scholarly journals K(+) currents in cultured neurones from a polyclad flatworm

2000 ◽  
Vol 203 (20) ◽  
pp. 3189-3198
Author(s):  
S.D. Buckingham ◽  
A.N. Spencer
Keyword(s):  
Steady State ◽  
Patch Clamp ◽  
K Channels ◽  
Type K ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation ◽  
Steady State Inactivation ◽  
The Brain ◽  
K Currents ◽  
Cultured Neurones

Cells from the brain of the polyclad flatworm Notoplana atomata were dispersed and maintained in primary culture for up to 3 weeks. Whole-cell patch-clamp of presumed neurones revealed outwardly directed K(+) currents that comprised, in varying proportions, a rapidly activating (time constant tau =0.94+/−0.79 ms; N=15) and inactivating (tau =26.1+/−1.9 ms; N=22) current and a second current that also activated rapidly (tau =1.1+/−0.2 ms; N=9) (means +/− s.e.m.) but did not inactivate within 100 ms. Both current types activated over similar voltage ranges. Activation and steady-state inactivation overlap and are markedly rightward-shifted compared with most Shaker-like currents (half-activation of 16.9+/−1. 9 mV, N=7, half-inactivation of −35.4+/−3.0 mV, N=5). Recovery from inactivation was rapid (50+/−2.5 ms at −90 mV). Both currents were unaffected by tetraethylammonium (25 mmol l(−1)), whereas 4-aminopyridine (10 mmol l(−1)) selectively blocked the inactivating current. The rapidly inactivating current, like cloned K(+) channels from cnidarians and certain cloned K(+) channels from molluscs and the Kv3 family of vertebrate channels, differed from most A-type K(+) currents reported to date. These findings suggest that K(+) currents in Notoplana atomata play novel roles in shaping excitability properties.

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 mechanism of the releasing action of amphetamine. Uptake, superfusion, and electrophysiological studies on transporter-transfected cells

2000 ◽  
Vol 72 (6) ◽  
pp. 1045-1050 ◽  
Author(s):  
C. Pifl ◽  
H. H Sitte ◽  
H. Reither ◽  
E. A. Singer
Keyword(s):  
Patch Clamp ◽  
Molecular Targets ◽  
Monoamine Transporters ◽  
Transfected Cells ◽  
Whole Cell Patch Clamp ◽  
Sodium Dependent ◽  
Electrophysiological Studies ◽  
Inward Currents ◽  
Vesicular Monoamine Transporters ◽  
The Brain

Amphetamine analogues are able to induce signs of neurotoxicity in the brain. In order to understand this type of neurotoxicity, the interaction of amphetamine with its molecular targets must be elucidated. These molecular targets are plasmalemmal and vesicular monoamine transporters. We investigated the interaction of amphetamine with these transporters in cells transfected with the respective cDNA. Superfusion and whole-cell, patch-clamp experiments were performed, and the toxicity of substrates of the transporters was studied. Amphetamine was taken up by dopamine transporter-expressing cells in a sodium-dependent and cocaine-blockable manner. Furthermore, it elicited inward currents in these cells concentration-dependently. Correlation of uptake, release, and patch-clamp experiments suggest that ion fluxes induced by substrate-gating on transporters may significantly contribute to the releasing action of amphetamine and of other transporter substrates. Dopamine accumulation into serotoninergic terminals depleted of serotonin by 3,4-methylenedioxymethamphetamine was discussed as a mechanism of Ecstasy-toxicity. This is in agreement with a toxic effect of intracellular dopamine which could be demonstrated on our transporter-overexpressing cells. These results, apart from their relevance for the toxicity by amphetamine analogues, may also have bearings on the mechanisms in neurodegenerative diseases affecting monoamine transmitters.

The TTX metabolite 4,9-anhydro-TTX is a highly specific blocker of the Nav1.6 voltage-dependent sodium channel

2007 ◽  
Vol 293 (2) ◽  
pp. C783-C789 ◽  
Author(s):  
Christian Rosker ◽  
Birgit Lohberger ◽  
Doris Hofer ◽  
Bibiane Steinecker ◽  
Stefan Quasthoff ◽  
...  
Keyword(s):  
Steady State ◽  
Sodium Channels ◽  
Therapeutic Intervention ◽  
Time Course ◽  
Specific Interaction ◽  
Xenopus Laevis Oocytes ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Invaluable Tool

