Correspondence to: Francisco Bezanilla, Dept. of Physiology, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095. Fax:310-794-9612 E-mail:fbezanil{at}ucla.edu.
Released online: 31 January 2000
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Abstract |
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In Shaker K+ channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be constrained by the length and flexibility of the S3S4 linker. To investigate the role of the S3S4 linker in the ShakerH4(646) (Shaker
) K+ channel activation, we constructed S3S4 linker deletion mutants. Using macropatches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K+ currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in Shaker
. The apparent number of gating charges was 12.6 ± 0.6 eo in Shaker
, 12.7 ± 0.5 in 10 aa linker, and 12.3 ± 0.9 in 5 aa linker channels, but it was only 5.6 ± 0.3 eo in the 0 aa linker mutant channel. The maximum probability of opening (Pomax) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89x and 45x slower than that of the Shaker
K+ channel, respectively. The initial lag of ionic currents when the prepulse was varied from -130 to -60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mutant channels, respectively. These results suggest that: (a) the S4 segment moves only a short distance during activation since an S3S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur, and (b) the length of the S3S4 linker plays an important role in setting Shaker
channel activation and deactivation kinetics.
Key Words: Shaker K+ channel, S3-S4 linker, S4 displacement
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INTRODUCTION |
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The opening of Shaker K+ channels is associated with the displacement of ~13 electron charge units (
All the evidence given above points to the fact that the S4 transmembrane domain is an important part of the voltage sensor and that this domain of the protein moves in the electric field when the channel is activated by voltage. According to some models, the S4 segment moves during channel activation across the membrane as a sliding helix (
The role of the S3S4 linker in Shaker K+ channel activation was previously studied by
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We found that after complete deletion of the S3S4 linker, fully functional channels are expressed, although the details of channel voltage-dependent activation are different from that of the wild type. We conclude that the S4 segment needs to move only a short distance to signal the ion pathway to open.
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MATERIALS AND METHODS |
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Mutations and Channel Expression
Constructs were prepared on ShakerH4(6-46) (ShakerH4
). The deletion of amino acid residues from 6 to 46 removes N-type inactivation. The ShakerH4
K+ channel cDNA was originally cloned into an engineered version of the pBSTA vector. The S3S4 deletion mutants were ShakerH4
-
(330-360), dubbed here 0 aa linker, ShakerH4
-
(330-355) (5 aa linker), and ShakerH4
-
(332-351) (10 aa linker), and were prepared by PCR-based mutagenesis (Expand Hi Fidelity PCR system; Boehringer Mannheim, or Pfu DNA polymerase; Stratagene). The S3S4 linker was defined as the segment comprised of residues 330360 (Figure 1;
Competent Escherichia Coli cells, DH5 or XLI Blue, were transformed with the ligation products. Colonies obtained after transformation were selected using HaeII (one cut at the S3S4 linker). cDNAs from preselected colonies were sequenced using T7 Sequenase version 2.0 (Amersham) to choose those having the expected sequence. The ~400-bp cassettes containing the mutations were cut out using the restriction enzymes XbaI and BsmI and were subcloned in the vector pBSTA with the original ShakerH4 DNA insert. The mutated cDNAs were transcribed in vitro using T7 RNA polymerase (mMessage Machine; Ambion). Transcription was done at 37°C in a volume of 10 µl, following the manufacturer's instructions. The cRNA products were extracted with ethanol, and suspended in double distilled water to a final concentration of 1 µg/µl.
