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 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 Mutations reveal voltage gating of CNGA1 channels in saturating cGMP

2009 ◽  
Vol 134 (2) ◽  
pp. 151-164 ◽  
Author(s):  
Juan Ramón Martínez-François ◽  
Yanping Xu ◽  
Zhe Lu
Keyword(s):  
Ion Selectivity ◽  
Selectivity Filter ◽  
Open Probability ◽  
K Channels ◽  
Voltage Gated ◽  
Open Conformation ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Low Probability ◽  
Inherent Voltage

Activity of cyclic nucleotide–gated (CNG) cation channels underlies signal transduction in vertebrate visual receptors. These highly specialized receptor channels open when they bind cyclic GMP (cGMP). Here, we find that certain mutations restricted to the region around the ion selectivity filter render the channels essentially fully voltage gated, in such a manner that the channels remain mostly closed at physiological voltages, even in the presence of saturating concentrations of cGMP. This voltage-dependent gating resembles the selectivity filter-based mechanism seen in KcsA K+ channels, not the S4-based mechanism of voltage-gated K+ channels. Mutations that render CNG channels gated by voltage loosen the attachment of the selectivity filter to its surrounding structure, thereby shifting the channel's gating equilibrium toward closed conformations. Significant pore opening in mutant channels occurs only when positive voltages drive the pore from a low-probability open conformation toward a second open conformation to increase the channels' open probability. Thus, the structure surrounding the selectivity filter has evolved to (nearly completely) suppress the expression of inherent voltage-dependent gating of CNGA1, ensuring that the binding of cGMP by itself suffices to open the channels at physiological voltages.

scholarly journals Heterologous expression of BI Ca2+ channels in dysgenic skeletal muscle.

1994 ◽  
Vol 104 (5) ◽  
pp. 985-996 ◽  
Author(s):  
B A Adams ◽  
Y Mori ◽  
M S Kim ◽  
T Tanabe ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Time Constant ◽  
Ionic Current ◽  
Ionic Currents ◽  
Rabbit Brain ◽  
Charge Movement ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Whole Cell Patch Clamp ◽  
Ca2 Channels

We have examined the ability of BI (class A) Ca2+ channels, cloned from rabbit brain, to mediate excitation-contraction (E-C) coupling in skeletal muscle. Expression plasmids carrying cDNA encoding BI channels were microinjected into the nuclei of dysgenic mouse myotubes grown in primary culture. Ionic currents and intramembrane charge movements produced by the BI channels were recorded using the whole-cell patch-clamp technique. Injected myotubes expressed high densities of ionic BI Ca2+ channel current (average 31 pA/pF) but did not display spontaneous contractions, and only very rarely displayed evoked contractions. The expressed ionic current was pharmacologically distinguished from the endogenous L-type current of dysgenic skeletal muscle (Idys) by its insensitivity to the dihydropyridine antagonist (+)-PN 200-110. Peak BI Ca2+ currents activated with a time constant (tau a) of approximately 2 ms and inactivated with a time constant (tau h) of approximately 260 ms (20-23 degrees C). The time constant of inactivation (tau h) was not increased by substituting Ba2+ for Ca2+ as charge carrier, demonstrating that BI channels expressed in dysgenic myotubes do not undergo Ca(2+)-dependent inactivation. The average maximal Ca2+ conductance (Gmax) produced by the BI channels was quite large (approximately 534 S/F). In contrast, the average maximal charge movement (Qmax) produced in the same myotubes (approximately 2.7 nC/microF) was quite small, being barely larger than Qmax in control dysgenic myotubes (approximately 2.3 nC/microF). Thus, the ratio Gmax/Qmax for the BI channels was considerably higher than previously found for cardiac or skeletal muscle L-type Ca2+ channels expressed in the same system, indicating that neuronal BI Ca2+ channels exhibit a much higher open probability than these L-type Ca2+ channels.

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 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 Mutations of Nonconserved Residues within the Calcium Channel α1-interaction Domain Inhibit β-Subunit Potentiation

2008 ◽  
Vol 132 (3) ◽  
pp. 383-395 ◽  
Author(s):  
Giovanni Gonzalez-Gutierrez ◽  
Erick Miranda-Laferte ◽  
David Naranjo ◽  
Patricia Hidalgo ◽  
Alan Neely
Keyword(s):  
Calcium Channels ◽  
Consensus Sequence ◽  
Ionic Current ◽  
Tryptophan Residue ◽  
Charge Movement ◽  
Β Subunit ◽  
Open Probability ◽  
Interaction Domain ◽  
Voltage Dependent ◽  
Voltage Dependent Calcium Channels

Voltage-dependent calcium channels consist of a pore-forming subunit (CaVα1) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary β-subunit (CaVβ) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. CaVβ binds reversibly to a conserved consensus sequence in CaVα1, the α1-interaction domain (AID), which forms an α-helix when complexed with CaVβ. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on CaVβ. Here, we studied the effect of mutating residues located opposite to the AID-CaVβ contact surface in CaV1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other CaVα1 subunits (E462R, K465N, D469S, and Q473K) hinders CaVβ's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for CaVβ. At the single channel level, these CaV1.2 mutants coexpressed with CaVβ2a visit high open probability mode less frequently than wild-type channels. On the other hand, CaV1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs CaVβ binding), or a deletion of the whole AID sequence, does not exhibit CaVβ-induced increase in I/Q. In addition, we observed a shift in the voltage dependence of activation by +12 mV in the AID-deleted channel in the absence of CaVβ, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that CaVβ-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that CaVβ spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by CaVβ.

