Correspondence to: Ehud Y. Isacoff, Department of Molecular and Cell Biology, Physical Biosciences Division, Lawrence Berkeley National Laboratory, 271 Life Science Addition, MC 3200, University of California, Berkeley, Berkeley, CA 94720. Fax:(510) 642-4968 E-mail:eisacoff{at}socrates.berkeley.edu.
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Abstract |
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The mechanism by which physiological signals regulate the conformation of molecular gates that open and close ion channels is poorly understood. Voltage clamp fluorometry was used to ask how the voltage-sensing S4 transmembrane domain is coupled to the slow inactivation gate in the pore domain of the Shaker K+ channel. Fluorophores attached at several sites in S4 indicate that the voltage-sensing rearrangements are followed by an additional inactivation motion. Fluorophores attached at the perimeter of the pore domain indicate that the inactivation rearrangement projects from the selectivity filter out to the interface with the voltage-sensing domain. Some of the pore domain sites also sense activation, and this appears to be due to a direct interaction with S4 based on the finding that S4 comes into close enough proximity to the pore domain for a pore mutation to alter the nanoenvironment of an S4-attached fluorophore. We propose that activation produces an S4pore domain interaction that disrupts a bond between the S4 contact site on the pore domain and the outer end of S6. Our results indicate that this bond holds the slow inactivation gate open and, therefore, we propose that this S4-induced bond disruption triggers inactivation.
Key Words: Shaker, rearrangement, voltage, gating, fluorescence
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INTRODUCTION |
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After depolarization of the membrane, voltage-dependent ion channels undergo a series of conformational changes that open and close several distinct molecular gates that regulate transmembrane ion flux. All of these gates must be open for ions to flow through the pore and cross the membrane. Depolarization first activates channels by displacing the positively charged S4, the fourth transmembrane segment, of each of the channels' four subunits. This displacement generates the gating current by carrying basic residues outward across the membrane electric field (
Inactivation occurs via two distinct mechanisms. Fast (N-type) inactivation occurs in milliseconds after the opening of the activation gate because of a block of the internal mouth of the pore by the NH2-terminal domain of the protein (
Slow inactivation is sensitive to mutation of the pore region (P) and S6, as well as S4 (
Although the mechanism of slow inactivation is inherently voltage-independent, it follows voltage because of its dependence on activation (
Recent work has used scanning mutagenesis to identify proteinprotein interaction surfaces in voltage-gated K+ channels via perturbation of the voltage dependence of activation (
Our present results provide two new classes of evidence that S4 and the pore domain interact directly and show that this is a dynamic interaction that changes with gating. The results demonstrate that fluorophores attached to both S4 and the pore domain change fluorescence in parallel with both activation and slow inactivation. To determine whether S4 and the pore domain interact directly, and where, we mutated residues in one domain to see if this influenced the environment of a fluorophore attached to the other domain, a method we call environment scanning. This technique is similar to that described by
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MATERIALS AND METHODS |
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Molecular Biology
Site-directed mutagenesis was done by the use of Quick Change mutagenesis kits from Stratagene and examined either by 35S or fluorescence sequencing. Unless otherwise denoted, the standard composition of the channel was Shaker H4 (6-46/W434/C245V/C462A) (
cRNA was transcribed using T7 Ambion mMessage mMachine. Injection of the oocytes (50 nl mRNA at 1 ng/nl), native cysteine blocking with tetraglycine maleimide, and the attachment of the fluorescent fluorophore 6'-tetramethylrhodamine maleimide (TMRM)1 were performed as previously described (
Voltage Clamp Fluorometry and Analysis
Two-electrode voltage clamp fluorometry was performed as described previously (
The bath solutions consisted of the following (in mM): 110 NaMes, 2 KMes, 2 CaMES2, 10 HEPES, pH 7.5; or 110 KMes, 2 CaMES2, and 10 HEPES, pH 7.5. Oocytes were washed before being placed in the bath, and the bath solution was constantly perfused through the chamber during recording if kinetics were required. The excitation light was reduced by neutral density filters (Carl Zeiss) with either a 5% transmission or with two 5% neutral density filters in series resulting in 1.25% transmission. Light was filtered with an HQ 535/50 excitor, an HQ 610/75 emitter, and a 565LP dichroic (Chroma Technology). The voltage output of the photomultiplier was low passfiltered at one quarter the sampling frequency with an 8-pole Bessel filter (Frequency Devices). A minimum of a 2-min rest interval at the holding potential of -80 mV was given between steps to +40 mV. Solutions were exchanged until currentvoltage relations indicated no further change in reversal potential (usually 10 min). All measurements on conducting (424C-TMRM/W434) channels were made under continuous perfusion. Data analysis was done with the Clampfit programs of PClamp6 and the Beta version of PClamp8 (Axon Instruments) and, in some cases, analyzed further and plotted with Origin (Microcal). Fits to the ionic current were made starting after the capacitive transient (1.5 ms after the start of the step) for current activation and partway into inactivation (
50100 ms after the start of the step). Fluorescence and current traces were normalized and were not averaged unless so noted. All values are mean ± SEM.