Translation of the cRNA's was done in Xenopus laevis oocytes. Xenopus were purchased from Nasco International, Inc. The protocol used to isolate Xenopus laevis oocytes received institutional approval and followed National Institutes of Health guidelines. Oocytes from Xenopus laevis were defolliculated by incubation with 2.5 mg/ml collagenase in filtered OR-2 medium. Oocytes were well separated after shaking 12 h on a clinical rotator at room temperature at 180 rpm (ROT-2; P+L Electrónica). Collagenase was removed by washing the separated oocytes five times with OR-2 medium containing (mM): 82.5 NaCl, 2.5 KCl, 1 MgCl2, 5 HEPES, pH 7.6, and five times with ND-96 medium containing (mM): 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 500 mg/ml gentamicin, 5 HEPES, pH 7.6. Collagenase was from GIBCO BRL Life Technologies, Inc. Stage V and VI oocytes were selected and maintained in ND-96 medium at 18°C for 1224 h before RNA injection. 50 nl of cRNA solution (0.10.2 µg/µl) was injected into either the vegetal or the animal poles of each oocyte using micropipettes (4878; World Precision Instruments) of 20-µm tip diameter attached to a microinjector (A203XVY; World Precision Instruments).
Electrophysiology
25 d after cRNA injection and after the manual removal of the vitelline membrane, currents elicited by the different mutants were recorded in cell-attached macropatches of oocyte membrane. Patch pipettes of 1030 µM in diameter were pulled from melting point tubes (9530-1; Corning Glass Works) and had a resistance of ~1 M. No series resistance compensation was used and the error due to uncompensated series resistance was never greater than 5 mV. Bath and pipette solutions contained (mM): 110 KMES, 10 HEPES, 2 CaCl2, pH 7.2. The acquisition and basic analysis of the data were performed with pClamp 6.0 software (Axon Instruments, Inc.) driving a 12-bit analogue interface card (Labmaster DMA; Scientific Solutions, Inc.).
Macroscopic Current Analysis
For macroscopic current relaxation experiments, the membrane was held at -100 mV. Each experimental episode consisted of an epoch at the holding potential, followed by a depolarization to different test voltages and ending with an epoch at -60 mV. The episode duration ranged from 140 to 1,400 ms, depending on the relaxation times of the various mutant channels. Each experiment consisted of 1050 episodes separated by 12 s. The test pulse was incremented by 310 mV on each subsequent episode. For each episode, 512 digital samples of current were acquired at equally spaced times. The analogue signal was filtered before digitization with an eight-pole low-pass Bessel filter with a cutoff frequency of 1/5 of the conversion frequency. Conductancevoltage relations were determined from the peak amplitude of the tail currents after repolarization to a fixed negative voltage.
Cole-Moore Analysis
Families of currents from the WT Shaker and linker mutant channels were obtained as follows: 100-ms-long prepulses to voltages between -130 and -40 mV preceded pulses to +50 mV. Each current record obtained during the 50-mV pulse was compared with the current record measured after the -130-mV prepulse. Records were displaced along the time axis and the differences were evaluated visually. The time displacement giving the best superposition of the traces was taken as the Cole-Moore shift.
Time Constants Calculation
A quantitative comparison of the activation kinetics was obtained from monoexponential fits of the upper 50% of the rising phase of the current. The mathematical expression used for the fit of the time course of channel activation was:
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(1) |
where d is the delay and t the time constant of the exponential. This simplified approach has been demonstrated to be efficient for assessing rate constants in multistep ramified activation pathways (a is a measure of the reciprocal value of the slowest rate constant in the activation path and d is a measure of the addition of the reciprocal values of all the rest of the constants.
Deactivation kinetics were obtained by fitting the early decay of the tail current to a sum of two monoexponential decays, leaving out the first 0.1 ms of the test pulse to allow for clamp settling. The fastest time constant of the decay of the tail currents was used as an estimation of the rate constant of closing (deactivation) of channels.
Variance Analysis
A series of identical records were recorded pulsing to a positive voltage from the holding potential. Pairs of M subsequent records Xi(t), Xi + 1(t) were subtracted to compute the experimental nonstationary ensemble variance. The average basal variance at the holding voltage was subtracted from the variance obtained during the test pulse. The subtracted variance (sigma}">2) was plotted versus mean current [I(t)] and the data was fitted using (
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(2) |
where i is the single channel current amplitude and N the number of channels. i was obtained from the initial slope, N was obtained from the nonlinear curve fitting analysis done under Microsoft Excel. The maximum open probability, Pomax was obtained according to the relation: Pomax = Imax/iN where Imax is the maximum mean current measured in the experiment.
