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 Separation of Q beta and Q gamma charge components in frog cut twitch fibers with tetracaine. Critical comparison with other methods.

1992 ◽  
Vol 99 (6) ◽  
pp. 985-1016 ◽  
Author(s):  
C S Hui ◽  
W Chen
Keyword(s):  
Boltzmann Distribution ◽  
Distribution Functions ◽  
Slow Inward Current ◽  
Total Charge ◽  
Charge Movement ◽  
Voltage Distribution ◽  
Advantages And Disadvantages ◽  
Control Current ◽  
Gap Technique ◽  
Voltage Dependent

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. 25 microM tetracaine had very little effect on the maximum amounts of Q beta and Q gamma but slowed the kinetics of the I gamma humps in the ON segments of TEST-minus-CONTROL current traces, giving rise to biphasic transients in the difference traces. This concentration of tetracaine also shifted V gamma 3.7 (SEM 0.7) mV in the depolarizing direction, resulting in a difference Q-V plot that was bell-shaped with a peak at approximately -50 mV. 0.5-1.0 mM tetracaine suppressed the total amount of charge. The suppressed component had a sigmoidal voltage distribution with V = -56.6 (SEM 1.1) mV, k = 2.5 (SEM 0.5) mV, and qmax/cm = 9.2 (SEM 1.5) nC/microF, suggesting that the tetracaine-sensitive charge had a steep voltage dependence, a characteristic of the Q gamma component. An intermediate concentration (0.1-0.5 mM) of tetracaine shifted V gamma and partially suppressed the tetracaine-sensitive charge, resulting in a difference Q-V plot that rose to a peak and then decayed to a plateau level. Following a TEST pulse to greater than -60 mV, the slow inward current component during a post-pulse to approximately -60 mV was also tetracaine sensitive. The voltage distribution of the charge separated by tetracaine (method 1) was compared with those separated by three other existing methods: (a) the charge associated with the hump component separated by a sum of two kinetic functions from the ON segment of a TEST-minus-CONTROL current trace (method 2), (b) the steeply voltage-dependent component separated from a Q-V plot of the total charge by fitting with a sum of two Boltzmann distribution functions (method 3), and (c) the sigmoidal component separated from the Q-V plot of the final OFF charge obtained with a two-pulse protocol (method 4). The steeply voltage-dependent components separated by all four methods are consistent with each other, and are therefore concluded to be equivalent to the same Q gamma component. The shortcomings of each separation method are critically discussed. Since each method has its own advantages and disadvantages, it is recommended that, as much as possible, Q gamma should be separated by more than one method to obtain more reliable results.

scholarly journals Comparison of charge movement components in intact and cut twitch fibers of the frog. Effects of stretch and temperature.

1991 ◽  
Vol 98 (2) ◽  
pp. 287-314 ◽  
Author(s):  
C S Hui
Keyword(s):  
Sarcomere Length ◽  
Boltzmann Distribution ◽  
Distribution Functions ◽  
Total Charge ◽  
Charge Movement ◽  
Time To Peak ◽  
Gap Technique ◽  
Different Temperatures ◽  
Charge Movements ◽  
Kinetics Of

Charge movements were measured in frog intact fibers with the three-microelectrode technique and in cut fibers with the double Vaseline gap technique. At 13-14 degrees C, the ON segments of charge movement records from both preparations showed an early I beta component and a late I gamma hump component. When an intact fiber was cooled to 4-7 degrees C, the time-to-peak of I gamma (tp,gamma) was prolonged, but I gamma still appeared as a hump. Q-V plots from intact fibers at 4-7 degrees C were fitted with a sum of two Boltzmann distribution functions (method 1). The more steeply voltage-dependent component, identified with Q gamma, accounted for 32.1% (SEM 2.2%) of the total charge. This fraction was larger than the 22.6% (SEM 1.5%) obtained by separating the ON currents with a sum of two kinetic functions (method 2). The total charge in cut fibers stretched to a sarcomere length of 3.5 microns at 13-14 degrees C was separated into Q beta and Q gamma by methods 1 and 2. The fraction of Q gamma in the total charge was 51.3% (SEM 1.7%) and 53.7% (SEM 1.8%), respectively, suggesting that cut fibers have a larger proportion of Q gamma:Q beta than intact fibers. When cut fibers were stretched to a sarcomere length of 4 microns, the proportion of Q gamma:Q beta was unchanged. Between 4 and 13 degrees C, the Q10 of l/tp,gamma in intact fibers was 2.33 (SEM 0.33) and that of 1/tau beta was less than 1.44 (SEM 0.04), implying that the kinetics of I gamma has a steeper temperature dependence than the kinetics of I beta. When cut fibers were cooled from 14 to 6 degrees C, I gamma in the ON segment generally became too broad to be manifested as a hump. In a cut fiber in which I gamma was manifested as a hump, the Q10 of l/tp,gamma was 2.08 and that of l/tau beta was less than 1.47. Separating the Q-V plots from cut fibers at different temperatures by method 1 showed that the proportion of Q gamma:Q beta was unaffected by temperature change. The appearance of I gamma humps at low temperatures in intact fibers but generally not in cut fibers suggests an intrinsic difference between the two fiber preparations.

scholarly journals Effects of conditioning depolarization and repetitive stimulation on Q beta and Q gamma charge components in frog cut twitch fibers.

