Divisions of 1 Biology and 2 Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125
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ABSTRACT |
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The nonsense codon
suppression technique was used to incorporate o-nitrobenzyl
cysteine or o-nitrobenzyl tyrosine (caged Cys or Tyr) into
the 9' position of the M2 transmembrane segment of the -subunit of
the muscle nicotinic ACh receptor expressed in Xenopus
oocytes. The caged amino acids replaced an endogenous Leu residue that
has been implicated in channel gating. ACh-induced current increased
substantially after ultraviolet (UV) irradiation to remove the caging
group. This represents the first successful incorporation of caged Cys
into a protein in vivo and the first incorporation of caged amino acids
within a transmembrane segment of a membrane protein. The bulky
nitrobenzyl group does not prevent the synthesis, assembly, or
trafficking of the ACh receptor. When side chains were decaged using
1-ms UV light flashes, the channels with caged Cys or caged Tyr
responded with strikingly different kinetics. The increase in current
upon photolysis of caged Cys was too rapid for resolution by the
voltage-clamp circuit [time constant (
) <10 ms], whereas the
increase in current upon photolysis of caged Tyr was dominated by a
phase with
~500 ms. Apparently, the presence of a bulky
o-nitrobenzyl Tyr residue distorts the receptor into an
abnormal conformation. Upon release of the caging group, the receptor
relaxes, with
~500 ms, into a conformation that allows the
channel to open. Tyr at the 9' position of the
-subunit greatly
increases the ability of ACh to block the channel by binding within the
channel pore. This is manifested in two ways. 1) A
"rebound," or increase in current, occurs upon removal of ACh from
the bathing medium; and 2) at ACh concentrations >400 µM,
inward currents are decreased through the mutated channel. The ability
to incorporate caged amino acids into proteins should have widespread utility.
nicotinic acetylcholine receptor; ion channel; site-directed mutagenesis; nonsense suppression; o-nitrobenzyl tyrosine; o-nitrobenzyl cysteine
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INTRODUCTION |
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A POWERFUL TECHNIQUE to investigate ion channels is site-directed mutagenesis, which allows naturally occurring amino acids to be substituted into any site of a protein. A recent extension of this approach is the nonsense suppression technique (21) for incorporating amino acids with unnatural side chains and backbones into proteins in intact cells (23). To apply this technique, the codon of interest of a cDNA is first mutated to a nonsense codon, TAG. cRNA is synthesized and coinjected into a Xenopus oocyte with a tRNA modified to include a nonsense anticodon (CUA) and charged with an unnatural amino acid. Extensive studies of the nicotinic acetylcholine receptor (nAChR) (e.g., Refs. 8, 23, and 34) establish the technique as having considerable potential for probing ion channels and related integral membrane proteins.
The nAChR is an extensively studied ligand-gated ion channel (for
reviews see Refs. 6 and 12). The nAChR is composed of five
homologous subunits (2
in the embryonic muscle
form used here) in a pentameric array around a central pore.
The extracellular NH2-terminal domains of the
-subunits
contribute to the two agonist binding sites with additional important
contributions from the
- and
-subunits. Each subunit possesses
four transmembrane segments, and the cation-selective pore is lined
primarily by the second transmembrane segment, M2, with some
contribution by M1. The nature of the interactions between ACh and its
binding sites on the
-subunit has been analyzed by the nonsense
suppression technique (11, 21, 30). The results implicated
the presence of a cation-
interaction between the quaternary
ammonium group of ACh and the aromatic ring of tryptophan 149 (34). Nonsense suppression has also been used to
investigate the role of backbone hydrogen bonding within
-helical
segments of the nAChR (8). Neither of these studies could
have been completed using conventional mutagenesis approaches.
Unnatural amino acids have also been employed to investigate the role
of a conserved leucine in channel gating (14). This leucine at the 9' position of M2 (numbered so that the putative NH2 terminus of M2 at the intracellular surface is assigned
the 1' position) is conserved in all nAChR subunits and has been
implicated in the gating of the nAChR channel (12, 30).
In addition, Miller et al. (20) used the nonsense
suppression technique to incorporate ortho-nitrobenzyl
tyrosine [Tyr(ONB), i.e., caged tyrosine; Fig.