Limiting Slope Analysis
The method developed by
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(3) |
where z is the number of effective charges per channel. The z value was also obtained from the relationship:
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(4) |
As pointed out by as a function of voltage (or Po) is a good test of having reached the true limiting slope. In this plot, at very negative voltages, z
will asymptotically approach a saturating value that is equal to the total number of charges per channel.
In the text, errors in all measured quantities are given as mean ± SD.
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RESULTS |
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Deletions at the Amino-Terminal End of the S3S4 Linker Slow Down the Rates of Channel Activation and Deactivation
Figure 2 shows ionic currents elicited by various depolarizing voltages from a holding voltage of -100 mV followed by a postpulse to -60 mV for the Shaker "wild type" (WT) (Figure 2 A) channel. Ionic currents records for the 10, 5, and 0 aa S3S4 linker channel mutants are shown in Figure 2B, Figure C, and Figure D, respectively. Robust K+ currents from each deletion mutant 1 or 2 d after the cRNA injection implied that none of the mutations in the S3S4 linker affected the synthesis and assembly of the functional Shaker
K+ channels in oocyte membranes. Complete (0 aa linker) or partial (5 aa linker) removal of the linker profoundly affected the rates of channel activation. The 10 aa linker mutant has biophysical properties very similar to those of the WT channels. Both activation and deactivation kinetics were slowed down in the 0 aa linker channel compared with the wild-type channel (Figure 2 D). In Figure 2 D, we can clearly see that in the 0 aa linker mutant the tail currents become very slow. The same phenomenon was observed in the case of the 5 aa linker mutant. However, in this case, the effect of the deletion is mainly on the activation kinetics. The time course of the ionic current in this case is very slow and at intermediate depolarizations the current increases only after a long time delay. This initial lag of ionic currents is also present in the 0 aa, but is particularly pronounced in the 5 aa linker mutant (Figure 1 C; see also Figure 4).
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Deletions in the S3S4 Linker Shift the VoltageActivation Curve
Information about the mutant and WT channels' behavior under equilibrium conditions was obtained from conductance versus voltage curves. Conductance versus voltage curves for the WT and mutant channels were obtained by directly plotting the peak tail current amplitude at a constant postpulse potential (-60 mV) and as a function of the test prepulse potential in high external K+. Notice that, at large depolarizations, the tail current saturates (e.g., Figure 2 D), indicating that the maximum channel open probability (Pomax) has been reached since the current at the peak of the tail [Itail(V)] determined with a test prepulse V is (Equation 5):
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(5) |
where i is the current amplitude of the single channel, N the number of channels, and Po(V) the voltage-dependent open probability. Therefore, Itail(V)/Itailmax(V) = Po(V)/Pomax, where Itailmax is the maximum amplitude reached by the tail currents (
Figure 3 shows the relationships Po(V)/Pomaxprepulse voltage for the WT and mutant channels.
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(6) |
with n equal to 1 or 4, where Kexp(-zF V)/RT represents the voltage-dependent equilibrium constant for each independent transition. In this case, the voltage-dependent activation mechanism involves n independent conformational changes before opening. The fit to the Po(V)/Pomaxvoltage curves using Equation 6 is poor for the WT channel (Figure 3 A, broken line) and is clearly inadequate for the 10 aa linker channel (Figure 3 B, broken line). In particular, the fit to the Po(V)/Pomax data, Equation 6 misses all data points at relative open probabilities >0.8. However, the fit to the Po(V)/Pomax data using Equation 6 is good for the 5 and 0 aa linker mutants (Figure 3C and Figure D; Table 1). Equation 6 is based in a model that requires only a single conformational change in each subunit to open the channel (
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Where C0 to C3 represent closed conformations after zero to three independent transitions, respectively, O represents the open conformation, K = K(0)exp(-z1F V/RT) is the voltage-dependent equilibrium constant, and K(0) is the equilibrium constant at zero voltage for each transition. Scheme 1 corresponds to class A of models in
A)4 _ O, where each subunit can be either in a resting (R) or an active (A) conformation; we defined K = R/A.
