Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor

Kenneth D. Philipson1, Justin P. Gallivan2, Gabriel S. Brandt2, Dennis A. Dougherty2, and Henry A. Lester1

Divisions of 1 Biology and 2 Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma -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 (tau ) <10 ms], whereas the increase in current upon photolysis of caged Tyr was dominated by a phase with tau  ~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 tau  ~500 ms, into a conformation that allows the channel to open. Tyr at the 9' position of the gamma -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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 (alpha 2beta gamma delta in the embryonic muscle form used here) in a pentameric array around a central pore. The extracellular NH2-terminal domains of the alpha -subunits contribute to the two agonist binding sites with additional important contributions from the gamma - and delta -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 alpha -subunit has been analyzed by the nonsense suppression technique (11, 21, 30). The results implicated the presence of a cation-pi 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 alpha -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 alpha -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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Fig. 1.   Photolysis of caged tyrosine and caged cysteine residues. Shown are the structures of ortho-nitrobenzyl Tyr [Tyr(ONB)], Cys(ONB), and photolysis products (Tyr, Cys, and nitrosobenzaldehyde). Also shown are the structures of 4-methylphenylalanine (4-Me-phe) and 4,5,6,7-tetrafluorotryptophan (F4-Trp).

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 gamma -subunit increases the affinity of ACh for a binding site within the pore of the channel.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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Molecular biology. All experiments used the mouse muscle nAChR alpha -, beta -, gamma -, and delta -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 gamma -subunit, the codon for leucine at position 260 was mutated to the stop signal TAG (gamma 9'TAG) (25). gamma 9'TAG cRNA (1.25 ng) plus 0.17 ng of cRNA encoding the alpha -, beta -, and delta -subunits in equal amounts were coinjected with 8.5 ng of the tRNA of interest. The alpha -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) delta  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) delta  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.

To prepare N-4-PO-cysteine-S-o-nitrobenzyl thioether cyanomethyl ester, N-4-PO-cysteine-S-o-nitrobenzyl thioether (360 mg, 1.06 mmol) was first dissolved in 2 ml of dry dimethylformamide, 2 ml ClCH2CN, and 0.45 ml Et3N. After 6 h, volatiles were removed, and the product was purified by flash chromatography (silica, 1:1 hexane-ethyl acetate to ethyl acetate) to give 341 mg of a pale yellow oil (85% yield): 1H NMR (300 MHz, CDCl3) delta  8.00 (d, J=8.2 Hz, 1 H), 7.60 (t, J=7.7 Hz, 1 H), 7.44 (m, 2 H), 6.8 (m - br, 1 H), 5.83 (m, 1 H), 5.10-4.95 (m, 2 H), 4.80 (s, 2 H), 4.11 (m, 2 H), 2.94 (m, 2 H), 2.36 (m - br, 4 H); 13C NMR (75 MHz, CDCl3) delta  172.7, 169.5, 148.3, 136.7, 133.4, 133.2, 132.1, 128.7, 125.6, 115.7, 114.2, 51.6, 49.4, 35.0, 34.0, 33.4, 29.3.

Finally, dCA-N-4-PO-cysteine-S-o-nitrobenzyl thioether was prepared as previously described (23), starting with 10 mg of N-4-PO-cysteine-S-o-nitrobenzyl thioether and 10 mg of dCA in 400 µl of dry DMF. Fast atom bombardment mass spectrometry (FAB-MS) [M-H]+ 957.2 calculated for C34H42N10O17P2S (MW 957.2). The dCA-N-4-PO-cysteine-S-o-nitrobenzyl thioether was coupled to tRNA as described previously (23).

For the syntheses, all reagents were purchased from Aldrich, and all chemical syntheses were performed under a positive pressure of argon. NMR spectra were recorded on a General Electric QE300 (300 MHz 1H, 75 MHz 13C) spectrometer and are referenced to residual solvent protons. FAB-MS was performed by the mass spectrometry facility at the University of California, Riverside. HPLC separations were performed on a Waters dual 510 pump liquid chromatograph system equipped with either a Waters 490E variable wavelength ultraviolet (UV) detector or a Waters 960 photo diode array detector. Analytical HPLC samples were separated using a Waters NovaPak C18 column (3.9 × 150 mm); semipreparative samples were separated using a Whatman Magnum 9 column (9.4 × 500 mm, Partisil 10, ODS-3).

