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 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 Electrostatics and the Gating Pore of Shaker Potassium Channels

2001 ◽  
Vol 117 (1) ◽  
pp. 69-90 ◽  
Author(s):  
Leon D. Islas ◽  
Fred J. Sigworth
Keyword(s):  
Charge Movement ◽  
Transmembrane Helices ◽  
Gating Currents ◽  
Conical Cavity ◽  
K Channels ◽  
Gating Charge ◽  
Voltage Dependent ◽  
Shaker Potassium Channels ◽  
Electrostatic Calculations ◽  
S4 Segment

Various experiments have suggested that the S4 segment in voltage-dependent Na+ and K+ channels is in contact with a solvent-accessible cavity. We explore the consequences of the existence of such a cavity through the electrostatic effects on the gating currents of Shaker K+ channels under conditions of reduced ionic strength S. We observe that ∼10-fold reductions of intracellular S produce reductions of the measured gating charge of ∼10%. These effects continue at even lower values of S. The reduction of gating charge when S is reduced by 10-fold at the extracellular surface is much smaller (∼2%). Shifts of the Q(V) curve because of a reduced S are small (<10 mV in size), which is consistent with very little fixed surface charge. Continuum electrostatic calculations show that the S effects on gating charge can be explained by the alteration of the local potential in an intracellular conical cavity of 20–24-Å depth and 12-Å aperture, and a smaller extracellular cavity of 3-Å depth and the same aperture. In this case, the attenuation of the membrane potential at low S leads to reduction of the apparent gating charge. We suggest that this cavity is made by a bundle of transmembrane helices, and that the gating charge movement occurs by translocation of charged residues across a thin septum of ∼3–7 Å thickness.

scholarly journals Factors affecting the appearance of the hump charge movement component in frog cut twitch fibers.

1991 ◽  
Vol 98 (2) ◽  
pp. 315-347 ◽  
Author(s):  
C S Hui
Keyword(s):  
Time Course ◽  
Extreme Case ◽  
Complete Recovery ◽  
Creatine Phosphate ◽  
Boltzmann Distribution ◽  
Distribution Functions ◽  
Charge Movement ◽  
Factors Affecting ◽  
Gap Technique ◽  
Voltage Dependent

Charge movement was measured in frog cut twitch fibers with the double Vaseline gap technique. Five manipulations listed below were applied to investigate their effects on the hump component (I gamma) in the ON segments of TEST minus CONTROL current traces. When external Cl-1 was replaced by MeSO3- to eliminate Cl current, I gamma peaked earlier due to a few millivolts shift of the voltage dependence of I gamma kinetics in the negative direction. The Q-V plots in the TEA.Cl and TEA.MeSO3 solutions were well fitted by a sum of two Boltzmann distribution functions. The more steeply voltage-dependent component (Q gamma) had a V approximately 6 mV more negative in the TEA.MeSO3 solution than in the TEA.Cl solution. These voltage shifts were partially reversible. When creatine phosphate in the end pool solution was removed, the I gamma hump disappeared slowly over the course of 20-30 min, partly due to a suppression of Q gamma. The hump reappeared when creatine phosphate was restored. When 0.2-1.0 mM Cd2+ was added to the center pool solution to block inward Ca current, the I gamma hump became less prominent due to a prolongation in the time course of I gamma but not to a suppression of Q gamma. When the holding potential was changed from -90 to -120 mV, the amplitude of I beta was increased, thereby obscuring the I gamma hump. Finally, when a cut fiber was stimulated repetitively, I gamma lost its hump appearance because its time course was prolonged. In an extreme case, a 5-min resting interval was insufficient for a complete recovery of the waveform. In general, a stimulation rate of once per minute had a negligible effect on the shape of I gamma. Of the five manipulations, MeSO3- has the least perturbation on the appearance of I gamma and is potentially a better substitute for Cl- than SO2-(4) in eliminating Cl current if the appearance of the I gamma hump is to be preserved.

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 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 Dissociation of charge movement from calcium release and calcium current in skeletal myotubes by gabapentin

2002 ◽  
Vol 283 (3) ◽  
pp. C941-C949 ◽  
Author(s):  
Kris J. Alden ◽  
Jesús Garcı́a
Keyword(s):  
Skeletal Muscle ◽  
Calcium Current ◽  
Calcium Release ◽  
Maximum Amplitude ◽  
Charge Movement ◽  
Initial Voltage ◽  
Extra Charge ◽  
Voltage Dependent ◽  
Integral Role ◽  
Skeletal Myotubes

The skeletal muscle L-type calcium channel or dihydropyridine receptor (DHPR) plays an integral role in excitation-contraction (E-C) coupling. Its activation initiates three sequential events: charge movement (Qr), calcium release, and calcium current ( I Ca,L). This relationship suggests that changes in Qr might affect release and I Ca,L. Here we studied the effect of gabapentin (GBP) on the three events generated by DHPRs in skeletal myotubes in culture. GBP specifically binds to the α2/δ1 subunit of the brain and skeletal muscle DHPR. Myotubes were stimulated with a protocol that included a depolarizing prepulse to inactivate voltage-dependent proteins other than DHPRs. Gabapentin (50 μM) significantly increased Qr while decreasing the rate of rise of calcium transients. Gabapentin also reduced the maximum amplitude of the I Ca,L (as we previously reported) without modifying the kinetics of activation. Exposure of GBP-treated myotubes to 10 μM nifedipine prevented the increase of Qr promoted by this drug, indicating that the extra charge recorded originated from DHPRs. Our data suggest that GBP dissociates the functions of the DHPR from the initial voltage-sensing step and implicates a role for the α2/δ1 subunit in E-C coupling.

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 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 The AMIGO1 adhesion protein modulates movement of Kv2.1 voltage sensors

2021 ◽  
Author(s):  
Rebecka J Sepela ◽  
Robert G Stewart ◽  
Luis Valencia ◽  
Parashar Thapa ◽  
Zeming Wang ◽  
...  
Keyword(s):  
Neuronal Excitability ◽  
Voltage Sensor ◽  
Charge Voltage ◽  
Adhesion Protein ◽  
Voltage Sensors ◽  
Kv Channels ◽  
Mammalian Cell Lines ◽  
Fluorescence Measurements ◽  
Gating Charge ◽  
And Shifts

Voltage-gated potassium (Kv) channels sense voltage and facilitate transmembrane flow of K+ to control the electrical excitability of cells. The Kv2.1 channel subtype is abundant in most brain neurons and its conductance is critical for homeostatic regulation of neuronal excitability. Many forms of regulation modulate Kv2.1 conductance, yet the biophysical mechanisms through which the conductance is modulated are unknown. Here, we investigate the mechanism by which the neuronal adhesion protein AMIGO1 modulates Kv2.1 channels. With voltage clamp recordings and spectroscopy of heterologously expressed Kv2.1 and AMIGO1 in mammalian cell lines, we show that AMIGO1 modulates Kv2.1 voltage sensor movement to change Kv2.1 conductance. AMIGO1 speeds early voltage sensor movements and shifts the gating charge-voltage relationship to more negative voltages. Fluorescence measurements from voltage sensor toxins bound to Kv2.1 indicate that the voltage sensors enter their earliest resting conformation, yet this conformation is less stable upon voltage stimulation. We conclude that AMIGO1 modulates the Kv2.1 conductance activation pathway by destabilizing the earliest resting state of the voltage sensors.

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