We found that the fit to the Po(V)/Pomax data improves notably for the wild type and the 10 aa linker mutant (Figure 3A and Figure B, solid lines) using a model in which the four independent and identical voltage-dependent transitions are followed by a final concerted transition that lead to the open state (
Where Ko represents the equilibrium constant for the concerted transition. Scheme 2 can be summarized as (R A)4
O. It is important to note here that, to fit the data for Po(V)/Pomax 0.8, the concerted transition must be voltage-dependent; i.e., Ko = Ko(0)exp(-z
2F V/RT) (Table 1). Recently,
For Scheme 2, the probability of opening is given by the relation:
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(7) |
The total gating charge per channel determines the slope at very low open probabilities (e.g.,
In the case of the 10 aa linker mutant, since the charge residues in the S4 segment were not modified, the increase in the slope (Table 1) can be due to an increase in positive cooperativity between subunits. The change in slope cannot be due to an increase in the number of gating charges per channel since the limiting slope in this mutant is not different from that of the WT Shaker channel (see below).
Why can the 5 and 0 aa linker mutants be described using a fourth power of a Boltzmann function? One possible explanation is to consider that the equilibrium R A is displaced to the left in an amount such that the last concerted transition is heavily biased to the open state at those voltages that are able to displace the R
A to the right (all subunits in their active configuration). In other words, if we assume that linker mutations do not affect the last transition, stabilization of the subunits in the R configuration brings as a consequence that the Po(V)/Pomax curves apparently behave according to Scheme 1.
Voltage Dependence of the Activation and Deactivation Kinetics
The voltage dependence of the activation kinetics in the wild type and mutant channels was characterized by fitting the upper half of the current record to a single exponential (Figure 4, inset) and a delay.
We fit the ionic currents obtained at depolarized voltages, where it is safe to assume that ßi = 0, using Equation 1. The activation time constant, a =
j-1 where
j-1 is the limiting rate in Scheme 3 and the delay, d =
i-1 (i
j to n). This approach is likely to be valid for branched models (
In the case of the 10 aa linker mutant, a at 0 mV was twofold slower than that of the WT channel. It also produced a 10-mV shift of the
a versus voltage curve to the right along the voltage axis (Figure 4 A, ). The 0 aa linker mutant resulted in a 45-fold slowing of
a at 0 mV compared with the WT channel and a positive shift of the
a versus voltage (Figure 4 A,
). On the other hand, the 5 aa linker mutant produced an 89-fold slowing down of activation at 0 mV (Figure 4 A,
). From the slopes of the solid lines in Figure 4 A, we calculated the number of apparent gating charges, za, displaced in the rate limiting transition. za was 1.01, 1.27, 0.71, and 0.45 for the WT, and the 10, 5, and 0 aa linker channels, respectively. Thus, shortening the S3S4 linker from 31 to 10 aa does not modify the voltage dependence of
a and the complete removal of the linker induces a decrease of 0.56 in za. This decrease in za promotes a crossover of the
a versus voltage curves for the 0 and 5 aa linker mutants (Figure 4 A).
Figure 4 B shows the delays (d) as a function of voltage for the 10 (), 5, and 0 aa linker mutant channels. Since d is the reciprocal value of the sum of all i (i
j), it is clear that one or more of the forward rate constants are affected by S3S4 linker shortening. Surprisingly, the voltage dependence of d for the 0 aa linker mutant channel was found to be larger than that of the WT Shaker
and the rest of the linker mutant channels. It is possible for this channel that a transition with a large voltage dependence has become much slower than the rest of the forward rates.
