scholarly journals Nile blue fluorescence signals from cut single muscle fibers under voltage or current clamp conditions.

1978 ◽  
Vol 72 (6) ◽  
pp. 775-800 ◽  
Author(s):  
J Vergara ◽  
F Bezanilla ◽  
B M Salzberg
Keyword(s):  
Voltage Dependence ◽  
Nile Blue ◽  
Surface Membrane ◽  
Muscle Fibers ◽  
Delayed Rectifier ◽  
Charge Movement ◽  
Blue Fluorescence ◽  
Optical Signals ◽  
Gating Charge ◽  
Physiological Properties

A method is presented for recording extrinsic optical signals from segments of single skeletal muscle fibers under current or voltage clamp conditions. Such segments, which are cut from intact fibers, are maintained in a relaxed state, while exhbiting otherwise normal physiological properties, including healthy delayed rectifier currents. Extrinsic fluorescence changes are demonstrated, using the permeant potentiometric probe, Nile Blue A. These changes vary nonlinearly with the controlled surface membrane potential, in a manner which suggests that they arise from potential changes in the sarcoplasmic reticulum. According to this interpretation, a simple model based on the gating charge movement implicated in excitation-contraction coupling, provides a self-consistent description of the voltage dependence of the signal that requires no additional parameters.

scholarly journals Components of gating charge movement and S4 voltage-sensor exposure during activation of hERG channels

2013 ◽  
Vol 141 (4) ◽  
pp. 431-443 ◽  
Author(s):  
Zhuren Wang ◽  
Ying Dou ◽  
Samuel J. Goodchild ◽  
Zeineb Es-Salah-Lamoureux ◽  
David Fedida
Keyword(s):  
Mammalian Cells ◽  
Voltage Sensor ◽  
Delayed Rectifier ◽  
Charge Movement ◽  
Α Subunit ◽  
Time Constants ◽  
Gating Charge ◽  
Activation Gating ◽  
Pore Opening ◽  
Herg Channels

The human ether-á-go-go–related gene (hERG) K+ channel encodes the pore-forming α subunit of the rapid delayed rectifier current, IKr, and has unique activation gating kinetics, in that the α subunit of the channel activates and deactivates very slowly, which focuses the role of IKr current to a critical period during action potential repolarization in the heart. Despite its physiological importance, fundamental mechanistic properties of hERG channel activation gating remain unclear, including how voltage-sensor movement rate limits pore opening. Here, we study this directly by recording voltage-sensor domain currents in mammalian cells for the first time and measuring the rates of voltage-sensor modification by [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET). Gating currents recorded from hERG channels expressed in mammalian tsA201 cells using low resistance pipettes show two charge systems, defined as Q1 and Q2, with V1/2’s of −55.7 (equivalent charge, z = 1.60) and −54.2 mV (z = 1.30), respectively, with the Q2 charge system carrying approximately two thirds of the overall gating charge. The time constants for charge movement at 0 mV were 2.5 and 36.2 ms for Q1 and Q2, decreasing to 4.3 ms for Q2 at +60 mV, an order of magnitude faster than the time constants of ionic current appearance at these potentials. The voltage and time dependence of Q2 movement closely correlated with the rate of MTSET modification of I521C in the outermost region of the S4 segment, which had a V1/2 of −64 mV and time constants of 36 ± 8.5 ms and 11.6 ± 6.3 ms at 0 and +60 mV, respectively. Modeling of Q1 and Q2 charge systems showed that a minimal scheme of three transitions is sufficient to account for the experimental findings. These data point to activation steps further downstream of voltage-sensor movement that provide the major delays to pore opening in hERG channels.

scholarly journals Optical signals from surface and T system membranes in skeletal muscle fibers. Experiments with the potentiometric dye NK2367.

1982 ◽  
Vol 80 (2) ◽  
pp. 203-230 ◽  
Author(s):  
J A Heiny ◽  
J Vergara
Keyword(s):  
Skeletal Muscle ◽  
Voltage Clamp ◽  
Surface Membrane ◽  
Muscle Fibers ◽  
Optical Signal ◽  
Intensity Change ◽  
Optical Signals ◽  
Potential Control ◽  
Skeletal Muscle Fibers ◽  
T System

