scholarly journals Calcium currents in the A7r5 smooth muscle-derived cell line. An allosteric model for calcium channel activation and dihydropyridine agonist action.

1992 ◽  
Vol 99 (3) ◽  
pp. 367-390 ◽  
Author(s):  
T N Marks ◽  
S W Jones
Keyword(s):  
Calcium Channel ◽  
Cell Line ◽  
Molecular Mechanisms ◽  
Calcium Currents ◽  
Single Channels ◽  
Channel Activation ◽  
Simple Assumption ◽  
Voltage Dependent ◽  
A7r5 Cell Line ◽  
Kinetics Of

We have investigated the gating kinetics of calcium channels in the A7r5 cell line at the level of single channels and whole cell currents, in the absence and presence of dihydropyridine (DHP) calcium channel agonists. Although latencies to first opening and macroscopic currents are strongly voltage dependent, analysis of amplitude histograms indicates that the primary open-closed transition is voltage independent. This suggests that the molecular mechanisms for voltage sensing and channel opening are distinct, but coupled. We propose a modified Monod-Wyman-Changeux (MWC) model for channel activation, where movement of a voltage sensor is analogous to ligand binding, and the closed and open channels correspond to inactive (T) and active (R) states. This model can account for the activation kinetics of the calcium channel, and is consistent with the existence of four homologous domains in the main subunit of the calcium channel protein. DHP agonists slow deactivation kinetics, shift the activation curve to more negative potentials with an increase in slope, induce intermingled fast and slow channel openings, and reduce the latency to first opening. These effects are predicted by the MWC model if we make the simple assumption that DHP agonists act as allosteric effectors to stabilize the open states of the channel.

G-Protein-Modulated Ca2+ Current With Slowed Activation Does Not Alter the Kinetics of Action Potential-Evoked Ca2+ Current

2000 ◽  
Vol 84 (5) ◽  
pp. 2417-2425 ◽  
Author(s):  
Debra E. Artim ◽  
Stephen D. Meriney
Keyword(s):  
Action Potential ◽  
Calcium Channel ◽  
G Protein ◽  
Calcium Current ◽  
Time Course ◽  
Channel Activation ◽  
Tail Current ◽  
Voltage Dependent ◽  
Dependent Inhibition ◽  
Kinetics Of

We have studied voltage-dependent inhibition of N-type calcium currents to investigate the effects of G-protein modulation-induced alterations in channel gating on action potential-evoked calcium current. In isolated chick ciliary ganglion neurons, GTPγS produced voltage-dependent inhibition that exhibited slowed activation kinetics and was partially relieved by a conditioning prepulse. Using step depolarizations to evoke calcium current, we measured tail current amplitudes on abrupt repolarization to estimate the time course of calcium channel activation from 1 to 30 ms. GTPγS prolonged significantly channel activation, consistent with the presence of kinetic slowing in the modulated whole cell current evoked by 100-ms steps. Since kinetic slowing is caused by an altered voltage dependence of channel activation (such that channels require stronger or longer duration depolarization to open), we asked if GTPγS-induced modulation would alter the time course of calcium channel activation during an action potential. Using an action potential waveform as a voltage command to evoke calcium current, we abruptly repolarized to −80 mV at various time points during the repolarization phase of the action potential. The resulting tail current was used to estimate the relative number of calcium channels that were open. Using action potential waveforms of either 2.2- or 6-ms duration at half-amplitude, there were no differences in the time course of calcium channel activation, or in the percent activation at any time point tested during the repolarization, when control and modulated currents were compared. It is also possible that modulated channels might open briefly and that these reluctant openings would effect the time course of action potential-evoked calcium current. However, when control and modulated currents were scaled to the same peak amplitude and superimposed, there was no difference in the kinetics of the two currents. Thus voltage-dependent inhibition did not alter the kinetics of action potential-evoked current. These results suggest that G-protein-modulated channels do not contribute significantly to calcium current evoked by a single action potential.

