An Extrasynaptic GABAA Receptor Mediates Tonic Inhibition in Thalamic VB Neurons

2005 ◽  
Vol 94 (6) ◽  
pp. 4491-4501 ◽  
Author(s):  
Fan Jia ◽  
Leonardo Pignataro ◽  
Claude M. Schofield ◽  
Minerva Yue ◽  
Neil L. Harrison ◽  
...  
Keyword(s):  
Critical Role ◽  
Brain Slices ◽  
Oscillatory Activity ◽  
Thalamic Neurons ◽  
Hek 293 ◽  
Subunit Protein ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Hypnotic Agent ◽  
Tonic Current

Whole cell patch-clamp recordings were obtained from thalamic ventrobasal (VB) and reticular (RTN) neurons in mouse brain slices. A bicuculline-sensitive tonic current was observed in VB, but not in RTN, neurons; this current was increased by the GABAA receptor agonist 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridine-3-ol (THIP; 0.1 μM) and decreased by Zn2+ (50 μM) but was unaffected by zolpidem (0.3 μM) or midazolam (0.2 μM). The pharmacological profile of the tonic current is consistent with its generation by activation of GABAA receptors that do not contain the α1 or γ2 subunits. GABAA receptors expressed in HEK 293 cells that contained α4β2δ subunits showed higher sensitivity to THIP (gaboxadol) and GABA than did receptors made up from α1β2δ, α4β2γ2s, or α1β2γ2s subunits. Western blot analysis revealed that there is little, if any, α3 or α5 subunit protein in VB. In addition, co-immunoprecipitation studies showed that antibodies to the δ subunit could precipitate α4, but not α1 subunit protein. Confocal microscopy of thalamic neurons grown in culture confirmed that α4 and δ subunits are extensively co-localized with one another and are found predominantly, but not exclusively, at extrasynaptic sites. We conclude that thalamic VB neurons express extrasynaptic GABAA receptors that are highly sensitive to GABA and THIP and that these receptors are most likely made up of α4β2δ subunits. In view of the critical role of thalamic neurons in the generation of oscillatory activity associated with sleep, these receptors may represent a principal site of action for the novel hypnotic agent gaboxadol.

scholarly journals Zn2+ Slows Down CaV3.3 Gating Kinetics: Implications for Thalamocortical Activity

2007 ◽  
Vol 98 (4) ◽  
pp. 2274-2284 ◽  
Author(s):  
M. Cataldi ◽  
V. Lariccia ◽  
V. Marzaioli ◽  
A. Cavaccini ◽  
G. Curia ◽  
...  
Keyword(s):  
Neuronal Excitability ◽  
Whole Cell ◽  
Hek 293 ◽  
Gating Kinetics ◽  
Epileptiform Discharges ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation ◽  
293 Cells ◽  
Channel Opening ◽  
Current Activation

We employed whole cell patch-clamp recordings to establish the effect of Zn2+ on the gating the brain specific, T-type channel isoform CaV3.3 expressed in HEK-293 cells. Zn2+ (300 μM) modified the gating kinetics of this channel without influencing its steady-state properties. When inward Ca2+ currents were elicited by step depolarizations at voltages above the threshold for channel opening, current inactivation was significantly slowed down while current activation was moderately affected. In addition, Zn2+ slowed down channel deactivation but channel recovery from inactivation was only modestly changed. Zn2+ also decreased whole cell Ca2+ permeability to 45% of control values. In the presence of Zn2+, Ca2+ currents evoked by mock action potentials were more persistent than in its absence. Furthermore, computer simulation of action potential generation in thalamic reticular cells performed to model the gating effect of Zn2+ on T-type channels (while leaving the kinetic parameters of voltage-gated Na+ and K+ unchanged) revealed that Zn2+ increased the frequency and the duration of burst firing, which is known to depend on T-type channel activity. In line with this finding, we discovered that chelation of endogenous Zn2+ decreased the frequency of occurrence of ictal-like epileptiform discharges in rat thalamocortical slices perfused with medium containing the convulsant 4-aminopyridine (50 μM). These data demonstrate that Zn2+ modulates CaV3.3 channel gating thus leading to increased neuronal excitability. We also propose that endogenous Zn2+ may have a role in controlling thalamocortical oscillations.