1992 ◽  
Vol 99 (6) ◽  
pp. 1017-1043 ◽  
Author(s):  
C S Hui ◽  
W Chen
Keyword(s):  
Boltzmann Distribution ◽  
Mirror Image ◽  
Distribution Functions ◽  
Repetitive Stimulation ◽  
Residual Fraction ◽  
Charge Movement ◽  
Gap Technique ◽  
Steady State Inactivation ◽  
Sigmoidal Curve ◽  
Time Courses

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. Steady-state inactivation of charge movement was studied by changing the holding potential from -90 mV to a level ranging from -70 to -30 mV. Q beta and Q gamma at each holding potential were separated by fitting the Q-V plot with a sum of two Boltzmann distribution functions. At -70 mV Q beta and Q gamma were inactivated to 54.0% (SEM 2.2) and 82.7% (SEM 3.0) of the amounts at -90 mV. At holding potentials greater than or equal to -60 mV, more Q gamma was inactivated than Q beta, and at -30 mV Q gamma was completely inactivated but Q beta was not. There was no holding potential at which Q beta was unaffected and Q gamma was completely inactivated. The differences between the residual fractions of Q beta and Q gamma are significant at all holding potentials (P less than 0.001-0.05). The plot of the residual fraction of Q beta or Q gamma versus holding potential can be fitted well by an inverted sigmoidal curve that is a mirror image of the activation curve of the respective charge component. The pair of curves for Q gamma correlates well with those for tension generation or Ca release obtained by other investigators. The time courses of the inactivation of Q beta and Q gamma were studied by obtaining several Q-V plots with conditioning depolarizations lasting 1-20 s and separating each Q-V plot into Q beta and Q gamma components by fitting with a sum of two Boltzmann distribution functions. The inactivation time constant of Q beta was found to be 5-10 times as large as that of Q gamma. During repetitive stimulation, prominent I gamma humps could be observed in TEST-minus-CONTROL current traces and normal Q gamma components could be separated from the Q-V plots, whether 20 or 50 mM EGTA was present in the internal solution, whether 2 or 10 stimulations were used, and whether the stimuli were separated by 400 ms or 6 s. Repetitive stimulation slowed the kinetics of the I gamma hump and could shift the Q-V curve slightly in the depolarizing direction in some cases, resulting in an apparent suppression of charge at the potentials that fall on the steep part of the Q-V curve.

scholarly journals Intramembranous charge movement in frog cut twitch fibers mounted in a double vaseline-gap chamber.

1990 ◽  
Vol 96 (2) ◽  
pp. 257-297 ◽  
Author(s):  
C S Hui ◽  
W K Chandler
Keyword(s):  
Voltage Dependence ◽  
Unit Length ◽  
Boltzmann Distribution ◽  
Distribution Functions ◽  
Charge Movement ◽  
Tetraethylammonium Chloride ◽  
Maximum Charge ◽  
Boltzmann Function ◽  
State Charge ◽  
Current Record