Dopamine Modulation of Calcium Currents in Pyloric Neurons of the Lobster Stomatogastric Ganglion

2003 ◽  
Vol 90 (2) ◽  
pp. 631-643 ◽  
Author(s):  
Bruce R. Johnson ◽  
Peter Kloppenburg ◽  
Ronald M. Harris-Warrick
Keyword(s):  
Calcium Channel ◽  
Transmitter Release ◽  
Stomatogastric Ganglion ◽  
Calcium Currents ◽  
Voltage Gated ◽  
Pyloric Network ◽  
Pyloric Dilator ◽  
Voltage Dependent ◽  
Inward Currents ◽  
Dopamine Modulation

We examined the dopamine (DA) modulation of calcium currents (ICa) that could contribute to the plasticity of the pyloric network in the lobster stomatogastric ganglion. Pyloric somata were voltage-clamped under conditions designed to block voltage-gated Na+, K+, and H currents. Depolarizing steps from –60 mV generated voltage-dependent, inward currents that appeared to originate in electrotonically distal, imperfectly clamped regions of the cell. These currents were blocked by Cd2+ and enhanced by Ba2+ but unaffected by Ni2+. Dopamine enhanced the peak ICa in the pyloric constrictor (PY), lateral pyloric (LP), and inferior cardiac (IC) neurons and reduced peak ICa in the ventricular dilator (VD), pyloric dilator (PD), and anterior burster (AB) neurons. All of these effects, except for the AB, are consistent with DA's excitation or inhibition of firing in the pyloric neurons. Enhancement of ICa in PY and LP neurons and reduction of ICa in VD and PD neurons are also consistent with DA-induced synaptic strength changes via modulation of presynaptic ICa. However, the reduction of ICa in AB suggests that DA's enhancement of AB transmitter release is not directly mediated through presynaptic ICa. ICa in PY and PD neurons was more sensitive to nifedipine block than in AB neurons. In addition, nifedipine blocked DA's effects on ICa in the PY and PD neurons but not in the AB neuron. Thus the contribution of specific calcium channel subtypes carrying the total ICa may vary between pyloric neuron classes, and DA may act on different calcium channel subtypes in the different pyloric neurons.

A background sodium conductance is necessary for spontaneous depolarizations in rat pituitary cell line GH3

1994 ◽  
Vol 266 (3) ◽  
pp. C709-C719 ◽  
Author(s):  
S. M. Simasko
Keyword(s):  
Cell Line ◽  
Calcium Current ◽  
Membrane Conductance ◽  
Potassium Currents ◽  
Pituitary Cell ◽  
Calcium Currents ◽  
Gh3 Cells ◽  
Sodium Conductance ◽  
Rat Pituitary ◽  
Voltage Dependent

The role of Na+ in the expression of membrane potential activity in the clonal rat pituitary cell line GH3 was investigated using the perforated patch variation of patch-clamp electrophysiological techniques. It was found that replacing bath Na+ with choline, tris(hydroxymethyl)aminomethane (Tris), or N-methyl-D-glucamine (NMG) caused the cells to hyperpolarize 20-30 mV. Tetrodotoxin had no effect. The effects of the Na+ substitutes could not be explained by effects on potassium or calcium currents. Although all three Na+ substitutes suppressed voltage-dependent calcium current by 10-20%, block of voltage-dependent calcium current by nifedipine or Co2+ did not result in hyperpolarization of the cells. There was no effect of the Na+ substitutes on voltage-dependent potassium currents. In contrast, all three Na+ substitutes influenced calcium-activated potassium currents [IK(Ca)], but only at depolarized potentials. Choline consistently suppressed IK(Ca), whereas Tris and NMG either had no effect or slightly increased IK(Ca). These effects on IK(Ca) also cannot explain the hyperpolarization induced by removing bath Na+. Choline always hyperpolarized cells yet suppressed IK(Ca). Furthermore, removing bath Na+ caused an increase in cell input resistance, an observation consistent with the loss of a membrane conductance as the basis of the hyperpolarization. Direct measurement of background currents revealed a 12-pA inward current at -84 mV that was lost upon removing bath Na+. These results suggest that this background sodium conductance provides the depolarizing drive for GH3 cells to reach the threshold for firing calcium-dependent action potentials.

scholarly journals Coupling of voltage-dependent gating and Ba++ block in the high-conductance, Ca++-activated K+ channel.

