scholarly journals Total Charge Movement per Channel

1997 ◽  
Vol 109 (1) ◽  
pp. 27-39 ◽  
Author(s):  
Daniel Sigg ◽  
Francisco Bezanilla
Keyword(s):  
Single Channel ◽  
Total Charge ◽  
Voltage Sensitivity ◽  
Charge Movement ◽  
Gating Charge ◽  
Experimental Resolution ◽  
Charge Displacement ◽  
Subconductance States ◽  
Activation Pathways ◽  
Sequential Models

One measure of the voltage dependence of ion channel conductance is the amount of gating charge that moves during activation and vice versa. The limiting slope method, introduced by Almers (Almers, W. 1978. Rev. Physiol. Biochem. Pharmacol. 82:96–190), exploits the relationship of charge movement and voltage sensitivity, yielding a lower limit to the range of single channel gating charge displacement. In practice, the technique is plagued by low experimental resolution due to the requirement that the logarithmic voltage sensitivity of activation be measured at very low probabilities of opening. In addition, the linear sequential models to which the original theory was restricted needed to be expanded to accommodate the complexity of mechanisms available for the activation of channels. In this communication, we refine the theory by developing a relationship between the mean activation charge displacement (a measure of the voltage sensitivity of activation) and the gating charge displacement (the integral of gating current). We demonstrate that recording the equilibrium gating charge displacement as an adjunct to the limiting slope technique greatly improves accuracy under conditions where the plots of mean activation charge displacement and gross gating charge displacement versus voltage can be superimposed. We explore this relationship for a wide variety of channel models, which include those having a continuous density of states, nonsequential activation pathways, and subconductance states. We introduce new criteria for the appropriate use of the limiting slope procedure and provide a practical example of the theory applied to low resolution simulation data.

scholarly journals Voltage Sensitivity and Gating Charge in Shaker and Shab Family Potassium Channels

1999 ◽  
Vol 114 (5) ◽  
pp. 723-742 ◽  
Author(s):  
Leon D. Islas ◽  
Fred J. Sigworth
Keyword(s):  
Single Channel ◽  
Delayed Rectifier ◽  
Voltage Sensitivity ◽  
Charge Movement ◽  
Gating Currents ◽  
Open Probability ◽  
Charge Voltage ◽  
Gating Charge ◽  
Charged Residues ◽  
Positively Charged

The members of the voltage-dependent potassium channel family subserve a variety of functions and are expected to have voltage sensors with different sensitivities. The Shaker channel of Drosophila, which underlies a transient potassium current, has a high voltage sensitivity that is conferred by a large gating charge movement, ∼13 elementary charges. A Shaker subunit's primary voltage-sensing (S4) region has seven positively charged residues. The Shab channel and its homologue Kv2.1 both carry a delayed-rectifier current, and their subunits have only five positively charged residues in S4; they would be expected to have smaller gating-charge movements and voltage sensitivities. We have characterized the gating currents and single-channel behavior of Shab channels and have estimated the charge movement in Shaker, Shab, and their rat homologues Kv1.1 and Kv2.1 by measuring the voltage dependence of open probability at very negative voltages and comparing this with the charge–voltage relationships. We find that Shab has a relatively small gating charge, ∼7.5 eo. Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 eo, essentially equal to that of Shaker and Kv1.1. Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10−9 in Shaker and below 4 × 10−8 in Kv2.1.

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 The gating charge should not be estimated by fitting a two-state model to a Q-V curve

2013 ◽  
Vol 142 (6) ◽  
pp. 575-578 ◽  
Author(s):  
Francisco Bezanilla ◽  
Carlos A. Villalba-Galea
Keyword(s):  
Infinite Number ◽  
Voltage Dependence ◽  
State Model ◽  
Total Charge ◽  
Gating Charge ◽  
Boltzmann Function ◽  
Sequential Models ◽  
Two State Model

The voltage dependence of charges in voltage-sensitive proteins, typically displayed as charge versus voltage (Q-V) curves, is often quantified by fitting it to a simple two-state Boltzmann function. This procedure overlooks the fact that the fitted parameters, including the total charge, may be incorrect if the charge is moving in multiple steps. We present here the derivation of a general formulation for Q-V curves from multistate sequential models, including the case of infinite number of states. We demonstrate that the commonly used method to estimate the charge per molecule using a simple Boltzmann fit is not only inadequate, but in most cases, it underestimates the moving charge times the fraction of the field.

scholarly journals Effects of D-600 on intramembrane charge movement of polarized and depolarized frog muscle fibers.