Absorbance signals were recorded from cut single skeletal muscle fibers stained with the nonpenetrating potentiometric dye NK2367 and mounted in a three-vaseline-gap voltage clamp. The characteristics of the optical signals recorded under current and voltage-clamp conditions were studied at various wavelengths between 500 and 800 nm using unpolarized light. Our results indicate that the absorbance signals recorded with this dye reflect potential changes across both the surface and T system membranes and that the relative contribution of each of these membrane compartments to the total optical change is strongly wavelength dependent. A peak intensity change was detected at 720 nm for the surface membrane signal and at 670 nm for the T system. Evidence for this wavelength-dependent separation derives from an analysis of the kinetics and voltage dependence of the optical signals at different wavelengths, and results obtained in detubulated fibers. The 670-nm optical signal was used to demonstrate the lack of potential control in the T system by the voltage clamp and the effect of a tetrodotoxin (TTX)-sensitive sodium conductance on tubular depolarization.

scholarly journals Shaker potassium channel gating. II: Transitions in the activation pathway.

1994 ◽  
Vol 103 (2) ◽  
pp. 279-319 ◽  
Author(s):  
W N Zagotta ◽  
T Hoshi ◽  
J Dittman ◽  
R W Aldrich
Keyword(s):  
Conformational Changes ◽  
Time Course ◽  
Voltage Dependence ◽  
Single Channel ◽  
Ionic Current ◽  
Activation Process ◽  
Charge Movement ◽  
Open Probability ◽  
Current Time ◽  
Gating Charge

Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single-channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.

scholarly journals Kinetic properties of intramembrane charge movement under depolarized conditions in frog skeletal muscle fibers.

1991 ◽  
Vol 98 (2) ◽  
pp. 365-378 ◽  
Author(s):  
G Szücs ◽  
Z Papp ◽  
L Csernoch ◽  
L Kovács
Keyword(s):  
Skeletal Muscle ◽  
Voltage Dependence ◽  
Slow Component ◽  
Muscle Fibers ◽  
Ionic Current ◽  
Kinetic Properties ◽  
Constant Field ◽  
Charge Movement ◽  
Skeletal Muscle Fibers ◽  
Intramembrane Charge Movement

Intramembrane charge movement was measured on skeletal muscle fibers of the frog in a single Vaseline-gap voltage clamp. Charge movements determined both under polarized conditions (holding potential, VH = -100 mV; Qmax = 30.4 +/- 4.7 nC/micro(F), V = -44.4 mV, k = 14.1 mV; charge 1) and in depolarized states (VH = 0 mV; Qmax = 50.0 +/- 6.7 nC/micro(F), V = -109.1 mV, k = 26.6 mV; charge 2) had properties as reported earlier. Linear capacitance (LC) of the polarized fibers was increased by 8.8 +/- 4.0% compared with that of the depolarized fibers. Using control pulses measured under depolarized conditions to calculate charge 1, a minor change in the voltage dependence (to V = -44.6 mV and k = 14.5 mV) and a small increase in the maximal charge (to Qmax = 31.4 +/- 5.5 nC/micro(F] were observed. While in most cases charge 1 transients seemed to decay with a single exponential time course, charge 2 currents showed a characteristic biexponential behavior at membrane potentials between -90 and -180 mV. The voltage dependence of the rate constant of the slower component was fitted with a simple constant field diffusion model (alpha m = 28.7 s-1, V = -124.0 mV, and k = 15.6 mV). The midpoint voltage (V) was similar to that obtained from the Q-V fit of charge 2, while the steepness factor (k) resembled that of charge 1. This slow component could also be isolated using a stepped OFF protocol; that is, by hyperpolarizing the membrane to -190 mV for 200 ms and then coming back to 0 mV in two steps. The faster component was identified as an ionic current insensitive to 20 mM Co2+ but blocked by large hyperpolarizing pulses. These findings are consistent with the model implying that charge 1 and the slower component of charge 2 interconvert when the holding potential is changed. They also explain the difference previously found when comparing the steepness factors of the voltage dependence of charge 1 and charge 2.

scholarly journals Inactivation of the sodium channel. II. Gating current experiments.

