Correspondence to: Fred J. Sigworth, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Fax:203-785-4951 E-mail:fred.sigworth{at}yale.edu.
Released online: 25 October 1999
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Abstract |
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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 chargevoltage 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 x 10-8 in Kv2.1.
Key Words: ion channels, patch-clamp, gating current, kinetics
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INTRODUCTION |
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Voltage-gated ion channels are remarkably sensitive to membrane potential changes. Large charge movements in the channel protein couple changes in membrane potential to the channel opening process. In the skeletal muscle sodium channel and the Shaker potassium channel, the size of this "gating charge" movement is seen to be equivalent to the displacement of 1214 elementary charges entirely across the membrane (
The various voltage-gated potassium channels contain four subunits; many of these subunits show differences in their S4 regions that are suggestive of differing amounts of gating charge. The S4 region is thought to be the primary voltage-sensing region (
Because of uncertainties in past measurements of charge movement based on voltage sensitivity, and to complement the studies in which the magnitude of gating charge has been changed through mutations, we report here measurements of the gating charge in four "native" voltage-gated potassium channel types using improved methods.
Gating charge movement is most directly measured by integrating the gating currents that are recorded from membranes having a high density of channels, under conditions in which no ionic current flows through the channels. To then evaluate the charge movement in a single channel, the number of channels contributing to the gating current must be estimated; this has been done for Shaker channels using either fluctuation analysis ( -
(and P
0), the apparent charge,
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(1) |
approaches the total charge movement qT (
The limiting-slope method has the advantage that it measures charge that is actually involved in opening the channel. Its asymptotic nature, on the other hand, poses a problem: how can one be sure that qs is evaluated at sufficiently negative V? Two recent advances now allow this problem to be addressed in a rigorous fashion. The first is the development by
The second advance is the theory of (V)be the macroscopic gating charge that is obtained by integrating the gating current, normalized such that
(-
)=0and
(+
)=1. Then, in the case of a channel that is found in its single open state at large depolarizations, the apparent charge qs is related to
by:
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(2) |
Thus, given experimentally determined values of qs and , a one-parameter curve fit can be used to estimate the value of qT.
In this paper, we incorporate these advances to the limiting-slope method and apply it to the determination of charge movements in homomeric channels encoded by the Drosophila potassium channel genes Shaker and Shab, and from the rat homologues Kv1.1 and Kv2.1. A comparison of these channel types is interesting in view of their differing amino acid sequences (Figure 1). Shaker and Kv1.1 both have seven basic residues in the S4 region, while Shab and Kv2.1 have five.
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MATERIALS AND METHODS |
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Channel Expression in Oocytes
Shaker H4 with N-type inactivation removed and the W434F mutant were subcloned into the vector pGEM, linearized with Not I and transcribed with T7 RNA polymerase. The final cRNA concentration was 1 µg/µl. Kv1.1 was subcloned into pGEMHE, linearized with PstI and transcribed using T7 polymerase. Shab was subcloned in a modified version of Bluescript for oocyte experiments. Linearization of cDNA was carried out with NotI and transcription with T3 polymerase.
The rat clone Kv2.1-7 (DRK1-
7), derived from DRK1 (
15 pM) of the channel blocker Agitoxin-1 (
7 in Bluescript was linearized with NotI, transcribed with T7 RNA polymerase and resuspended in water to a final concentration of 0.30.5 µg/µl.
Xenopus laevis oocytes were harvested under anesthesia and defolliculated by incubation in Ca+2-free OR2 solution supplemented with 2 mg/ml collagenase 1A for 1 h. Oocytes were incubated at 20°C in a solution containing (mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, and injected 1 or 2 d after harvesting with 50 nl of cRNA solution with a Nanostepper (Drummond Scientific Co.) using pipettes of 20-µm opening diameter. Oocytes were used for experiments 17 d after injection.
In some cases, oocytes expressing Kv2.1-7 were incubated in the presence of Agitoxin-1 at a concentration of 13 µM for the period of channel expression. This resulted in an increase in expression of two- to threefold.
