scholarly journals Differential Occurrence of Reluctant Openings in G-Protein–Inhibited N- and P/Q-Type Calcium Channels

2000 ◽  
Vol 115 (2) ◽  
pp. 175-192 ◽  
Author(s):  
Henry M. Colecraft ◽  
Parag G. Patil ◽  
David T. Yue
Keyword(s):  
Calcium Channels ◽  
G Protein ◽  
Action Potentials ◽  
Qualitative Difference ◽  
Hek 293 ◽  
Protein Inhibition ◽  
Voltage Dependent ◽  
Dependent Inhibition ◽  
Time Courses ◽  
Prepulse Facilitation

Voltage-dependent inhibition of N- and P/Q-type calcium channels by G proteins is crucial for presynaptic inhibition of neurotransmitter release, and may contribute importantly to short-term synaptic plasticity. Such calcium-channel modulation could thereby impact significantly the neuro-computational repertoire of neural networks. The differential modulation of N and P/Q channels could even further enrich their impact upon synaptic tuning. Here, we performed in-depth comparison of the G-protein inhibition of recombinant N and P/Q channels, expressed in HEK 293 cells with the m2 muscarinic receptor. While both channel types display classic features of G-protein modulation (kinetic slowing of activation, prepulse facilitation, and voltage dependence of inhibition), we confirmed previously reported quantitative differences, with N channels displaying stronger inhibition and greater relief of inhibition by prepulses. A more fundamental, qualitative difference in the modulation of these two channels was revealed by a modified tail-activation paradigm, as well as by a novel “slope” analysis method comparing time courses of slow activation and prepulse facilitation. The stark contrast in modulatory behavior can be understood within the context of the “willing–reluctant” model, in which binding of G-protein βγ subunits to channels induces a reluctant mode of gating, where stronger depolarization is required for opening. Our experiments suggest that only N channels could be opened in the reluctant mode, at voltages normally spanned by neuronal action potentials. By contrast, P/Q channels appear to remain closed, especially over these physiological voltages. Further, the differential occurrence of reluctant openings is not explained by differences in the rate of G-protein unbinding from the two channels. These two scenarios predict very different effects of G-protein inhibition on the waveform of Ca2+ entry during action potentials, with potentially important consequences for the timing and efficacy of synaptic transmission.

Relative contributions of G protein, channel, and receptor to voltage-dependent inhibition of neuronal N-type and P/Q-type calcium channels in HEK 293 cell lines

1997 ◽  
Vol 239 (2-3) ◽  
pp. 89-92 ◽  
Author(s):  
Brian A McCool ◽  
Michael M Harpold ◽  
Kenneth A Stauderman ◽  
Paul F Brust ◽  
David M Lovinger
Keyword(s):  
Calcium Channels ◽  
G Protein ◽  
Cell Lines ◽  
Hek 293 ◽  
Voltage Dependent ◽  
Dependent Inhibition ◽  
Protein Channel

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.

Neurotransmitters acting via different G proteins inhibit N-type calcium current by an identical mechanism in rat sympathetic neurons

1995 ◽  
Vol 74 (6) ◽  
pp. 2251-2257 ◽  
Author(s):  
I. Ehrlich ◽  
K. S. Elmslie
Keyword(s):  
Calcium Channels ◽  
G Protein ◽  
G Proteins ◽  
Calcium Current ◽  
Voltage Dependence ◽  
Sympathetic Neurons ◽  
Patch Clamp Technique ◽  
Voltage Dependent ◽  
Dependent Inhibition ◽  
Kinetics Of

1. We studied the mechanism of voltage-dependent inhibition of N-type calcium current by norepinephrine (NE) and vasoactive intestinal peptide (VIP) in adult rat superior cervical ganglion (SCG) neurons using the whole cell patch-clamp technique. 2. The voltage dependence of inhibition is manifest in the reversal of inhibition by strong depolarization. We tested the hypothesis that this voltage dependence results from disruption of G proteins binding to calcium channels. According to this hypothesis, the kinetics of calcium current reinhibition following a strong depolarization should become faster for higher concentrations of active G proteins. 3. Assuming that larger inhibitions result from higher concentrations of active G proteins, we used different concentrations of NE to alter the amplitude of inhibition and, thus, the active G protein concentration. We found that the kinetics of reinhibition at -80 mV following a depolarizing pulse to +80 mV were faster for larger inhibitions. 4. VIP induces voltage-dependent inhibition of N-current via a different G protein (Gs) than that of NE (Go). We found that the effect of VIP on reinhibition kinetics was identical to that produced by NE. 5. Combined application of NE and VIP did not greatly increase the amplitude of the inhibition but significantly increased the rate of reinhibition. Thus NE plus VIP appear to greatly increase the concentration of the molecule binding to the channel (G protein according to the hypothesis). 6. The kinetics of calcium current disinhibition during strong depolarization (step to +80 mV) did not change with the size of the inhibition induced by NE, VIP or application of NE and VIP together. 7. Both the concentration-dependent reinhibition kinetics and concentration-independent disinhibition kinetics are consistent with the hypothesis that active G proteins bind directly to N-type calcium channels to modulate their activity in rat sympathetic neurons.

scholarly journals Elementary events underlying voltage-dependent G-protein inhibition of N-type calcium channels

1996 ◽  
Vol 71 (5) ◽  
pp. 2509-2521 ◽  
Author(s):  
P.G. Patil ◽  
M. de Leon ◽  
R.R. Reed ◽  
S. Dubel ◽  
T.P. Snutch ◽  
...  
Keyword(s):  
Calcium Channels ◽  
G Protein ◽  
Protein Inhibition ◽  
Voltage Dependent

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.

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