scholarly journals Synaptic and extrasynaptic transmission of kidney-related neurons in the rostral ventrolateral medulla

2013 ◽  
Vol 110 (11) ◽  
pp. 2637-2647 ◽  
Author(s):  
Hong Gao ◽  
Andrei V. Derbenev
Keyword(s):  
Large Scale ◽  
Critical Role ◽  
Input Resistance ◽  
Rostral Ventrolateral Medulla ◽  
Kainate Receptors ◽  
Ventrolateral Medulla ◽  
Resting Membrane Potential ◽  
Arterial Blood ◽  
Whole Cell Patch Clamp ◽  
Tonic Current

The rostral ventrolateral medulla (RVLM) is a critical component of the sympathetic nervous system regulating homeostatic functions including arterial blood pressure. Using the transsynaptic retrograde viral tracer PRV-152, we identified kidney-related neurons in the RVLM. We found that PRV-152-labeled RVLM neurons displayed an unusually large persistent, tonic current to both glutamate, via N-methyl-d-aspartate (NMDA) and 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)/kainate receptors, and to γ-aminobutyric acid (GABA), via GABAAreceptors, in the absence of large-scale phasic neurotransmission with whole cell patch-clamp recordings. A cocktail of potent NMDA and AMPA/kainate ionotropic glutamate receptor antagonists AP-5 (50 μM) and CNQX (10 μM) revealed a two-component somatic tonic excitatory current with an overall amplitude of 42.6 ± 13.4 pA. Moreover, application of the GABAAreceptor blockers gabazine (15 μM) and bicuculline (30 μM) revealed a robust somatic tonic inhibitory current with an average amplitude of 196.3 ± 39.3 pA. These findings suggest that the tonic current plays a role in determining the resting membrane potential, input resistance, and firing rate of RVLM neurons. The magnitude of the tonic inhibitory current demonstrates that GABAergic inhibition plays a critical role in regulation of kidney-related RVLM neurons. Our results indicate that the GABAergic tonic current may determine the basal tone of firing activity in kidney-related RVLM neurons.

scholarly journals Expression of tachykinin receptors (tacr1a and tacr1b) in zebrafish: influence of cocaine and opioid receptors

2012 ◽  
Vol 50 (2) ◽  
pp. 115-129 ◽  
Author(s):  
Roger López-Bellido ◽  
Katherine Barreto-Valer ◽  
Raquel E Rodríguez
Keyword(s):  
Embryonic Development ◽  
Opioid Receptors ◽  
Phylogenetic Analyses ◽  
Critical Role ◽  
Cocaine Exposure ◽  
Peripheral Tissues ◽  
Tachykinin Receptors ◽  
Hek 293 ◽  
293 Cells ◽  
Gene Study

Opioid and tachykinin receptors (TACRs) are closely related in addiction and pain processes. In zebrafish, opioid receptors have been cloned and characterized both biochemically and pharmacologically. However, thetacr1gene has not yet been described in zebrafish. The aim of this research was to identify thetacr1gene, study the effects of cocaine ontacr1, and analyze the interaction betweentacr1and opioid receptors. We have identified a duplicate oftacr1gene in zebrafish, designated astacr1aandtacr1b. Phylogenetic analyses revealed an alignment of these receptors in the Tacr1 fish cluster, with a clear distinction from other TACR1s of amphibians, birds, and mammals. Our qPCR results showed thattacr1aandtacr1bmRNAs are expressed during embryonic development. Whole-mountin situhybridization showedtacr1expression in the CNS and in the peripheral tissues. Cocaine (1.5 μM) induced an upregulation oftacr1aandtacr1bat 24 and 48 h post-fertilization (hpf; except fortacr1aat 48 hpf, which was downregulated). By contrast, HEK-293 cells transfected withtacr1aandtacr1band exposed to cocaine showed a downregulation oftacr1s. The knockdown of ZfDOR2 and ZfMOR, opioid receptors, induced a down- and upregulation oftacr1aandtacr1brespectively. In conclusion,tacr1aandtacr1bin zebrafish are widely expressed throughout the CNS and peripherally, suggesting a critical role of thesetacr1sduring embryogenesis.tacr1aandtacr1bmRNA expression is altered by cocaine exposure and by the knockdown of opioid receptors. Thus, zebrafish can provide clues for a better understanding of the relationship between tachykinin and opioid receptors in pain and addiction during embryonic development.