Figure 5 shows the voltage dependence of the deactivation kinetics obtained using the pulse protocol illustrated in the inset. In the range between -100 and -50 mV, the deactivation time constants are 6-, 3-, and 1.5-fold larger in the 0, 5, and 10 aa linker mutants than in the WT channel, respectively. The voltage dependence of the 0 aa linker mutant is essentially indistinguishable from the WT channel, and the equivalent number of gating charges related to channel deactivation, zd, for the 5 aa linker mutant is 0.58 compared with 0.53 in the WT channel.
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A Large Cole-Moore Shift Is Present in the 5 aa Linker Mutant
K+ channels, an ~1-ms time shift of the ionic current onset is induced when the prepulse is varied from -150 to -50 mV (
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Change in Gating Valence Induced by Complete S3S4 Linker Removal
The next step was to determine whether the linker deletions modify the apparent number of gating charges per channel. If the S4 domain undergoes a large displacement during channel activation, this movement should be constrained by the linker length. Since the apparent number of charges per channel is the product of the valence z times the displacement that each charge undergoes, an abridgment of the S4 motion imposed by the shorter linker should be manifested as a decrease in z
.
To calculate the number of effective charges per channel, z, we have applied the limiting slope method (
. To increase the voltage resolution for the WT Shaker
K+ and the linker mutant channels, we used slow ramp stimulation from -100 to 0 mV (Figure 7;
obtained in previous studies for the WT Shaker K+ channel (
0; Figure 7 A) gave a z
= 12.1. To verify the validity of our assumption Po
0, we determined the slope of the logarithm of the G-V curve at low open probabilities, and z
at the different voltages was obtained using Equation 4. When z
is plotted as a function of V, it reaches a plateau that is equal to the actual value of the limiting slope (Figure 7 E), giving a value very close to that obtained by the exponential fit (straight line). For the WT Shaker K+ channels, we obtained a z
= 12.5 ± 0.62 (n = 5), in reasonable agreement to the z
= 12.3 found by
= 12.8 found by Noceti et al. (1997) and 12.6 found by
was found for the 10 aa linker mutant, Figure 7B and Figure F (12.7 ± 0.54).
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We used the ramp method to determine the z's of the 5 aa (Figure 7C and Figure G) and the 0 aa (D and H) linker mutants. Figure 7 C is the conductance corresponding to the ionic current flowing through 5 aa linker mutant channels in response to a ramp from -90 to -20 mV (1.2 mV/s). The conductance values obtained for very negative voltages (-70 to -50) were fitted to Equation 3 to approximate to the limit Po
0. The calculated parameters of the fit were A = 5 x 1013 nS and z
= 12.0. We also calculated z
at different voltages using Equation 4 (Figure 7 G) to obtain a z(V) versus V curve. Figure 7 C shows that z
approaches a limiting value close to the fitted value obtained in Figure 7 G (straight line). The average value for z
in the 5 aa linker mutant was 12.3 ± 0.87.
Figure 7D and Figure H, illustrates a limiting slope experiment in the 0 aa linker mutant channel with the same strategy as the experiments shown in Figure 7AG. In this case, a slow ramp from -90 to -20 mV (1.2 mV/s) was used to induce the ionic current that was then converted into conductance (Figure 7 D). Notice that for both the 5 and 0 aa linker mutants, very slow ramps were used to attain a quasi steady state G-V curve, due to the slow activation kinetics showed by these two mutants (Figure 2). The monoexponential fit to the initial part of the curve (-89 to -67 mV) produced the following parameters: A = 4 x 107 nS and z = 5.21. As for the wild type and 5 aa linker mutant channels, a clear plateau in the curve z
versus voltage was observed, providing a good estimate of the total number of charges per channel displaced during activation. For the 0 aa linker mutant, the average z
obtained from seven experiments was 5.6 ± 0.32.