Normalization of Fluorescence Change
Normalizing to account for differing levels of channel expression is a potentially difficult process. To take into account differences in the level of expression of various mutants, changes in the F (
Fs) because of mutations in the protein environment of the fluorophore were normalized by two different methods. The methods gave similar and consistent
Fs. All channel constructs were examined in the W434F background (
Fs were normalized by their gating charge (integrated IgOFF). Normalization by the conductance of low numbers of W434 conducting channels yielded similar numbers (data not shown).
Fs were also normalized by comparing the baseline fluorescence at 80 mV after subtracting the background fluorescence of an oocyte that had been injected with conducting wild-type Shaker channels without any introduced cysteines. Normalizing by gating charge (or low level of conducting channels) for a fluorophore at position 359C (359C-TMRM) yielded a change of fluorescence of 28 ± 8.2%, 22 ± 2.2%, 6.98 ± 3.2%, and 16.9 ± 2.3% for E418Q, E422Q, D431N, and D447N, respectively; normalization by baseline fluorescence yielded
Fs of 23 ± 4.7%, 23 ± 4.8%, 6.3 ± 0.37%, and 11.6 ± 1.6%. Normalization by the gating charge of nonconducting (W434F) versions of 424C-TMRM yielded
Fs of 6.25 ± 1.1%, 10.2 ± 5.9%, 0.8 ± 0.18%, and 7.63 ± 0.5% for E418,Q E422Q, D431N, and D447N, respectively, whereas normalizing by baseline fluorescence gave
Fs of 5.98 ± 1.4%, 8.06 ± 3.5%, 0.42 ± 0.2%, and 5.68 ± 1.1%, respectively.
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RESULTS |
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We set out to understand how voltage sensing and gating are coupled at the molecular level. We used fluorescence to examine protein motions in S4 and the pore domain (S5, P-region, and S6). We reasoned that if a site in S4 couples to inactivation then it may experience a change in its local environment not only when S4 moves through the surrounding protein during its transmembrane charge translocation (voltage sensing) step, but also during the structural changes that underlie the slow closure of the inactivation gate. Although previous studies have suggested a role for S4 in slow inactivation, they have been restricted to either functional effects of mutations (
All of the experiments were carried out in the NH2-terminal ball-deleted and external cysteine-removed channel, so that the only form of inactivation was slow inactivation, and where the only TMRM attachment site was the introduced cysteine (see MATERIALS AND METHODS). The depolarizing steps were long enough to close the inactivation gate (P-type inactivation), but not long enough to stabilize the closed state (C-type inactivation; see MATERIALS AND METHODS).
