scholarly journals Inactivation of Gating Currents of L-Type Calcium Channels

1998 ◽  
Vol 111 (6) ◽  
pp. 807-823 ◽  
Author(s):  
Roman Shirokov ◽  
Gonzalo Ferreira ◽  
Jianxun Yi ◽  
Eduardo Ríos
Keyword(s):  
Charge Movement ◽  
Ca Channels ◽  
Mobile Charge ◽  
Gating Currents ◽  
Recovery From Inactivation ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Separate Channel ◽  
Α2δ Subunit ◽  
Modal Change

In studies of gating currents of rabbit cardiac Ca channels expressed as α1C/β2a or α1C/β2a/α2δ subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489–517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863–895). Charge 1 (voltage of half-maximal transfer, V1/2 ≃ 0 mV) gates noninactivated channels, while charge 2 (V1/2 ≃ −90 mV) is generated in inactivated channels. In α1C/β2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant τ ≃ 8, while the available charge 2 decreased upon recovery from inactivation (at −200 mV) with τ ≃ 0.3 s. These processes therefore are much slower than charge movement, which takes <50 ms. This separation between the time scale of measurable charge movement and that of changes in their availability, which was even wider in the presence of α2δ, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the α2δ subunit did not change the V1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to τ ≃ 2 s), while slowing the reduction of charge 2 upon recovery (τ ≃ 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489–498) and inactivation as discrete modal change. The effects of α2δ are reproduced assuming that the subunit lowers the free energy of the inactivated mode.

scholarly journals Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels

1997 ◽  
Vol 110 (5) ◽  
pp. 579-589 ◽  
Author(s):  
Riccardo Olcese ◽  
Ramón Latorre ◽  
Ligia Toro ◽  
Francisco Bezanilla ◽  
Enrico Stefani
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Ionic Current ◽  
K Channel ◽  
Charge Movement ◽  
Slow Inactivation ◽  
Gating Currents ◽  
Similar Time ◽  
K Channels ◽  
Recovery From Inactivation

Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K+ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.

scholarly journals Effective gating charges per channel in voltage-dependent K+ and Ca2+ channels.

1996 ◽  
Vol 108 (3) ◽  
pp. 143-155 ◽  
Author(s):  
F Noceti ◽  
P Baldelli ◽  
X Wei ◽  
N Qin ◽  
L Toro ◽  
...  
Keyword(s):  
Ionic Current ◽  
Variance Analysis ◽  
Beta Subunit ◽  
Charge Movement ◽  
Gating Currents ◽  
Open Probability ◽  
K Channels ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Ca2 Channels

In voltage-dependent ion channels, the gating of the channels is determined by the movement of the voltage sensor. This movement reflects the rearrangement of the protein in response to a voltage stimulus, and it can be thought of as a net displacement of elementary charges (e0) through the membrane (z: effective number of elementary charges). In this paper, we measured z in Shaker IR (inactivation removed) K+ channels, neuronal alpha 1E and alpha 1A, and cardiac alpha 1C Ca2+ channels using two methods: (a) limiting slope analysis of the conductance-voltage relationship and (b) variance analysis, to evaluate the number of active channels in a patch, combined with the measurement of charge movement in the same patch. We found that in Shaker IR K+ channels the two methods agreed with a z congruent to 13. This suggests that all the channels that gate can open and that all the measured charge is coupled to pore opening in a strictly sequential kinetic model. For all Ca2+ channels the limiting slope method gave consistent results regardless of the presence or type of beta subunit tested (z = 8.6). However, as seen with alpha 1E, the variance analysis gave different results depending on the beta subunit used. alpha 1E and alpha 1E beta 1a gave higher z values (z = 14.77 and z = 15.13 respectively) than alpha 1E beta 2a (z = 9.50, which is similar to the limiting slope results). Both the beta 1a and beta 2a subunits, coexpressed with alpha 1E Ca2+ channels facilitated channel opening by shifting the activation curve to more negative potentials, but only the beta 2a subunit increased the maximum open probability. The higher z using variance analysis in alpha 1E and alpha 1E beta 1a can be explained by a set of charges not coupled to pore opening. This set of charges moves in transitions leading to nulls thus not contributing to the ionic current fluctuations but eliciting gating currents. Coexpression of the beta 2a subunit would minimize the fraction of nulls leading to the correct estimation of the number of channels and z.