Expression of Channels in Sf9 Cells
The Shab channel was expressed in Sf9 cells for the recording of gating currents in whole cells. The Bac-to-BacTM recombinant baculovirus system (GIBCO BRL) was used. After transfer of the Shab gene to a bacmid vector, Sf9 cells at 70% confluence were transfected with lipofectin in Sf900 medium (GIBCO BRL). The culture medium containing the Shab baculovirus was collected after 48 h of culture and stored at 4°C. After infection, expression of channels in cells was assessed by electrophysiological recording. Sf9 cells were maintained in an incubator at 26°C in 1% CO2.
Patch Recording
Patch-clamp recordings were obtained using standard techniques. All recordings reported here were made in the cell-attached configuration, for maximum patch stability. Oocytes were bathed in a depolarizing solution that effectively zeroed the membrane potential as checked with a two-microelectrode voltage clamp. The bath solution contained (mM): 120 K-aspartate, 20 KCl, 1.8 CaCl2, 10 HEPES, adjusted to pH 7.4. Unless otherwise stated in the figure legends, pipettes were filled with a solution of the following composition (mM): 40 K-aspartate, 20 KCl, 1.8 CaCl2, 60 N-methyl-D-glucamine (NMDG)1-aspartate, 10 HEPES, pH 7.4. Gating currents of Kv2.1-7 were recorded using a similar solution that contained 120 mM NMDG-aspartate and no K+, and included 1 µM Agitoxin-1. The same solution was used for Shaker gating currents without the Agitoxin-1. For gating currents, 20 sweeps were averaged at every voltage. A P/5 subtraction protocol was used to subtract linear capacity and leak currents, using a leak-holding potential of -120 mV.
Pipettes were pulled from soft borosilicate glass capillaries (Kimax-51) and had resistance in the range 1.53 M. Currents were recorded with an EPC-9 amplifier running the Pulse software (HEKA Elektronik) on a Macintosh computer. In macroscopic current recordings, the linear current components were subtracted by the use of a P/5 protocol and were filtered at 2.5 kHz (-3 dB) with a Bessel filter and sampled at 15 kHz. Single-channel recordings were obtained in the same patches as macroscopic currents, with the same filter cut-off frequency and a sample frequency of 10 kHz. Null-trace averages were used to subtract the linear capacitive and leak currents. All recordings were obtained at room temperature (22°C).
Whole-cell gating currents in Sf9 cells were recorded using the following solutions (mM): Pipette: 150 NMDG-HCl, 10 MOPS, 5 EGTA, 1 MgCl2, pH 7.2; Bath: 150 NMDG-HCl, 10 MOPS, 10 CaCl2, 1 MgCl2, pH 6.7. Recording electrodes had a resistance of 0.51 M and were coated with Sylgard® to reduce capacitance. No series resistance compensation was used. The capacitance of the Sf9 cells used for gating current recordings ranged from 20 to 42 pF. The settling time of capacity transients was ~100 µs. The subtraction protocol was similar to that used for Kv2.1.
Data Analysis
For macroscopic currents, the voltage dependence of the open probability was estimated from tail current measurements; the size of the tail current is proportional to the channel open probability at the end of the preceding depolarization. We averaged the first millisecond of tail current at -100 mV in the case of Kv2.1 channels. For Shaker and Shab, the first 300 µs of tail currents at -90 mV were averaged and for Kv1.1, 400 µs was averaged. The values obtained were in very close agreement to the more traditional method of measuring P from the steady state currents and normalizing by the instantaneous currentvoltage curve. Contamination of the ionic tail currents by gating currents was taken to be negligible because OFF gating current amplitudes were <0.5% of the maximal tail current amplitude.
Estimation of the number of channels in a patch of membrane was done by nonstationary noise analysis (
where sigma}">2 is the variance, I is the mean current, i is the single channel current, and n is the number of channels. The maximal open probability was then obtained as Pmax = Imax/Ni.
The analysis of the single channel traces to obtain the time-dependent NP values was carried out as follows. The current traces were leak subtracted using a template of averaged null sweeps (sweeps that contain no channel openings), digitally filtered with a Gaussian filter to 1.5 kHz and analyzed with the 50% threshold crossing method (
The apparent charge qs was estimated from experimental data by discretizing Equation 1 in the following way. Given open probabilities P1 and P2 obtained at adjacent voltages V1 and V2, and letting V' = (V1 + V2)/2, we computed qs as:
The equilibrium voltage dependence of state occupancies was computed according to Equation 3:
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(3) |
where Pn is the probability of occupancy of state n, Kj is the equilibrium constant for transition from state j - 1 to state j, and the M states are linearly connected.