1], in place of tyrosine, into three sites (positions 93, 127, and 198) in the extracellular domain of the
-subunit of the nAChR. The presence of the caging group interfered
with ACh binding. Upon flash photolysis, the wild-type tyrosine was
revealed, agonist binding sites were generated, and channel activation
occurred. The time course of channel activation at some sites was
surprisingly slow (several seconds in some cases) and implied that the
receptor needed to undergo a conformational rearrangement before
channel opening could occur; that is, the presence of the caged
compound induced an abnormal conformation of the receptor. In another
study, Tyr(ONB) was decaged to reveal a tyrosine-based signal
transduction pathway at an intracellular site in an inward rectifier
K+ channel (29).
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In this study, we extended the use of caged amino acids to further
study the nAChR. Both caged cysteine [Cys(ONB); Fig. 1] and caged
tyrosine are incorporated into a site within a transmembrane segment.
Cysteine modification has been a major tool in the study of ion
channels. The ability to decage a specific cysteine residue at a
specified time during an experiment could have substantial utility. We
found that the kinetics of channel activation after flash photolysis
strikingly differ for caged tyrosine and caged cysteine at this
position. Unexpectedly, we also found that introducing a tyrosine at
the 9' position of the M2 segment of the -subunit increases the
affinity of ACh for a binding site within the pore of the channel.
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METHODS |
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Molecular biology.
All experiments used the mouse muscle nAChR -,
-,
-, and
-subunits. cRNA was synthesized using the Ambion T7 mMessage mMachine kit. For the wild-type channel, 1.25 ng of cRNA, containing equal amounts of each subunit, were injected in 50 nl per
Xenopus oocyte. For nonsense suppression experiments at the
9' position of M2 of the
-subunit, the codon for leucine at position
260 was mutated to the stop signal TAG (
9'TAG) (25).
9'TAG cRNA (1.25 ng) plus 0.17 ng of cRNA encoding the
-,
-,
and
-subunits in equal amounts were coinjected with 8.5 ng of the
tRNA of interest. The
-amino group of the aminoacylated tRNA was
protected with 4-pentenoyl (4-PO) until just before injection into
oocytes (20). Deprotection was accomplished by incubating
an aliquot of the 4-PO-Tyr(ONB) tRNA or 4-PO-Cys(ONB) tRNA with an
equal volume of saturated I2 solution for 10 min. The
deprotected tRNA was then added to an equal volume of cRNA to produce a
final concentration as described above. These procedures have been
described in detail previously (20, 22, 25).
Chemical synthesis.
The production and use of caged tyrosine has been described previously
(20, 29). To prepare caged cysteine coupled to tRNA,
N-4-PO-cysteine-S-o-nitrobenzyl thioether was
first synthesized. Sodium spheres (49 mg, 4.1 mmol) were dissolved in 4 ml of dry MeOH at 0°C, and the mixture was warmed to room temperature
over 10 min. L-Cysteine (250 mg, 2.06 mmol) was added,
followed after 5 min by o-nitrobenzyl bromide (356 mg, 1.65 mmol). After 1 h, the mixture had turned to a gel, and the
volatiles were removed by rotary evaporation. The remaining residue was
dissolved in water (10 ml) and dioxane (10 ml).
Na2CO3 was added (436 mg, 4.1 mmol), followed
by 4-pentenoic anhydride (4-PO-anhydride, 380 mg, 2.1 mmol) (4,
7). After 3 h, an additional 50 mg of 4-PO-anhydride were
added. After an additional 1 h, CH2Cl2 (25 ml) and 1 N NaHSO4 (25 ml) were added, the mixture was
extracted with CH2Cl2 (3 × 25 ml), and
the combined organics were dried over Na2SO4.