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -subunit. Some suppression was achieved at the 9' position of the beta -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 gamma -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 gamma -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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Fig. 2.   Light-induced decaging of Cys(ONB). Current was first activated by the application of ACh (20 µM) to an oocyte expressing the ACh receptor. The oocyte had been injected with cRNA coding for wild-type, alpha -, beta -, and delta -subunits and for the gamma -subunit with a Leu260TAG (gamma 9'TAG) mutation. Suppressor tRNA charged with Cys(ONB) was coinjected. Methanethiosulfonate ethylammonium (MTSEA; 0.35 mM) was included in the bathing medium as indicated. Three pulses of ultraviolet (UV) light of 3 s duration each are indicated by arrows. Spikes in current traces are artifacts due to switching of the bathing medium.

Methanethiosulfonate ethylammonium (MTSEA) is a positively charged sulfhydryl reagent that has previously been used as a tool to study the nAChR in cysteine-scanning mutagenesis experiments (e.g., Refs. 2 and 33). We found that MTSEA (0.3 mM) had no effect on ACh-induced current before UV light exposure (Fig. 2). This concentration of MTSEA also had no effect on the wild-type nAChR (not shown). However, after decaging of a cysteine residue at the 9' position of the gamma -subunit, the current was irreversibly blocked by MTSEA treatment (Fig. 2). Over a series of similar experiments, MTSEA inhibited, on average, 66.7 ± 12.6% (n = 6) of the light-induced current. These data provide unequivocal evidence that caged cysteine has been successfully incorporated into the nAChR. Light-induced sensitivity to MTSEA has no other obvious interpretation. In a few cases, a single pulse of light increased the background ACh-induced current by >2.5 µA, and the increase was >= 200% of the background current. Large responses of this type were seen in only three oocytes. Thus, although the data in Fig. 2 are typical, the potential exists for larger responses upon the decaging of cysteine.

After decaging of the Cys(ONB), the EC50(ACh) was 10.4 ± 4.4 µM, and the Hill coefficient was 0.9 ± 0.1 (n = 5). In a parallel series of experiments, the wild-type channel had an EC50(ACh) of 36.9 ± 5.4 µM with a Hill coefficient of 1.6 ± 0.1 (n = 3). In agreement with these observations, an approximately fourfold ratio of EC50 has recently been measured for the mouse wild type vs. gamma -subunit 9' Cys mutation expressed in mammalian cells, although the absolute values were about twofold lower than measured here (15). The significance of a decrease in the Hill coefficient is unknown but has precedent in the literature. Akabas et al. (2) noted a Hill coefficient <1 after a cysteine substitution within M2 of the alpha -subunit. Likewise, Kosolapov et al. (15) observed decreased Hill coefficients after substitution of various amino acids at the 9' position of M2 of the gamma -subunit, and M. White (Dept. of Pharmacology and Physiology, MCP Hahnemann University, Philadelphia, PA, personal communication) has confirmed this observation.

Decaging of Tyr(ONB). A typical response to photolysis of caged tyrosine at the 9' position of M2 of the gamma -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 gamma -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 gamma 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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Fig. 3.   Light-induced decaging of Tyr(ONB). Current was activated by the application of ACh (20 µM) to oocytes expressing the ACh receptor. A Leu260TAG mutation was present in the gamma -subunit (9' position of M2). The stop codon was suppressed by the coinjection of suppressor tRNA charged with Tyr(ONB). Two pulses of UV light of 3 s duration each are indicated by arrows. The current transient induced by the second flash is artifactual, as similar transients were sometimes also seen with the wild-type channel.

The photolytic efficiency is conveniently expressed by the parameter k, where e-kt is the fraction of caged side chains remaining after photolysis for a time t (20). The present results are consistent with the previous estimate that k = ~0.7/s for Tyr(ONB) (20, 29). However, this contrasts with the situation with caged cysteine, in which each 3-s light pulse produced much less fractional decaging (Fig. 2), and we estimate that k = ~0.15/s for Cys(ONB). Because there were partial responses to ACh before photolysis in the case of both Cys(ONB) and Tyr(ONB), the most accurate calculations of k would require knowledge of the complete dose-response relations both before and after decaging. We did not systematically measure the dose-response relations before decaging.

Block of the channel by ACh. After decaging of tyrosine at the 9' position of M2 of the gamma -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 gamma 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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Fig. 4.   "Rebound" current upon removal of ACh. ACh receptors with Tyr(ONB) incorporated at the gamma 9' position were expressed in oocytes. Current was induced by the application of 100 µM ACh, and current was substantially increased by pulse (3 s) decaging. ACh was removed, and the effects of different concentrations of ACh were tested. At higher concentrations of ACh, removal of the agonist induced an increase in current.