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)

Slow inward Ca current in frog heart: theoretical evidence against a voltage-dependent inactivation

1982 ◽  
Vol 60 (9) ◽  
pp. 1185-1192 ◽  
Author(s):  
Rodolphe Fischmeister ◽  
Magda Horackova
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Frog Heart ◽  
Type Model ◽  
Actual Behavior ◽  
Ca Current ◽  
Recovery From Inactivation ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Theoretical Evidence

The validity of a Hodgkin–Huxley type voltage-dependent inactivation of slow inward Ca current (Isi) was tested in frog heart using a computer simulation. The time course of Isi, was calculated during the development of a frog atrial action potential (AP). With a time constant of inactivation (τf) of 55 ms at a membrane potential (Em) of –15 mV, the variation of Isi was biphasic; after a transient increase followed by a decrease to zero, Isi partially "reactivated" (at the beginning of the AP repolarization phase) and then fully deactivated. The "reactivation" phase of Isi developed whether τf was an increasing, decreasing, U-shaped, or bell-shaped function of Em. The addition of an independent and slower process responsible for the recovery from inactivation only partly suppressed the "reactivation" phase. However, until now there was no experimental evidence supporting such a biphasic variation of Isi during AP repolarization. Thus our results indicate that the Hodgkin–Huxley type model of the voltage-dependence of Isi-inactivation process may not correctly represent the actual behavior of frog cardiac muscle.

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.

scholarly journals Ion-dependent Inactivation of Barium Current through L-type Calcium Channels

1997 ◽  
Vol 109 (4) ◽  
pp. 449-461 ◽  
Author(s):  
Gonzalo Ferreira ◽  
Jianxun Yi ◽  
Eduardo Ríos ◽  
Roman Shirokov
Keyword(s):  
Ionic Current ◽  
Reversal Potential ◽  
Slow Phase ◽  
Gating Currents ◽  
Current Decay ◽  
Gating Charge ◽  
Long Duration ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Ca2 Channels

It is widely believed that Ba2+ currents carried through L-type Ca2+ channels inactivate by a voltage- dependent mechanism similar to that described for other voltage-dependent channels. Studying ionic and gating currents of rabbit cardiac Ca2+ channels expressed in different subunit combinations in tsA201 cells, we found a phase of Ba2+ current decay with characteristics of ion-dependent inactivation. Upon a long duration (20 s) depolarizing pulse, IBa decayed as the sum of two exponentials. The slow phase (τ ≈ 6 s, 21°C) was parallel to a reduction of gating charge mobile at positive voltages, which was determined in the same cells. The fast phase of current decay (τ ≈ 600 ms), involving about 50% of total decay, was not accompanied by decrease of gating currents. Its amplitude depended on voltage with a characteristic U-shape, reflecting reduction of inactivation at positive voltages. When Na+ was used as the charge carrier, decay of ionic current followed a single exponential, of rate similar to that of the slow decay of Ba2+ current. The reduction of Ba2+ current during a depolarizing pulse was not due to changes in the concentration gradients driving ion movement, because Ba2+ entry during the pulse did not change the reversal potential for Ba2+. A simple model of Ca2+-dependent inactivation (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1993. J. Gen. Physiol. 102:1005–1030) robustly accounts for fast Ba2+ current decay assuming the affinity of the inactivation site on the α1 subunit to be 100 times lower for Ba2+ than Ca2+.

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.

scholarly journals Mechanism of gating of T-type calcium channels.

1990 ◽  
Vol 96 (3) ◽  
pp. 603-630 ◽  
Author(s):  
C F Chen ◽  
P Hess
Keyword(s):  
Single Channel ◽  
Ca Channels ◽  
3T3 Fibroblasts ◽  
Channel Inactivation ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Single Burst ◽  
Opening And Closing ◽  
Rate Limiting ◽  
Kinetics Of

We have analyzed the gating kinetics of T-type Ca channels in 3T3 fibroblasts. Our results show that channel closing, inactivation, and recovery from inactivation each include a voltage-independent step which becomes rate limiting at extreme potentials. The data require a cyclic model with a minimum of two closed, one open, and two inactivated states. Such a model can produce good fits to our data even if the transitions between closed states are the only voltage-dependent steps in the activating pathway leading from closed to inactivated states. Our analysis suggests that the channel inactivation step, as well as the direct opening and closing transitions, are not intrinsically voltage sensitive. Single-channel recordings are consistent with this scheme. As expected, each channel produces a single burst per opening and then inactivates. Comparison of the kinetics of T-type Ca current in fibroblasts and neuronal cells reveals significant differences which suggest that different subtypes of T-type Ca channels are expressed differentially in a tissue specific manner.

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