The volatiles were removed, and the product was purified by flash
chromatography (silica, 1:1 hexane-ethyl acetate to ethyl acetate + 1% AcOH) to give 472 mg of a pale yellow solid (85% yield for 2 steps): 1H nuclear magnetic resonance (NMR; 300 MHz,
CD3OD) 7.93 (AB, J = 9.0 Hz,
J = 1.3 Hz), 7.62-7.40 (m, 3 H), 5.90-5.75
(m, 1 H), 4.97 (dd, J = 32.0 Hz, J = 1.8 Hz, 2 H), 4.94 (dd, J = 32.0 Hz, J = 1.8 Hz, 2 H), 4.59 (dd, J = 4.8 Hz, 1 H), 4.08 (m, 2 H), 2.90-2.75 (m, 2 H), 2.34 (m, 4 H); 13C NMR
(75 MHz, CD3OD)
175.4, 173.5, 149.7, 138.1, 134.9, 134.3, 133.3, 129.7, 126.5, 116.0, 53.0, 36.0, 34.4, 34.3, 30.7.
Optics. Amino acids were decaged either with 3-s pulses of continuous light from a 300-W Hg arc lamp or by 1-ms flashes from a xenon short-arc flashlamp charged to 450 V through a capacitor bank (2,600 µf). In both cases, light was filtered to provide wavelengths of 300-350 nm and focused onto a liquid light guide for delivery to an oocyte in the electrophysiological chamber. Details of the optical arrangement have been described previously (20, 29).
Electrophysiology.
ACh-induced currents were measured in oocytes 18-48 h
postinjection. Whole cell currents were measured in the two-electrode voltage clamp configuration by use of a Gene Clamp 500 amplifier (Axon
Instruments). The bathing solution contained 96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, and 5 mM HEPES (pH 7.5). The membrane potential was
held at 80 mV in all experiments unless otherwise noted. Data are
presented as means ± SE.
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RESULTS |
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Nonsense suppression.
In the nonsense suppression technique, a TAG stop codon is first
introduced into a cDNA at the site of interest, and cRNA is
synthesized. cRNA is then coinjected into a Xenopus oocyte with a tRNA mutated to recognize and bind to the stop codon and is
synthetically charged with a natural or unnatural amino acid. The
primary purpose of the experiments described here was to incorporate caged cysteine and caged tyrosine into the pore-lining segment, M2,
of the nAChR. Thus tRNAs charged with either Cys(ONB) or
Tyr(ONB) were coinjected with the cRNA. In initial experiments, we were unsuccessful in suppressing a nonsense codon at the 9' position of M2
of the -subunit. Some suppression was achieved at the 9' position of
the
-subunit. It is usually not possible to identify the cause of
unsuccessful suppression (i.e., the absence of channel activity). The
incorporation of an unnatural amino acid into a protein may cause
inappropriate folding or instability. Alternatively, incorporation of
the unnatural amino acid may be inefficient due, for example, to an
inappropriate context of the nucleotide sequence. Suppression was
greatest at the 9' position (L260) of the
-subunit, and all results
presented here involve suppression of a TAG codon at this site. As
mentioned above, the 9' position of M2 may have a special role in the
gating of the nAChR. To first order, the 9' Leu of each subunit makes a
comparable contribution to gating (20, 29).
Decaging of Cys(ONB).
We used two light sources to photolyze the caged amino acids: 3-s
pulses of continuous light or ~1-ms flashes from a short-arc flashlamp. Results from the use of continuous UV light will be presented first. Figure 2 shows typical
results illustrating the effects of light on an oocyte expressing nAChR
in which Cys(ONB) had been incorporated at the 9' position of the M2
transmembrane segment of the -subunit. In this experiment, ACh (20 µM) induced a current even before exposure to UV light. An analysis
of this "background" ACh-induced current will be presented in
DISCUSSION. A 3-s UV light pulse increased the ACh-induced
current by ~1 µA. Subsequent light pulses induced further
increases, each roughly two-thirds as large as the previous increase.
Typically, little further increase in current was seen after the fourth
pulse. In this experiment, the magnitude of the cumulative
light-induced current was about equal to the background current. The
current induced by the decaging of cysteine ranged from 50 to 150% of the background current. No current was induced by light in the absence
of ACh (not shown). With the wild-type channel, light exposure
sometimes induced a small increase in current only during the 3-s light
exposure itself, but no sustained increase in current was ever seen
[see also Fig. 8B in Millisecond decaging of
Tyr(ONB) and Cys(ONB)].
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Decaging of Tyr(ONB).