Statistical quantities are given as mean ± SEM
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RESULTS |
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Channel Activation Properties
To extend the previous studies of Shab channel currents (7, that was engineered to bind the pore blocker Agitoxin-1 with high affinity (
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Activation time constants were estimated from exponential fits to the second half of the activation time course of macroscopic currents (
was also obtained. Shaker channels show a small activation time constant that is rather weakly voltage dependent with an effective charge of 0.51 eo; the delay is roughly equal to the time constant and has similar voltage dependence (Figure 2D and Figure E) over the positive voltage range. The kinetic features of Kv1.1 channels are nearly identical to those of Shaker, having almost identical activation time constants and delay values, with the main difference being a slightly steeper activation curve.
Like Shaker, Shab channels activate and deactivate quickly, with time constants similar to those of Shaker. Shab channels activate, however, with a smaller delay that is more steeply voltage dependent (Figure 2 E); this means that at positive voltages these channels show less sigmoidicity in their activation time course than Shaker (;
and
having values that are larger by an order of magnitude.
Gating Currents
The time course of charge movement of Shaker, Kv2.1, and Shab channels was directly measured in the form of gating currents. Shaker gating currents were obtained from cell-attached patches in oocytes expressing the nonconducting mutant W434F (
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Kv2.1 gating currents were also measured in cell-attached patches in oocytes, using Agitoxin-1 to block the ionic currents. The general characteristics are similar to those previously reported (
The Kv2.1 charge movement curve lies to the left of the P(V) curve in the voltage axis, as is expected for a channel that has multiple closed states before the open state. The voltage dependence of charge can be fitted to a Boltzmann function with a charge of 2 eo (Figure 3 B).
Shab gating currents were recorded in the whole-cell configuration from baculovirus-infected Sf9 insect cells, with ionic currents eliminated through the use of impermeant N-methyl glucamine (NMG) cations. Figure 3B and Figure C, compares the voltage dependence of ON charge and the voltage dependence of the time constant of ON currents in these channels. Shab ON gating currents are less voltage dependent than those of Shaker or Kv2.1, and their kinetics are intermediate. Shab OFF gating currents are, however, very different in having a much faster time course than the others. The slow OFF time course in Shaker channels arises from slow initial closing transitions (
Limiting-Slope Measurements in Shaker Channels
With the high expression obtained with Shaker in oocytes, it was possible to record from patches containing hundreds or thousands of channels. The number N of channels in a patch was estimated by nonstationary fluctuation analysis, using depolarizations to +70 mV. Subsequent recordings using small depolarizing pulses allowed NP to be estimated from the statistics of single-channel events in the same patch. Recordings from a representative patch containing 2,250 channels are shown in Figure 4 A. The channel openings observed during the small depolarizations were identified as Shaker channels based on their conductance and kinetics.
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To obtain the steady state open probability from pulsed data sweeps, we first performed an idealization of the single channel events for each sweep, and then averaged these idealized records. This ensemble average (Figure 4 B) represents the product NP of the open probability and the number of channels that contribute to the record. No inactivation is visible in these time courses, and because of the high external K+ concentration the degree of slow "C-type" inactivation on these channels is expected to be very small (
At the most negative voltages (-70 to -55 mV), the value of NP changed by about a factor of 10 for each 5-mV change in the depolarization. No openings were detected during the total 450 s of recording at the holding potential of -80 mV. At -80 mV, the mean burst duration is expected to be ~1.5 ms so that, were a single opening to occur, it would correspond to NP 3.3 x 10-6. The fact that no events were observed at -80 mV in this patch or in two others having similar numbers of channels implies that at -80 mV P is smaller than ~10-9.