scholarly journals Characterization of α3 Glycine Receptors with Ginkgolide B and Picrotoxin

2018 ◽  
Author(s):  
Sampurna Chakrabarti ◽  
Anil Neelakantan ◽  
Malcolm M. Slaughter
Keyword(s):  
Beta Subunits ◽  
Ginkgolide B ◽  
Glycine Receptors ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Voltage Dependent ◽  
The Central Nervous System ◽  
Subunit Isoforms

AbstractGinkgolide B (GB) and picrotoxin (PTX) are antagonists of the major inhibitory receptors of the central nervous system: GABA and glycine receptors (GlyRs). GlyRs contain one or more of the four alpha subunit isoforms of which α1 and α2 have been extensively studied. This report compares GB and PTX block of α3 GlyRs expressed in HEK 293 cells, using whole-cell patch clamp techniques. In CNS, α3 exists as a heteropentamer in conjunction with beta subunits in a 2α:3β ratio. Thus, the nature of block was also tested in α3β heteromeric glycine receptors. GB and PTX blocked α3 GlyRs both in the presence (liganded state) and absence of glycine (unliganded state). This property is unique to α3 subunits; α1 and α2 subunits are only blocked in the liganded state. The GB block of α3 GlyRs is voltage-dependent (more effective when the cell is depolarized) and non-competitive, while the PTX block is competitive and not voltage-dependent. The heteromeric and homomeric α3 GlyRs recovered significantly faster from unliganded GB block compared to liganded GB block, but no such distinction was found for PTX block suggesting more than one binding site for GB. This study sheds light on features of the α3 GlyR that distinguish it from the more widely studied α1 and α2 subunits. Understanding these properties can help decipher the physiological functioning of GlyRs in the CNS and may permit development of subunit specific drugs.

Abstract 355: Modulation of Cardiac L-type Calcium Channels by Phospholemman

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Xianming Wang ◽  
Guofeng Gao ◽  
Blaise Z Peterson
Keyword(s):  
Plasma Membranes ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Ion Homeostasis ◽  
Functional Coupling ◽  
Mouse Heart ◽  
Channel Activation ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Ca 2+ entry through L-type Ca 2+ channels plays a critical role in shaping the action potential and is the initial trigger for EC-coupling. The gating, expression and targeting Ca 2+ channels are tightly regulated by auxiliary subunits and second messenger signaling mechanisms. Here, we report that cardiac Ca 2+ channels are directly modulated by phospholemman (PLM), a single transmembrane protein that is important for regulating ion homeostasis in the heart through its interactions with the Na,K-ATPase and Na/Ca Exchanger (NCX). Experiments using confocal immunofluorescence microscopy indiate that PLM and the Ca 2+ channel alpha-1 subunit, Ca V 1.2, co-localize to the plasma membranes of HEK 293 and COS-7 cells. Recipricol co-immunoprecipitation studies demonstrate that PLM and Ca V 1.2 are specifically associated in the mouse heart (see Figure ) and HEK 293 cells expressing the two proteins (not shown). Whole-cell patch-clamp was used to assess the functional consequences of the interaction between PLM and the Ca V 1.2 subunit using HEK 293 cells transfected with PLM ((+)PLM) or empty PLM vector ((−)PLM). These studies demonstrate that PLM substantially slows the activation kinetics of Ca V 1.2 channels (see Figure ), but has no effect on neuronal Ca V 2.1 Ca 2+ channels (not shown). As a result, the level of Ca 2+ entry during the first 50 msec of channel activation is decreased by up to 32%. Since PLM is upregulated in post-ischemic rat hearts and due to the tight functional coupling between the cardiac Ca 2+ channel and NCX, we propose that PLM-induced slowing channel activation and PLM-dependent inhibition of NCX combine synergistically to reduce peak [Ca 2+ ]i in infacted myocytes.

Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland

1999 ◽  
Vol 276 (6) ◽  
pp. C1261-C1270 ◽  
Author(s):  
Achim D. Gruber ◽  
Kevin D. Schreur ◽  
Hong-Long Ji ◽  
Catherine M. Fuller ◽  
Bendicht U. Pauli
Keyword(s):  
Mammary Gland ◽  
Human Lung ◽  
Glycosylation Site ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Amino Terminal ◽  
Anion Conductance ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Human Trachea

The CLCA family of Ca2+-activated Cl− channels has recently been discovered, with an increasing number of closely related members isolated from different species. Here we report the cloning of the second human homolog, hCLCA2, from a human lung cDNA library. Northern blot and RT-PCR analyses revealed additional expression in trachea and mammary gland. A primary translation product of 120 kDa was cleaved into two cell surface-associated glycoproteins of 86 and 34 kDa in transfected HEK-293 cells. hCLCA2 is the first CLCA homolog for which the transmembrane structure has been systematically studied. Glycosylation site scanning and protease protection assays revealed five transmembrane domains with a large, cysteine-rich, amino-terminal extracellular domain. Whole cell patch-clamp recordings of hCLCA2-transfected HEK-293 cells detected a slightly outwardly rectifying anion conductance that was increased in the presence of the Ca2+ ionophore ionomycin and inhibited by DIDS, dithiothreitol, niflumic acid, and tamoxifen. Expression in human trachea and lung suggests that hCLCA2 may play a role in the complex pathogenesis of cystic fibrosis.

ClC-2 chloride channels contribute to HTC cell volume homeostasis

2001 ◽  
Vol 280 (3) ◽  
pp. G344-G353 ◽  
Author(s):  
Richard M. Roman ◽  
Roderic L. Smith ◽  
Andrew P. Feranchak ◽  
Gerald H. Clayton ◽  
R. Brian Doctor ◽  
...  
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Chloride Channels ◽  
Purinergic Receptor ◽  
Cell Volume Regulation ◽  
Rat Hepatocytes ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Htc Cells

Membrane Cl−channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl−currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl−currents when non-ClC-2 currents were blocked by DIDS (100 μM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl−currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl−permeability and cell volume homeostasis.

Temperature dependence of early and late currents in human cardiac wild-type and long Q-T ΔKPQ Na+ channels

1998 ◽  
Vol 275 (6) ◽  
pp. H2016-H2024 ◽  
Author(s):  
Toshihisa Nagatomo ◽  
Zheng Fan ◽  
Bin Ye ◽  
Gayle S. Tonkovich ◽  
Craig T. January ◽  
...  
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Inactivation Kinetics ◽  
Wild Type ◽  
Positive Direction ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation ◽  
293 Cells ◽  
Steady State Inactivation

Na+current ( I Na) through wild-type human heart Na+channels (hH1) is important for normal cardiac excitability and conduction, and it participates in the control of repolarization and refractoriness. I Na kinetics depend strongly on temperature, but I Na for hH1 has been studied previously only at room temperature. We characterized early I Na (the peak and initial decay) and late I Na of the wild-type hH1 channel and a mutant channel (ΔKPQ) associated with congenital long Q-T syndrome. Channels were stably transfected in HEK-293 cells and studied at 23 and 33°C using whole cell patch clamp. Activation and inactivation kinetics for early I Na were twofold faster at higher temperature for both channels and shifted activation and steady-state inactivation in the positive direction, especially for ΔKPQ. For early I Na (<24 ms), ΔKPQ decayed faster than the wild type for voltages negative to −20 mV but slower for more positive voltages, suggesting a reduced voltage dependence of fast inactivation. Late I Na at 240 ms was significantly greater for ΔKPQ than for the wild type at both temperatures. The majority of late I Na for ΔKPQ was not persistent; rather, it decayed slowly, and this late component exhibited slower recovery from inactivation compared with peak I Na. Additional kinetic changes for early and peak I Na for ΔKPQ compared with the wild type at both temperatures were 1) reduced voltage dependence of steady-state inactivation with no difference in midpoint, 2) positive shift for activation kinetics, and 3) more rapid recovery from inactivation. This study represents the first description of human Na+ channel kinetics near physiological temperature and also demonstrates complex gating changes in the ΔKPQ that are present at 33°C and that may underlie the electrophysiological and clinical phenotype of congenital long Q-T Na+ channel syndromes.