1987 ◽  
Vol 90 (3) ◽  
pp. 427-449 ◽  
Author(s):  
C Miller ◽  
R Latorre ◽  
I Reisin
Keyword(s):  
Lipid Bilayers ◽  
Dissociation Rate ◽  
K Channel ◽  
Single Channels ◽  
Closed Channel ◽  
Association Rate ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
Kinetics Of ◽  
High Conductance

Voltage-dependent Ca++-activated K+ channels from rat skeletal muscle were reconstituted into planar lipid bilayers, and the kinetics of block of single channels by Ba++ were studied. The Ba++ association rate varies linearly with the probability of the channel being open, while the dissociation rate follows a rectangular hyperbolic relationship with open-state probability. Ba ions can be occluded within the channel by closing the channel with a strongly hyperpolarizing voltage applied during a Ba++-blocked interval. Occluded Ba ions cannot dissociate from the blocking site until after the channel opens. The ability of the closed channel to occlude Ba++ is used as an assay to study the channel's gating equilibrium in the blocked state. The blocked channel opens and closes in a voltage-dependent process similar to that of the unblocked channel. The presence of a Ba ion destabilizes the closed state of the blocked channel, however, by 1.5 kcal/mol. The results confirm that Ba ions block this channel by binding in the K+-conduction pathway. They further show that the blocking site is inaccessible to Ba++ from both the cytoplasmic and external solutions when the channel is closed.

scholarly journals Amino acid substitutions in the FXYD motif enhance phospholemman-induced modulation of cardiac L-type calcium channels

2010 ◽  
Vol 299 (5) ◽  
pp. C1203-C1211 ◽  
Author(s):  
Kai Guo ◽  
Xianming Wang ◽  
Guofeng Gao ◽  
Congxin Huang ◽  
Keith S. Elmslie ◽  
...  
Keyword(s):  
Amino Acid ◽  
Calcium Channels ◽  
Molecular Mechanisms ◽  
Channel Gating ◽  
Fine Tuning ◽  
Amino Acid Substitutions ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Kinetics Of

We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149–1159, 2010). The short 17 amino acid extracellular NH2-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate CaV1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with CaV1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on CaV1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca2+ dynamics in the heart.

scholarly journals Calcium currents in the A7r5 smooth muscle-derived cell line. Calcium-dependent and voltage-dependent inactivation.

1991 ◽  
Vol 98 (5) ◽  
pp. 987-1003 ◽  
Author(s):  
B Giannattasio ◽  
S W Jones ◽  
A Scarpa
Keyword(s):  
Smooth Muscle ◽  
Cell Line ◽  
Short Pulse ◽  
Reversal Potential ◽  
Calcium Currents ◽  
Recovery From Inactivation ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Rat Thoracic Aorta ◽  
Rapid Current

Inactivation of a dihydropyridine-sensitive calcium current was studied in a cell line (A7r5) derived from smooth muscle of the rat thoracic aorta. Inactivation is faster with extracellular Ca2+ than with Ba2+. In Ba2+, inactivation increases monotonically with depolarization. In Ca2+, inactivation is related to the amount of inward current, so that little inactivation is seen in Ca2+ for brief depolarizations approaching the reversal potential. Longer depolarizations in Ca2+ reveal two components of inactivation, the slower component behaving like that observed in Ba2+. Furthermore, lowering extracellular Ca2+ slows inactivation. These results are consistent with the coexistence of two inactivation processes, a slow voltage-dependent inactivation, and a more rapid current-dependent inactivation which is observable only with Ca2+. Ca(2+)-dependent inactivation is decreased but not eliminated when intracellular Ca2+ is buffered by 10 mM BAPTA, suggesting that Ca2+ acts at a site on or near the channel. We also studied recovery from inactivation after either a short pulse (able to produce significant inactivation only in Ca2+) or a long pulse (giving similar inactivation with either cation). Surprisingly, recovery from Ca(2+)-dependent inactivation was voltage dependent. This suggests that the pathways for recovery from inactivation are similar regardless of how inactivation is generated. We propose a model where Ca(2+)- and voltage-dependent inactivation occur independently.

Multiple calcium currents in a thyroid C-cell line: biophysical properties and pharmacology

1991 ◽  
Vol 260 (6) ◽  
pp. C1253-C1263 ◽  
Author(s):  
B. A. Biagi ◽  
J. J. Enyeart
Keyword(s):  
Cell Line ◽  
Time Constant ◽  
Calcium Currents ◽  
C Cells ◽  
Patch Clamp Technique ◽  
C Cell ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Rat Thyroid ◽  
Ca2 Channels