Fluorescence Analysis of the Outer End of S4
We began with an examination of a series of positions (359362) at the outer end of S4. The most NH2-terminal (and external) of these sites (359) is buried in the gating canal in the resting state when S4 is retracted into the cell (
A previous characterization of the fluorescence of TMRM attached to one position (359C) demonstrated that the fluorophore's environment undergoes two very different phases of change after membrane depolarization: one that correlates with fast gating charge movement and a second that correlates with the slow closure of the external inactivation gate (
Fluorescence traces for the four consecutive TMRM attachment sites are shown in Fig 1. At each of the sites, a depolarizing step evoked a large fluorescence change (F) with an onset (
Fon) that paralleled the onset of inactivation of the ionic current (Fig 1; also see legend). At 359 and 362, the inactivation component of the
Fon was preceded by a prominent fast component that paralleled the opening of the activation gate (Fig 1 B). In nonconducting channels, the fast
Fon for these sites has been shown to follow the gating current (
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Surprisingly, at the intervening sites (360 and 361), Fon was dominated by an inactivation-coupled component. The activation component was less than signal noise in a single sweep and was difficult to detect without averaging. In addition, at 361, the foot of the
Fon was distorted by a component with intermediate kinetics, revealing an intermediary conformational step between activation and closure of the gate.
The common feature of all of the sites is that TMRM experiences a change in environment with closure of the inactivation gate. The detection by the fluorophore of the inactivation motion in all directions projecting from S4 is consistent with either a rigid body motion of S4 occurring during inactivation or a cooperative inactivation rearrangement of the entire protein surrounding S4.
The difference between the Fs for these four neighboring sites indicates that TMRM occupies a distinct molecular environment at each residue. The rough resemblance in kinetics between 359 and 362, on one hand, and 360 and 361, on the other (Fig 1 A), suggests that TMRM faces the same direction, which is consistent with an
-helical structure for S4.
Fluorescence Analysis of an Outer Edge of the Pore Domain
Is there a functional tertiary interaction between S4 and the perimeter of the pore domain? We examined as a possible contact region the outer end of S5. Based on the KcsA crystal structure (
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All four sites displayed a F (Fig 2). The magnitude of the
F was largest at 418 and 419 and smallest at 416. As with S4, the fluorescence behavior of TMRM at each position was unique, confirming the very local nature of the environmental detection seen at consecutive positions in S4. At 416 and 417, the
Fon was slow (Fig 2 A) and paralleled the onset of inactivation (Fig 2 legend). At 418 and 419, the
Fon had a fast component that paralleled activation, and 419 also had a slow inactivation component (Fig 2A and Fig D, and legend). This is the first detection of an inactivation-correlated protein motion in the pore domain at a location so far from the selectivity filter (the site where closure of the pore is thought to occur), indicating that the slow inactivation rearrangement is more widespread than previously known. Equally striking is the detection of an activation motion by a fluorophore at pore domain residue 419, but not at neighboring residues 416 and 417 (418 also follows activation, see DISCUSSION). This finding suggests that 419 interacts with S4 during its transmembrane motion, in keeping with earlier work (
Environmental Scanning Detects Direct S4Pore Domain Interaction
The kinetics of the F that we observe for TMRM attached to both S4 and the pore domain suggest that these domains are both involved in slow inactivation gating. Could this match between the fluorescence kinetics be due to a direct interaction between the domains? To address this question, we took advantage of the evidence, described above (Fig 1 and Fig 2), that the spatial extent over which the fluorophore senses its environment is so confined that even neighboring residues have distinct fluorescence reports of protein motion. This means that a selective change in the protein nanoenvironment surrounding the fluorophore attachment site should alter the fluorescence report. We illustrate this idea in Fig 3 A, where we show how the fluorophore (represented by an asterisk) is confined to a small space around its site of attachment (represented by a small circle). Mutation of a residue that is in the nanoenvironment in one state of the channel (illustrated as a change in the circle from black to white) will alter both the fluorescence of that state and the change in fluorescence that occurs upon transition to other states (in this case shown as when the inactivation gate closes and the fluorophore moves into another environment). The significance is that this effect can be used to identify amino acids in one part of the protein that are located so close to a fluorophore attached elsewhere in the protein as to contribute to it's nanoenvironment and, thus, influence its fluorescence (see
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With this idea in mind, we carried out an environment scan in the pore region for two fluorophore attachment sites, one at position 359 in S4 and another at position 424 in the pore region (Fig 3 A). To limit the number of permutations in the scan to manageable proportions, we confined ourselves to one possible cause of fluorescence change in a fluorophore's nanoenvironment, changing proximity to a charged residue. The possibility of interaction with acidic residues was particularly attractive to explore since the F of TMRM at both 359 and 424 was found to be pH-dependent, with a midpoint at approximately pH 45 (data not shown;
We first examined the effect of neutralizations on the fluorescence of TMRM attached to position 424 in the pore region so that the results could be interpreted in light of the KcsA crystal structure (Fig 2 E). Two of the neutralizations, E418Q and D447N, eliminated ionic current but retained gating current (Fig 3 B). This effect could be explained by the stabilization of the closed conformation of the inactivation gate, as described below (Fig 4 and Fig 6) and as shown earlier for D447N by F was examined. We found that three of the four neutralizations (E418Q, E422Q, and D447N) had no effect on the magnitude of the percent
F for a fluorophore attached to 424C (Fig 3B and Fig C). However, the neutralization D431N virtually eliminated the
F of 424-TMRM (Fig 3B and Fig C).
