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.

Comparison of the effects of BAY K 8644 on cardiac Ca2+ current and Ca2+ channel gating current

1992 ◽  
Vol 262 (2) ◽  
pp. H472-H477 ◽  
Author(s):  
R. W. Hadley ◽  
W. J. Lederer
Keyword(s):  
Ventricular Myocytes ◽  
Channel Gating ◽  
Bay K 8644 ◽  
Charge Movement ◽  
Gating Current ◽  
Inhibitory Effects ◽  
Open Probability ◽  
Charge Voltage ◽  
Gating Charge ◽  
Voltage Dependent

Effects of (-)-BAY K 8644 on Ca2+ channel function were studied in guinea pig ventricular myocytes. It was found that the compound has both voltage-dependent stimulatory and inhibitory effects on the Ca2+ current (ICa), in agreement with prior studies. The basis for these effects was studied by evaluating the effects of (-)-BAY K 8644 on the Ca2+ channel gating current. It was found that the voltage-dependent inhibitory effects of the drug on ICa could be well explained by similar reductions in the amount of gating charge moved. However, the stimulatory effect of (-)-BAY K 8644 on ICa could not be simply correlated with changes in the amount of gating charge moved. Although the drug produced a shift of the charge-voltage relationship to more negative potentials, the drug actually reduced the total amount of movable gating charge. Thus it could be demonstrated that there are membrane potentials where (-)-BAY K 8644 reduced the Ca2+ channel gating current while enhancing ICa. In addition, the drug was found to slow the decay of the gating current during repolarization. It seems likely that (-)-BAY K 8644 has a dual effect on Ca2+ channels: affecting both the voltage dependence of gating charge and the relationship between open probability and charge movement.

scholarly journals Nonlinear charge movement in mammalian cardiac ventricular cells. Components from Na and Ca channel gating.

1989 ◽  
Vol 94 (1) ◽  
pp. 65-93 ◽  
Author(s):  
B P Bean ◽  
E Rios
Keyword(s):  
Channel Gating ◽  
Cell Voltage ◽  
Na Channel ◽  
Charge Movement ◽  
Ca Channel ◽  
Gating Current ◽  
Channel Inactivation ◽  
Gating Charge ◽  
Ventricular Cells ◽  
Extra Charge

Intramembrane charge movement was recorded in rat and rabbit ventricular cells using the whole-cell voltage clamp technique. Na and K currents were eliminated by using tetraethylammonium as the main cation internally and externally, and Ca channel current was blocked by Cd and La. With steps in the range of -110 to -150 used to define linear capacitance, extra charge moves during steps positive to approximately -70 mV. With holding potentials near -100 mV, the extra charge moving outward on depolarization (ON charge) is roughly equal to the extra charge moving inward on repolarization (OFF charge) after 50-100 ms. Both ON and OFF charge saturate above approximately +20 mV; saturating charge movement is approximately 1,100 fC (approximately 11 nC/muF of linear capacitance). When the holding potential is depolarized to -50 mV, ON charge is reduced by approximately 40%, with little change in OFF charge. The reduction of ON charge by holding potential in this range matches inactivation of Na current measured in the same cells, suggesting that this component might arise from Na channel gating. The ON charge remaining at a holding potential of -50 mV has properties expected of Ca channel gating current: it is greatly reduced by application of 10 muM D600 when accompanied by long depolarizations and it is reduced at more positive holding potentials with a voltage dependence similar to that of Ca channel inactivation. However, the D600-sensitive charge movement is much larger than the Ca channel gating current that would be expected if the movement of channel gating charge were always accompanied by complete opening of the channel.

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 S4-based voltage sensors have three major conformations

2008 ◽  
Vol 105 (46) ◽  
pp. 17600-17607 ◽  
Author(s):  
Carlos A. Villalba-Galea ◽  
Walter Sandtner ◽  
Dorine M. Starace ◽  
Francisco Bezanilla
Keyword(s):  
Time Course ◽  
Stable State ◽  
Charge Movement ◽  
Voltage Sensors ◽  
Initial Voltage ◽  
Fluorescence Measurements ◽  
Gating Charge ◽  
Voltage Dependent ◽  
Α Helix ◽  
S4 Segment

Voltage sensors containing the charged S4 membrane segment display a gating charge vs. voltage (Q–V) curve that depends on the initial voltage. The voltage-dependent phosphatase (Ci-VSP), which does not have a conducting pore, shows the same phenomenon and the Q–V recorded with a depolarized initial voltage is more stable by at least 3RT. The leftward shift of the Q–V curve under prolonged depolarization was studied in the Ci-VSP by using electrophysiological and site-directed fluorescence measurements. The fluorescence shows two components: one that traces the time course of the charge movement between the resting and active states and a slower component that traces the transition between the active state and a more stable state we call the relaxed state. Temperature dependence shows a large negative enthalpic change when going from the active to the relaxed state that is almost compensated by a large negative entropic change. The Q–V curve midpoint measured for pulses that move the sensor between the resting and active states, but not long enough to evolve into the relaxed states, show a periodicity of 120°, indicating a 310 secondary structure of the S4 segment when determined under histidine scanning. We hypothesize that the S4 segment moves as a 310 helix between the resting and active states and that it converts to an α-helix when evolving into the relaxed state, which is most likely to be the state captured in the crystal structures.

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 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.

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 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 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.

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