Correspondence to: F. 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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When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (F) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanilla. 1998. J. Gen. Physiol. 112:391408.). We attached TMRM at the same sites [corresponding to M356C and A359C in the wild-type (wt) channel] in a deletion mutant of Shaker where all but the five amino acids closest to S4 had been removed from the S3S4 linker. In the deletion mutant, the maximal
F/F seen was diminished 10-fold, and the
F at M356C became pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3S4 linker. The residual
F showed that the probe still interacted with two putative quenching groups near the S4 segment. One group was detected by M356C-TMRM (located outside of S3 in the deletion mutant) and reported on deactivation gating charge movement when applying hyperpolarizing voltage steps from a holding potential of 0 mV. During activating voltage steps from a holding potential of -90 mV, the fluorescence lagged considerably behind the movement of gating charge over a range of potentials. Another putative quenching group was seen by probes attached closer to the S4 and caused a
F at extreme hyperpolarizations (more negative than -90 mV) only. A signal from the interaction with this group in the wt S3S4 linker channel (at L361C) correlated with gating charge moving in the hyperpolarized part of the Q-V curve. Probe attached at A359C in the deletion mutant and at L361C in wt channel showed a biphasic
F as the probe oscillated between the two groups, revealing that there is a transient state of the voltage sensor in between, where the probe has maximal fluorescence. We conclude that the voltage sensor undergoes two distinct conformational changes as seen from probes attached outside the S4 segment.
Key Words: Shaker K+ channel, S3S4 linker, S4 displacement, fluorescence quenching, gating currents
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INTRODUCTION |
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The development of the technique of site-directed fluorescent labeling has made it possible to track movements in specific regions of the protein whether or not these rearrangements carry gating charge (
Initially, the mechanism of fluorescence quenching near the S4 segment was hypothesized to result from changes in the hydrophobicity of the probe's environment in response to voltage (
From the sequence of Shaker channel protein, a likely candidate for the putative protein vestibule is the ~31 amino acid extracellular S3S4 linker. We tested this hypothesis by examining properties of fluorescence quenching near the S4 segment in a deletion mutant where 26 amino acids in the linker had been removed (
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MATERIALS AND METHODS |
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Electrophysiology and Fluorescence Measurements
The modified cut-open oocyte epifluorescence setup used for simultaneous measurement of gating currents and fluorescence signals has been presented before ( resistor to minimize integration resetting spikes from the traces. The excitation light from the lamp was interrupted with a TTL-triggered VS25 shutter (Vincent Associates) between measurements.
Gating and fluorescence currents were digitized by two analogue-to-digital converters at a resolution of 16 bits and transferred onto two different channels of a PC44 board (Innovative Technologies) interfacing with a computer via an AT-slot. The acquisition program running the PC44 board was developed in our laboratory and runs under MS-DOS. When data were sampled at intervals longer than 5 µs (all traces presented in this paper), the program acquired the data at 5 µs per point, digitally filtered them to the new Nyquist frequency, and decimated them to the new sampling frequency. The data analysis program was also developed in our research group and runs in Windows 95. The fluorescence signal, F, is displayed as -F; i.e., positive deflections indicate a decrease in fluorescence or an increase in fluorescence quenching.
