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.

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.

scholarly journals Inhibition of Small-Conductance, Ca2+-Activated K+ Current by Ondansetron

2021 ◽  
Vol 12 ◽  
Author(s):  
Shuai Guo ◽  
Zhenhui Chen ◽  
Peng-Sheng Chen ◽  
Michael Rubart
Keyword(s):  
Inhibitory Effect ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Inwardly Rectifying ◽  
Small Conductance

Background: Small-conductance Ca2+-activated K+ channels (SK channels) have been proposed as antiarrhythmic targets for the treatment of atrial fibrillation. We previously demonstrated that the 5-HT3 receptor antagonist ondansetron inhibits heterologously expressed, human SK2 (hSK2) currents as well as native cardiac SK currents in a physiological extra-/intracellular [K+] gradient at therapeutic (i.e., sub-micromolar) concentrations. A recent study, using symmetrical [K+] conditions, challenged this result. The goal of the present study was to revisit the inhibitory effect of ondansetron on hSK2-mediated currents in symmetrical [K+] conditions.Experimental Approach: The whole-cell patch clamp technique was used to investigate the effects of ondansetron and apamin on hSK2-mediated currents expressed in HEK 293 cells. Currents were measured in symmetrical [K+] conditions in the presence of 100 nM [Ca2+]o.Results: Expression of hSK2 produced inwardly rectifying whole-cell currents in the presence of 400 nM free cytosolic Ca2+. Ondansetron inhibited whole-cell hSK2 currents with IC50 values of 154 and 113 nM at −80 and 40 mV, respectively. Macroscopic current inhibited by ondansetron and current inhibited by apamin exhibited inwardly rectifying current-voltage relationships with similar reversal potentials (apamin, ∼5 mV and ondansetron, ∼2 mV). Ondansetron (1 μM) in the continuing presence of apamin (100 nM) had no effect on hSK2-mediated whole-cell currents. Wild-type HEK 293 cells did not express ondansetron- or apamin-sensitive currents.Conclusion: Ondansetron in sub-micromolar concentrations inhibits hSK2 currents even under altered ionic conditions.

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 Inhibitory effects of cannabidiol on voltage-dependent sodium currents

2018 ◽  
Vol 293 (43) ◽  
pp. 16546-16558 ◽  
Author(s):  
Mohammad-Reza Ghovanloo ◽  
Noah Gregory Shuart ◽  
Janette Mezeyova ◽  
Richard A. Dean ◽  
Peter C. Ruben ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Cannabis Sativa ◽  
Case Reports ◽  
Hek 293 ◽  
Recovery From Inactivation ◽  
293 Cells ◽  
Hill Slope ◽  
Voltage Dependent ◽  
Nav Channels ◽  
Therapeutic Mechanisms

Cannabis sativa contains many related compounds known as phytocannabinoids. The main psychoactive and nonpsychoactive compounds are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively. Much of the evidence for clinical efficacy of CBD-mediated antiepileptic effects has been from case reports or smaller surveys. The mechanisms for CBD's anticonvulsant effects are unclear and likely involve noncannabinoid receptor pathways. CBD is reported to modulate several ion channels, including sodium channels (Nav). Evaluating the therapeutic mechanisms and safety of CBD demands a richer understanding of its interactions with central nervous system targets. Here, we used voltage-clamp electrophysiology of HEK-293 cells and iPSC neurons to characterize the effects of CBD on Nav channels. Our results show that CBD inhibits hNav1.1–1.7 currents, with an IC50 of 1.9–3.8 μm, suggesting that this inhibition could occur at therapeutically relevant concentrations. A steep Hill slope of ∼3 suggested multiple interactions of CBD with Nav channels. CBD exhibited resting-state blockade, became more potent at depolarized potentials, and also slowed recovery from inactivation, supporting the idea that CBD binding preferentially stabilizes inactivated Nav channel states. We also found that CBD inhibits other voltage-dependent currents from diverse channels, including bacterial homomeric Nav channel (NaChBac) and voltage-gated potassium channel subunit Kv2.1. Lastly, the CBD block of Nav was temperature-dependent, with potency increasing at lower temperatures. We conclude that CBD's mode of action likely involves 1) compound partitioning in lipid membranes, which alters membrane fluidity affecting gating, and 2) undetermined direct interactions with sodium and potassium channels, whose combined effects are loss of channel excitability.

