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 Gating charge movement precedes ionic current activation in hERG channels

Channels ◽  
2013 ◽  
Vol 8 (1) ◽  
pp. 84-89 ◽  
Author(s):  
Samuel J Goodchild ◽  
David Fedida
Keyword(s):  
Ionic Current ◽  
Charge Movement ◽  
Gating Charge ◽  
Current Activation ◽  
Herg Channels

scholarly journals Mode shift of the voltage sensors in Shaker K+ channels is caused by energetic coupling to the pore domain

2011 ◽  
Vol 137 (5) ◽  
pp. 455-472 ◽  
Author(s):  
Georges A. Haddad ◽  
Rikard Blunck
Keyword(s):  
Conformational Change ◽  
Mechanical Load ◽  
Voltage Sensor ◽  
Charge Movement ◽  
Gating Currents ◽  
Mode Shift ◽  
Voltage Sensors ◽  
Pore Opening ◽  
Voltage Gated Ion Channels ◽  
Pore Domain

The voltage sensors of voltage-gated ion channels undergo a conformational change upon depolarization of the membrane that leads to pore opening. This conformational change can be measured as gating currents and is thought to be transferred to the pore domain via an annealing of the covalent link between voltage sensor and pore (S4-S5 linker) and the C terminus of the pore domain (S6). Upon prolonged depolarizations, the voltage dependence of the charge movement shifts to more hyperpolarized potentials. This mode shift had been linked to C-type inactivation but has recently been suggested to be caused by a relaxation of the voltage sensor itself. In this study, we identified two ShakerIR mutations in the S4-S5 linker (I384N) and S6 (F484G) that, when mutated, completely uncouple voltage sensor movement from pore opening. Using these mutants, we show that the pore transfers energy onto the voltage sensor and that uncoupling the pore from the voltage sensor leads the voltage sensors to be activated at more negative potentials. This uncoupling also eliminates the mode shift occurring during prolonged depolarizations, indicating that the pore influences entry into the mode shift. Using voltage-clamp fluorometry, we identified that the slow conformational change of the S4 previously correlated with the mode shift disappears when uncoupling the pore. The effects can be explained by a mechanical load that is imposed upon the voltage sensors by the pore domain and allosterically modulates its conformation. Mode shift is caused by the stabilization of the open state but leads to a conformational change in the voltage sensor.

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 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 Specificity of Charge-carrying Residues in the Voltage Sensor of Potassium Channels

2004 ◽  
Vol 123 (3) ◽  
pp. 205-216 ◽  
Author(s):  
Christopher A. Ahern ◽  
Richard Horn
Keyword(s):  
Potassium Channels ◽  
Electric Field ◽  
Protein A ◽  
Membrane Lipid ◽  
Voltage Sensor ◽  
Charge Movement ◽  
Gating Currents ◽  
Gating Charge ◽  
Channel Opening ◽  
Positively Charged

Positively charged voltage sensors of sodium and potassium channels are driven outward through the membrane's electric field upon depolarization. This movement is coupled to channel opening. A recent model based on studies of the KvAP channel proposes that the positively charged voltage sensor, christened the “voltage-sensor paddle”, is a peripheral domain that shuttles its charged cargo through membrane lipid like a hydrophobic cation. We tested this idea by attaching charged adducts to cysteines introduced into the putative voltage-sensor paddle of Shaker potassium channels and measuring fractional changes in the total gating charge from gating currents. The only residues capable of translocating attached charges through the membrane-electric field are those that serve this function in the native channel. This remarkable specificity indicates that charge movement involves highly specialized interactions between the voltage sensor and other regions of the protein, a mechanism inconsistent with the paddle model.

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.

scholarly journals Extracellular protons accelerate hERG channel deactivation by destabilizing voltage sensor relaxation