The blocking efficacy of 4,9-anhydro-TTX (4,9-ah-TTX) and TTX on several isoforms of voltage-dependent sodium channels, expressed in Xenopus laevis oocytes, was tested (Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, and Nav1.8). Generally, TTX was 40–231 times more effective, when compared with 4,9-ah-TTX, on a given isoform. An exception was Nav1.6, where 4,9-ah-TTX in nanomole per liter concentrations sufficed to result in substantial block, indicating that 4,9-ah-TTX acts specifically at this peculiar isoform. The IC50 values for TTX/4,9-ah-TTX were as follows (in nmol/l): 7.8 ± 1.3/1,260 ± 121 (Nav1.2), 2.8 ± 2.3/341 ± 36 (Nav1.3), 4.5 ± 1.0/988 ± 62 (Nav1.4), 1,970 ± 565/78,500 ± 11,600 (Nav1.5), 3.8 ± 1.5/7.8 ± 2.3 (Nav1.6), 5.5 ± 1.4/1,270 ± 251 (Nav1.7), and 1,330 ± 459/>30,000 (Nav1.8). Analysis of approximal half-maximal doses of both compounds revealed minor effects on voltage-dependent activation only, whereas steady-state inactivation was shifted to more negative potentials by both TTX and 4,9-ah-TTX in the case of the Nav1.6 subunit, but not in the case of other TTX-sensitive ones. TTX shifted steady-state inactivation also to more negative potentials in case of the TTX-insensitive Nav1.5 subunit, where it also exerted profound effects on the time course of recovery from inactivation. Isoform-specific interaction of toxins with ion channels is frequently observed in the case of proteinaceous toxins. Although the sensitivity of Nav1.1 to 4,9-ah-TTX is not known, here we report evidence on a highly isoform-specific TTX analog that may well turn out to be an invaluable tool in research for the identification of Nav1.6-mediated function, but also for therapeutic intervention.

ANG II blocks potassium currents in zona glomerulosa cells from rat, bovine, and human adrenals

1991 ◽  
Vol 260 (5) ◽  
pp. E772-E779 ◽  
Author(s):  
U. Brauneis ◽  
P. M. Vassilev ◽  
S. J. Quinn ◽  
G. H. Williams ◽  
D. L. Tillotson
Keyword(s):  
Single Channel ◽  
Calcium Influx ◽  
Membrane Resistance ◽  
Zona Glomerulosa ◽  
Ang Ii ◽  
K Channels ◽  
Channel Interaction ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
K Currents

Angiotensin II (ANG II) is a principal secretagogue of adrenal zona glomerulosa (ZG) cells. The transduction process includes a depolarization of the plasma membrane and the activation of calcium influx. The ANG II-induced depolarization is associated with an increase in total membrane resistance. To directly address the mechanism underlying these observations, we examined the effect of ANG II on K+ currents of rat, bovine, and human ZG cells, using whole cell patch clamp. Although some differences were seen in the characteristics of K+ currents between species, ANG II consistently blocked outward currents in ZG cells [rat: 47.1 +/- 4.5% (SE), n = 17; bovine: 38.6 +/- 3.3%, n = 21; and human: 13-63%, n = 3]. With the use of the cell-attached mode, single-channel recordings in bovine ZG cells demonstrated K+ channels that were reversibly blocked when ANG II was added to the bath solution. This indicates that the block of K+ channels by ANG II involves a diffusible intracellular messenger rather than a direct receptor-channel interaction. The decreased conductance of K+ can account for the ANG II-induced membrane depolarization.

Electrophysiological characteristics of cloned skeletal and cardiac muscle sodium channels

1996 ◽  
Vol 271 (2) ◽  
pp. H498-H506 ◽  
Author(s):  
M. Chahine ◽  
I. Deschene ◽  
L. Q. Chen ◽  
R. G. Kallen
Keyword(s):  
Steady State ◽  
Sodium Channels ◽  
H Infinity ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Current Decay ◽  
Voltage Gated Sodium Channels ◽  
Recovery From Inactivation ◽  
Steady State Inactivation ◽  
Kinetics Of

The alpha-subunit encoding for voltage-gated sodium channels rSkM1 (rat skeletal muscle subtype 1) and hH1 (human heart subtype 1) has been cloned and expressed by various groups under various conditions in Xenopus oocytes and the tsA201 (HEK 293) mammalian cell line derived from human embryonic kidney cells. In this study, we have expressed hH1 and rSkM1 in tsA201 cells for comparison under the same conditions using patch-clamp methods. Our results show significant differences in the current-voltage (I-V) relationship, kinetics of current decay, voltage dependence of steady-state inactivation, and the time constant for recovery from inactivation. We studied several rSkM1/hH1 chimeric sodium channels to identify the structural regions responsible for the different biophysical behavior of the two channel subtypes. Exchanging the interdomain (ID3-4) loops, thought to contain the inactivation particle, between rSkM1 and hH1 had no effect on the electrophysiological behaviors, including inactivation, indicating that the differences in channel subtype characteristics are determined by parts of the channel other than the ID3-4 segment. The data on a chimeric channel in which D1 and D4 are derived from hH1 while D2 and D3 and the ID1-2, ID2-3, and ID3-4 loops are from rSkM1 show that D1 and/or D4 seem to be responsible for the slower kinetics of inactivation of hH1 while D2 and/or D3 appear to contain the determinants for the differences in the I-V relationship, steady-state inactivation (h infinity) curve, and the kinetics of the recovery from inactivation.