Molecular Biology, Expression, and Labeling
A noninactivating version of the Shaker H4 channel [H4(6-46)] with pore mutation W434F in a pBSTA vector background was used for measurements of gating currents with maximal expression (
(6-46) W434F vector, the PCR insert was sequenced to exclude the possibility of unwanted mutations. The constructs are designated by the original amino acid, residue number, and substituted amino acid (i.e., M356C designates the construct where cysteine was substituted for methionine at residue 356). The cRNA was transcribed in vitro with the T7 mMessage machine kit (Ambion Inc.), and 50 nl cRNA at a concentration of 100 ng/ml were injected into each Xenopus oocyte. Experiments were performed from 27 d after injection. The sterile oocyte incubation solution consisted of 100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 10 µM EDTA, and 100 µM DTT. The oocytes were stained in depolarizing solution containing 5 µM tetramethylrhodamine-5-maleimide (
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RESULTS |
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The technique of site-directed fluorescent labeling involves attaching an extrinsic fluorescent probe (i.e., TMRM) to specific regions in the protein. TMRM attached to cysteines in the S3S4 linker just outside of S4 (at M356C or A359C) demonstrated a voltage-dependent fluorescence signal displaying features of gating charge movement (
A Rearrangement Outside the S3 and S4 Segments Tracked by a Fluorophore in the Deletion Mutant
Even with the linker deleted, TMRM attached to the inserted cysteine M356C in the 5 amino acid (5-aa) residual S3S4 linker exhibited fluorescence changes with voltage (Figure 1 A). The changes in the M356C 5-aa linker were in the same direction (i.e., an increase in quenching with depolarization), but about one order of magnitude smaller (~1.5% of background in Figure 1 A) than those reported by
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By making the cysteine substitutions in the W434F background, we should be able to simultaneously measure gating currents and fluorescence and compare the two. In our case, the situation is complicated by the fact that the 5-aa linker mutant presents an activation kinetic more than 200-fold slower than the wt channel (at 0 mV), whereas deactivation is slowed down only threefold (
We then estimated the activation kinetics of the charge by measuring gating currents at negative potentials, taking advantage of the fact that at those potentials the kinetics are fast enough to detect gating currents reliably, as shown in Figure 1 B. The Q-V curves for the M356C mutant are given in Figure 1 C, for HP = 0 and -90 mV. For the case of the HP = -90 mV, the charge was estimated at -90 mV after a test pulse of duration of 40, 150, and 400 ms. In this situation, since gating charge movement is being measured during the return to -90 mV after the activation pulse (OFF gating charge), more and more total gating charge is being recruited with longer pulse durations. By comparing with the total gating charge measured from HP = 0 mV, it is clear that activation pulses of 400-ms duration are required to move ~90% of the total gating charge at 50 mV membrane potential. By using the fraction of the total gating charge moving with pulses of different length, the approximate time constant of activation gating charge movement can be determined as a function of voltage (Figure 1 D). The result verifies the extremely slow movement of the voltage sensor during activating voltage pulses; e.g., at 0 mV, the time constant for activation gating charge movement was ~570 ms, which is even slower than the extrapolated time constant of activation of the ionic current at 0 mV (~218 ms) in the 5-aa linker mutant without the cysteine and without the TMRM dye attached (
The time constants of fluorescence changes during activating voltage steps (Figure 1 A) were likewise measured and displayed in Figure 1 D. At lower activation potentials, fluorescence moved even slower than gating charge movement, whereas at higher potentials the opposite appears to be true. Reinspecting Figure 1 B, the same holds true when deactivation gating charge movement is being compared with fluorescence: for larger voltage steps (-170 mV) the fluorescence (jagged traces) is faster than gating charge movement (smooth traces), whereas the opposite is true for smaller voltage steps (-80 mV). This contrasts to the situation for the M356C position in the wt channel, where the fluorescence was always found to lag behind the movement of the gating charge (
A more detailed comparison of activation gating charge movement and fluorescence changes in the M356C 5-aa linker mutant is shown in Figure 2. In this case, activation gating charge movement was estimated by applying a pulse to 0 mV for variable durations (t), followed by a step to -180 mV to measure (deactivation) gating charge. Figure 2 A shows gating current and fluorescence during the -180-mV step. By integrating the current traces at -180 mV, the total gating charge moving at -180 mV is measured. For short durations at 0 mV, this represents the gating charge moved between -90 and 0 mV during the voltage step to 0 mV, since little gating charge moves between -180 and -90 mV, when holding at -90 mV (Figure 1 C). However, for longer durations at 0 mV, the channel becomes progressively C-type inactivated, resulting in a leftward shift of the Q-V, with a significant part of the gating charge now being moved between -90 and -180 mV (Figure 1 C;
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The special feature of the 5-aa linker channel is the slow activation and relatively fast deactivation kinetics (
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The deactivation Q-Vs obtained for the M356C W434F 5-aa linker mutation show that there is a rightward shift in the Q-V (Table 1), with the second Boltzmann distribution shifting from -44 (W434F wt linker) to +5 mV, when holding at -90 mV. On the other hand, when holding at 0 mV (C-type inactivated channel), the Q-V gets shifted to the left by deletion of the linker, indicating that the interaction that stabilizes the voltage sensor at 0 mV is stronger in the deletion mutant.