A typical response to photolysis of caged tyrosine at the 9' position
of M2 of the -subunit of the nAChR is shown in Fig. 3. ACh (20 µM) induces a background
current of ~1 µA before photolysis. The current is increased
substantially after a single light pulse (3 s). In one series of
experiments, increases were 4.1 ± 0.4-fold (n = 5). A second light pulse produces a small artifact (similar to that
seen with the wild-type channel) during the light exposure but little
sustained increase in current. Tyrosine at the 9' position of M2 of the
-subunit greatly decreased the EC50 for ACh to activate the channel. After decaging of the Tyr(ONB), the EC50(ACh)
was 1.0 ± 0.1 µM, and the Hill coefficient was 1.3 ± 0.2. This 36-fold lower EC50 for the
9'Tyr mutation (from 36 µM for the wild type) is somewhat more dramatic than the eightfold
decrease (from 17.5 to 2.2 µM) reported recently for the same
mutation expressed in mammalian cells (15).
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Block of the channel by ACh.
After decaging of tyrosine at the 9' position of M2 of the -subunit,
current transiently increased after removal of ACh. We will refer to
this transient increase as a "rebound." Higher ACh concentrations
produced rebound phases that were larger fractions of the ACh-induced
current. This point is exemplified by the data shown in Fig.
4. The current induced by ACh (100 µM)
was increased 3.5-fold by UV light. The channel then partially
desensitized. Then, when the ACh was removed, a small increase in
current was observed. This rebound was much larger upon removal of 400 µM ACh but was not evident at 20 µM ACh. As presented in detail in the DISCUSSION, we interpret the rebound effect as being
due to direct blocking of the channel pore by ACh. Upon removal of ACh, ACh leaves the pore and current increases. Rebound was never seen with
the wild-type nAChR with an endogenous leucine at the
9' position.
Rebound was sometimes, but not always, present in the ACh-induced
current before photolysis of the caged tyrosine. In eight experiments
at 200 µM ACh, rebound was seen in three cases before photolysis.
Quantitative analysis of the rebound is complicated, because at the
time of ACh removal, at least two phenomena are occurring.
1) Current decreases as ACh dissociates from activation sites on the extracellular surface. 2) Current increases as
ACh diffuses from a blocking site within the channel pore. Because of
this complexity, we assessed the blocking effect of ACh through the use
of current-voltage (I-V) relationships during steady
exposure to ACh.
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Millisecond decaging of Tyr(ONB) and Cys(ONB).
We also used ~1-ms UV flashes for decaging of tyrosine and cysteine.
This approach has been used previously in this laboratory to study the
kinetics of decaging of Tyr(ONB) incorporated into the agonist binding
site of the nAChR (20). Typical results using caged
tyrosine incorporated at the 9' position are shown in Fig.
7. Application of ACh induces a modest
inward current. The inward current is then enhanced by a series of
flashes spaced at 12-s intervals. The increment of current is greatest
with the initial flash and decreases with each subsequent flash. In
this experiment, each light flash activated ~5% of the pool of caged receptors. This fraction varied but could be increased up to ~15% by
careful optimization of the optics. These observations are consistent
with previous measurements that the flashlamp decages tyrosine with an
efficiency k between 0.05 and 0.15/flash (20). In the experiment of Fig. 7, ACh was then removed from the bath to
remove cumulative desensitization of the receptors. Reapplication of
ACh produced a current about threefold larger than that induced by the
initial application of ACh before flash activation.
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DISCUSSION |
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We have successfully incorporated caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor. Caged tyrosine has been successfully used previously at extracellular sites of the nAChR (20) and at an intracellular site in an inward rectifier K+ channel (29). There are no previous reports on the translational incorporation of caged cysteine into a protein. It is remarkable that the incorporation of a bulky nitrobenzyl group in the interior of a polytopic membrane protein does not inhibit synthesis, assembly, or trafficking. An advantage of using caged amino acids in suppressor experiments is that the protein can be activated by UV light. Activation of the protein by irradiation cannot occur unless the caged compound has been incorporated into the protein as intended.