2018 ◽  
Vol 151 (2) ◽  
pp. 231-246 ◽  
Author(s):  
Yu Patrick Shi ◽  
Samrat Thouta ◽  
Yen May Cheng ◽  
Tom W. Claydon
Keyword(s):  
Ph Dependence ◽  
Voltage Sensor ◽  
Herg Channel ◽  
K Channel ◽  
Delayed Rectifier ◽  
Mode Shift ◽  
Deactivation Kinetics ◽  
Shift Behavior ◽  
Activated State ◽  
Herg Channels

hERG channels underlie the delayed-rectifier K+ channel current (IKr), which is crucial for membrane repolarization and therefore termination of the cardiac action potential. hERG channels display unusually slow deactivation gating, which contributes to a resurgent current upon repolarization and may protect against post-depolarization–induced arrhythmias. hERG channels also exhibit robust mode shift behavior, which reflects the energetic separation of activation and deactivation pathways due to voltage sensor relaxation into a stable activated state. The mechanism of relaxation is unknown and likely contributes to slow hERG channel deactivation. Here, we use extracellular acidification to probe the structural determinants of voltage sensor relaxation and its influence on the deactivation gating pathway. Using gating current recordings and voltage clamp fluorimetry measurements of voltage sensor domain dynamics, we show that voltage sensor relaxation is destabilized at pH 6.5, causing an ∼20-mV shift in the voltage dependence of deactivation. We show that the pH dependence of the resultant loss of mode shift behavior is similar to that of the deactivation kinetics acceleration, suggesting that voltage sensor relaxation correlates with slower pore gate closure. Neutralization of D509 in S3 also destabilizes the relaxed state of the voltage sensor, mimicking the effect of protons, suggesting that acidic residues on S3, which act as countercharges to S4 basic residues, are involved in stabilizing the relaxed state and slowing deactivation kinetics. Our findings identify the mechanistic determinants of voltage sensor relaxation and define the long-sought mechanism by which protons accelerate hERG deactivation.

The moving parts of voltage-gated ion channels

1998 ◽  
Vol 31 (3) ◽  
pp. 239-295 ◽  
Author(s):  
GARY YELLEN
Keyword(s):  
Ion Channels ◽  
Basic Structure ◽  
Voltage Sensor ◽  
Site Directed Mutagenesis ◽  
Charge Movement ◽  
Voltage Gated ◽  
Activation Gate ◽  
Activation Gating ◽  
Voltage Gated Ion Channels ◽  
Moving Parts

Ion channels, like many other proteins, have moving parts that perform useful functions. The channel proteins contain an aqueous, ion-selective pore that crosses the plasma membrane, and they use a number of distinct ‘gating’ mechanisms to open and close this pore in response to biological stimuli such as the binding of a ligand or a change in the transmembrane voltage.This review is written at a watershed in our understanding of ion channels.1. INTRODUCTION 2401.1 Basic structure of voltage-activated channels 2411.2 What are the physical motions of the channel protein during gating? 2431.3 Gating involves several distinct mechanisms of activation and inactivation 2462. ACTIVATION GATING 2462.1 Early evidence for an activation gate at the intracellular mouth 2472.1.1 Open channel blockade 2472.1.2 The ‘ foot-in-the-door’ effect 2492.1.3 Trapping of blockers behind closed activation gates 2492.2 Site-directed mutagenesis and the difficulty of inferring structural roles from functional effects 2502.3 State-dependent cysteine modification as a reporter of position and motion 2512.4 Localization of activation gating 2542.4.1 The trapping cavity 2542.4.2 The activation gate 2552.4.3 Is there more than one site of activation gating? 2583. INACTIVATION GATING 2593.1 Ball-and-chain (N-type) inactivation 2613.1.1 Nature of the ‘ball’ – a tethered blocking particle 2623.1.2 The ball receptor 2633.1.3 The chain 2643.1.4 Variations on the N-type inactivation theme: multiple balls, foreign balls, anti-balls 2653.2 C-type inactivation 2663.2.1 C-type inactivation and the outer mouth of the K+channel 2663.2.2 The selectivity filter participates in C-type inactivation 2673.2.3 A consistent structural picture of C-type inactivation 2693.3 By what mechanism do other voltage-gated channels inactivate? 2724. THE VOLTAGE SENSOR 2734.1 Quantitative principles of voltage-dependent gating 2764.2 S4 (and its neighbours) as the principal voltage sensor 2774.2.1 Mutational effects on voltage-dependence and charge movement 2774.2.2 Evidence for the translocation of S4 2794.2.3 Real-time monitoring of S4motion by fluorescence 2824.3 Coupling between the voltage sensor and gating 2835. CONCLUSION 2846. ACKNOWLEDGEMENTS 2877. REFERENCES 287

Sign in / Sign up

Export Citation Format

Share Document