Characterization of a swelling-induced chloride conductance in cultured rat epididymal cells

1993 ◽  
Vol 265 (4) ◽  
pp. C997-C1005 ◽  
Author(s):  
H. C. Chan ◽  
W. O. Fu ◽  
Y. W. Chung ◽  
S. J. Huang ◽  
T. S. Zhou ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Induced Current ◽  
Whole Cell ◽  
Cyclic Monophosphate ◽  
Whole Cell Patch Clamp ◽  
Current Activation ◽  
Ethanesulfonic Acid ◽  
Cl Conductance

Swelling-induced Cl- conductance in cultured rat epididymal cells was characterized using whole cell patch-clamp techniques. Activation of whole cell current with an outwardly rectifying current-potential relationship was observed in cells exposed to hyposmotic solutions. This current was determined, from the observed current-reversal potentials at different Cl- concentrations, to be Cl- selective. The anion selectivity sequence of the swelling-induced Cl- conductance was I- approximately NO3- approximately Br- > Cl- > 2-(N-morpholino)ethanesulfonic acid. The swelling-induced Cl- conductance was reversibly inhibited by different Cl- channel blockers. Unlike diphenylamine-2-carboxylate or 5-nitro-2-(3-phenylpropylamino)-benzoate, which showed voltage-independent blockade, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid showed a marked voltage-dependent blockade of the volume-sensitive Cl- current, with a greater effect at depolarizing voltages. The swelling-induced Cl- conductance appeared to be different from the Ca(2+)- or adenosine 3',5'-cyclic monophosphate-activated Cl- conductances on the basis of the following observations: 1) swelling-induced current activation was seen even in the presence of kinase inhibitor (H-8) or absence of external free Ca2+, and 2) further increase in current activation could be produced by swelling after Ca(2+)- or adenosine 3',5'-cyclic monophosphate-induced current activation. The swelling-induced Cl- conductance may be involved in regulating epithelial cell volume as well as serving other important epididymal functions such as facilitating transepithelial secretion of organic compounds.

scholarly journals Acidification of Rat TRPV1 Alters the Kinetics of Capsaicin Responses

2005 ◽  
Vol 1 ◽  
pp. 1744-8069-1-28 ◽  
Author(s):  
Torben R Neelands ◽  
Michael F Jarvis ◽  
Ping Han ◽  
Connie R Faltynek ◽  
Carol S Surowy
Keyword(s):  
Acidic Ph ◽  
Hek 293 ◽  
Deactivation Rate ◽  
Vanilloid Receptor 1 ◽  
293 Cells ◽  
Activation Rate ◽  
Current Activation ◽  
Acidic Conditions ◽  
Tissue Acidosis ◽  
Trpv1 Antagonist

TRPV1 (vanilloid receptor 1) receptors are activated by a variety of ligands such as capsaicin, as well as by acidic conditions and temperatures above 42°C. These activators can enhance the potency of one another, shifting the activation curve for TRPV1 to the left. In this study, for example, we observed an approximately 10-fold shift in the capsaicin EC50 (640 nM to 45 nM) for rat TRPV1 receptors expressed in HEK-293 cells when the pH was lowered from 7.4 to 5.5. To investigate potential causes for this shift in capsaicin potency, the rates of current activation and deactivation of whole-cell currents were measured in individual cells exposed to treatments of pH 5.5, 1 μM capsaicin or in combination. Acidic pH was found to both increase the activation rate and decrease the deactivation rate of capsaicin-activated currents providing a possible mechanism for the enhanced potency of capsaicin under acidic conditions. Utilizing a paired-pulse protocol, acidic pH slowed the capsaicin deactivation rate and was readily reversible. Moreover, the effect could occur under modestly acidic conditions (pH 6.5) that did not directly activate TRPV1. When TRPV1 was maximally activated by capsaicin and acidic pH, the apparent affinity of the novel and selective capsaicin-site competitive TRPV1 antagonist, A-425619, was reduced ∼35 fold. This shift was overcome by reducing the capsaicin concentration co-applied with acidic pH. Since inflammation is associated with tissue acidosis, these findings enhance understanding of TRPV1 receptor responses in inflammatory pain where tissue acidosis is prevalent.