The dihydropyridine analogue cerebrocrast blocks both T-type and L-type calcium currents

2009 ◽  
Vol 87 (11) ◽  
pp. 923-932 ◽  
Author(s):  
Mária Drígeľová ◽  
Bohumila Tarabová ◽  
Gunars Duburs ◽  
Ľubica Lacinová
Keyword(s):  
Calcium Currents ◽  
Hek 293 ◽  
State Dependent ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Voltage Dependent ◽  
Dependent Inhibition ◽  
Partial Interaction ◽  
Dihydropyridine Derivative ◽  
Dependent Interaction

Cerebrocrast is a novel lipophilic dihydropyridine derivative with potential neuroprotective and antidiabetic properties. We have analyzed its interaction with L-type (CaV1.2b) and T-type (CaV3.1) calcium channels using a whole-cell patch clamp in HEK 293 cells. Cerebrocrast inhibited current flux through both CaV1.2b and CaV3.1 channels. In both cases, the drug was about 10-fold less effective than neutral dihydropyridines, but more efficient than the charged dihydropyridine amlodipine. IC50 values for the CaV1.2b channel were 586 ± 96 nmol/L and 178 ± 78 nmol/L at holding potentials of –80 mV and –50 mV, respectively. Approximately 50 µmol/L of cerebrocrast was needed to block 50% of the current amplitude in the CaV3.1 channel, but this inhibition was not facilitated by shifting the holding potential from –100 mV to –70 mV. Cerebrocrast did not alter current kinetics in either investigated channel, and the inhibition of calcium current was partly reversible or irreversible. In conclusion, the interaction of cerebrocrast with CaV3.1 lacked the typical characteristics of a state-dependent interaction, and voltage-dependent inhibition of CaV1.2b was consistent with partial interaction with the inactivated state of the channel.

scholarly journals Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis

2013 ◽  
Vol 305 (4) ◽  
pp. C392-C405 ◽  
Author(s):  
Quansheng Zhu ◽  
Xuesi M. Shao ◽  
Liyo Kao ◽  
Rustam Azimov ◽  
Alan M. Weinstein ◽  
...  
Keyword(s):  
Renal Tubular Acidosis ◽  
Reverse Direction ◽  
Gene Encoding ◽  
Proximal Tubule Cells ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Proximal Renal Tubular Acidosis ◽  
Renal Tubular ◽  
Teeth Abnormalities

Mutations in SLC4A4, the gene encoding the electrogenic Na+-HCO3− cotransporter NBCe1, cause severe proximal renal tubular acidosis (pRTA), growth retardation, decreased IQ, and eye and teeth abnormalities. Among the known NBCe1 mutations, the disease-causing mechanism of the T485S (NBCe1-A numbering) mutation is intriguing because the substituted amino acid, serine, is structurally and chemically similar to threonine. In this study, we performed intracellular pH and whole cell patch-clamp measurements to investigate the base transport and electrogenic properties of NBCe1-A-T485S in mammalian HEK 293 cells. Our results demonstrated that Ser substitution of Thr485 decreased base transport by ∼50%, and importantly, converted NBCe1-A from an electrogenic to an electroneutral transporter. Aqueous accessibility analysis using sulfhydryl reactive reagents indicated that Thr485 likely resides in an NBCe1-A ion interaction site. This critical location is also supported by the finding that G486R (a pRTA causing mutation) alters the position of Thr485 in NBCe1-A thereby impairing its transport function. By using NO3− as a surrogate ion for CO32−, our result indicated that NBCe1-A mediates electrogenic Na+-CO32− cotransport when functioning with a 1:2 charge transport stoichiometry. In contrast, electroneutral NBCe1-T485S is unable to transport NO3−, compatible with the hypothesis that it mediates Na+-HCO3− cotransport. In patients, NBCe1-A-T485S is predicted to transport Na+-HCO3− in the reverse direction from blood into proximal tubule cells thereby impairing transepithelial HCO3− absorption, possibly representing a new pathogenic mechanism for generating human pRTA.

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