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 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 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 Molecular Action of Lidocaine on the Voltage Sensors of Sodium Channels

2003 ◽  
Vol 121 (2) ◽  
pp. 163-175 ◽  
Author(s):  
Michael F. Sheets ◽  
Dorothy A. Hanck
Keyword(s):  
Channel Gating ◽  
Ionic Current ◽  
Na Channel ◽  
Relative Reduction ◽  
Charge Movement ◽  
Charge Voltage ◽  
Voltage Sensors ◽  
Gating Charge ◽  
Voltage Dependent ◽  
Domain Iii

Block of sodium ionic current by lidocaine is associated with alteration of the gating charge-voltage (Q-V) relationship characterized by a 38% reduction in maximal gating charge (Qmax) and by the appearance of additional gating charge at negative test potentials. We investigated the molecular basis of the lidocaine-induced reduction in cardiac Na channel–gating charge by sequentially neutralizing basic residues in each of the voltage sensors (S4 segments) in the four domains of the human heart Na channel (hH1a). By determining the relative reduction in the Qmax of each mutant channel modified by lidocaine we identified those S4 segments that contributed to a reduction in gating charge. No interaction of lidocaine was found with the voltage sensors in domains I or II. The largest inhibition of charge movement was found for the S4 of domain III consistent with lidocaine completely inhibiting its movement. Protection experiments with intracellular MTSET (a charged sulfhydryl reagent) in a Na channel with the fourth outermost arginine in the S4 of domain III mutated to a cysteine demonstrated that lidocaine stabilized the S4 in domain III in a depolarized configuration. Lidocaine also partially inhibited movement of the S4 in domain IV, but lidocaine's most dramatic effect was to alter the voltage-dependent charge movement of the S4 in domain IV such that it accounted for the appearance of additional gating charge at potentials near −100 mV. These findings suggest that lidocaine's actions on Na channel gating charge result from allosteric coupling of the binding site(s) of lidocaine to the voltage sensors formed by the S4 segments in domains III and IV.

scholarly journals Influence of Permeant Ions on Voltage Sensor Function in the Kv2.1 Potassium Channel

2004 ◽  
Vol 123 (4) ◽  
pp. 387-400 ◽  
Author(s):  
Joseph F. Consiglio ◽  
Stephen J. Korn
Keyword(s):  
Potassium Channel ◽  
Ionic Current ◽  
Ionic Currents ◽  
Voltage Sensor ◽  
Selectivity Filter ◽  
Charge Movement ◽  
Channel Activation ◽  
Outer Vestibule ◽  
Specific Selectivity ◽  
Current Activation

We previously demonstrated that the outer vestibule of activated Kv2.1 potassium channels can be in one of two conformations, and that K+occupancy of a specific selectivity filter site determines which conformation the outer vestibule is in. These different outer vestibule conformations result in different sensitivities to internal and external TEA, different inactivation rates, and different macroscopic conductances. The [K+]-dependent switch in outer vestibule conformation is also associated with a change in rate of channel activation. In this paper, we examined the mechanism by which changes in [K+] modulate the rate of channel activation. Elevation of symmetrical [K+] or [Rb+] from 0 to 3 mM doubled the rate of on-gating charge movement (Qon), measured at 0 mV. Cs+produced an identical effect, but required 40-fold higher concentrations. All three permeant ions occupied the selectivity filter over the 0.03–3 mM range, so simple occupancy of the selectivity filter was not sufficient to produce the change in Qon. However, for each of these permeant ions, the speeding of Qonoccurred with the same concentration dependence as the switch between outer vestibule conformations. Neutralization of an amino acid (K356) in the outer vestibule, which abolishes the modulation of channel pharmacology and ionic currents by the K+-dependent reorientation of the outer vestibule, also abolished the K+-dependence of Qon. Together, the data indicate that the K+-dependent reorientation in the outer vestibule was responsible for the change in Qon. Moreover, similar [K+]-dependence and effects of mutagenesis indicate that the K+-dependent change in rate of Qoncan account for the modulation of ionic current activation rate. Simple kinetic analysis suggested that K+reduced an energy barrier for voltage sensor movement. These results provide strong evidence for a direct functional interaction, which is modulated by permeant ions acting at the selectivity filter, between the outer vestibule of the Kv2.1 potassium channel and the voltage sensor.