1977 ◽  
Vol 70 (5) ◽  
pp. 567-590 ◽  
Author(s):  
C M Armstrong ◽  
F Bezanilla
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Slow Component ◽  
Base Line ◽  
Charge Movement ◽  
Gating Current ◽  
Small Current ◽  
Gating Charge ◽  
Recovery From Inactivation ◽  
Activation Gate

Gating current (Ig) has been studied in relation to inactivation of Na channels. No component of Ig has the time course of inactivation; apparently little or no charge movement is associated with this step. Inactivation nonetheless affects Ig by immobilizing about two-thirds of gating charge. Immobilization can be followed by measuring ON charge movement during a pulse and comparing it to OFF charge after the pulse. The OFF:ON ratio is near 1 for a pulse so short that no inactivation occurs, and the ratio drops to about one-third with a time course that parallels inactivation. Other correlations between inactivation and immobilization are that: (a) they have the same voltage dependence; (b) charge movement recovers with the time coures of recovery from inactivation. We interpret this to mean that the immobilized charge returns slowly to "off" position with the time course of recovery from inactivation, and that the small current generated is lost in base-line noise. At -150 mV recover is very rapid, and the immobilized charge forms a distinct slow component of current as it returns to off position. After destruction of inactivation by pronase, there is no immobilization of charge. A model is presented in which inactivation gains its voltage dependence by coupling to the activation gate.

scholarly journals Uncoupling Charge Movement from Channel Opening in Voltage-gated Potassium Channels by Ruthenium Complexes

2011 ◽  
Vol 286 (18) ◽  
pp. 16414-16425 ◽  
Author(s):  
Andrés Jara-Oseguera ◽  
Itzel G. Ishida ◽  
Gisela E. Rangel-Yescas ◽  
Noel Espinosa-Jalapa ◽  
José A. Pérez-Guzmán ◽  
...  
Keyword(s):  
Electromechanical Coupling ◽  
Voltage Dependence ◽  
Voltage Sensor ◽  
Delayed Rectifier ◽  
Charge Movement ◽  
Β Cells ◽  
Delayed Rectifier Current ◽  
Mechanism Of Inhibition ◽  
Channel Opening ◽  
Sensor Activation

The Kv2.1 channel generates a delayed-rectifier current in neurons and is responsible for modulation of neuronal spike frequency and membrane repolarization in pancreatic β-cells and cardiomyocytes. As with other tetrameric voltage-activated K+-channels, it has been proposed that each of the four Kv2.1 voltage-sensing domains activates independently upon depolarization, leading to a final concerted transition that causes channel opening. The mechanism by which voltage-sensor activation is coupled to the gating of the pore is still not understood. Here we show that the carbon-monoxide releasing molecule 2 (CORM-2) is an allosteric inhibitor of the Kv2.1 channel and that its inhibitory properties derive from the CORM-2 ability to largely reduce the voltage dependence of the opening transition, uncoupling voltage-sensor activation from the concerted opening transition. We additionally demonstrate that CORM-2 modulates Shaker K+-channels in a similar manner. Our data suggest that the mechanism of inhibition by CORM-2 may be common to voltage-activated channels and that this compound should be a useful tool for understanding the mechanisms of electromechanical coupling.

scholarly journals Modulation of the Voltage Sensor of L-type Ca2+ Channels by Intracellular Ca2+

2004 ◽  
Vol 123 (5) ◽  
pp. 555-571 ◽  
Author(s):  
Dmytro Isaev ◽  
Karisa Solt ◽  
Oksana Gurtovaya ◽  
John P. Reeves ◽  
Roman Shirokov
Keyword(s):  
Intracellular Calcium ◽  
Calcium Binding ◽  
Voltage Dependence ◽  
Voltage Sensor ◽  
Charge Movement ◽  
Wild Type ◽  
Calcium Binding Site ◽  
Gating Charge ◽  
Voltage Dependent ◽  
Charge Movements

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (≥100 μM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from ∼0.1 to 100–300 μM sped up the conversion of the gating charge into the negatively distributed mode 10–100-fold. Since the “IQ-AA” mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the “IQ” motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Δ1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation.

scholarly journals Phosphorylation modulates potassium conductance and gating current of perfused giant axons of squid.

1990 ◽  
Vol 95 (2) ◽  
pp. 245-271 ◽  
Author(s):  
C K Augustine ◽  
F Bezanilla
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Potassium Conductance ◽  
Charge Movement ◽  
Gating Current ◽  
Gating Currents ◽  
Internal Solution ◽  
Giant Axons ◽  
Gating Charge ◽  
Phosphorylation Reaction