1989 ◽  
Vol 94 (1) ◽  
pp. 43-64 ◽  
Author(s):  
C Caputo ◽  
P Bolaños
Keyword(s):  
Muscle Fibers ◽  
External Solution ◽  
Ionic Currents ◽  
Membrane Depolarization ◽  
Total Charge ◽  
Voltage Sensitivity ◽  
Charge Movement ◽  
Internal Solution ◽  
Intramembrane Charge Movement ◽  
Movement Parameters

Intramembrane charge movement has been measured in frog cut skeletal muscle fibers using the triple vaseline gap voltage-clamp technique. Ionic currents were reduced using an external solution prepared with tetraethylammonium to block potassium currents, and O sodium + tetrodotoxin to abolish sodium currents. The internal solution contained 10 mM EGTA to prevent contractions. Both the internal and external solutions were prepared with impermeant anions. Linear capacitive currents were subtracted using the P-P/4 procedure, with the control pulses being subtracted either at very negative potentials, for the case of polarized fibers, or at positive potentials, for the case of depolarized fibers. In 63 polarized fibers dissected from Rana pipiens or Leptodactylus insularis frogs the following values were obtained for charge movement parameters: Qmax = 39 nC/microF, V = 36 mV, k = 18.5 mV. After depolarization we found that the total amount of movable charge was not appreciably reduced, while the voltage sensitivity was much changed. For 10 fibers, in which charge movement was measured at -100 and at 0 mV, Qmax changed from 46 to 41 nC/microF, while V changed from -41 to -103 mV and k changed from 20.5 to 30 mV. Thus membrane depolarization to 0 mV produces a shift of greater than 50 mV in the Q-V relationship and a decrease of the slope. Membrane depolarization to -20 and -30 mV, caused a smaller shift of the Q-V relationship. In normally polarized fibers addition of D-600 at concentrations of 50-100 microM, does not cause important changes in charge movement parameters. However, the drug appears to have a use-dependent effect after depolarization. Thus in depolarized fibers, total charge is reduced by approximately 20%. D-600 causes no further changes in the voltage sensitivity of charge movement in fibers depolarized to 0 mV, while in fibers depolarized to -20 and -30 mV it causes the same effects as that obtained with depolarization to 0 mV. These results are compatible with the idea that after depolarization charge 1 is transformed into charge 2. D-600 appears to favor the conversion of charge 1 into charge 2. Since D-600 also favors contractile inactivation, charge 2 could represent the state of the voltage sensor for excitation-contraction coupling in the inactivated state.

scholarly journals Activation of Shaker Potassium Channels

1998 ◽  
Vol 111 (2) ◽  
pp. 313-342 ◽  
Author(s):  
N.E. Schoppa ◽  
F.J. Sigworth
Keyword(s):  
Single Channel ◽  
Conformational Stability ◽  
Ionic Currents ◽  
Total Charge ◽  
Charge Movement ◽  
Gating Currents ◽  
Shaker Potassium Channel ◽  
Channel Opening ◽  
Tetrameric Structure ◽  
Charge Movements

A functional kinetic model is developed to describe the activation gating process of the Shaker potassium channel. The modeling in this paper is constrained by measurements described in the preceding two papers, including macroscopic ionic and gating currents and single channel ionic currents. These data were obtained from the normally activating wild-type channel as well as a mutant channel V2, in which the leucine at position 382 has been mutated to a valine. Different classes of models that incorporate Shaker's symmetrical tetrameric structure are systematically examined. Many simple gating models are clearly inadequate, but a model that can account for all of the qualitative features of the data has the channel open after its four subunits undergo three transitions in sequence, and two final transitions that reflect the concerted action of the four subunits. In this model, which we call Scheme 3+2′, the channel can also close to several states that are not part of the activation path. Channel opening involves a large total charge movement (10.8 e0), which is distributed among a large number of small steps each with rather small charge movements (between 0.6 and 1.05 e0). The final two transitions are different from earlier steps by having slow backward rates. These steps confer a cooperative mechanism of channel opening at Shaker's activation voltages. In the context of Scheme 3+2′, significant effects of the V2 mutation are limited to the backward rates of the final two transitions, implying that L382 plays an important role in the conformational stability of the final two states.

scholarly journals α-Actinin-1 promotes activity of the L-type Ca2+ Channel CaV1.2

2019 ◽  
Author(s):  
Matthew Turner ◽  
David E. Anderson ◽  
Madeline Nieves-Cintron ◽  
Peter Bartels ◽  
Andrea M. Coleman ◽  
...  
Keyword(s):  
Single Channel ◽  
Cardiac Contraction ◽  
Charge Movement ◽  
Open Probability ◽  
Iq Motif ◽  
Nmr Structures ◽  
Gating Charge ◽  
Channel Opening ◽  
Target Sites ◽  
Surface Localization

ABSTRACTThe L-type Ca2+ channel CaV1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1646-1664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits CaV1.2 to defined surface regions and simultaneously boosts its open probability so that CaV1.2 is mostly active when appropriately localized.

scholarly journals Role of Transmembrane Segment S5 on Gating of Voltage-dependent K+ Channels