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A359C: Two Quenching Groups and a Transient State of the Voltage Sensor in the 5-aa Linker Mutant
From the above results and those of
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We first asked whether the biphasic fluorescence signal was affected by C-type inactivation, since it was primarily observed from potentials where the channel is likely to be in the C-type inactivated state(s). We therefore prepulsed the potential from HP = -90 mV (where the channel is noninactivated) to -40 mV (where this mutation is C-type inactivated, data not shown) for variable durations and looked at the signal during a subsequent test pulse to -180 mV (Figure 5). The result showed that the biphasic fluorescence signal persisted even for quite small dwell times at -40 mV, where the channel would be noninactivated. The biphasic fluorescence signal is therefore an obligatory feature of the gating process for this mutant and is not exclusive of the channels being in the inactivated state(s).
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The biphasic nature of the fluorescence signal suggests that the TMRM probe oscillates between two quenching groups with an intermediate nonquenched state (see Figure 9). In this view, when holding at 0 mV, the probe is close to one quenching group (e.g., QG1). Stepping to potentials less than -90 mV, the probe moves through an intermediate state of minimum quenching (maximum fluorescence) before reaching another state where it is being quenched by the other group (QG2). Thus, QG1 would interact with the probe at depolarized and QG2 at hyperpolarized potentials. Adopting the model of the S4 movement as a rotation (
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L361C: A Transient State of the Voltage Sensor in Shaker with wt S3S4 Linker
The proposed location of the putative quenching group QG2, and the fact that such a group was not detected when placing probes further out into the S3S4 linker (M356C, see above) suggests that QG2 might be located deep in the inner crevice. If so, it should be detectable using probes placed further towards the S4 segment in the wt linker channel. We therefore tested the L361C W434F (wt S3S4 linker) mutation.
The first interesting feature of the signal at L361C (Figure 6 A) is the absence of substantial quenching at depolarized potentials in this mutant even though the S3S4 linker is intact. Thus, the linker is not quenching the signal at this position, which agrees with the demonstration that the protein vestibule becomes more constrained further away from S4 (
To test the hypothesis that the two components of the fluorescence are associated with separate movements of the voltage sensor, we tested whether they would kinetically follow the movement of different components of gating charge. The time constants of each of the two different phases of fluorescence were measured and compared with the two time constants present in the gating charge movement in the A359C 5-aa linker mutation (Figure 6 D). The result showed that the fast part of the gating charge movement correlated with the fast, decreasing part of the fluorescence signal. However, the second and slower part of the gating charge movement seemed to be considerably faster than the slow part of the fluorescence signal. In the L361C wt linker mutation, the fast, decreasing part of the fluorescence signal also followed the time constant of the gating charge movement (Figure 6 B), but in this case no part of the gating charge movement kinetics was found to be as slow as the slow part of the fluorescence trace.
Thus, when recasting our data into the model of two interconversions between three states (see Figure 9), we found only evidence that the interconversion that brings the fluorophore into the vicinity of QG1 carries gating charge. However, a small component of gating charge moving with the slow time constant of fluorescence could be unmeasurable under these conditions. We therefore explored the origin of the slow fluorescence component further. The L361C wt linker mutant was better suited for this than the A359C 5-aa linker, firstly because the slow component of fluorescence was the dominant signal and secondly because the faster kinetics of gating charge movement in wt S3S4 linker channels facilitated the kinetic correlation of fluorescence and gating charge movement. Figure 7 A shows Q-V and F-V for an oocyte expressing L361C-TMRM held at 0 or -90 mV. The gating charge in this mutant revealed a shallow voltage dependence (Figure 7 A, and Table 1), even in the absence of staining. Thus, the second Boltzmann (denoted Q2L361C) had an estimated charge movement of only 2.1 eo in the unstained and ~1.5 eo in the TMRM-stained L361C, significantly different from the value of 4.4 estimated for wt W434F (
To match the fluorescence signal kinetically with the gating charge movement, we designed a prepulse experiment to evaluate at extreme hyperpolarizations gating charge that moves slowly. In L361 stained with TMRM, the charge movement is faster at 0 than at -160 mV. Therefore, we evaluated the time course of the charge movement at -160 mV by measuring gating currents at 0 mV after a variable interval at -160 mV (Figure 7 B, inset). The result showed that a slow component of gating charge movement was recruited as the prepulse duration was extended (Figure 7 B). The fit through the points (solid line) is a single exponential fitted by varying the amplitude, but with a fixed time constant of 75.7 ms (mean = 71 ± 2 ms, n = 4), which was the value obtained by fitting a single exponential to the fluorescence trace measured during a 400-ms pulse to -160 mV. Thus, gating charge is being recruited with the same time constant as the fluorescence changes at -160 mV, meaning that the slow fluorescence signal indeed tracks gating charge movement at very hyperpolarized potentials. Therefore, in our scheme (see Figure 9), both interconversions carry gating charge and correspond to separate movements of the voltage sensor.