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We next examined the effect of the same four pore domain neutralizations on the fluorescence of TMRM attached at position 359 in S4. Again, there was no significant effect of E418Q and D422N on the F, and again D431N had the greatest reduction of the
F (Fig 3B and Fig C). In addition, D447N, which had no effect on 424C-TMRM, significantly reduced the
F of 359C-TMRM.
The effect of the neutralization mutations could be explained in one of two ways. The COOH group of the side chain may directly contribute to the nanoenvironment of fluorophore, so that its removal alters the fluorescence report. Alternatively, the mutation could disrupt channel structure in a manner that propagates for some distance from the site of mutation, and, thus, indirectly alters the nanoenvironment of the fluorophore. The perturbation of function by the D447N mutation indicates that this mutation may indeed produce a global distortion of channel structure, although it may also maintain normal structure, but simply favor the native inactivated conformation. Therefore, to be safe, we refrain from interpreting the effect of this mutation.
By contrast, the case of D431N is much clearer. Two sets of factors argue against a large-scale disruption of channel structure for D431N and in favor of a direct interaction of D431 with both 424C-TMRM and 359C-TMRM. First, in KcsA, the residue at the 431 position is located at the outermost extremity of the protein surface, where the side chain points out into the water ( = 45 ± 0.8 ms; and 431D,
= 51 ± 2 ms for oocytes with equal expression). Activation kinetics remained normal, and there was no detectable effect on voltage dependence, sensitivity of inactivation to external K+, or ionic selectivity (data not shown). The lack of a significant functional effect of D431N is consistent with an absence of structural perturbation of the mutation.
To further test the idea that side chain substitution at D431 does not perturb channel structure, we examined the effect on channel function of other, far less conservative substitutions at D431. Substitutions with H, C, and C-TMRM, which changed the charge and bulk of the side chain considerably, had no significant effect on either the conductancevoltage relation or the kinetics of channel activation (data not shown), adding further support to the idea that the side chain points away from the protein and into the external solution, so that the effect of substitution remains confined to a local effect on the chemical nanoenvironment of 431.
The results indicate that the influence of the D431N mutation on the fluorescence of TMRM at 359C and 424C is due to the entry of the fluorophore into the 431 side chain environment. This leads us to the functional conclusion that site 359 in S4 comes into close proximity to the pore domain during inactivation. This is consistent with direct S4pore domain interaction, supporting recent work by
S4Pore Interaction May Trigger Inactivation: The Role of E418
Could interaction of S4 with the pore domain be responsible for triggering inactivation? Stabilization of the closed conformation of the inactivation gate by W434F and D447N (
One residue that may play such a role is E418. Like W434F and D447N, we found the E418Q mutant to be nonconducting (Fig 3 B), to have normal gating currents (data not shown), and to convert the F of 359-TMRM and 424-TMRM from slow inactivation into fast activation kinetics while maintaining the size of the
F (Fig 3 B). All of these features are consistent with closure of the inactivation gate by the E418Q mutation (
The implication of these results is that E418 forms a bond that contributes energy to the open conformation of the inactivation gate, leading to the prediction that other changes of the side-chain at this position would also alter the rate of closure of the gate. Results consistent with this were obtained. First, the E418C mutation inactivated more quickly than wild type and destabilized the open state (Fig 5 A). Second, conjugation of TMRM to 418C completely abolished ionic current, leaving only gating current (Fig 5 B), indicating an even larger destabilization of the open conformation of the inactivation gate. Interestingly, while this point mutation dramatically alters the conducting properties of the pore, it doesn't seem to alter the structure of the outer mouth of the protein since AgTx2 is still able to readily block K+ conduction. (Fig 5 C).