Figure 4 C shows the result of combining the single channel data with macroscopic activation measurements and the value of n obtained from nonstationary noise analysis. The smallest open probability measured in these patches was ~10-7, and the maximum was 0.79. At values of P between 10-7 and 10-4 the P(V) relation is very nearly exponential and can be well fitted to the relationship
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(4) |
The charge estimate ql from this linear fit to the log-transformed P values represents a local average of qs as defined in Equation 1; for these channels it had the value 12.7 ± 0.3 eo (n = 3).
We performed two tests to determine whether these experiments can provide estimates of the apparent charge that approach the true asymptotic value. First, we computed qs from Equation 1 at each voltage and compared its behavior with that of two simple models in which P is given by a Boltzmann function or by the fourth power of a Boltzmann function. The comparison is done by plotting the logarithmic slope variable qs as a function of P, with V being the independent variable (
In the second test, qs(V) is compared with the function, which from Equation 2 should yield identical values. Figure 4 E makes this comparison for the case qT = 13 eo, where it is seen that the voltage dependences are very similar. Most importantly, the charge movement shows very little change below V = ~70 mV, implying that qs will remain essentially constant below this membrane potential. On the basis of this fit, we extrapolate the qs values to obtain an estimate of 13.0 eo for the total charge qT.
The Gating Charge of Kv1.1 Is Similar to that of Shaker
Rat Kv1.1 (previously called RCK1) is a potassium channel that does not have fast inactivation. Its general gating characteristics are very similar to those of inactivation-removed Shaker and the amino acid sequence in the S4 region is identical (
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Because of the low channel density, we were unable to record gating currents from patches containing Kv1.1 channels; therefore, we were unable to provide an independent check on the approach to the limiting charge movement. We conclude that qT is at least 11.5 eo and is probably near 13 eo, as is the case with Shaker.
Although no inactivation is apparent in the reconstructed time courses of Figure 5 B, it is conceivable that slow inactivation or rundown could introduce errors into our estimates for P. We therefore compared the N values obtained from noise analysis before and immediately after delivering the 600 depolarizing pulses to -60 and -50 mV for single-channel analysis of one patch; the values of N were 14 and 12, respectively. If the reduction in the number of channels is caused by inactivation, this indicates that there was at most 14% long-term inactivation. In our experiments, this small amount of inactivation at negative voltages would, if anything, have led to an underestimation of the limiting slope.
Limiting-Slope Measurements in Kv2.1 Channels
Previous estimates of the gating charge of the rat Kv2.1 channel (
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At the most negative voltages (-45 to -65 mV), the value of NP decreased by a factor of ~10 per 5-mV decrease in depolarization (Figure 6 B). No openings that could be assigned to Kv2.1 channels were detected at the holding potential of -80 mV in two experiments where the total time of recording at -80 mV was 118 and 180 s and the number of channels N was 967 and 1,250, respectively. The channel burst duration, extrapolated to -80 mV, is ~10 ms. Were a single burst of openings to occur in one of these records, this would correspond to the open probability P 4 x 10-8. Therefore, we take the value of P at -80 mV to be less than ~4 x 10-8.
The result of combining the single channel data with the values of N and Pmax is shown in Figure 6 C. The minimum measured value of the absolute open probability in this patch is ~10-7 and the maximum, from noise analysis at 70 mV, was 0.71. At values of P between 10-7 and 10-4 exponential fits of the P(V) relation yield an apparent charge qs of 12.1 ± 0.5 eo. A fourth power Boltzmann function with 12 or 13 eo total charge (continuous curves) superimposes fairly well on the data (Figure 6 C) when qs is plotted as a function of P.
The same charge estimate results in comparing qs with the macroscopic charge movement. The quantity superimposes well on the qs data (Figure 6 D) with the fitted value qT = 12.5 eo. Thus, this channel behaves as expected from Equation 2 and the charge movement in the voltage range below that accessible to our qs measurements is seen to be small. Our best estimate of the total charge for Kv2.1 is therefore 12.5 eo.
It is unlikely that this estimate of total charge is in error due to inactivation or missed channel events. Inactivation in the Kv2.1 channels was measured with 500-ms prepulses (Figure 7 A) and was found to be negligible at potentials below -50 mV (Figure 7 B). Single-channel events at negative voltages show a single open-time component with a mean duration of 8 ms (Figure 7 C), consistent with the presence of only one open state of the channel. The duration of bursts of openings, ~15 ms (Figure 7 D), is essentially voltage independent, always much greater than the detection dead-time of 120 µs; thus, a very small and voltage-independent fraction of events is expected to be missed in the evaluation of NP.