Noise Analysis Reveals the Same Pomax for WT Shaker and Mutant Channels
The maximum probability of opening for the WT Shaker K+ channel was determined using nonstationary fluctuation analysis (
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The maximum open probability for the 5 aa linker mutant was also estimated by nonstationary fluctuation analysis (Figure 8, DF). As with the 10 aa linker mutant, a parabolic relationship between the variance and the mean current was seen for the 5 aa linker mutant (Figure 8 F), leading to accurate estimates of N and Pomax. The solid line in Figure 8 F is a fit to the variancemean current data with N = 10,000 channels, i = 0.82 pA (average i = 0.730.2, n = 8), and the calculated Pomax was 0.75 (average Pomax = 0.76 ± 0.05). In the case of the 0 aa linker mutant channel, Figure 8G and Figure H, shows the time course of the average current for a +120-mV pulse and the corresponding variance versus time plot. The solid line in Figure 8 I is a fit to the variancemean current data with N = 890 channels, i = 0.36 pA, (average i = 0.540.3, n = 5), and the calculated Pomax was 0.73.
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DISCUSSION |
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General
By inserting an epitope at different positions in the S3S4 linker,
The initial studies on the role of the S3S4 linker performed by by the introduction of point mutations in the Shaker
S3S4 linker. Replacement of the Shaker
S3S4 linker by the shorter (7 aa) linker of Shaw resulted in a three- to fourfold slowing down in the activation kinetics and in voltage shifts of the G-V curves of -13 mV. The linker definition of
in almost every respect except for slower speed of activation and deactivation (approximately twofold) and an increase in the slope (Table 1), possibly due to an increase in positive cooperativity between subunits. Our results obtained using the 5 and 0 aa linker mutants are interesting in four respects. First, the main effect of the mutations was to slow down dramatically the activation kinetics and, to a much lesser extent, the speed of deactivation. Second, we expected to find an inverse relationship between speed of activation and linker length. Contrary to this expectation, we found that for voltages smaller than 60 mV the speed of activation was much slower in the 5 aa mutant linker than in the 0 aa mutant linker. However, for voltages >60 mV, the speed of activation of the 0 aa linker channel became slower than that of the 5 aa linker channel mutant. On the other hand, the speed of deactivation decreased inversely with S3S4 linker length. Third, we found no evidence for a change in the gating valence for the 5 aa linker mutant, but in the 0 aa linker mutant the valence was halved. Fourth, the Pomax was the same for Shaker
and the three mutants studied here.
The results obtained with the 5 aa S3S4 linker mutant suggest that the linker length determines to a great extent the activation and deactivation channel kinetics. Moreover, the fact that in this mutant z and Pomax are not altered suggests that a 5 aa linker is enough to allow for the full displacement of the voltage sensor and for normal coupling between voltage-dependent gating and pore opening.
Complete deletion of the S3S4 linker in Shaker K+ channels greatly slows down the activation (~45-fold) and deactivation (~6-fold) kinetics compared with the wild-type Shaker
K+ channel. These changes in channel gating kinetics are accompanied by a shift toward depolarizing voltages of the G-V curve. The shift to the right, measured at Po = 0.5, induced by complete removal of the S3S4 linker, is 45 mV. The change in free energy,
G, due to the linker deletion can be calculated using the expression:
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(8) |
where the values of the equilibrium constants for the 0 aa linker [K(0)0aa] and the WT Shaker [K(0)WT] channel are those of Table 1. K(0) values were obtained using Equation 6 for the 0 aa linker and Equation 7 for the wild type. Comparison of the K(0) values is meaningful since K(0) determines the equilibrium between resting or an active conformation of subunits in both cases. Different equations are needed since, for the WT Shaker K+ channel, an extra process is required to reach the open state. Using Equation 8, we obtain a G = 1.2 kcal/mol. This is a relatively small free energy change considering that the whole linker was deleted, and suggests that the conformational change of the S4 domain during activation does not involve extensive changes in sensor structure (see below). Most notably, deletion of the S3S4 linker in Shaker
K+ channels decreases the apparent gating valence from 13 eo in Shaker
K+ to 5.6 eo, with no change in the Pomax. The Pomax is determined by transitions that have little or no voltage dependence; such transitions account for Shaker's single channel data at depolarized voltages and are outside of the activation path (
Possible Interpretations of the Decrease in z of the 0 aa Linker Mutant Channel
The studies of ) traversed by some of the gating charges as pictured in Figure 9 since after linker deletion they would be "inside" the dielectric region.