The presence of internal Mg-ATP produced a number of changes in the K conductance of perfused giant axons of squid. For holding potentials between -40 and -50 mV, steady-state K conductance increased for depolarizations to potentials more positive than approximately -15 mV and decreased for smaller depolarizations. The voltage dependencies of both steady-state activation and inactivation also appears shifted toward more positive potentials. Gating kinetics were affected by internal ATP, with the activation time constant slowed and the characteristic delay in K conductance markedly enhanced. The rate of deactivation also was hastened during perfusion with ATP. Internal ATP affected potassium channel gating currents in similar ways. The voltage dependence of gating charge movement was shifted toward more positive potentials and the time constants of ON and OFF gating current also were slowed and hastened, respectively, in the presence of ATP. These effects of ATP on the K conductance occurred when no exogenous protein kinases were added to the internal solution and persisted even after removing ATP from the internal perfusate. Perfusion with a solution containing exogenous alkaline phosphatase reversed the effects of ATP. These results provide further evidence that the effects of ATP on the K conductance are a consequence of a phosphorylation reaction mediated by a kinase present and active in perfused axons. Phosphorylation appears to alter the K conductance of squid giant axons via a minimum of two mechanisms. First, the voltage dependence of gating parameters are shifted toward positive potentials. Second, there is an increase in the number of functional closed states and/or a decrease in the rates of transition between these states of the K channels.

scholarly journals Voltage Sensitivity and Gating Charge in Shaker and Shab Family Potassium Channels

1999 ◽  
Vol 114 (5) ◽  
pp. 723-742 ◽  
Author(s):  
Leon D. Islas ◽  
Fred J. Sigworth
Keyword(s):  
Single Channel ◽  
Delayed Rectifier ◽  
Voltage Sensitivity ◽  
Charge Movement ◽  
Gating Currents ◽  
Open Probability ◽  
Charge Voltage ◽  
Gating Charge ◽  
Charged Residues ◽  
Positively Charged

The members of the voltage-dependent potassium channel family subserve a variety of functions and are expected to have voltage sensors with different sensitivities. The Shaker channel of Drosophila, which underlies a transient potassium current, has a high voltage sensitivity that is conferred by a large gating charge movement, ∼13 elementary charges. A Shaker subunit's primary voltage-sensing (S4) region has seven positively charged residues. The Shab channel and its homologue Kv2.1 both carry a delayed-rectifier current, and their subunits have only five positively charged residues in S4; they would be expected to have smaller gating-charge movements and voltage sensitivities. We have characterized the gating currents and single-channel behavior of Shab channels and have estimated the charge movement in Shaker, Shab, and their rat homologues Kv1.1 and Kv2.1 by measuring the voltage dependence of open probability at very negative voltages and comparing this with the charge–voltage relationships. We find that Shab has a relatively small gating charge, ∼7.5 eo. Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 eo, essentially equal to that of Shaker and Kv1.1. Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10−9 in Shaker and below 4 × 10−8 in Kv2.1.

Sequential gating in the human heart K+ channel Kv1.5 incorporatesQ 1 andQ 2 charge components

1999 ◽  
Vol 277 (5) ◽  
pp. H1956-H1966 ◽  
Author(s):  
J. Christian Hesketh ◽  
David Fedida
Keyword(s):  
K Channel ◽  
Delayed Rectifier ◽  
Potential Range ◽  
Charge Movement ◽  
Gating Current ◽  
Gating Currents ◽  
Charge Voltage ◽  
Hek 293 ◽  
Gating Charge ◽  
State Dependent

On-gating current from the Kv1.5 cardiac delayed rectifier K+ channel expressed in HEK-293 cells was separated into two distinct charge systems, Q 1 and Q 2, obtained from double Boltzmann fits to the charge-voltage relationship. Q 1 and Q 2 had characteristic voltage dependence and sensitivity with half-activation potentials of −29.6 ± 1.6 and −2.19 ± 2.09 mV and effective valences of 1.87 ± 0.15 and 5.53 ± 0.27 e −, respectively. The contribution to total gating charge was 0.20 ± 0.04 for Q 1 and 0.80 ± 0.04 ( n = 5) for Q 2. At intermediate depolarizations, heteromorphic gating current waveforms resulted from relatively equal contributions from Q 1 and Q 2, but with widely different kinetics. Prepulses to −20 mV moved only Q 1, simplified on-gating currents, and allowed rapid Q 2 movement. Voltage-dependent on-gating current recovery in the presence of 4-aminopyridine (1 mM) suggested a sequentially coupled movement of the two charge systems during channel activation. This allowed the construction of a linear five-state model of Q 1 and Q 2 gating charge movement, which predicted experimental on-gating currents over a wide potential range. Such models are useful in determining state-dependent mechanisms of open and closed channel block of cardiac K+ channels.

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