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Limiting-Slope Measurements in Shab Channels
In Xenopus oocytes, the expression of Shab channels was low, producing maximal whole-cell currents of ~10 µA at +40 mV. To obtain higher channel densities and to allow the possibility of whole-cell recording of Shab gating currents, we established a baculovirus expression system using Sf9 insect cells. The currents obtained in cell-attached patches from Sf9 cells infected with the Shab baculovirus have very similar characteristics to those recorded in patches in oocytes. No voltage-dependent currents were observed in whole-cell recordings from five uninfected Sf9 cells.
Subconductance levels. Interestingly, single Shab channels in single-channel patches and in recordings from multiple channel patches show a tendency to open to subconductance states. These events arise from Shab channels since direct transitions are observed between the subconductance and the fully open states. Figure 8 A shows representative current recordings at -70 mV. Indicated are the fully open state and the substate level at 40% of the full open current level. It is evident that the channel can dwell only in the substate or make transitions to the open state with or without having to traverse the substate level.
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Subconductance openings are more prominent at negative voltages. At -90 mV, less than one percent of the time is spent in the subconductance level, but 98% of the observed events are subconductance openings. This is illustrated in Figure 8 B, where all-points histograms of current recordings from multichannel patches at the indicated potentials are shown. The intermediate Gaussian component fitted to the histogram corresponds to the substate and its integral is proportional to the probability of occupancy of the substate. At potentials more negative than -70 mV, the probability of the substate Ps is greater than the probability of the fully open state, P. These probabilities are plotted in Figure 8 C; it is seen that while P increases monotonically, Ps increases and then decreases with depolarization. This characteristic of Ps, along with its smaller voltage dependence (the apparent charge is only 2.2 e0 between -90 and -70 mV) is consistent with the sublevel representing an intermediate state in the activation pathway.
The voltage dependence of occupancies of the substate and the fully open state can be described by specific schemes. The steady state occupancy curves in Figure 8 C were computed from the model shown in that figure in which sequential transitions bring the channel to a substate (S), and then the fully open state. In the model, a parallel pathway also allows direct openings without passing through the substate; this provision is necessary because the substate is traversed in only ~6% of full openings of the channel. The model yields a limiting slope corresponding to a total charge of 7.2 eo for the open state and 5 eo for the substate.
Kinetic analysis of the substate was carried out to characterize the transitions near the open and subconductance states at negative potentials. The rate constants for transitions away from the substate were obtained from the dwell-time distribution in the substate and the fraction of transitions occurring in each particular path. For example, at negative voltages, dwells in the substate were seen to be terminated by transitions to the open state, to a closed state, or transitions to a closed state followed, after a delay of ~50 ms, by channel opening. The rate constants kij were obtained:
where i is the mean dwell time in state i and fij is the fraction of all transitions leaving state i that end in state j (Zheng and Sigworth, 1997). Dwell-time distributions in the fully open state were used to obtain rates for transitions to the closed state in the activation pathway identified as Cn, and to the substate, using the same method. Values of the rate constants for transitions near the open state obtained in this way are shown in Figure 8 D, which is presented as an example of a scheme that can account for the kinetic phenomena we observe. In conclusion, both the steady state and kinetic behaviors are consistent with the subconductance level being associated with a state that is a kinetic intermediate and that is reached with less than the full charge movement.
Magnitude of charge movement. For the estimation of total charge movement, we ignore dwells at the sublevel because these reflect an intermediate state. Instead, we make use of only the probability P of the fully open state, which is the predominant state at large depolarizations. Sublevel dwells will not affect the estimation of N by noise analysis because they are brief and very rare at the test voltage of +60 mV.