Intramembranous charge movement was measured in cut twitch fibers mounted in a double Vaseline-gap chamber with either a tetraethylammonium chloride (TEA.Cl) or a TEA2.SO4 solution (13-14 degrees C) in the central pool. Charge vs. voltage data were fitted by a single two-state Boltzmann distribution function. The average values of V (the voltage at which steady-state charge is equally distributed between the two Boltzmann states), k (the voltage dependence factor), and qmax/cm (the maximum charge divided by the linear capacitance, both per unit length of fiber) were V = -53.3 mV (SEM, 1.1 mV), k = 6.3 mV (SEM, 0.3 mV), qmax/cm = 18.0 nC/microF (SEM, 1.1 nC/microF) in the TEA.Cl solution; and V = -35.1 mV (SEM, 1.8 mV), k = 10.5 mV (SEM, 0.9 mV), qmax/cm = 36.3 nC/microF (SEM, 3.2 nC/microF) in the TEA2.SO4 solution. These values of k are smaller than those previously reported for cut twitch fibers and are as small as those reported for intact fibers. If a correction is made for the contributions of currents from under the Vaseline seals, V = -51.2 mV (SEM, 1.1 mV), k = 7.2 mV (SEM, 0.4 mV), qmax/cm = 22.9 nC/microF (SEM, 1.4 nC/microF) in the TEA.Cl solution; and V = -34.0 mV (SEM, 1.9 mV), k = 10.1 mV (SEM, 1.1 mV), qmax/cm = 38.8 nC/microF (SEM, 3.2 nC/microF) in the TEA2.SO4 solution. With this correction, however, the fit of the theoretical curve to the data is poor. A good fit with this correction can be obtained with a sum of two Boltzmann distribution functions. The first has average values V = -33.0 mV (SEM, 2.8 mV), k = 11.0 mV (SEM, 0.5 mV), qmax/cm = 10.6 nC/microF (SEM, 1.0 nC/microF) in the TEA.Cl solution; and V = -20.0 mV (SEM, 3.3 mV), k = 17.0 mV (SEM, 2.0 mV), qmax/cm = 36.4 nC/microF (SEM, 2.3 nC/microF) in the TEA2.SO4 solution. The second has average values V = -56.5 mV (SEM, 1.3 mV), k = 2.9 mV (SEM, 0.4 mV), qmax/cm = 13.2 nC/microF (SEM, 1.0 nC/microF) in the TEA.Cl solution; and V = -41.6 mV (SEM, 1.4 mV), k = 2.5 mV (SEM, 0.8 mV), qmax/cm = 11.8 nC/microF (SEM, 1.7 nC/microF) in the TEA2.SO4 solution. When a fiber is depolarized to near V of the second Boltzmann function, a slowly developing "hump" appears in the ON-segment of the current record.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Q beta and Q gamma components of intramembranous charge movement in frog cut twitch fibers.

1991 ◽  
Vol 98 (3) ◽  
pp. 429-464 ◽  
Author(s):  
C S Hui ◽  
W K Chandler
Keyword(s):  
Boltzmann Distribution ◽  
Active State ◽  
Charge Movement ◽  
Mean Values ◽  
Pulse Charge ◽  
Boltzmann Function ◽  
Voltage Dependent ◽  
The Mean ◽  
The Difference ◽  
The One

Intramembranous charge movement was measured in frog cut twitch fibers mounted in a double Vaseline-gap chamber with a TEA.Cl solution at 13-14 degrees C in the central pool. When a fiber was depolarized from a holding potential of -90 mV to a potential near -60 mV, the current from intramembranous charge movement was outward in direction and had an early, rapid component and a late, more slowly developing component, referred to as I beta and I gamma, respectively (1979. J. Physiol. [Lond.]. 289:83-97). When the pulse to -60 mV was preceded by a 100-600-ms pulse to -40 mV, early I beta and late I gamma components were also observed, but in the inward direction. The shape of the Q gamma vs. voltage curve can be estimated with this two-pulse protocol. The first pulse to voltage V allows the amounts of Q beta and Q gamma charge in the active state to change from their respective resting levels, Q beta (-90) and Q gamma (-90), to new steady levels, Q beta (V) and Q gamma (V). A second 100-120-ms pulse, usually to -60 mV, allows the amount of Q beta charge in the active state to change from Q beta (V) to Q beta (-60) but is not sufficiently long for the amount of Q gamma charge to change completely from Q gamma (V) to Q gamma (-60). The difference between the amount of Q gamma charge at the end of the second pulse and Q gamma (-60) is estimated from the OFF charge that is observed on repolarization to -90 mV. The OFF charge vs. voltage data were fitted, with gap corrections, with a Boltzmann distribution function plus a constant. The mean values of V (the potential at which, in the steady state, charge is distributed equally between the resting and active states) and k (the voltage dependence factor) were -59.2 mV (SEM, 1.1 mV) and 1.2 mV (SEM, 0.6 mV), respectively. The one-pulse charge vs. voltage data from the same fibers were fitted with a sum of two Boltzmann functions (1990. J. Gen. Physiol. 96:257-297). The mean values of V and k for the steeply voltage-dependent Boltzmann function, which is likely to be associated with the Q gamma component of charge, were -55.3 mV (SEM, 1.3 mV) and 3.3 mV (SEM, 0.6 mV), respectively, similar to the corresponding values obtained with the two-pulse protocol.(ABSTRACT TRUNCATED AT 400 WORDS)

IgG from amyotrophic lateral sclerosis affects tubular calcium channels of skeletal muscle

