scholarly journals The Role of the Putative Inactivation Lid in Sodium Channel Gating Current Immobilization

2000 ◽  
Vol 115 (5) ◽  
pp. 609-620 ◽  
Author(s):  
Michael F. Sheets ◽  
John W. Kyle ◽  
Dorothy A. Hanck
Keyword(s):  
Sodium Channel ◽  
Time Constant ◽  
Time Course ◽  
Gating Current ◽  
Similar Time ◽  
Charge Voltage ◽  
Voltage Gated Sodium Channels ◽  
Gating Charge ◽  
Domain Iii ◽  
A Site

We investigated the contribution of the putative inactivation lid in voltage-gated sodium channels to gating charge immobilization (i.e., the slow return of gating charge during repolarization) by studying a lid-modified mutant of the human heart sodium channel (hH1a) that had the phenylalanine at position 1485 in the isoleucine, phenylalanine, and methionine (IFM) region of the domain III–IV linker mutated to a cysteine (ICM-hH1a). Residual fast inactivation of ICM-hH1a in fused tsA201 cells was abolished by intracellular perfusion with 2.5 mM 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The time constants of gating current relaxations in response to step depolarizations and gating charge–voltage relationships were not different between wild-type hH1a and ICM-hH1aMTSET. The time constant of the development of charge immobilization assayed at −180 mV after depolarization to 0 mV was similar to the time constant of inactivation of INa at 0 mV for hH1a. By 44 ms, 53% of the gating charge during repolarization returned slowly; i.e., became immobilized. In ICM-hH1aMTSET, immobilization occurred with a similar time course, although only 31% of gating charge upon repolarization (OFF charge) immobilized. After modification of hH1a and ICM-hH1aMTSET with Anthopleurin-A toxin, a site-3 peptide toxin that inhibits movement of the domain IV-S4, charge immobilization did not occur for conditioning durations up to 44 ms. OFF charge for both hH1a and ICM-hH1aMTSET modified with Anthopleurin-A toxin were similar in time course and in magnitude to the fast component of OFF charge in ICM-hH1aMTSET in control. We conclude that movement of domain IV-S4 is the rate-limiting step during repolarization, and it contributes to charge immobilization regardless of whether the inactivation lid is bound. Taken together with previous reports, these data also suggest that S4 in domain III contributes to charge immobilization only after binding of the inactivation lid.

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 Altered sodium and gating current kinetics in frog skeletal muscle caused by low external pH.

1984 ◽  
Vol 84 (5) ◽  
pp. 771-788 ◽  
Author(s):  
D T Campbell ◽  
R Hahin
Keyword(s):  
Skeletal Muscle ◽  
Time Constant ◽  
Time Course ◽  
Voltage Dependence ◽  
Low Ph ◽  
Na Channel ◽  
Frog Skeletal Muscle ◽  
Gating Current ◽  
Gating Charge ◽  
External Ph

The effect of low pH on the kinetics of Na channel ionic and gating currents was studied in frog skeletal muscle fibers. Lowering external pH from 7.4 to 5.0 slows the time course of Na current consistent with about a +25-mV shift in the voltage dependence of activation and inactivation time constants. Similar shifts in voltage dependence adequately describe the effects of low pH on the tail current time constant (+23.3 mV) and the gating charge vs. voltage relationship (+22.1 mV). A significantly smaller shift of +13.3 mV described the effect of pH 5.0 solution on the voltage dependence of steady state inactivation. Changes in the time course of gating current at low pH were complex and could not be described as a shift in voltage dependence. tau g, the time constant that describes the time course of the major component of gating charge movement, was slowed in pH 5.0 solution by a factor of approximately 3.5 for potentials from -60 to +45 mV. We conclude that the effects of low pH on Na channel gating cannot be attributed simply to a change in surface potential. Therefore, although it may be appropriate to describe the effect of low pH on some Na channel kinetic properties as a "shift" in voltage dependence, it is not appropriate to interpret such shifts as a measure of changes in surface potential. The maximum gating charge elicited from a holding potential of -150 mV was little affected by low pH.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

scholarly journals α-Scorpion Toxin Impairs a Conformational Change that Leads to Fast Inactivation of Muscle Sodium Channels