ACh is positively charged, and blockage of the pore of the nAChR by ACh is expected to change with transmembrane voltage if the binding site for ACh in the pore is within the membrane field. As the membrane potential becomes more negative, ACh is attracted into the pore, and blocking efficacy increases (1, 17). Figure 5 demonstrates the protocol that we used to test this hypothesis. Voltage was jumped from a holding potential of -80 mV to test potentials ranging from -120 to +60 mV for 250 ms. This procedure was carried out at ACh concentrations of 10, 100, 400, and 2,000 µM. ACh-induced currents were isolated by subtracting the resistive and capacitive currents measured before ACh application. Figure 5A shows representative I-V relations for the wild-type nAChR at an ACh concentration of 2,000 µM. The I-V relation is roughly linear. The mutant channel, after decaging of tyrosine, shows a markedly different response (Fig. 5B). The current shows marked outward rectification. The results of a series of such experiments are shown in Fig. 5, C and D. The I-V relationships of the wild-type channel are linear at all ACh concentrations (Fig. 5C). The I-V relationships of the gamma 9'Y mutant, in contrast, are linear at 10 and 100 µM ACh, show slight outward rectification at 400 µM ACh, and are markedly outwardly rectifying at 2,000 µM ACh (Fig. 5D). At 2,000 µM ACh, the current of the wild-type channel more than doubles upon increasing membrane potential from -60 to -120 mV, whereas the mutant channel barely shows any change in current in this voltage range. Also noticeable for the mutant channel is the decrease in current as ACh concentration increases, consistent with a pore-blocking effect of the agonist.


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Fig. 5.   Current-voltage (I-V) relationships of ACh receptors. Shown are results for the wild-type receptor (A) and for a mutant receptor with decaged Tyr at the 9' position of the gamma -subunit (B). Voltage was held at -80 mV and jumped from -120 to +60 mV in 10-mV steps of 250 ms duration. Currents before the application of ACh (2,000 µM) have been subtracted. C and D: summary of I-V relationships. Averaged results from experiments (n = 3) at different ACh concentrations. Data for wild-type (C) and mutant (D; decaged Tyr at the 9' position of the gamma -subunit) channels are shown. ACh concentrations were 10 (), 100 (), 400 (black-triangle), and 2,000 (black-down-triangle ) µM.

To validate the fidelity of the nonsense suppression and decaging protocols, we also incorporated tyrosine directly at the gamma 9' position by use of nonsense expression. As expected, a rebound effect was observed upon ACh removal, just as is seen when a tyrosine is inserted by first incorporating Tyr(ONB) and then photolyzing (Fig. 6B).


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Fig. 6.   Rebound current in mutant ACh receptors. Shown are results with 4-Me-Phe (A), Tyr (B), and Ser (C). 4-Me-Phe and Tyr were incorporated at the 9' position of the gamma -subunit in place of Leu by the nonsense suppression technique. Ser was incorporated at the 262 position (9' position) of the beta -subunit in place of Leu by conventional mutagenesis. The ACh concentration was 200 µM in all cases.

Aromatic residues can contribute significantly to the binding cations through a cation-pi interaction (5); indeed, binding sites for ACh are known to involve cation-pi interactions (28, 34). The increased affinity of ACh for a binding site within the pore of the channel after the decaging of an aromatic residue, tyrosine, suggested that we could be creating a pore-binding site for ACh involving a cation-pi interaction. To test this hypothesis, we utilized nonsense suppression to incorporate alternative residues at the 9' position of the gamma -subunit. First, as shown in Fig. 6A, we introduced a 4-methylphenylalanine (4-Me-Phe; Fig. 1) residue (13, 23). In multiple attempts, we were unable to evoke a rebound effect with 4-Me-Phe under conditions that induced a rebound when a tyrosine residue was present. We also incorporated both tryptophan and 4,5,6,7-tetrafluorotryptophan (F4-Trp; Fig. 1) at the gamma 9' position. Upon removal of 200 µM ACh, a substantial rebound was seen in both cases (n = 2; not shown).

As mentioned above, the EC50 for ACh was significantly diminished when tyrosine was decaged at the 9' position compared with the wild-type leucine. We tested whether the rebound effect seen with Tyr could somehow be related to the decreased EC50. We evaluated another mutant known to have a large effect on EC50. We have previously shown that serine at the 9' position of the beta -subunit significantly lowers EC50 (EC50 = ~0.5 µM) for ACh (16). However, no rebound effect was seen when ACh was removed from the beta L9'S mutant (Fig. 6C). Thus the rebound effect seen with a tyrosine residue at the 9' position of the gamma -subunit is unlikely to be an artifact due to the decreased EC50.