S4Pore Interaction May Trigger Inactivation: Possible Bonding Partner of E418
We attempted to identify the bonding partner of E418. By analogy with the KcsA crystal structure, it appears that E418 may interact with the backbone amino groups of two residues at the beginning of S6: V451 and G452 (Fig 6 A). Although the resolution of the structure is not refined enough to reveal many of the possible hydrogen bonds, these hydrogen bonds are the only ones that appear to connect S5 to S6 in this area of the crystal structure.
Mutations of positions 451 and 452 would not be expected to alter the protein backbone, except in the case of a proline substitution. Therefore, we tested the idea that E418 interacts with the backbone amino of V451 and G452 by making several substitutions including proline. We found that substitution with glutamate or cysteine labeled with fluorophore (V451C-TMRM, G452E, and G452C-TMRM), which are side chains of very distinct bulk and polarity, had no effect on the conductancevoltage relation or activation kinetics (data not shown). We substituted proline, whose side chain protects the backbone amino group. V451, G452, and V453 were individually mutated to proline with the prediction that if 451 and 452 interact with E418 then the proline mutations should disrupt the bond and shut the inactivation gate. The mutation V453P served as a control for the effect of a proline substitution at this general location on the global structure. G452P did not express as functional channels, however, other point mutations at this residue (452E, 452C-TMRM) showed no effect on channel activation or inactivation kinetics arguing for the importance of the amino group for bond formation (data not shown). However, V451P channels did express. They lacked ionic current, which is consistent with the stabilization of the closed conformation of the inactivation gate. To test this, V451P channels were labeled with TMRM at 424C and measured photometrically. These channels showed a fast F, typical of permanent P-type inactivation (Fig 6). V453P channels were fully functional, conducting ions, inactivating slowly, and generating
F from 424C-TMRM that correlated with inactivation in the manner of wild-type channels (Fig 6). This wild-type behavior of V453P suggests that kinking of the S6
-helix, which could displace it along most of its length from its wild-type angle, is probably not responsible for effect of the V451P mutation. Instead, we propose that closure of the inactivation gate by the V451P mutation is due to a block of the backbone amino, which prevents formation of the hydrogen bond with E418 that normally holds the inactivation gate open.
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DISCUSSION |
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Membrane depolarization drives voltage-gated ion channels through a series of functional transitions. The process for the Shaker K+ channel starts with transmembrane displacement of charged residues on the voltage sensor in each of the four subunits, followed by the opening of the internal activation gate, finally followed by the closure of the external slow inactivation gate. This is not an obligatory sequence. The slow inactivation gate can close before the activation gate opens, and even in channels where slow inactivation follows opening, it can take place more rapidly from activated closed states (
To determine how the voltage-sensing apparatus could control the inactivation gate, we studied the interaction between S4 and the pore domain, where the inactivation gate appears to reside (
Fluorescence Evidence for Direct Interaction Between S4 and the Pore Domain
We obtained several pieces of evidence that S4 and the pore domain interact. The interpretation of these findings depends on how big the environment of a fluorophore really is. If the fluorophore is large, or is attached by a long flexible linker, it could sweep out an extensive volume of space and interact with many residues scattered over large parts of the protein. In this case, we would expect there to be little difference in fluorescence between nearby attachment sites since they would occupy virtually the same large space. In fact, our observations indicate that the contrary is true, and that a fluorophore senses only a very confined local environment specific to its attachment site.