scholarly journals Mechanism of Auxiliary Subunit Modulation of Neuronal α1E Calcium Channels

1998 ◽  
Vol 112 (2) ◽  
pp. 125-143 ◽  
Author(s):  
Lisa P. Jones ◽  
Shao-kui Wei ◽  
David T. Yue
Keyword(s):  
Calcium Channels ◽  
Ionic Current ◽  
Charge Movement ◽  
Open Probability ◽  
Hek 293 ◽  
Gating Model ◽  
Auxiliary Subunit ◽  
Activation Gating ◽  
Α2δ Subunit ◽  
Current Activation

Voltage-gated calcium channels are composed of a main pore-forming α1 moiety, and one or more auxiliary subunits (β, α2δ) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of α1E calcium channels in combination with a β (β1–β4) and/or the α2δ subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting α2δ with β subunits, of transfecting two different β subunits simultaneously, and of COOH-terminal truncation of α1E to remove a second β binding site. The main results are as follows. (a) The α2δ and β subunits modulate α1E in fundamentally different ways. The sole effect of α2δ is to increase current density by elevating channel density. By contrast, though β subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different β isoforms produce nearly indistinguishable effects on activation. However, β subunits produced clear, isoform-specific effects on inactivation properties. (b) All the β subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different β subunits. (d) The modulatory features found here for α1E do not generalize uniformly to other α1 channel types, as α1C activation gating shows marked β isoform dependence that is absent for α1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of α1E calcium channels.

scholarly journals Gating of the Bacterial Sodium Channel, NaChBac

2004 ◽  
Vol 124 (4) ◽  
pp. 349-356 ◽  
Author(s):  
Alexey Kuzmenkin ◽  
Francisco Bezanilla ◽  
Ana M. Correa
Keyword(s):  
Sodium Channel ◽  
Mammalian Cells ◽  
Ionic Current ◽  
Bacillus Halodurans ◽  
Kinetic Properties ◽  
Charge Movement ◽  
Gating Current ◽  
Gating Currents ◽  
Charge Voltage ◽  
Gating Charge

The bacterial sodium channel, NaChBac, from Bacillus halodurans provides an excellent model to study structure–function relationships of voltage-gated ion channels. It can be expressed in mammalian cells for functional studies as well as in bacterial cultures as starting material for protein purification for fine biochemical and biophysical studies. Macroscopic functional properties of NaChBac have been described previously (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001. Science. 294:2372–2375). In this study, we report gating current properties of NaChBac expressed in COS-1 cells. Upon depolarization of the membrane, gating currents appeared as upward inflections preceding the ionic currents. Gating currents were detectable at −90 mV while holding at −150 mV. Charge–voltage (Q–V) curves showed sigmoidal dependence on voltage with gating charge saturating at −10 mV. Charge movement was shifted by −22 mV relative to the conductance–voltage curve, indicating the presence of more than one closed state. Consistent with this was the Cole-Moore shift of 533 μs observed for a change in preconditioning voltage from −160 to −80 mV. The total gating charge was estimated to be 16 elementary charges per channel. Charge immobilization caused by prolonged depolarization was also observed; Q–V curves were shifted by approximately −60 mV to hyperpolarized potentials when cells were held at 0 mV. The kinetic properties of NaChBac were simulated by simultaneous fit of sodium currents at various voltages to a sequential kinetic model. Gating current kinetics predicted from ionic current experiments resembled the experimental data, indicating that gating currents are coupled to activation of NaChBac and confirming the assertion that this channel undergoes several transitions between closed states before channel opening. The results indicate that NaChBac has several closed states with voltage-dependent transitions between them realized by translocation of gating charge that causes activation of the channel.

scholarly journals A Dipeptidyl Aminopeptidase–like Protein Remodels Gating Charge Dynamics in Kv4.2 Channels