Successful incorporation of caged cysteine into the 9' position of M2
of the -subunit of the nAChR was demonstrated through the use of
MTSEA. Before cysteine decaging, ACh-induced current was impervious to
this sulfhydryl-modifying reagent. After exposure to UV light, a
portion of the current was irreversibly inhibited by MTSEA (Fig. 2).
Conventional mutagenesis has previously been used to replace the
leucine at the 9'position of M2 of both the
- (2) and
-subunits (32) with a cysteine residue. In these cases,
MTSEA inhibition approached 70%, a value similar to that obtained here
using the
-subunit. The earlier studies attributed the effects of
MTSEA to direct blockage of the channel pore. It is probable that the
nitrobenzyl caging group also obstructs the pore and induces an
unfavorable conformation for channel function before light activation.
ACh-induced current was often present in our experiments, even before
decaging. This current was substantial with caged cysteine but much
less so with caged tyrosine (see RESULTS). This current could have several sources. First, some photolysis of the caged amino
acids may have occurred before our experimental protocols were begun.
This does not seem to be the case, at least for the caged cysteine, as
indicated by the absence of MTSEA effects before intentional
photolysis. Second, it is possible that caged cysteine or caged
tyrosine at the 9' position blocks the channel pore incompletely or
transiently. Some channel function may exist before the decaging event.
Third, endogenous tRNA synthetases may reacylate the suppressor tRNA.
This would result in the incorporation of a natural amino acid at the
mutated site. Although the suppressor tRNA has been engineered to
minimize reacylation (25), such events may occur. The
background current with caged cysteine may limit some applications of
the approach.
After decaging of tyrosine at the 9' position of the nAChR, removal
of high concentrations of ACh from the chamber led to a transient
increase in current, followed by the expected decline in current. The
simplest interpretation of this "rebound" effect is that ACh itself
blocks the mutant channel. Indeed, blockage of the nAChR by ACh has
been described previously. Initial studies used single-channel
recordings to characterize blockage of the receptor by ACh (24,
27). Analysis of the effects of ACh on channel lifetimes
indicated that ACh was an open channel blocker. Detailed studies of the
rebound effect are reported by Maconochie and Steinbach
(18). We observe channel blockage at relatively low ACh
concentrations when the
9' residue is Tyr. Rebound was always
evident at 100 µM ACh (Fig. 4) and was sometimes discernible at
concentrations as low as 20 µM. Maconochie and Steinbach reported that the dissociation constant (Kd) for
blockage of the fetal murine muscle ACh receptor (as used here) was
12.7 mM. We conclude that the leucine-to-tyrosine mutation at the
9'
position within the channel pore increases the binding affinity of ACh
within the pore. We cannot specify the new Kd
for blockage of the channel. Upon removal of ACh, both activation and
inhibition are occurring simultaneously, and the kinetics of these
effects are also dependent upon flow rate and fluid exchange. However,
we estimate that the decrease in Kd is at least
50-fold. Mutations increasing the efficacy of ACh to act as a pore
blocker have not been previously described.
If a binding site for ACh is located in the channel pore within the
membrane electric field, then blockage of the channel by ACh should be
voltage dependent. That this is the case is demonstrated in Fig. 5.
High concentrations of ACh cause outward rectification at the
-Leu9'Tyr mutant channel. It can also be seen that, with the mutant
channel, steady-state current decreases as ACh increases (Fig.
5D).
The fact that the introduction of an aromatic residue, tyrosine, into
the pore increases the binding affinity of cationic ACh raises the
possibility that a cation- interaction has been introduced. In
cation-
interactions, cations are attracted to the negative
electrostatic potential associated with the quadrupole moment of an
aromatic residue. Cation-
interactions have been implicated in the
binding of ACh to acetylcholinesterase (28) and in the
binding of ACh to its activation site on the nAChR (34).
To test the possibility of a cation-
interaction, we introduced
other amino acids besides tyrosine at the 9' position. No rebound
effect was seen when 4-Me-Phe was introduced into the receptor by
nonsense suppression (Fig. 6A). This amino acid is sterically very similar to tyrosine. In terms of the cation-
interaction, gas phase calculations suggest that 4-Me-Phe and tyrosine
should be very similar (19). However, in aqueous solution, hydrogen bonding to the OH of tyrosine can enhance its cation-
binding ability, making it more potent than 4-Me-Phe. (10,
19). Still, 4-Me-Phe is certainly more potent at binding cations
than is the wild-type leucine; therefore, if cation-
interactions were involved, a rebound effect might have been expected with this
residue. In contrast to 4-Me-Phe, a rebound effect is seen when either
Trp or F4-Trp is incorporated at
9'. Trp is a potent cation-
binder, but F4-Trp most definitely is not
(34).