1991 ◽  
Vol 260 (6) ◽  
pp. C1347-C1351 ◽  
Author(s):  
O. Delbono ◽  
J. Garcia ◽  
S. H. Appel ◽  
E. Stefani
Keyword(s):  
Skeletal Muscle ◽  
Amyotrophic Lateral Sclerosis ◽  
Charge Movement ◽  
Mammalian Skeletal Muscle ◽  
Gap Technique ◽  
Voltage Dependent ◽  
Als Patients ◽  
Tubular Membranes ◽  
Ca2 Channels ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating human disease of upper and lower motoneurons. We studied the action of the immunoglobulin G (IgG) from ALS and disease control patients on dihydropyridine (DHP)-sensitive Ca2+ channels in single mammalian skeletal muscle fibers with the double Vaseline gap technique. The peak of the Ca2+ current (ICa) and the charge movement were reduced when the fibers were incubated in ALS IgG. These effects were lost when the IgG was boiled or adsorbed with skeletal tubular membranes. ALS IgG reduced skeletal muscle ICa in a similar fashion as nifedipine; the ICa blockade was voltage dependent, and the associated charge movement was reduced. These observations suggest that IgG from ALS patients reacts with the skeletal muscle DHP-sensitive Ca2+ channels or some associated regulatory moiety.

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 State dependent effects on the frequency response of prestin’s real and imaginary components of nonlinear capacitance

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Joseph Santos-Sacchi ◽  
Dhasakumar Navaratnam ◽  
Winston J. T. Tan
Keyword(s):  
Frequency Response ◽  
Outer Hair Cell ◽  
Absolute Magnitude ◽  
Total Charge ◽  
Charge Movement ◽  
Membrane Tension ◽  
Nonlinear Capacitance ◽  
Complex Component ◽  
Trade Offs ◽  
Voltage Dependent

AbstractThe outer hair cell (OHC) membrane harbors a voltage-dependent protein, prestin (SLC26a5), in high density, whose charge movement is evidenced as a nonlinear capacitance (NLC). NLC is bell-shaped, with its peak occurring at a voltage, Vh, where sensor charge is equally distributed across the plasma membrane. Thus, Vh provides information on the conformational state of prestin. Vh is sensitive to membrane tension, shifting to positive voltage as tension increases and is the basis for considering prestin piezoelectric (PZE). NLC can be deconstructed into real and imaginary components that report on charge movements in phase or 90 degrees out of phase with AC voltage. Here we show in membrane macro-patches of the OHC that there is a partial trade-off in the magnitude of real and imaginary components as interrogation frequency increases, as predicted by a recent PZE model (Rabbitt in Proc Natl Acad Sci USA 17:21880–21888, 2020). However, we find similar behavior in a simple 2-state voltage-dependent kinetic model of prestin that lacks piezoelectric coupling. At a particular frequency, Fis, the complex component magnitudes intersect. Using this metric, Fis, which depends on the frequency response of each complex component, we find that initial Vh influences Fis; thus, by categorizing patches into groups of different Vh, (above and below − 30 mV) we find that Fis is lower for the negative Vh group. We also find that the effect of membrane tension on complex NLC is dependent, but differentially so, on initial Vh. Whereas the negative group exhibits shifts to higher frequencies for increasing tension, the opposite occurs for the positive group. Despite complex component trade-offs, the low-pass roll-off in absolute magnitude of NLC, which varies little with our perturbations and is indicative of diminishing total charge movement, poses a challenge for a role of voltage-driven prestin in cochlear amplification at very high frequencies.

Acute effects of repetitive depolarization on sodium current in chick myocytes

1991 ◽  
Vol 260 (6) ◽  
pp. H1810-H1818
Author(s):  
M. R. Gold ◽  
G. R. Strichartz
Keyword(s):  
Time Course ◽  
Sodium Current ◽  
Complete Recovery ◽  
Acute Effects ◽  
Na Current ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Atrial Cells ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation

Acute effects of repetitive depolarization on the inward Na+ current (INa) of cultured embryonic chick atrial cells were studied using the whole cell patch-clamp technique. Stimulation rates of 1 Hz or greater produced a progressive decrement of peak INa. With depolarizations to 0 mV of 150-ms duration, applied at 2 Hz from a holding potential of -100 mV, the steady-state decrement was approximately 20%. The magnitude of this effect increased with stimulation frequency and with test potential depolarization and decreased with membrane hyperpolarization. Analysis of INa kinetics revealed that reactivation was sufficiently slow to preclude complete recovery from inactivation with interpulse intervals less than 1,000 ms. Moreover, reactivation accelerated markedly with membrane hyperpolarization, in parallel with the response to repetitive stimulation. The multiexponential time course of recovery of peak INa from repetitive depolarization was similar to that observed after single stimuli; however, there was a shift toward a greater proportion of current recovering with the slower of two time constants. It is concluded that incomplete recovery from inactivation is responsible for the decrement in INa observed with short interpulse intervals.

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.

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