scholarly journals Permeation and Gating in CaV3.1 (α1G) T-type Calcium Channels Effects of Ca2+, Ba2+, Mg2+, and Na+

2008 ◽  
Vol 132 (2) ◽  
pp. 223-238 ◽  
Author(s):  
Nilofar Khan ◽  
I. Patrick Gray ◽  
Carlos A. Obejero-Paz ◽  
Stephen W. Jones
Keyword(s):  
Calcium Channels ◽  
Surface Charge ◽  
Concentration Dependence ◽  
Divalent Cations ◽  
Hek 293 ◽  
Voltage Range ◽  
293 Cells ◽  
Instantaneous Current ◽  
Voltage Dependent ◽  
Inward Currents

We examined the concentration dependence of currents through CaV3.1 T-type calcium channels, varying Ca2+ and Ba2+ over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current–voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by PCa/PNa = 87 and PCa/PBa = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent Kd values were similar for Ca2+ and Ba2+, both for block of currents carried by Na+ (3 μM for Ca2+ vs. 4 μM for Ba2+, at −30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca2+ vs. 2.5 mM for Ba2+; nearly voltage independent). Block by 3–10 μM Ca2+ was time dependent, described by bimolecular kinetics with binding at ∼3 × 108 M−1s−1 and voltage-dependent exit. Ca2+o, Ba2+o, and Mg2+o also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e− per 98 Å2 from Gouy-Chapman theory. Additionally, inward currents inactivated ∼35% faster in Ba2+o (vs. Ca2+o or Na+o). The accelerated inactivation in Ba2+o correlated with the transition from Na+ to Ba2+ permeation, suggesting that Ba2+o speeds inactivation by occupying the pore. We conclude that the selectivity of the “surface charge” among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca2+ or Ba2+.

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

2005 ◽  
Vol 103 (6) ◽  
pp. 1156-1166 ◽  
Author(s):  
Kevin J. Gingrich ◽  
Son Tran ◽  
Igor M. Nikonorov ◽  
Thomas J. Blanck
Keyword(s):  
Charge Transfer ◽  
Calcium Channels ◽  
Dependent Manner ◽  
Current Time ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Dependent Inactivation ◽  
Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

Determination of Pure Voltage-Dependent Ca2+ Current in Paramecium Caudatum and its Inhibition by Divalent Cations

1985 ◽  
Vol 119 (1) ◽  
pp. 321-334
Author(s):  
FRANK WEHNER ◽  
EILO HILDEBRAND
Keyword(s):  
Dissociation Constants ◽  
Divalent Cations ◽  
Equilibrium Potential ◽  
Ca Channel ◽  
Paramecium Caudatum ◽  
Voltage Range ◽  
Outward Currents ◽  
Voltage Dependent ◽  
Inward Currents

Voltage-dependent Ca2+ currents in Paramecium caudatum were studied under voltage clamp conditions. To separate Ca2+ inward currents from concomitant K+ outward currents, the voltage-dependent Ca2+ conductance was temporarily inactivated by a preceding depolarization. The remaining currents were then subtracted from the overall currents measured in the absence of a prepulse. In this way pure Ca2+ currents could be obtained up to a depolarization of 100 mV, which is about 50 mV below the theoretical Ca2+ equilibrium potential (Eca). Ca2+ currents were maximal at a depolarization of 35 mV and declined with further approach to Eca, but they did not reverse sign in the voltage range tested. In the presence of Mg2+, Co2+, Mn2+ or Ni2+, the Ca2+ inward currents decreased to a different extent. From experiments where these cations were added at different concentrations and from measurements at different Ca2+ concentrations in the absence of other divalent cations the following ratio of apparent dissociation constants could be derived: kNi: kco: kca: kMg = 1:3:4.3-4.7:5:6.5. With a confidence of 95% the absolute value of kca lies between 40 and 130μmol l−1. These results indicate that Ca2+ and other divalent cations compete for binding sites at the Ca-channel and thus determine excitability. Indirect effects due to changes of the surface potential are discussed.

scholarly journals Ca2+ entry through NaV channels generates submillisecond axonal Ca2+ signaling

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Naomi AK Hanemaaijer ◽  
Marko A Popovic ◽  
Xante Wilders ◽  
Sara Grasman ◽  
Oriol Pavón Arocas ◽  
...  
Keyword(s):  
Calcium Channels ◽  
High Speed ◽  
Initial Segment ◽  
Hek 293 ◽  
Voltage Gated ◽  
Voltage Gated Calcium Channels ◽  
293 Cells ◽  
Axonal Initial Segment ◽  
Nav Channels ◽  
Activity Dependent