2006 ◽  
Vol 128 (6) ◽  
pp. 745-753 ◽  
Author(s):  
Kevin Dougherty ◽  
Manuel Covarrubias
Keyword(s):  
Membrane Potentials ◽  
Charge Movement ◽  
Transmembrane Segment ◽  
Gating Currents ◽  
Voltage Sensing ◽  
Charge Voltage ◽  
Charge Dynamics ◽  
Gating Charge ◽  
Kv4 Channels ◽  
Dipeptidyl Aminopeptidase

Dipeptidyl aminopeptidase–like proteins (DPLPs) interact with Kv4 channels and thereby induce a profound remodeling of activation and inactivation gating. DPLPs are constitutive components of the neuronal Kv4 channel complex, and recent observations have suggested the critical functional role of the single transmembrane segment of these proteins (Zagha, E., A. Ozaita, S.Y. Chang, M.S. Nadal, U. Lin, M.J. Saganich, T. McCormack, K.O. Akinsanya, S.Y. Qi, and B. Rudy. 2005. J. Biol. Chem. 280:18853–18861). However, the underlying mechanism of action is unknown. We hypothesized that a unique interaction between the Kv4.2 channel and a DPLP found in brain (DPPX-S) may remodel the channel's voltage-sensing domain. To test this hypothesis, we implemented a robust experimental system to measure Kv4.2 gating currents and study gating charge dynamics in the absence and presence of DPPX-S. The results demonstrated that coexpression of Kv4.2 and DPPX-S causes a −26 mV parallel shift in the gating charge-voltage (Q-V) relationship. This shift is associated with faster outward movements of the gating charge over a broad range of relevant membrane potentials and accelerated gating charge return upon repolarization. In sharp contrast, DPPX-S had no effect on gating charge movements of the Shaker B Kv channel. We propose that DPPX-S destabilizes resting and intermediate states in the voltage-dependent activation pathway, which promotes the outward gating charge movement. The remodeling of gating charge dynamics may involve specific protein–protein interactions of the DPPX-S's transmembrane segment with the voltage-sensing and pore domains of the Kv4.2 channel. This mechanism may determine the characteristic fast operation of neuronal Kv4 channels in the subthreshold range of membrane potentials.

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 A gating charge interaction required for late slow inactivation of the bacterial sodium channel NavAb

2013 ◽  
Vol 142 (3) ◽  
pp. 181-190 ◽  
Author(s):  
Tamer M. Gamal El-Din ◽  
Gilbert Q. Martinez ◽  
Jian Payandeh ◽  
Todd Scheuer ◽  
William A. Catterall
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Late Phase ◽  
Membrane Potentials ◽  
Charge Movement ◽  
Slow Inactivation ◽  
Functional Studies ◽  
Voltage Gated Sodium Channels ◽  
Gating Charge ◽  
Charge Interaction

Voltage-gated sodium channels undergo slow inactivation during repetitive depolarizations, which controls the frequency and duration of bursts of action potentials and prevents excitotoxic cell death. Although homotetrameric bacterial sodium channels lack the intracellular linker-connecting homologous domains III and IV that causes fast inactivation of eukaryotic sodium channels, they retain the molecular mechanism for slow inactivation. Here, we examine the functional properties and slow inactivation of the bacterial sodium channel NavAb expressed in insect cells under conditions used for structural studies. NavAb activates at very negative membrane potentials (V1/2 of approximately −98 mV), and it has both an early phase of slow inactivation that arises during single depolarizations and reverses rapidly, and a late use-dependent phase of slow inactivation that reverses very slowly. Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ∼75 mV to more positive potentials and abolishes the late phase of slow inactivation. The gating charge R3 interacts with Asn49 in the crystal structure of NavAb, and mutation of this residue to Cys causes a similar positive shift in the voltage dependence of activation and block of the late phase of slow inactivation as mutation N49K. Prolonged depolarizations that induce slow inactivation also cause hysteresis of gating charge movement, which results in a requirement for very negative membrane potentials to return gating charges to their resting state. Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation. Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.

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