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.

A Novel Gain-of-Function KCND3 Variant Associated with Brugada Syndrome

Cardiology ◽  
2020 ◽  
Vol 145 (10) ◽  
pp. 623-632
Author(s):  
Xianqing Li ◽  
Zongzhe Li ◽  
Dao Wen Wen Wang ◽  
Dao Wu Wang ◽  
Yan Wang
Keyword(s):  
Patch Clamp ◽  
Brugada Syndrome ◽  
Critical Role ◽  
Gain Of Function ◽  
Α Subunit ◽  
Whole Cell ◽  
Electrophysiological Properties ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation

Brugada syndrome (BrS) is a known cause of sudden cardiac death (SCD) characterized by abnormal electrocardiograms and fatal arrhythmias. The variants in KCND3 encoding the KV4.3 potassium-channel (the α-subunit of the Ito) have seldom been reported in BrS. This study aimed to identify novel KCND3 variants associated with BrS and elucidate BrS pathogenesis. High-depth targeted sequencing was performed and the electrophysiological properties of the variants were detected by whole-cell patch-clamp methods in a cultured-cell expressing system. The transcriptional levels of KV4.3 in different genotypes were studied by real-time PCR. Western blot was used to assess channel protein expression. A novel KCND3heterozygous variant, c.1292G>A (Arg431His, R431H), was found in the proband. Whole-cell patch-clamp results revealed a gain-of-function phenotype in the variant, with peak Ito current density increased and faster recovery from inactivation. The expression of mutant Kv4.3 membrane protein increased and the cytoplasmic protein decreased, demonstrating that the membrane/cytoplasm ratio was significantly different. In conclusion, a novel KCND3 heterozygous variant was associated with BrS. The increased Ito current explained the critical role of KCND3 in the pathogenesis of BrS. Genetic screening for KCND3 could be useful for understanding the pathogenesis of BrS and providing effective risk stratification in the clinic.

Functional and pharmacological properties of canine ERG potassium channels

2003 ◽  
Vol 284 (1) ◽  
pp. H256-H267 ◽  
Author(s):  
Jixin Wang ◽  
Kimberly Della Penna ◽  
Hao Wang ◽  
Jerzy Karczewski ◽  
Thomas M. Connolly ◽  
...  
Keyword(s):  
Inward Rectification ◽  
Delayed Rectifier ◽  
Pharmacological Properties ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
Channel Opening ◽  
Maximum Activation ◽  
Voltage Dependent ◽  
Cardiac Delayed Rectifier ◽  
Action Potential Repolarization

We established HEK-293 cell lines that stably express functional canine ether-à-go-go-related gene (cERG) K+ channels and examined their biophysical and pharmacological properties with whole cell patch clamp and35S-labeled MK-499 ([35S]MK-499) binding displacement. Functionally, cERG current had the hallmarks of cardiac delayed rectifier K+ current ( I Kr). Channel opening was time- and voltage dependent with threshold near −40 mV. The half-maximum activation voltage was −7.8 ± 2.4 mV at 23°C, shifting to −31.9 ± 1.2 mV at 36°C. Channels activated with a time constant of 13 ± 1 ms at +20 mV, showed prominent inward rectification at depolarized potentials, were highly K+ selective (Na+-to-K+permeability ratio = 0.007), and were potently inhibited by I Kr blockers. Astemizole, terfenadine, cisapride, and MK-499 inhibited cERG and human ERG (hERG) currents with IC50 values of 1.3, 13, 19, and 15 nM and 1.2, 9, 14, and 21 nM, respectively, and competitively displaced [35S]MK-499 binding from cERG and hERG with IC50 values of 0.4, 12, 35, and 0.6 nM and 0.8, 5, 47, and 0.7 nM, respectively. cERG channels had biophysical properties appropriate for canine action potential repolarization and were pharmacologically sensitive to agents known to prolong QT. A novel MK-499 binding assay provides a new tool to detect agents affecting ERG channels.

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