In the normal W434F channel, a very small fraction of gating charge movement has been reported between -90 and -160 mV when holding at -90 mV (
The biphasic fluorescence signal present in the A359C 5-aa linker and L361C wt mutation illustrates an interaction with a quenching group at extremely negative potentials. This interaction results in a fluorescence signal, as well as a displacement of the Q-V, so that part of the gating charge moves at extremely hyperpolarized potentials (Figure 7 B), creating a very large Cole-Moore shift (see
Properties of the Remaining Protein Vestibule: pH Titration
Since the quantum yield of TMRM is insensitive to pH, pH titration may be used as a tool to investigate properties of the protein residues interacting with the fluorophore. Thus, if pH titration changes the fluorescence intensity, this indicates a change in interaction between protein and fluorophore, rather than an effect of pH on the fluorophore itself. Accordingly, properties of the external protein vestibule in Shaker have been described in terms of pH titration, which revealed that at positions M356C and A359C in the wt linker W434F channel, acidic pH caused a larger total F (
In the TMRM-stained M356C 5-aa linker mutant, a monophasic fluorescence signal was detected, pointing to the interaction with a group outside the S4. The ability of this group to interact with the fluorophore was hardly affected by pH changes between 5 and 9 (Figure 8). The absence of titration in the deletion mutant suggests that the group responsible for modulating the fluorescence in the M356C W434F wt linker is no longer interacting with the fluorophore. Furthermore, it was found that there was only a very small rightward displacement of the F-V at low pH, in contrast to the marked displacement seen in the wt linker channel (
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DISCUSSION |
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Origin of the Protein Vestibule at the Outside of S4
A pH-titratable protein vestibule outside the S4 has been held responsible for voltage-dependent quenching of TMRM attached near the S4 (
With p
Two Quenching Groups and an Intermediate State of the Voltage Sensor
With the A359C-TMRM probe, it became clear that not one, but two groups were available for quenching the signal near the outside S4 segment in the deletion mutant. Furthermore, on oscillating between these two groups, the population of A359C-TMRM probes traversed a state of minimal quenching when moving in the hyperpolarizing direction, but apparently not when moving in the depolarizing direction (but see below). The first, fast downward deflection in signal (increase in fluorescence) was present when holding at positive potentials and applying hyperpolarizing voltage steps, and may arise from the probe moving away from a group (QG1) placed at the outside of the S4 segment, close to the activated state of the voltage sensor (Figure 9 C). The second, slower upward deflection in signal (decrease in fluorescence) was present when going to extreme hyperpolarizations, and the other quenching group (QG2) must therefore reside close to the fully retracted state of the S4 segment (Figure 9 A).
The interaction of the A359C-TMRM probe with a quenching group (QG2) at hyperpolarized potentials is also present at this position (A359C) in the wt linker W434F channel, giving rise to a slight increase in signal at hyperpolarized potentials, as described previously (see Figure 3B and Figure E, in
In the L361C W434F wt linker channel, the activation is faster than in the deletion mutant, and therefore gating currents are easier to measure, which made it possible to determine that both parts of the fluorescence signal track conformational changes carrying gating charge (Figure 6 and Figure 7). Although the signal in L361C was biphasic, the difference in fluorescence between the intermediate state and the two other states was clearly much larger when the linker was deleted. This places the linker close to the S2 state of the A359C probe.