The local nature of the fluorescence report means that we have three pieces of evidence for direct dynamic interaction between S4 and the pore domain. First, fluorophores attached to S4 sense a molecular motion that closes the inactivation gate (an event known to involve the pore domain); however, this change in environment occurs at sites that point in every direction from S4, which is consistent with an inactivation rearrangement of S4 itself, and suggestive of an integral role for S4 in slow inactivation, an idea consistent with earlier evidence that mutation of S4 alters inactivation (
Proposed Mechanism by which S4 Movement Triggers Inactivation
Among the sites in the pore domain that have been examined with fluorescence, sites 416, 417, and 419 near the outer end of S5, which we examined here, as well as turret residues 424 and 425, which were examined earlier (
How could an interaction between S4 and 418/419 trigger inactivation? We observe here, in agreement with recent work from two other groups (
Our finding that the E418C mutation produces a weaker destabilization of the open conformation than E418Q indicates that glutamine is less well accommodated than the smaller cysteine, and suggests that there is a steric hindrance to the packing of the native glutamate, which is compensated for by the greater ability of its charged oxygen to hydrogen bond. Together, these results are consistent with an S4pore domain interaction that triggers inactivation with E418 acting as a latch. In this model, S4 extrusion and/or twist breaks the 418451 bond, leading to a rearrangement that propagates from the perimeter of the pore domain to the central pore axis, where it closes the gate.
The Inactivation Rearrangement Involves Multiple Bonds
The propagation of the inactivation rearrangement from the perimeter of the pore domain, at the S4 contact site, to the central axis of the pore appears to involve at least three residues other than 418451. Residues W434 in the pore helix, D447 and T449 just outside the selectivity filter, and Y445 in the selectivity filter were shown here, and earlier, to stabilize the open conformation of the gate (
We suggest that the role of K+ in stabilizing the open state of the slow inactivation gate derives from a stabilization by K+ occupancy of the selectivity filter of the backbone orientation of the outer end of S6, favoring the 418451 interaction even when S4 is in its activated conformation, thus, slowing the onset of inactivation. Thus, external K+ may accelerate recovery from inactivation (
A model for the mechanism of how S4's activation movement may be linked to slow inactivation is presented in Fig 7. Underlying the model is the finding that S4 appears to have an intimate dynamic interaction with the pore domain, based on fluorescence reports from both the pore domain and S4, and supported by the present environment scan, as well as by recent work by
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Conclusion
In summary, our results show that the inactivation rearrangement projects much further than previously known from the central axis, all the way to the edge of the pore domain and into the voltage-sensing domain to S4. We also find that the edge of the pore domain senses the activation rearrangement, providing evidence for direct interaction between the pore domain and S4, which is supported by our environmental scanning mutagenesis. These findings support recent evidence by others for contact between S4 and the pore domain, provide a first indication of the location on the pore domain of the S4 contact site, and demonstrate that this contact is dynamic, changing during gating. We explore the possible functional significance of the S4pore domain interaction and find that the open conformation of the slow inactivation gate is destabilized by mutation of a pore domain edge residue, E418, near a site of apparent S4 contact. In addition, the open conformation of the slow inactivation gate is also destabilized by a mutation of residue V451 in S6, whose backbone amino group is predicted to interact with E418 based on the KcsA crystal structure. This destabilization only occurs when V451 is substituted with a proline, which eliminates the ability of its backbone amino group to form a hydrogen bond. Together, the results lead to an explicit structural model for how the voltage-sensing apparatus controls the conformation of the inactivation gate: S4 activation motion changes S4 contact with the edge of the pore domain, and breaks a key interaction of the edge of the pore domain with S6, which is required to hold the inactivation gate open, thus triggering the slow inactivation closure.
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Footnotes |
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1 Abbreviation used in this paper: TMRM, 6'-tetramethylrhodamine maleimide.
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Acknowledgements |
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We would like to thank Chris Gandhi, Lidia Mannuzzu and other members of our lab, as well as John Ngai and members of his lab for helpful discussion.
This study was funded by the National Institutes of Health (grant No. NS35549), by the Department of Energy, and by a Laboratory Directed Research and Development Grant from the Lawrence Berkeley National Lab.
Submitted: 7 July 2000
Revised: 11 September 2000
Accepted: 13 September 2000
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