Calcium ions (Ca2+) are essential for many cellular signaling mechanisms and enter the cytosol mostly through voltage-gated calcium channels. Here, using high-speed Ca2+ imaging up to 20 kHz in the rat layer five pyramidal neuron axon we found that activity-dependent intracellular calcium concentration ([Ca2+]i) in the axonal initial segment was only partially dependent on voltage-gated calcium channels. Instead, [Ca2+]i changes were sensitive to the specific voltage-gated sodium (NaV) channel blocker tetrodotoxin. Consistent with the conjecture that Ca2+ enters through the NaV channel pore, the optically resolved ICa in the axon initial segment overlapped with the activation kinetics of NaV channels and heterologous expression of NaV1.2 in HEK-293 cells revealed a tetrodotoxin-sensitive [Ca2+]i rise. Finally, computational simulations predicted that axonal [Ca2+]i transients reflect a 0.4% Ca2+ conductivity of NaV channels. The findings indicate that Ca2+ permeation through NaV channels provides a submillisecond rapid entry route in NaV-enriched domains of mammalian axons.

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.

scholarly journals Overexpression of T-type calcium channels in HEK-293 cells increases intracellular calcium without affecting cellular proliferation

FEBS Letters ◽  
2000 ◽  
Vol 478 (1-2) ◽  
pp. 166-172 ◽  
Author(s):  
Jean Chemin ◽  
Arnaud Monteil ◽  
Christelle Briquaire ◽  
Sylvain Richard ◽  
Edward Perez-Reyes ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Intracellular Calcium ◽  
Cellular Proliferation ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Identification of PDZ Domain Containing Proteins Interacting with 1.2 and PMCA4b

2013 ◽  
Vol 2013 ◽  
pp. 1-16 ◽  
Author(s):  
Doreen Korb ◽  
Priscilla Y. Tng ◽  
Vladimir M. Milenkovic ◽  
Nadine Reichhart ◽  
Olaf Strauss ◽  
...  
Keyword(s):  
Pdz Domain ◽  
Pdz Domains ◽  
Hek 293 ◽  
Zonula Occludens ◽  
C Terminus ◽  
293 Cells ◽  
Interaction Partners ◽  
Voltage Dependent ◽  
Interaction Domains ◽  
Zonula Occludens 1

PDZ (PSD-95/Disc large/Zonula occludens-1) protein interaction domains bind to cytoplasmic protein C-termini of transmembrane proteins. In order to identify new interaction partners of the voltage-gated L-type Ca2+ channel 1.2 and the plasma membrane Ca2+ ATPase 4b (PMCA4b), we used PDZ domain arrays probing for 124 PDZ domains. We confirmed this by GST pull-downs and immunoprecipitations. In PDZ arrays, strongest interactions with 1.2 and PMCA4b were found for the PDZ domains of SAP-102, MAST-205, MAGI-1, MAGI-2, MAGI-3, and ZO-1. We observed binding of the 1.2 C-terminus to PDZ domains of NHERF1/2, Mint-2, and CASK. PMCA4b was observed to interact with Mint-2 and its known interactions with Chapsyn-110 and CASK were confirmed. Furthermore, we validated interaction of 1.2 and PMCA4b with NHERF1/2, CASK, MAST-205 and MAGI-3 via immunoprecipitation. We also verified the interaction of 1.2 and nNOS and hypothesized that nNOS overexpression might reduce Ca2+ influx through 1.2. To address this, we measured Ca2+ currents in HEK 293 cells co-expressing 1.2 and nNOS and observed reduced voltage-dependent 1.2 activation. Taken together, we conclude that 1.2 and PMCA4b bind promiscuously to various PDZ domains, and that our data provides the basis for further investigation of the physiological consequences of these interactions.

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.

Voltage-dependent facilitation of cardiac L-type Ca channels expressed in HEK-293 cells requires β-subunit

2000 ◽  
Vol 278 (1) ◽  
pp. H126-H136 ◽  
Author(s):  
Timothy J. Kamp ◽  
Hai Hu ◽  
Eduardo Marban
Keyword(s):  
G Protein ◽  
Somatostatin Receptors ◽  
Ca Channel ◽  
Β Subunit ◽  
Ca Channels ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Protein Inhibition ◽  
293 Cells ◽  
Voltage Dependent