Extensive studies on K+ channels over three decades show
that the binding of quaternary ammonium blockers is sensitive to the structure of side chains as well as to the state of the channel (31). Tyrosine side chains can either increase
(11) or decrease (26) the affinity of
quaternary ammonium blockers. Quaternary ammonium ions are also
sensitive probes of the M2 pore-forming region in nAChR channels
(3), and the present paper is the first to show that an
introduced tyrosine side chain in the conducting pathway can increase
the affinity of a binding site for a quaternary ammonium ion (ACh
itself). There is not yet an atomic-scale structure for a quaternary
ammonium ion in an ion channel, and we do not, therefore, appreciate
the details of the interactions. For the specific case of nAChR
channels, the present data rule out a cation- interaction. Other
possibilities include polar interactions with the hydroxyl group and
nonpolar interactions with the aromatic ring.
Direct introduction of tyrosine at the 9' position results in a
channel that exhibits rebound (Fig. 6B). That is, the same result is obtained whether tyrosine is introduced by photolysis of
caged tyrosine or by direct introduction of tyrosine. This serves as a
positive control; a negative result would have cast doubt on the use of
photolysis to liberate tyrosine from the caging nitrobenzyl group. We
also tested the effect of a serine at the 9' position (although in this
case the substitution was in the
-subunit). No rebound of current
upon removal of ACh was seen in this case (Fig. 6C). Serine
at the 9' position of any of the subunits of the ACh receptor
substantially increases the apparent affinity of the receptor for ACh
(16). As mentioned above, we found that tyrosine at the 9'
position of the
-subunit also increases ACh apparent affinity. The
serine mutant serves as a control to indicate that the introduction of
the rebound effect is not simply an artifact of an increased apparent affinity.
We also examined the ability of brief pulses (1 ms) of UV light to
decage tyrosine and cysteine (Figs. 7-9). The increase in current
upon decaging of tyrosine occurred with a surprisingly slow time course
( ~ 0.5 s). The wild-type channel would be expected to
respond much more rapidly to a sudden change in ACh concentration or
voltage (1, 9). The present result is reminiscent of the
results of Miller et al. (20), who examined the effects of
decaging tyrosine at the agonist binding site. Miller et al. suggested
that the caged tyrosine induced an abnormal, closed-state conformation
of the channel. After tyrosine decaging, the receptor needed to relax
back to the native conformation before agonist binding could induce an
open state. This rearrangement would be the rate-limiting step and
would determine the relaxation time. We propose an analogous mechanism
to explain the relatively slow activation of the channel when tyrosine
is decaged within a transmembrane segment. The perturbation in
structure caused by the presence of a bulky Tyr(ONB) group within the
pore apparently prevents rapid opening of the channel upon flash
photolysis. The small, rapid component of current relaxation upon
illumination (Fig. 8A) may be due to a population of
channels that retain a more native conformation, even in the presence
of the caged tyrosine.
A striking difference between our results and those of Miller et al.
(20) is the response of current to a series of flashes. In
the earlier study, current increments were initially small, increased
in magnitude, and then declined. The interpretation of these results
followed directly from the fact that the two agonist binding sites must
be occupied to induce channel opening. With a caged tyrosine
incorporated at each of the two ACh binding sites, an initial flash
would be unlikely to activate both binding sites. A response to a
series of flashes would be expected to be sigmoidal, and this, indeed,
was seen. In contrast, we found in the present experiments that the
first flash of light always induced the largest response (Fig.
7A) and that the increments in current decreased
exponentially. This result is consistent with the presence of only 1 -subunit/channel and demonstrates the sensitivity of the technique
to subunit number.