scholarly journals Electrostatics and the Gating Pore of Shaker Potassium Channels

2001 ◽  
Vol 117 (1) ◽  
pp. 69-90 ◽  
Author(s):  
Leon D. Islas ◽  
Fred J. Sigworth
Keyword(s):  
Charge Movement ◽  
Transmembrane Helices ◽  
Gating Currents ◽  
Conical Cavity ◽  
K Channels ◽  
Gating Charge ◽  
Voltage Dependent ◽  
Shaker Potassium Channels ◽  
Electrostatic Calculations ◽  
S4 Segment

Various experiments have suggested that the S4 segment in voltage-dependent Na+ and K+ channels is in contact with a solvent-accessible cavity. We explore the consequences of the existence of such a cavity through the electrostatic effects on the gating currents of Shaker K+ channels under conditions of reduced ionic strength S. We observe that ∼10-fold reductions of intracellular S produce reductions of the measured gating charge of ∼10%. These effects continue at even lower values of S. The reduction of gating charge when S is reduced by 10-fold at the extracellular surface is much smaller (∼2%). Shifts of the Q(V) curve because of a reduced S are small (<10 mV in size), which is consistent with very little fixed surface charge. Continuum electrostatic calculations show that the S effects on gating charge can be explained by the alteration of the local potential in an intracellular conical cavity of 20–24-Å depth and 12-Å aperture, and a smaller extracellular cavity of 3-Å depth and the same aperture. In this case, the attenuation of the membrane potential at low S leads to reduction of the apparent gating charge. We suggest that this cavity is made by a bundle of transmembrane helices, and that the gating charge movement occurs by translocation of charged residues across a thin septum of ∼3–7 Å thickness.

scholarly journals Allosteric Voltage Gating of Potassium Channels II

1999 ◽  
Vol 114 (2) ◽  
pp. 305-336 ◽  
Author(s):  
Frank T. Horrigan ◽  
Richard W. Aldrich
Keyword(s):  
Preceding Paper ◽  
Voltage Sensor ◽  
Voltage Gating ◽  
Total Charge ◽  
Charge Movement ◽  
Gating Currents ◽  
Open Probability ◽  
K Channels ◽  
Virtual Absence ◽  
Gating Charge

Large-conductance Ca2+-activated K+ channels can be activated by membrane voltage in the absence of Ca2+ binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca2+-activated K+ channels in the virtual absence of Ca2+ (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge–voltage relationship (Q–V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G–V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (τON = 60 μs at +200 mV, τOFF = 16 μs at −80 mV). However, QOFF increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of IK activation. The slow onset of this gating charge prevents its detection as a component of IgON, although it represents ∼40% of the total charge moved at +140 mV. The decay of IgOFF is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277–304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C–C, O–O, and C–O transitions.

scholarly journals Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

1992 ◽  
Vol 100 (3) ◽  
pp. 401-426 ◽  
Author(s):  
M D Ganfornina ◽  
J López-Barneo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Glomus Cells ◽  
Control Value ◽  
Voltage Dependent ◽  
K Current ◽  
Chemoreceptor Cells

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.

scholarly journals Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons.

1987 ◽  
Vol 90 (1) ◽  
pp. 27-47 ◽  
Author(s):  
A Hermann ◽  
C Erxleben
Keyword(s):  
Single Channel ◽  
Delayed Rectifier ◽  
Pacemaker Activity ◽  
Dependent Manner ◽  
Open Probability ◽  
Apparent Dissociation Constant ◽  
External Application ◽  
K Channels ◽  
Voltage Dependent ◽  
K Current

The action of charybdotoxin (ChTX), a peptide component isolated from the venom of the scorpion Leiurus quinquestriatus, was investigated on membrane currents of identified neurons from the marine mollusk, Aplysia californica. Macroscopic current recordings showed that the external application of ChTX blocks the Ca-activated K current in a dose- and voltage-dependent manner. The apparent dissociation constant is 30 nM at V = -30 mV and increases e-fold for a +50- to +70-mV change in membrane potential, which indicates that the toxin molecule is sensitive to approximately 35% of the transmembrane electric field. The toxin is bound to the receptor with a 1:1 stoichiometry and its effect is reversible after washout. The toxin also suppresses the membrane leakage conductance and a resting K conductance activated by internal Ca ions. The toxin has no significant effect on the inward Na or Ca currents, the transient K current, or the delayed rectifier K current. Records from Ca-activated K channels revealed a single channel conductance of 35 +/- 5 pS at V = 0 mV in asymmetrical K solution. The channel open probability increased with the internal Ca concentration and with membrane voltage. The K channels were blocked by submillimolar concentrations of tetraethylammonium ions and by nanomolar concentrations of ChTX, but were not blocked by 4-aminopyridine if applied externally on outside-out patches. From the effects of ChTX on K current and on bursting pacemaker activity, it is concluded that the termination of bursts is in part controlled by a Ca-activated K conductance.

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