A Two-Step Activation Model Describes the Fluorescence Change
Both the fast and slow parts of the fluorescence trace track conformational changes related to charge carrying transitions near the S4 segment. This makes it possible to further explore the three-state model in Figure 9 by directly fitting the kinetic equivalent of this model (Figure 10 A) to fluorescence data. With the above data, it became clear that all rate parameters (Figure 10 A, , ß,
, and
) must be modeled as being voltage dependent. Furthermore, each state has assigned to it its own fluorescence intensity, which adds another three free parameters to the model in this case. We chose to fit the model to the A359C 5-aa linker data, since in this mutant the fast and slow fluorescence components had comparable magnitudes, thereby furnishing the two interconversions in the kinetic scheme with similar weights during the fit. The model was fitted simultaneously to seven traces from two holding potentials, showing the characteristic features of the signal. The resulting fit represents the data well (Figure 10 B). The fit is most satisfactory for extreme voltage steps, which is expected when considering that at least eight states in a linear scheme are required to fit the gating charge movement of the Shaker channel (
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The fitted parameters of the model confirms the interpretation suggested above: the middle state (S2) has the highest fluorescence intensity (lowest quenching; -12.2 vs. 0.9 for S1 and -4.8 for S2). The hysteresis of the fluorescence signal at large voltage steps is explained by the difference in kinetics between hyperpolarizing and depolarizing voltage steps. Thus, when stepping, for instance from -110 to 100 mV, the rate constant for entry into state S2 is much smaller than from exit ( <
). During the return of the voltage sensor (from 100 to -110 mV), the rate constant for entry into state S2 is now larger than that of exit (ß >
). As a consequence, S2 is heavily populated and the fluorescence signal is biphasic only when the potential is stepped in a hyperpolarizing direction, not when it is stepped in a depolarizing direction.
The demonstration that both A359C-TMRM and M356C-TMRM interact with a group outside the S4 segment at depolarized potentials has been economically combined in the model in Figure 9 by assuming that the same group is involved. The model in Figure 9 pictures the activation as a rotation of the S4 segment (
The consequence of applying Figure 9 or Figure 10 A to the W434F channel is that there is an intermediate state of the voltage sensor and that activation takes place in (at least) two separate movements. When considering the position of the two parts of the Q-V in the TMRM-labeled L361C (Figure 7 A), it is most likely that the gating charge moving at -160 mV (Figure 7 B) moves within the leftward Boltzmann in this mutant, which we denote as Q1L361C. The two phases of the fluorescence signal when stepping to negative potentials from HP = 0 mV, therefore, most likely correspond to Q2L361C (the fast part of the fluorescence signal) and Q1L361C (the slow fluorescence signal), respectively. This conclusion was made possible by the identification of a mutant (L361C) that in the TMRM-stained situation has a better separation between Q1 and Q2 than the wt channel, combined with a fluorescence signal correlating with Q1L361C. The same conclusion was reached by
Whereas our data agree with those of
Conclusions
The results presented here confirm the hypothesis that a large fraction of the protein-lined vestibule outside the S4 segment is made up by the S3S4 linker. The acidic residues of this linker may be responsible for the pH effects on fluorescence changes. After the removal of 22 amino acids of the linker, the probe at M356, placed close to the outside of the S3 segment, still tracked deactivation gating charge movement, but displayed a markedly slower fluorescence change than expected from the gating charge movement during activating voltage steps. Position A359 in the same deletion interacts with two different quenching groups, revealing an intermediate state of the sensor movement. This intermediate state was also seen in the Shaker with wt linker when the leucine at position 361 was replaced with cysteine, suggesting that these mutations stabilize the sensor movement after about half of the charge has translocated.
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Footnotes |
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Portions of this work were previously published in abstract form (Sørensen, J.B., A. Cha, R. Latorre, E. Rosenmann, and F. Bezanilla. 1999. Biophys. J. 76:A411).
1 Abbreviations used in this paper: aa, amino acid; HP, holding potential; wt, wild type; TMRM, tetramethylrhodamine.
2 Due to false saturation of the Q-V, it was not possible to identify the exact magnitude of the displacement.
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Acknowledgements |
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This work was supported by National Institutes of Health (NIH) grant GM30376 (F. Bezanilla) and Chilean grants FONDECYT 197-0739 (R. Latorre) and Cátedra Presidencial (R. Latorre). J.B. Sørensen was supported by a university scholarship from University of Copenhagen. A. Cha was also supported by NIH grant GM08042, a National Research Service Award from the National Institute of Mental Health, and the UCLA Medical Scientist Training Program.
Submitted: 2 July 1999
Revised: 5 January 2000
Accepted: 6 January 2000
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References |
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