2008 ◽  
Vol 132 (2) ◽  
pp. 251-263 ◽  
Author(s):  
Fabiana V. Campos ◽  
Baron Chanda ◽  
Paulo S.L. Beirão ◽  
Francisco Bezanilla
Keyword(s):  
Sodium Channels ◽  
Slow Component ◽  
Scorpion Toxin ◽  
Fast Inactivation ◽  
Gating Current ◽  
Voltage Sensors ◽  
Current Decay ◽  
Fluorescence Measurements ◽  
Gating Charge ◽  
Domain Iii

α-Scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an α-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence–voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

A model of sodium channel inactivation based upon the modulated blocker

1983 ◽  
Vol 219 (1217) ◽  
pp. 423-438 ◽  
Keyword(s):  
Sodium Channel ◽  
Time Constant ◽  
Voltage Dependence ◽  
Physical Principle ◽  
Membrane Voltage ◽  
Displacement Current ◽  
Channel Inactivation ◽  
Gating Charge ◽  
Sodium Channel Inactivation ◽  
Activation Characteristic

An attempt is made to model sodium channel inactivation based upon real physical processes. The principle involved, which is supported by calculation and by direct appeal to experimental results, is that the gating dipole reversal or gating charge transfer that occurs when the channel is activated, markedly modulates the electrical properties of charged groups at the channel ends. Four examples of possible mechanisms that lead to channel inactivation are described. The simple four-state model that results is able to predict: ( а ) the steep voltage dependence of the equilibrium inactivation characteristic without the presence of any appreciable displacement current associated with inactivation; ( b ) the negative shift in membrane voltage of the equilibrium inactivation characteristic relative to the activation characteristic; ( c ) the bell-shaped dependence of inactivation time constant on membrane voltage; ( d ) the similarity of the membrane voltage dependence of the time constant of recovery from inactivation, to that of inactivation itself. A brief discussion of a model for sodium channel activation based upon the same physical principle is included.

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 Protonation state of inhibitors determines interaction sites within voltage-gated sodium channels

2018 ◽  
Vol 115 (14) ◽  
pp. E3135-E3144 ◽  
Author(s):  
Amanda Buyan ◽  
Delin Sun ◽  
Ben Corry
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
The Body ◽  
Voltage Gated ◽  
Interaction Sites ◽  
Voltage Gated Sodium Channels ◽  
Neutral Compounds ◽  
Dynamics Simulations ◽  
A Site ◽  
Subtype Selective

Voltage-gated sodium channels are essential for carrying electrical signals throughout the body, and mutations in these proteins are responsible for a variety of disorders, including epilepsy and pain syndromes. As such, they are the target of a number of drugs used for reducing pain or combatting arrhythmias and seizures. However, these drugs affect all sodium channel subtypes found in the body. Designing compounds to target select sodium channel subtypes will provide a new therapeutic pathway and would maximize treatment efficacy while minimizing side effects. Here, we examine the binding preferences of nine compounds known to be sodium channel pore blockers in molecular dynamics simulations. We use the approach of replica exchange solute tempering (REST) to gain a more complete understanding of the inhibitors’ behavior inside the pore of NavMs, a bacterial sodium channel, and NavPas, a eukaryotic sodium channel. Using these simulations, we are able to show that both charged and neutral compounds partition into the bilayer, but neutral forms more readily cross it. We show that there are two possible binding sites for the compounds: (i) a site on helix 6, which has been previously determined by many experimental and computational studies, and (ii) an additional site, occupied by protonated compounds in which the positively charged part of the drug is attracted into the selectivity filter. Distinguishing distinct binding poses for neutral and charged compounds is essential for understanding the nature of pore block and will aid the design of subtype-selective sodium channel inhibitors.

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 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.

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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