scholarly journals p.L1612P, a Novel Voltage-gated Sodium Channel Nav1.7 Mutation Inducing a Cold Sensitive Paroxysmal Extreme Pain Disorder

2015 ◽  
Vol 122 (2) ◽  
pp. 414-423 ◽  
Author(s):  
Marc R. Suter ◽  
Zahurul A. Bhuiyan ◽  
Cédric J. Laedermann ◽  
Thierry Kuntzer ◽  
Muriel Schaller ◽  
...  
Keyword(s):  
Steady State ◽  
Sodium Channel ◽  
Cold Exposure ◽  
Pain Disorder ◽  
Clinical Genetic ◽  
Voltage Gated Sodium Channel ◽  
Voltage Gated ◽  
Recovery From Inactivation ◽  
Steady State Inactivation ◽  
Extreme Pain

Abstract Background: Mutations in the SCN9A gene cause chronic pain and pain insensitivity syndromes. We aimed to study clinical, genetic, and electrophysiological features of paroxysmal extreme pain disorder (PEPD) caused by a novel SCN9A mutation. Methods: Description of a 4-generation family suffering from PEPD with clinical, genetic and electrophysiological studies including patch clamp experiments assessing response to drug and temperature. Results: The family was clinically comparable to those reported previously with the exception of a favorable effect of cold exposure and a lack of drug efficacy including with carbamazepine, a proposed treatment for PEPD. A novel p.L1612P mutation in the Nav1.7 voltage-gated sodium channel was found in the four affected family members tested. Electrophysiologically the mutation substantially depolarized the steady–state inactivation curve (V1/2 from −61.8 ± 4.5 mV to −30.9 ± 2.2 mV, n = 4 and 7, P &lt; 0.001), significantly increased ramp current (from 1.8% to 3.4%, n = 10 and 12) and shortened recovery from inactivation (from 7.2 ± 5.6 ms to 2.2 ± 1.5 ms, n = 11 and 10). However, there was no persistent current. Cold exposure reduced peak current and prolonged recovery from inactivation in wild-type and mutated channels. Amitriptyline only slightly corrected the steady–state inactivation shift of the mutated channel, which is consistent with the lack of clinical benefit. Conclusions: The novel p.L1612P Nav1.7 mutation expands the PEPD spectrum with a unique combination of clinical symptoms and electrophysiological properties. Symptoms are partially responsive to temperature but not to drug therapy. In vitro trials of sodium channel blockers or temperature dependence might help predict treatment efficacy in PEPD.

scholarly journals The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. II. Closed state reverse use-dependent block by 4-aminopyridine.

1993 ◽  
Vol 101 (4) ◽  
pp. 603-626 ◽  
Author(s):  
D L Campbell ◽  
Y Qu ◽  
R L Rasmusson ◽  
H C Strauss
Keyword(s):  
Steady State ◽  
Right Ventricular ◽  
Ventricular Myocytes ◽  
Patch Clamp Technique ◽  
Time Constants ◽  
Closed State ◽  
Whole Cell Patch Clamp ◽  
Dependent Behavior ◽  
Steady State Inactivation ◽  
K Current

Block of the calcium-independent transient outward K+ current, I(to), by 4-aminopyridine (4-AP) was studied in ferret right ventricular myocytes using the whole cell patch clamp technique. 4-AP reduces I(to) through a closed state blocking mechanism displaying "reverse use-dependent" behavior that was inferred from: (a) development of tonic block at hyperpolarized potentials; (b) inhibition of development of tonic block at depolarized potentials; (c) appearance of "crossover phenomena" in which the peak current is delayed in the presence of 4-AP at depolarized potentials; (d) relief of block at depolarized potentials which is concentration dependent and parallels steady-state inactivation for low 4-AP concentrations (V1/2 approximately -10 mV in 0.1 mM 4-AP) and steady-state activation at higher concentrations (V1/2 = +7 mV in 1 mM 4-AP, +15 mV in 10 mM 4-AP); and (e) reassociation of 4-AP at hyperpolarized potentials. No evidence for interaction of 4-AP with either the open or inactivated state of the I(to) channel was obtained from measurements of kinetics of recovery and deactivation in the presence of 0.5-1.0 mM 4-AP. At hyperpolarized potentials (-30 to -90 mV) 10 mM 4-AP associates slowly (time constants ranging from approximately 800 to 1,300 ms) with the closed states of the channel (apparent Kd approximately 0.2 mM). From -90 to -20 mV the affinity of the I(to) channel for 4-AP appears to be voltage insensitive; however, at depolarized potentials (+20 to +100 mV) 4-AP dissociates with time constants ranging from approximately 350 to 150 ms. Consequently, the properties of 4-AP binding to the I(to) channel undergo a transition in the range of potentials over which channel activation and inactivation occurs (-30 to +20 mV). We propose a closed state model of I(to) channel gating and 4-AP binding kinetics, in which 4-AP binds to three closed states. In this model 4-AP has a progressively lower affinity as the channel approaches the open state, but has no intrinsic voltage dependence of binding.