Even ignoring the sublevels, activation of Shab channels is not as voltage dependent as that of Kv2.1. At a holding potential of -90 mV it is possible to observe channel openings, and NP for the fully open state is seen to change only 10-fold for 10-mV depolarizations (Figure 9A and Figure B); this is about half the sensitivity seen for the other channel types. In constructing idealized traces, we took care not to confuse overlapping sublevel currents with open-state events; such overlapping events were very rare except at relatively depolarized voltages such as -70 mV. The resulting apparent charge estimate, based on P values between 10-5 and 10-3 was ql = 7.1 ± 0.3 eo (n = 2; Figure 9 C). Plotting qs against P, the asymptotic value appears to be 7.5 eo (Figure 9 D). Similarly, low apparent charge values were seen in recordings from Shab channels in oocytes, where ql = 7.5 ± 0.3 eo (n = 3) when evaluated over the range of P values from 10-6 to 10-4. Whole-cell recordings of gating currents in Sf9 cells allow the comparison of macroscopic charge movements with the voltage dependence of qs. The quantity superimposes fairly well on qs values with qT = 7.5 eo (Figure 9 E) and demonstrates that very little charge movement occurs at voltages negative to -90 mV. We therefore take 7.5 e0 to be the estimate for the total charge in this channel.
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A comparison of the voltage dependence of qs with that predicted from Q(V) indeed shows a small discrepancy of this sort: a minor component of the gating charge Q(V) moves at potentials above -40 mV, where the qs curve is essentially flat. This component is, however, much smaller than that expected from Scheme 1 and therefore is unlikely to arise from an additional open state. The discrepancy between qs and Q(V) is in fact similar to the discrepancies seen in Shaker and Kv2.1 channels (Figure 4 E and 6 D). Because the discrepancy occurs in the voltage range of channel opening, we believe that it arises from an interaction of permeant ions with the open channel. Effects of permeating ions on activation gating have been studied in sodium channels (
We expect that, in the presence of permeant ions, the equilibrium of the final opening transition is shifted toward the open state, tending to increase the cooperativity of channel opening and resulting in a steeper voltage dependence of qs. In our gating-current experiments, permeant ions were either absent or prevented from entering the pore, so the cooperative effect would be absent, yielding the shallower Q(V) curves that are observed. We conclude that there is no evidence for a large, additional charge movement that occurs while the channel is open.
As a further check for the possible existence of multiple open states, we examined the single-channel kinetics of Shab channels. Figure 10 A shows recordings from a patch containing a single Shab channel. Over the range of voltages from -30 to +40 mV, the open time distribution showed a single exponential component (Figure 10 B). Data from multichannel patches also show no evidence for a second open-time component at voltages down to -80 mV. The mean burst duration at -60 mV is 3.7 ± 0.1 ms (n = 3) and has a very weak voltage dependence corresponding to a partial charge of 0.1 eo. The single-exponential open-time distributions support the idea that Shab has only one fully open state. Thus again, we find no reason to expect multiple open states, and find the simplest conclusion to be that Shab channels have a reduced gating charge of ~7.5 e0.
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Ideally, this estimate of gating charge should be confirmed with an independent measure in which the total integrated gating current is normalized by the number of channels. Unfortunately, the relatively low expression level of Shab channels has precluded this sort of measurement, which requires either the ability to measure gating currents in a patch membrane or the ability to measure radioligand binding to a single cell.
Single-channel kinetics.
The Shab single-channel closed-time distributions were well described by three exponential components (Figure 10 B). The fastest closures are most frequent at the largest depolarization of +40 mV. The dwell time in this closed state is essentially voltage independent (Figure 10 C, ). This closed state may be the result of block by intracellular components or it could be a conformational state that is entered after the channel has opened, as is the case in Shaker channels (
Transitions to this fast closed state from the open state accounts for the fact that P does not approach unity at large depolarizations. The value of Pmax is 0.72, as obtained from noise analysis of macroscopic currents at +50 to +70 mV. From the single-channel recordings, we calculate an open probability of 0.68 at +60 mV, very close to the estimate from noise analysis.