The whole cell version of the patch-clamp technique was used to characterize voltage-gated Ca2+ channels in the calcitonin-secreting rat thyroid C-cell line 6-23 (clone 6). Three types of Ca2+ channels could be distinguished based on differences in voltage dependence, kinetics, and pharmacological sensitivity. T-type current was half-maximal at -31 mV, showed steady-state voltage-dependent inactivation that was half-maximal at -57 mV, inactivated with a voltage-dependent time constant that reached a minimum of 20 ms at potentials positive to -20 mV, and deactivated with a single time constant of approximately 2 ms at -80 mV. Reactivation of inactivated channels occurred with a time constant of 1.26 s at -90 mV. T current was selectively blocked by Ni2+ at concentrations between 5 and 50 microM. La3+ and Y3+ blocked the T current at 10- to 20-fold lower concentrations. Dihydropyridine-sensitive L-type current was half-maximal at a test potential of -3 mV and was approximately doubled in size when Ba2+ replaced Ca2+ as the charge carrier. Unlike L-type Ca2+ current in many cells, this current in C-cells displayed little Ca(2+)-dependent inactivation. N-type current was composed of inactivating and sustained components that were inhibited by omega-conotoxin. The inactivating component was half-maximal at +9 mV and could be fitted by two exponentials with time constants of 22 and 142 ms. A slow inactivation of N current with a time constant of 24.9 s was observed upon switching the holding potential from -80 to -40 mV. These results demonstrate that, similar to other neural crest derived cells, thyroid C-cells express multiple Ca2+ channels, including one previously observed only in neurons.

Altered Voltage Dependent Calcium Currents in a Neuronal Cell Line Derived From the Cerebral Cortex of a Trisomy 16 Fetal Mouse, an Animal Model of Down Syndrome

2011 ◽  
Vol 22 (1) ◽  
pp. 59-68 ◽  
Author(s):  
Mario A. Acuña ◽  
Ramón Pérez-Nuñez ◽  
Jorge Noriega ◽  
Ana María Cárdenas ◽  
Juan Bacigalupo ◽  
...  
Keyword(s):  
Animal Model ◽  
Down Syndrome ◽  
Cerebral Cortex ◽  
Cell Line ◽  
Neuronal Cell ◽  
Calcium Currents ◽  
Trisomy 16 ◽  
Fetal Mouse ◽  
Neuronal Cell Line ◽  
Voltage Dependent

Functional Expression of L-, N-, P/Q-, and R-Type Calcium Channels in the Human NT2-N Cell Line

2000 ◽  
Vol 84 (6) ◽  
pp. 2933-2944 ◽  
Author(s):  
Torben R. Neelands ◽  
Anthony P. J. King ◽  
Robert L. Macdonald
Keyword(s):  
Calcium Channel ◽  
Cell Line ◽  
Functional Expression ◽  
Calcium Currents ◽  
Channel Blockers ◽  
Patch Clamp Technique ◽  
Teratocarcinoma Cell ◽  
Inactivation Curve ◽  
Whole Cell ◽  
Channel Currents

The biophysical and pharmacological properties of voltage-gated calcium channel currents in the human teratocarcinoma cell line NT2-N were studied using the whole cell patch-clamp technique. When held at −80 mV, barium currents ( I Bas) were evoked by voltage commands to above −35 mV that peaked at +5 mV. When holding potentials were reduced to −20 mV or 5 mM barium was substituted for 5 mM calcium, there was a reduction in peak currents and a right shift in the current-voltage curve. A steady-state inactivation curve for I Ba was fit with a Boltzmann curve ( V 1/2 = −43.3 mV; slope = −17.7 mV). Maximal current amplitude increased from 1-wk (232 pA) to 9-wk (1025 pA) postdifferentiation. Whole cell I Bas were partially blocked by specific channel blockers to a similar extent in 1- to 3-wk and 7- to 9-wk postdifferentiation NT2-N cells: 10 μM nifedipine (19 vs. 25%), 10 μM conotoxin GVIA (27 vs. 25%), 10 μM conotoxin MVIIC (15 vs. 16%), and 1.75 μM SNX-482 (31 vs. 33%). Currents were completely blocked by 300 μM cadmium. In the presence of nifedipine, GVIA, and MVIIC, ∼35% of current remained, which was reduced further by SNX-482 (7–14% of current remained), consistent with functional expression of L-, N-, and P/Q-calcium channel types and one or more R-type channel. The presence of multiple calcium currents in this human neuronal-type cell line provides a potentially useful model for study of the regulation, expression and cellular function of human derived calcium channel currents; in particular the R-type current(s).

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