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 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.

scholarly journals Fast Inactivation in Shaker K+ Channels

1998 ◽  
Vol 111 (5) ◽  
pp. 625-638 ◽  
Author(s):  
Michel J. Roux ◽  
Riccardo Olcese ◽  
Ligia Toro ◽  
Francisco Bezanilla ◽  
Enrico Stefani
Keyword(s):  
Time Course ◽  
Ionic Current ◽  
Fast Inactivation ◽  
Gating Current ◽  
Gating Currents ◽  
Current Decay ◽  
Gating Charge ◽  
External Potassium ◽  
Kinetics Of ◽  
Full Charge

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as “charge immobilization” (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567–590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at −90 mV return potential changed from a single fast component to at least two components, the slower requiring ∼200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at −120 and −90 mV. In contrast, at higher potentials (−70 and −50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of “parallel” inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH2-terminus region.

scholarly journals Extracellular Mg2+ Modulates Slow Gating Transitions and the Opening of Drosophila Ether-à-Go-Go Potassium Channels

2000 ◽  
Vol 115 (3) ◽  
pp. 319-338 ◽  
Author(s):  
Chih-Yung Tang ◽  
Francisco Bezanilla ◽  
Diane M. Papazian
Keyword(s):  
Time Course ◽  
Ionic Current ◽  
Ionic Currents ◽  
K Channel ◽  
Gating Currents ◽  
Gating Charge ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Sequential Activation

We have characterized the effects of prepulse hyperpolarization and extracellular Mg2+ on the ionic and gating currents of the Drosophila ether-à-go-go K+ channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg2+ dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg2+ modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg2+ slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg2+ modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg2+. We have also identified mutations in the S3–S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg2+, indicating an important role for this region in the voltage-dependent activation of eag.

scholarly journals A Complex of RIM2alpha and RIM-Binding Protein 2 Stabilizes Slow Voltage-Dependent Inactivation of Cochlear Inner Hair Cell Cav1.3 L-Type Ca2+ Channels

2018 ◽  
Vol 114 (3) ◽  
pp. 637a-638a
Author(s):  
Nadine J. Ortner ◽  
Alexandra Pinggera ◽  
Anita Siller ◽  
Nadja Hofer ◽  
Niels Brandt ◽  
...  
Keyword(s):  
Hair Cell ◽  
Binding Protein ◽  
Inner Hair Cell ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Ca2 Channels

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.

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 The Voltage Sensor in Voltage-Dependent Ion Channels

2000 ◽  
Vol 80 (2) ◽  
pp. 555-592 ◽  
Author(s):  
Francisco Bezanilla
Keyword(s):  
Membrane Potential ◽  
Conformational Changes ◽  
Structural Information ◽  
Noise Analysis ◽  
Voltage Sensor ◽  
Fluorescence Labeling ◽  
Gating Current ◽  
Gating Currents ◽  
Gating Charge ◽  
Voltage Dependent

In voltage-dependent Na, K, or Ca channels, the probability of opening is modified by the membrane potential. This is achieved through a voltage sensor that detects the voltage and transfers its energy to the pore to control its gate. We present here the theoretical basis of the energy coupling between the electric field and the voltage, which allows the interpretation of the gating charge that moves in one channel. Movement of the gating charge constitutes the gating current. The properties are described, along with macroscopic data and gating current noise analysis, in relation to the operation of the voltage sensor and the opening of the channel. Structural details of the voltage sensor operation were resolved initially by locating the residues that make up the voltage sensor using mutagenesis experiments and determining the number of charges per channel. The changes in conformation are then analyzed based on the differential exposure of cysteine or histidine-substituted residues. Site-directed fluorescence labeling is then analyzed as another powerful indicator of conformational changes that allows time and voltage correlation of local changes seen by the fluorophores with the global change seen by the electrophysiology of gating currents and ionic currents. Finally, we describe the novel results on lanthanide-based resonance energy transfer that show small distance changes between residues in the channel molecule. All of the electrophysiological and the structural information are finally summarized in a physical model of a voltage-dependent channel in which a change in membrane potential causes rotation of the S4 segment that changes the exposure of the basic residues from an internally connected aqueous crevice at hyperpolarized potentials to an externally connected aqueous crevice at depolarized potentials.

scholarly journals Fast and slow voltage sensor rearrangements during activation gating in Kv1.2 channels detected using tetramethylrhodamine fluorescence

2010 ◽  
Vol 136 (1) ◽  
pp. 83-99 ◽  
Author(s):  
Andrew James Horne ◽  
Christian Joseph Peters ◽  
Thomas William Claydon ◽  
David Fedida
Keyword(s):  
Conformational Changes ◽  
Time Course ◽  
Slow Component ◽  
Ionic Current ◽  
Voltage Sensor ◽  
Activation Process ◽  
Dependent Manner ◽  
Gating Currents ◽  
Kv Channel ◽  
Voltage Dependent

The Kv1.2 channel, with its high resolution crystal structure, provides an ideal model for investigating conformational changes associated with channel gating, and fluorescent probes attached at the extracellular end of S4 are a powerful way to gain a more complete understanding of the voltage-dependent activity of these dynamic proteins. Tetramethylrhodamine-5-maleimide (TMRM) attached at A291C reports two distinct rearrangements of the voltage sensor domains, and a comparative fluorescence scan of the S4 and S3–S4 linker residues in Shaker and Kv1.2 shows important differences in their emission at other homologous residues. Kv1.2 shows a rapid decrease in A291C emission with a time constant of 1.5 ± 0.1 ms at 60 mV (n = 11) that correlates with gating currents and reports on translocation of the S4 and S3–S4 linker. However, unlike any Kv channel studied to date, this fast component is dwarfed by a larger, slower quenching of TMRM emission during depolarizations between −120 and −50 mV (τ = 21.4 ± 2.1 ms at 60 mV, V1/2 of −73.9 ± 1.4 mV) that is not seen in either Shaker or Kv1.5 and that comprises >60% of the total signal at all activating potentials. The slow fluorescence relaxes after repolarization in a voltage-dependent manner that matches the time course of Kv1.2 ionic current deactivation. Fluorophores placed directly in S1 and S2 at I187 and T219 recapitulate the time course and voltage dependence of slow quenching. The slow component is lost when the extracellular S1–S2 linker of Kv1.2 is replaced with that of Kv1.5 or Shaker, suggesting that it arises from a continuous internal rearrangement within the voltage sensor, initiated at negative potentials but prevalent throughout the activation process, and which must be reversed for the channel to close.

Sign in / Sign up

Export Citation Format

Share Document