Although we interpret the slow response to photolysis of caged tyrosine in terms of a slow conformational change, there is at least one alternative explanation. The nitrosobenzaldehyde byproduct of photolysis (Fig. 1), after being released from tyrosine within the pore, may exert an inhibitory effect on channel function. In this case, the current relaxation time may be determined by the rate of diffusion of the byproduct from the pore. This would seem unlikely, because diffusion of a small molecule should be many times too rapid to account for the slow kinetics observed here. In addition, the results obtained with the rapid decaging of cysteine (Fig. 9) strongly argue against this possibility. When cysteine is decaged, the same group, nitrosobenzaldehyde, is released. If the released nitrosobenzaldehyde were somehow responsible for the slow relaxation kinetics, then we would expect the same slow kinetics whether cysteine or tyrosine was being decaged. The difference in the kinetics of current change upon decaging of cysteine and tyrosine is quite striking (Figs. 8A and 9B). Apparently, the more bulky Tyr(ONB) has a larger effect on channel structure. Upon decaging of tyrosine, time on the order of 1 s is required for rearrangement of the protein. This extended time period is not required when the less bulky Cys(ONB) is photolyzed.
We found that caged tyrosine was easier to use than caged cysteine for the incorporation of an unnatural amino acid into the nAChR. First, caged tyrosine was more efficiently decaged than caged cysteine. We estimated that k was four to five times greater for caged tyrosine than for caged cysteine. A 3-s pulse of light decaged 90-95% of caged tyrosine but only ~45% of the caged cysteine (compare Figs. 2A and 3). This presumably reflects an intrinsic reactivity difference between nitrobenzyls attached to a phenol vs. a thiol, although some aspect of the environment at the receptor may also contribute. Second, large currents were seen consistently after photolysis of caged tyrosine but not with the use of caged cysteine. With caged cysteine, the increase in current upon photolysis was often modest (Fig. 2), although large currents were seen occasionally. The overall trend suggests that suppression efficiency is greater with Tyr(ONB) than with Cys(ONB). The reason for this variability is unknown, but there is certainly considerable variability in the efficiency of incorporation of differing unnatural amino acids by nonsense suppression both in vitro and in vivo. It is formally possible that the thiol liberated from decaging reacts with the nitrosobenzaldehyde. This might explain the differential results in decaging the tyrosine and cysteine, and also the incomplete block by MTSEA, because the cysteine has reacted with the nitrosobenzaldehyde to produce a new species. In any case, sufficient quantities of Cys(ONB) can be incorporated into the nAChR to give observable signals upon photolysis.
The techniques developed here have a number of potential applications. Decaging of tyrosine, as used here, or of serine or threonine, should be useful in phosphorylation experiments. Acute decaging of one of these residues would allow kinases to act as demonstrated by Tong et al. (29). Also, comparison of the time courses of channel activation after the decaging of different amino acid residues at different locations within a transmembrane segment should provide information on the strain induced in the protein by the caging group. Although this concept was applied by Miller et al. (20) and here, an extended study should greatly expand interpretations and perhaps provide an additional kinetic signal for mapping the conformational wave of receptor gating by linear free energy analysis. Sudden exposure of cysteine may be useful in studying the kinetics and the effects of farnesylation reactions. The combination of the use of caged cysteine with the versatility of sulfhydryl reagents may be especially productive for structure-function studies. For example, for fluorescence resonance energy transfer experiments, a protein could be engineered to have one cysteine residue and one caged cysteine residue. The first cysteine could be labeled with one fluorescent sulfhydryl reagent, and then, after decaging, the second cysteine could be labeled with a different fluorescent reagent. The application and optimization of the use of caged cysteine will be the subject of further investigations.
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ACKNOWLEDGEMENTS |
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We thank Dr. B. Khakh for helpful discussions.
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FOOTNOTES |
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This research was supported by grants from the National Institutes of Health (NS-11756, NS-34407, and HL-49101).
Present address of K. D. Philipson: Dept. of Physiology, UCLA School of Medicine, Los Angeles, CA 90095.
Address for reprint requests and other correspondence: H. A. Lester, Division of Biology, California Institute of Technology, Pasadena, CA 91125 (E-mail: lester{at}caltech.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 October 2000; accepted in final form 28 February 2001.
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