Shab channels inactivate on a time scale of seconds and with a half-inactivation voltage of -30 mV (
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DISCUSSION |
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In the experiments described in this report, we have estimated the effective gating charge of four members of the voltage-gated K+ channel superfamily. We find that Shaker and Kv1.1 have an effective gating charge of ~13 eo, as does the Kv2.1 channel. Shab channels, on the other hand, have an effective gating charge of only 7.5 eo. The estimates of gating charge are based on measurements of the channel open probability P at negative membrane potentials where P is very small. The voltage dependence of P provides a lower-bound estimate on the total charge movement (
In measuring the open probability, we are confronted with several possible sources of error. First, an artifactually large voltage dependence of P would result if the probability of missing brief channel openings increased substantially as the membrane potential was made more negative. We find, however, that the channel burst durations are weakly dependent on voltage in the range of potentials examined for each of the four channel types so that most bursts of openings are sufficiently long to be detected (Table 1). The measurements in Shaker and Shab channels were the most susceptible to this effect, since the mean burst duration becomes <4 ms at -70 mV. This is nevertheless considerably longer than the minimum detectable event duration, 120 µs at our analysis bandwidth of 1.5 kHz, so that the error is negligible.
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A second source of error would arise from voltage-dependent inactivation of channels, resulting in an artifactually reduced apparent voltage dependence. The known properties of Shaker channels, and tests for inactivation in Kv1.1, Kv2.1, and Shab channels, however, showed that inactivation effects were negligible under the conditions of our measurements.
Finally, our estimates of P to very low values make use of the assumption of a homogeneous population of channels; if a fraction of channels has a shifted voltage dependence, then the estimated voltage dependence of P would be distorted. Heterogeneity in the voltage dependence of single, mutant Shaker channels has been observed (as evidence that errors from heterogeneity are small.
S4 Charges and Gating Charge Movements
Our experiments, which provide limiting-slope estimates of charge to much lower open probabilities and which distinguish between the main conductance level and a subconductance state in Shab, nevertheless confirm the view that Shab's total charge movement is much smaller, only 7.5 eo, compared with 13 eo in Kv1.1.
Other evidence from mutation studies is equivocal about the role of residues R1 and K7 in the voltage sensor. Early studies showed moderate effects on the voltage dependence of gating from the neutralization of these residues in Shaker ( = 0.5. Meanwhile, near K7, no voltage-dependent accessibility change is seen (
In view of these results, the large charge movement in Kv2.1 channels, indistinguishable from that in Shaker channels, comes as a surprise. Like Shab, Kv2.1 has only five basic residues in S4, and serves as a counterexample to the requirement of R1 for full charge movement. Instead, this result is consistent with the idea originally proposed by
Previous estimates of the gating charge of Kv2.1 are much smaller than ours, in the range of 46 eo. They were obtained from the steepness of channel activation over the range of open probabilities from 0.005 to 0.1 (
Why is the behavior of Shab so different from its mammalian homologue Kv2.1? In these channels, the sequences of S2, S3, and S4 are very well conserved, with identical charged residues. Since the gating charge movement arises from charges within the protein (
Strict Coupling of Charge Movement to Channel Opening
Ligand-gated ion channels show a finite open probability even in the absence of agonists. The mouse muscle acetylcholine receptor channel has a spontaneous open probability of ~5 x 10-6 (
The same strict coupling between charge movement and channel opening is seen in Kv2.1 channels, where we have estimated the voltage-insensitive open probability to be <4 x 10-8, and in sodium channels, where the open probability has been measured down to 10-7 (
As might be expected for the reduced voltage sensitivity requirement of delayed-rectifier potassium channels, Shab channels have a much lower voltage dependence of channel activation. They also have less gating charge movement than the A-type Shaker channel and its homologue, Kv1.1, which also produces transient currents when coexpressed with beta subunits (
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Acknowledgements |
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We thank Dr. R. MacKinnon for providing the Kv2.1 construct and Agitoxin, and Dr. D. Logothetis (Mount Sinai Medical Center, New York, NY) for Kv1.1 and Shab DNA. We also thank Y.-Y. Yan for expert technical assistance and Y. Yang, J. Zheng, Q.-X. Jiang, T. Rosenbaum, and S. Basavappa for helpful comments.
This work was supported by National Institute of Health grant NS 21501.
Submitted: 18 May 1999
Revised: 17 September 1999
Accepted: 20 September 1999
1used in this paper: NMDG, N-methyl-D-glucamine
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