1997 ◽  
Vol 109 (6) ◽  
pp. 767-778 ◽  
Author(s):  
Char-Chang Shieh ◽  
Kathryn G. Klemic ◽  
Glenn E. Kirsch
Keyword(s):  
Single Channel ◽  
Expression System ◽  
Charge Movement ◽  
Gating Charge ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
The Stability

The cytoplasmic half of S5 (5′S5) has been identified as part of the inner mouth of the pore based on evidence that mutations in this region greatly alter single channel conductance, 4-aminopyridine (4-AP) block and the rate of channel closing upon repolarization (deactivation). The latter effect, suggestive of a role for 5′S5 in channel gating was investigated in the present study. The biophysical properties of chimeric channels, in which the 5′S5 regions were exchanged between two host channels (Kv2.1 and Kv3.1) that differ in 4-AP sensitivity and deactivation rate, were examined in a Xenopus oocyte expression system. Exchange of 5′S5 between Kv2.1 and Kv3.1 confers steady-state voltage dependence of activation and rates of channel deactivation similar to those of the donor channel. The involvement of voltage-dependent gating was confirmed by the observation that exchanging the 5′S5 segment of Kv2.1 with that of Kv3.1 confers a change from slow to fast deactivation kinetics by accelerating the decay of off-gating charge movement. We suggest that a conformational change that extends from the voltage-sensor in S4 to the region of the pore lined by S5 regulates the stability of the open state. Therefore, the cytoplasmic end of S5, in addition to forming part of the conduction pathway near the inner mouth of the pore, also participates in the conformational rearrangements associated with late steps in channel activation and early steps in deactivation.

scholarly journals Allosteric Voltage Gating of Potassium Channels II

1999 ◽  
Vol 114 (2) ◽  
pp. 305-336 ◽  
Author(s):  
Frank T. Horrigan ◽  
Richard W. Aldrich
Keyword(s):  
Preceding Paper ◽  
Voltage Sensor ◽  
Voltage Gating ◽  
Total Charge ◽  
Charge Movement ◽  
Gating Currents ◽  
Open Probability ◽  
K Channels ◽  
Virtual Absence ◽  
Gating Charge

Large-conductance Ca2+-activated K+ channels can be activated by membrane voltage in the absence of Ca2+ binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca2+-activated K+ channels in the virtual absence of Ca2+ (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge–voltage relationship (Q–V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G–V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (τON = 60 μs at +200 mV, τOFF = 16 μs at −80 mV). However, QOFF increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of IK activation. The slow onset of this gating charge prevents its detection as a component of IgON, although it represents ∼40% of the total charge moved at +140 mV. The decay of IgOFF is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277–304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C–C, O–O, and C–O transitions.

scholarly journals Effective Activation of BKCa Channels by QO-40 (5-(Chloromethyl)-3-(Naphthalen-1-yl)-2-(Trifluoromethyl)Pyrazolo [1,5-a]pyrimidin-7(4H)-one), Known to Be an Opener of KCNQ2/Q3 Channels

2021 ◽  
Vol 14 (5) ◽  
pp. 388
Author(s):  
Wei-Ting Chang ◽  
Sheng-Nan Wu
Keyword(s):  
Single Channel ◽  
Slow Component ◽  
Ionic Channel ◽  
Gh3 Cells ◽  
Bkca Channel ◽  
Bkca Channels ◽  
Gating Charge ◽  
Ramp Voltage ◽  
K Current ◽  
The Mean

QO-40 (5-(chloromethyl)-3-(naphthalene-1-yl)-2-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-7(4H)-one) is a novel and selective activator of KCNQ2/KCNQ3 K+ channels. However, it remains largely unknown whether this compound can modify any other type of plasmalemmal ionic channel. The effects of QO-40 on ion channels in pituitary GH3 lactotrophs were investigated in this study. QO-40 stimulated Ca2+-activated K+ current (IK(Ca)) with an EC50 value of 2.3 μM in these cells. QO-40-stimulated IK(Ca) was attenuated by the further addition of GAL-021 or paxilline but not by linopirdine or TRAM-34. In inside-out mode, this compound added to the intracellular leaflet of the detached patches stimulated large-conductance Ca2+-activated K+ (BKCa) channels with no change in single-channel conductance; however, there was a decrease in the slow component of the mean closed time of BKCa channels. The KD value required for the QO-40-mediated decrease in the slow component at the mean closure time was 1.96 μM. This compound shifted the steady-state activation curve of BKCa channels to a less positive voltage and decreased the gating charge of the channel. The application of QO-40 also increased the hysteretic strength of BKCa channels elicited by a long-lasting isosceles-triangular ramp voltage. In HEK293T cells expressing α-hSlo, QO-40 stimulated BKCa channel activity. Overall, these findings demonstrate that QO-40 can interact directly with the BKCa channel to increase the amplitude of IK(Ca) in GH3 cells.

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.

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