The activity of native L-type Ca channels can be facilitated by strong depolarizations. The cardiac Ca channel α1C-subunit was transiently expressed in human embryonic kidney (HEK-293) cells, but these channels did not exhibit voltage-dependent facilitation. Coexpression of the Ca channel β1a- or β2a-subunit with the α1C-subunit enabled voltage-dependent facilitation in 40% of cells tested. The onset of facilitation in α1C + β1a-expressing HEK-293 cells was rapid after a depolarization to +100 mV (τ = 7.0 ms). The kinetic features of the facilitated currents were comparable to those observed for voltage-dependent relief of G protein inhibition demonstrated for many neuronal Ca channels; however, intracellular dialysis with guanosine 5′- O-(2-thiodiphosphate) and guanosine 5′- O-(3-thiotriphosphate) in the patch pipette had no effect on facilitation. Stimulation of G protein-coupled receptors, either endogenous (somatostatin receptors) or coexpressed (adenosine A1receptors), did not affect voltage-dependent facilitation. These results indicate that the cardiac Ca channel α1C-subunit can exhibit voltage-dependent facilitation in HEK-293 cells only when coexpressed with an auxiliary β-subunit and that this facilitation is independent of G protein pathways.

KCNQ4 channels expressed in mammalian cells: functional characteristics and pharmacology

2001 ◽  
Vol 280 (4) ◽  
pp. C859-C866 ◽  
Author(s):  
Rikke Søgaard ◽  
Trine Ljungstrøm ◽  
Kamilla Angelo Pedersen ◽  
Søren-Peter Olesen ◽  
Bo Skaaning Jensen
Keyword(s):  
Mammalian Cells ◽  
Inward Rectification ◽  
Excitable Cells ◽  
Independent Manner ◽  
Hek 293 ◽  
M Current ◽  
Gating Charge ◽  
293 Cells ◽  
Boltzmann Function ◽  
Instantaneous Current

Human cloned KCNQ4 channels were stably expressed in HEK-293 cells and characterized with respect to function and pharmacology. Patch-clamp measurements showed that the KCNQ4 channels conducted slowly activating currents at potentials more positive than −60 mV. From the Boltzmann function fitted to the activation curve, a half-activation potential of −32 mV and an equivalent gating charge of 1.4 elementary charges was determined. The instantaneous current-voltage relationship revealed strong inward rectification. The KCNQ4 channels were blocked in a voltage-independent manner by the memory-enhancing M current blockers XE-991 and linopirdine with IC50 values of 5.5 and 14 μM, respectively. The antiarrhythmic KCNQ1 channel blocker bepridil inhibited KCNQ4 with an IC50 value of 9.4 μM, whereas clofilium was without significant effect at 100 μM. The KCNQ4-expressing cells exhibited average resting membrane potentials of −56 mV in contrast to −12 mV recorded in the nontransfected cells. In conclusion, the activation and pharmacology of KCNQ4 channels resemble those of M currents, and it is likely that the function of the KCNQ4 channel is to regulate the subthreshold electrical activity of excitable cells.

scholarly journals TRPM7 Provides an Ion Channel Mechanism for Cellular Entry of Trace Metal Ions

2002 ◽  
Vol 121 (1) ◽  
pp. 49-60 ◽  
Author(s):  
Mahealani K. Monteilh-Zoller ◽  
Meredith C. Hermosura ◽  
Monica J.S. Nadler ◽  
Andrew M. Scharenberg ◽  
Reinhold Penner ◽  
...  
Keyword(s):  
Trace Metal ◽  
Ion Channel ◽  
Metal Ions ◽  
Metal Ion ◽  
Divalent Cations ◽  
Divalent Metal Ions ◽  
Hek 293 ◽  
Trace Metal Ions ◽  
293 Cells ◽  
Better Than

Trace metal ions such as Zn2+, Fe2+, Cu2+, Mn2+, and Co2+ are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca2+- and Mg2+-permeable cation channel, whose activity is regulated by intracellular Mg2+ and Mg2+·ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide–regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn2+ and Ni2+, which both permeate TRPM7 up to four times better than Ca2+. Similarly, native MagNuM currents are also able to support Zn2+ entry. Furthermore, TRPM7 allows other essential metals such as Mn2+ and Co2+ to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd2+, Ba2+, and Sr2+. Equimolar replacement studies substituting 10 mM Ca2+ with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn2+ ≈ Ni2+ >> Ba2+ > Co2+ > Mg2+ ≥ Mn2+ ≥ Sr2+ ≥ Cd2+ ≥ Ca2+, while trivalent ions such as La3+ and Gd3+ are not measurably permeable. With the exception of Mg2+, which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn2+, Co2+, or Ni2+ suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca2+ and Mg2+, suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.

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