain slices, including analysis of short-term, long-term, and tonic eCB signaling modes, and the unique considerations for working with eCBs and TRPV1/cannabinoid ligands in acute brain slices.
Copyright © 2016 John Wiley & Sons, Inc.
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10 MeSH Terms
Reduction of thalamic and cortical Ih by deletion of TRIP8b produces a mouse model of human absence epilepsy.
Heuermann RJ, Jaramillo TC, Ying SW, Suter BA, Lyman KA, Han Y, Lewis AS, Hampton TG, Shepherd GMG, Goldstein PA, Chetkovich DM
(2016) Neurobiol Dis 85: 81-92
MeSH Terms: Animals, Blotting, Western, Cerebral Cortex, Disease Models, Animal, Electrocardiography, Electrocorticography, Electrodes, Implanted, Epilepsy, Absence, Immunohistochemistry, Male, Membrane Potentials, Membrane Proteins, Mice, Knockout, Motor Activity, Neurons, Patch-Clamp Techniques, Peroxins, Rotarod Performance Test, Sequence Deletion, Thalamus, Tissue Culture Techniques
Show Abstract · Added April 2, 2019
Absence seizures occur in several types of human epilepsy and result from widespread, synchronous feedback between the cortex and thalamus that produces brief episodes of loss of consciousness. Genetic rodent models have been invaluable for investigating the pathophysiological basis of these seizures. Here, we identify tetratricopeptide-containing Rab8b-interacting protein (TRIP8b) knockout mice as a new model of absence epilepsy, featuring spontaneous spike-wave discharges on electroencephalography (EEG) that are the electrographic hallmark of absence seizures. TRIP8b is an auxiliary subunit of the hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels, which have previously been implicated in the pathogenesis of absence seizures. In contrast to mice lacking the pore-forming HCN channel subunit HCN2, TRIP8b knockout mice exhibited normal cardiac and motor function and a less severe seizure phenotype. Evaluating the circuit that underlies absence seizures, we found that TRIP8b knockout mice had significantly reduced HCN channel expression and function in thalamic-projecting cortical layer 5b neurons and thalamic relay neurons, but preserved function in inhibitory neurons of the reticular thalamic nucleus. Our results expand the known roles of TRIP8b and provide new insight into the region-specific functions of TRIP8b and HCN channels in constraining cortico-thalamo-cortical excitability.
Copyright © 2015 Elsevier Inc. All rights reserved.
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"Slow" Voltage-Dependent Inactivation of CaV2.2 Calcium Channels Is Modulated by the PKC Activator Phorbol 12-Myristate 13-Acetate (PMA).
Zhu L, McDavid S, Currie KP
(2015) PLoS One 10: e0134117
MeSH Terms: Animals, Calcium Channel Blockers, Calcium Channels, N-Type, Calcium Signaling, Cattle, Cells, Cultured, Chromaffin Cells, Enzyme Activation, Guanosine Diphosphate, HEK293 Cells, Humans, Kinetics, Patch-Clamp Techniques, Protein Kinase C, Recombinant Proteins, Tetradecanoylphorbol Acetate, Thionucleotides
Show Abstract · Added November 10, 2015
CaV2.2 (N-type) voltage-gated calcium channels (Ca2+ channels) play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA) had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.
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17 MeSH Terms
Potentiation of M1 Muscarinic Receptor Reverses Plasticity Deficits and Negative and Cognitive Symptoms in a Schizophrenia Mouse Model.
Ghoshal A, Rook JM, Dickerson JW, Roop GN, Morrison RD, Jalan-Sakrikar N, Lamsal A, Noetzel MJ, Poslusney MS, Wood MR, Melancon BJ, Stauffer SR, Xiang Z, Daniels JS, Niswender CM, Jones CK, Lindsley CW, Conn PJ
(2016) Neuropsychopharmacology 41: 598-610
MeSH Terms: Animals, Antipsychotic Agents, Cognition, Disease Models, Animal, Long-Term Synaptic Depression, Male, Mice, Inbred C57BL, Mice, Knockout, Patch-Clamp Techniques, Phencyclidine, Pyridines, Pyrroles, Receptor, Muscarinic M1, Schizophrenia, Schizophrenic Psychology, Social Behavior
Show Abstract · Added February 18, 2016
Schizophrenia patients exhibit deficits in signaling of the M1 subtype of muscarinic acetylcholine receptor (mAChR) in the prefrontal cortex (PFC) and also display impaired cortical long-term depression (LTD). We report that selective activation of the M1 mAChR subtype induces LTD in PFC and that this response is completely lost after repeated administration of phencyclidine (PCP), a mouse model of schizophrenia. Furthermore, discovery of a novel, systemically active M1 positive allosteric modulator (PAM), VU0453595, allowed us to evaluate the impact of selective potentiation of M1 on induction of LTD and behavioral deficits in PCP-treated mice. Interestingly, VU0453595 fully restored impaired LTD as well as deficits in cognitive function and social interaction in these mice. These results provide critical new insights into synaptic changes that may contribute to behavioral deficits in this mouse model and support a role for selective M1 PAMs as a novel approach for the treatment of schizophrenia.
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16 MeSH Terms