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.

scholarly journals The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. I. Basic characterization and kinetic analysis.

1993 ◽  
Vol 101 (4) ◽  
pp. 571-601 ◽  
Author(s):  
D L Campbell ◽  
R L Rasmusson ◽  
Y Qu ◽  
H C Strauss
Keyword(s):  
Steady State ◽  
Ventricular Myocytes ◽  
Nonlinear Least Squares ◽  
Patch Clamp Technique ◽  
Time Constants ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
K Current ◽  
Necessary And Sufficient

Enzymatically isolated myocytes from ferret right ventricles (12-16 wk, male) were studied using the whole cell patch clamp technique. The macroscopic properties of a transient outward K+ current I(to) were quantified. I(to) is selective for K+, with a PNa/PK of 0.082. Activation of I(to) is a voltage-dependent process, with both activation and inactivation being independent of Na+ or Ca2+ influx. Steady-state inactivation is well described by a single Boltzmann relationship (V1/2 = -13.5 mV; k = 5.6 mV). Substantial inactivation can occur during a subthreshold depolarization without any measurable macroscopic current. Both development of and recovery from inactivation are well described by single exponential processes. Ensemble averages of single I(to) channel currents recorded in cell-attached patches reproduce macroscopic I(to) and indicate that inactivation is complete at depolarized potentials. The overall inactivation/recovery time constant curve has a bell-shaped potential dependence that peaks between -10 and -20 mV, with time constants (22 degrees C) ranging from 23 ms (-90 mV) to 304 ms (-10 mV). Steady-state activation displays a sigmoidal dependence on membrane potential, with a net aggregate half-activation potential of +22.5 mV. Activation kinetics (0 to +70 mV, 22 degrees C) are rapid, with I(to) peaking in approximately 5-15 ms at +50 mV. Experiments conducted at reduced temperatures (12 degrees C) demonstrate that activation occurs with a time delay. A nonlinear least-squares analysis indicates that three closed kinetic states are necessary and sufficient to model activation. Derived time constants of activation (22 degrees C) ranged from 10 ms (+10 mV) to 2 ms (+70 mV). Within the framework of Hodgkin-Huxley formalism, Ito gating can be described using an a3i formulation.

scholarly journals Lamin-A/C variants found in patients with cardiac conduction disease reduce sodium currents

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Michael A. Olaopa ◽  
Katherine G. Spoonamore ◽  
Deepak Bhakta ◽  
Zhenhui Chen ◽  
Patricia B.S. Celestino-Soper ◽  
...  
Keyword(s):  
Steady State ◽  
Patch Clamp ◽  
Missense Variant ◽  
Cardiac Conduction ◽  
Lamin A ◽  
Lmna Gene ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Steady State Inactivation

Variants in the LMNA gene, which encodes Lamin-A/C, have been commonly associated with cardiac conduction system diseases usually accompanying cardiomyopathy. We have seen two unrelated patients who presented with atrioventricular block (AVB) with or without cardiomyopathy. Genetic testing identified the LMNA missense variant c.1634G>A (p.R545H) and the single nucleotide deletion c.859delG (p.A287Lfs*193). The deletion leads to a shift in the reading frame and subsequent protein truncation. Since impaired Nav1.5 function has been reported to cause AVB, we sought to investigate the effects of abnormal Lamins on Nav1.5 in HEK-293 cells using patch-clamp methods. Patch-clamp studies showed that p.R545H decreased the peak INa by approximately 70%. The voltage-dependency of steady state inactivation was rightward shifted in the cells transfected with p.R545H. The p.A287Lfs*193 also decreased the peak INa by approximately 62%. The voltagedependency of steady state inactivation was rightward shifted in the cells transfected with p.A287Lfs*193. Variants of the LMNA gene caused significant reduction of the peak INa in HEK-293 cells, which may account for the patients’ AVB.

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