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However, the decrease in z can also be due to the appearance of new open states connected by voltage-dependent rates induced by the linker deletion. The limiting slope method gives an estimate of the number of gating charges displaced between closed states and the first open state (
It is also possible that the correct value of the total charge is not estimated properly because the applied voltages were not negative enough to reach a Po low enough ( vs. Po, where it is possible to visualize whether the estimate shows signs of reaching a limiting value. Figure 7 H shows that z
has reached a saturating value and that, with the caveat of the possible presence of several open states, this represents the total charge movement. In principle, it is possible to determine z
by determining the total charge (Q) displaced and the number of channels (N) in the membrane area considered (
Kinetic Effects of Mutations
The analysis of the Cole-Moore shift of the ionic currents indicates that shortening the S3S4 linker slows down transitions between closed states further removed from the open state. A quantitative comparison of the kinetics of the rate-limiting step of activation was obtained for Shaker and the S3S4 linker mutants. This was done by determining activation time constants from monoexponential fits of the rising phase of the test pulse currents. The analysis of the data shows that transitions in the activation pathway and near the open state are also greatly affected by shortening the linker to 5 or 0 amino acids. The 5 aa linker has an 88-fold slower activation than the Shaker
channel with no difference in the voltage dependence (Figure 4 A). In the case of the 0 aa linker mutant channel, the difference in activation speed with the Shaker
is 45-fold at 0 mV. The experimental protocols we used do not allow conclusions about the effect of the mutations on the backward rates in the activation pathway. However,
If the ratio of activation time constants between the 5 aa linker and the Shaker channel is assumed to be related to the difference in the energy necessary to displace the voltage sensor,
G, this energy amounts to 2.7 kcal/mol. In both the 5 and 0 aa linker channel mutants, the deactivation rate constant is much less affected than the activation process. Thus, a difference of sevenfold in deactivation rates between Shaker
and the 0 aa linker implies a
G = 1.2 kcal/mol. The interpretation of this difference is obscured by the fact that the forward rate of the last transition,
N, (Scheme 3) is much faster than the rates of the previous forward rates, and even at -100 mV can have values comparable with the backward rate, ßN (
In summary, shortening the S3S4 linker to <10 amino acids appears to increase all the energy barriers separating the various kinetic transitions leading to channel opening. It is interesting to note that, with the exception of the extreme case of the 0 aa linker channel, these kinetic effects occurred in the absence of large changes in the voltage dependence of activation.
A Model Consistent with Results
The total charge per channel of the WT Shaker estimated by three different methods is between 12.6 eo (
indicates that all the nonlinear charge in Shaker
K+ channel is involved in channel opening (
-helical S4 region (
Our results show that a very short linker is sufficient for the full displacement of the S4 segment. This result is inconsistent with the sliding helix model of
All the above evidence suggests that the S4 segment undergoes a rotation (
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Footnotes |
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1 Abbreviations used in this paper: aa, amino acid; WT, wild type.
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Acknowledgements |
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We thank Dr. Albert Cha for critical reading of the manuscript and Luisa Soto for technical assistance.
This work was supported by Chilean grants FONDECYT 197-0739 (R. Latorre) and Cátedra Presidencial, a Human Frontier in Science Program grant (R. Latorre), and a group of Chilean companies (AFP Protection, CODELCO, Empresas CMPC, CGE, Gener S.A., Minera Escondida, Minera Collahuasi, NOVAGAS, Business Design Ass., and XEROX Chile) (R. Latorre), by National Institutes of Health grant GM30376 (F. Bezanilla), and a TWAS 1997 grant (C. Gonzalez).
Submitted: 2 July 1999
Revised: 5 January 2000
Accepted: 6 January 2000
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