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 gamma 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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Fig. 7.   Millisecond decaging of Tyr(ONB). ACh (100 µM) was used to activate a mutant ACh receptor with Tyr(ONB) incorporated at the gamma 9' position. After ACh application, Tyr was decaged by a series of 24, 1-ms flashes at 12-s intervals. For clarity, only the first light flash is marked with an arrow. The small increase in current upon the removal of ACh is a rebound effect and was not due to a light flash.

Figure 8A shows details from a separate experiment of two flash-induced increases in current at higher time resolution. The first flash induced an increase of current of ~0.4 µA. The increase in current occurred in two phases. An initial phase, constituting about 20% of the signal, had a time constant of <5 ms and was not resolved by the voltage-clamp circuit. The larger component of the flash-induced current was well fit to a single exponential with a time constant tau  ranging from 0.3 to 1.2 s. In the example shown, current increased after the first two flashes, with tau  values of 1.0 s. No significant differences between the relaxation times induced by the first flash and by subsequent flashes were ever seen. In a series of experiments, the current induced by decaging of tyrosine at the gamma 9' position of the nACh receptor had a time constant tau  = 0.52 ± 0.09 s. The time constant was independent of the ACh concentration. Membrane potential had a modest effect on the time constant. At -80 mV, the ACh-induced current was inward; tau  was 0.40 ± 0.02 s, whereas at +60 mV, outward current relaxations displayed tau  = 0.28 ± 0.05 s (n = 4; P < 0.05).


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Fig. 8.   A: kinetics of current increases induced by flash decaging of Tyr(ONB). ACh (3.3 µM) was applied to an oocyte expressing the ACh receptor with caged Tyr incorporated at the gamma 9' position. Shown are the responses to the first 2 brief flashes of light in an experiment similar to that shown in Fig. 7. B: lack of effect of flashes on the wild-type ACh receptor. ACh (100 µM) was applied to an oocyte expressing the wild-type ACh receptor. ACh induced an inward current of ~6 µA. When current peaked, 2 UV light flashes were applied as indicated.

Figure 8B demonstrates the responses of an oocyte expressing the wild-type nAChR to UV flashes. ACh was applied to the oocyte, and when the maximal inward current occurred, the oocyte was exposed to two pulses of UV light. There was a brief spike artifact, presumably caused by local heating by photoelectric effects at the Ag electrodes, or by electromagnetic coupling of the 12-kV trigger pulse at the flashlamp. However, no sustained changes in current were ever observed. This experiment rules out the possibility that either phase of the relaxations is caused by artifacts.

Figure 9 shows the response of an oocyte expressing caged cysteine at the gamma 9' position of the nAChR to ~1-ms flashes. As in the experiments using 3-s exposures to UV light (Fig. 2), Cys(ONB) was not decaged so efficiently as Tyr(ONB). Nevertheless, flash-induced current increases were readily discernible. In this experiment, 10 flashes of 1-ms duration increased current by a total of ~0.5 µA. Reapplication of ACh (50 µM) induced a larger current than the initial application of ACh. The responses to the first two flashes are shown with an expanded time scale in Fig. 9B. In sharp contrast to the slow increase of current observed when tyrosine was decaged at this position (Fig. 9), there was typically a rapid increase in current (tau  < 10 ms) upon decaging of cysteine. In some cases, as seen here, there was a brief, modest overshoot in the current response.


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Fig. 9.   Rapid decaging of Cys(ONB). ACh (50 µM) was used to activate a mutant ACh receptor with Cys(ONB) incorporated at the gamma 9' position. After ACh application, Cys was decaged by a series of 10, 1-ms flashes at 5- to 8-s intervals. For clarity, only the first light flash is marked with an arrow.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma -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 alpha - (2) and beta -subunits (32) with a cysteine residue. In these cases, MTSEA inhibition approached 70%, a value similar to that obtained here using the gamma -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 gamma 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 gamma 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 gamma 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 gamma 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 gamma -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-pi interaction has been introduced. In cation-pi interactions, cations are attracted to the negative electrostatic potential associated with the quadrupole moment of an aromatic residue. Cation-pi 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-pi 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-pi 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-pi 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-pi 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 gamma 9'. Trp is a potent cation-pi 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-pi interaction. Other possibilities include polar interactions with the hydroxyl group and nonpolar interactions with the aromatic ring.

Direct introduction of tyrosine at the gamma 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 beta -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 gamma -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 (tau  ~ 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 gamma -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.


    ACKNOWLEDGEMENTS

We thank Dr. B. Khakh for helpful discussions.


    FOOTNOTES

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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