scholarly journals State-Dependent Inactivation of the α1g T-Type Calcium Channel

1999 ◽  
Vol 114 (2) ◽  
pp. 185-202 ◽  
Author(s):  
Jose R. Serrano ◽  
Edward Perez-Reyes ◽  
Stephen W. Jones
Keyword(s):  
Kinetic Model ◽  
Hek 293 ◽  
Voltage Sensors ◽  
State Dependent ◽  
State Recovery ◽  
Recovery From Inactivation ◽  
293 Cells ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Kinetics Of

We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the α1G channel, with symmetrical Na+i and Na+o and 2 mM Ca2+o. After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential −100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from −120 to −70 mV \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}({\mathrm{e-fold\;for}}\;31\;{\mathrm{mV}};\;{\mathrm{{\tau}}}\;=\;2.5\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation*}\end{document}, but \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}{\mathrm{{\tau}}}\;=\;12{\raisebox{1mm}{\line(1,0){6}}}17\;{\mathrm{ms\;from}}-40\;{\mathrm{to}}\;+60\;{\mathrm{mV}}\end{equation*}\end{document}. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100–300 ms, inactivation was strong but incomplete (∼98%). Inactivation was also produced by long, weak depolarizations \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}({\mathrm{{\tau}}}\;=\;220\;{\mathrm{ms\;at}}\;-80\;{\mathrm{mV}};\;{\mathrm{V}}_{1/2}\;=\;-82\;{\mathrm{mV}})\end{equation*}\end{document}, which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}({\mathrm{{\tau}}}\;=\;85\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation*}\end{document}, but weakly voltage dependent. Recovery was similar after 60-ms steps to −20 mV or 600-ms steps to −70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at −100 mV during recovery from inactivation, consistent with ≤8% of the channels recovering through the open state. The results are well described by a kinetic model where inactivation is allosterically coupled to the movement of the first three voltage sensors to activate. One consequence of state-dependent inactivation is that α1G channels continue to inactivate after repolarization, primarily from the open state, which leads to cumulative inactivation during repetitive pulses.

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.

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 Arachidonic acid inhibition of L-type calcium (CaV1.3b) channels varies with accessory CaVβ subunits

2009 ◽  
Vol 133 (4) ◽  
pp. 387-403 ◽  
Author(s):  
Mandy L. Roberts-Crowley ◽  
Ann R. Rittenhouse
Keyword(s):  
Arachidonic Acid ◽  
Enzyme Activation ◽  
Membrane Excitability ◽  
Recovery From Inactivation ◽  
293 Cells ◽  
Dependent Inactivation ◽  
Accessory Subunits ◽  
Kinetics Of ◽  
Ca2 Channels

Arachidonic acid (AA) inhibits the activity of several different voltage-gated Ca2+ channels by an unknown mechanism at an unknown site. The Ca2+ channel pore-forming subunit (CaVα1) is a candidate for the site of AA inhibition because T-type Ca2+ channels, which do not require accessory subunits for expression, are inhibited by AA. Here, we report the unanticipated role of accessory CaVβ subunits on the inhibition of CaV1.3b L-type (L-) current by AA. Whole cell Ba2+ currents were measured from recombinant channels expressed in human embryonic kidney 293 cells at a test potential of −10 mV from a holding potential of −90 mV. A one-minute exposure to 10 µM AA inhibited currents with β1b, β3, or β4 58, 51, or 44%, respectively, but with β2a only 31%. At a more depolarized holding potential of −60 mV, currents were inhibited to a lesser degree. These data are best explained by a simple model where AA stabilizes CaV1.3b in a deep closed-channel conformation, resulting in current inhibition. Consistent with this hypothesis, inhibition by AA occurred in the absence of test pulses, indicating that channels do not need to open to become inhibited. AA had no effect on the voltage dependence of holding potential–dependent inactivation or on recovery from inactivation regardless of CaVβ subunit. Unexpectedly, kinetic analysis revealed evidence for two populations of L-channels that exhibit willing and reluctant gating previously described for CaV2 channels. AA preferentially inhibited reluctant gating channels, revealing the accelerated kinetics of willing channels. Additionally, we discovered that the palmitoyl groups of β2a interfere with inhibition by AA. Our novel findings that the CaVβ subunit alters kinetic changes and magnitude of inhibition by AA suggest that CaVβ expression may regulate how AA modulates Ca2+-dependent processes that rely on L-channels, such as gene expression, enzyme activation, secretion, and membrane excitability.

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 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.

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 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.

Characterization of the Ca2+ current in freshly dissociated crustacean peptidergic neuronal somata

1995 ◽  
Vol 73 (6) ◽  
pp. 2357-2368 ◽  
Author(s):  
J. E. Richmond ◽  
E. Sher ◽  
I. M. Cooke
Keyword(s):  
Slow Component ◽  
Neuronal Somata ◽  
Current Increase ◽  
Inactivation Curve ◽  
Test Pulse ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation ◽  
Dependent Inactivation ◽  
Na Currents ◽  
Voltage Dependent

1. Freshly dissociated neuronal somata of the crab (Cardisoma carnifex) X-organ were studied in the whole cell patch-clamp configuration. To characterize the Ca2+ currents in these somata, recordings were made under conditions designed to suppress K+ and Na+ currents. 2. In 52 mM external Ca2+ the threshold for activation of Ca2+ currents was above -40 mV, with peak amplitudes occurring around +10 to +20 mV. The full component of the current was available for activation at -50 mV because no current increase was observed when the holding potential was increased to -90 mV. These characteristics of the current characterize it as a high-voltage activated (HVA) current. 3. The Ca2+ current was almost completely (60-90%) inactivated within 200 ms at maximal current potentials (+10 to +20 mV). The decay was best described by a double-exponential function with a fast and slow component of inactivation (tau f = 12 ms and tau s = 64 ms). Both Sr2+ and Ba2+ substitutions reduced the rates of inactivation. 4. In double-pulse experiments, plots of variable prepulse potential versus test pulse current produced a U-shaped curve with test pulse currents showing maximal inactivation at potentials that produced maximal Ca2+ influx during the prepulse. Tail currents also displayed a U-shaped inactivation curve. The extent of current-dependent inactivation was sequentially reduced by Sr2+ and Ba2+ substitutions. These data suggest that inactivation in crab somata is predominantly Ca2+ dependent. The remaining inactivation of Ba2+ currents suggests that there is also a component of voltage-dependent inactivation in the somata. 5. Part of the inactivated Ca2+ current could be recovered during short (4-10 ms) hyperpolarizing pulses to -130 mV. The absolute extent of recovery from inactivation was greatest for currents carried by Ca2+ rather than Sr2+ or Ba2+. When voltage-dependent inactivation was dominant (Ba2+ currents), the relative amount of current recovered was greater. The data suggest that hyperpolarizing pulses are more effective in removing voltage-dependent inactivation, but also allow some recovery from Ca(2+)-dependent inactivation. 6. In the crab saline, which contained 24 mM Mg2+, the amplitudes of currents carried by 52 mM Ca2+, Sr2+ and Ba2+ were similar. Removing the Mg2+ from the saline augmented both the Ba2+ and Sr2+ currents relative to the Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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