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Previous Article | Next Article
The Journal of Neuroscience, March 1, 2002, 22(5):1629-1639
Evidence for a Centrally Located Gate in the Pore of a Serotonin-Gated Ion Channel
Sandip Panicker2, *, Hans Cruz1, *, Christine Arrabit1, and Paul A. Slesinger1, 2 1 The Salk Institute for Biological Studies, La Jolla, California 92037, and 2 Neurosciences Graduate Program, University of California, San Diego, La Jolla, California 92093
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Serotonin-gated ion channels (5-HT3) are members of the ligand-gated channel family, which includes channels that are opened directly by the neurotransmitter acetylcholine, GABA, glycine, or glutamate. Although there is general agreement that the second transmembrane domain (M2) lines the pore, the position of the gate in the M2 is less certain. Here, we used substituted cysteine accessibility method (SCAM) to provide new evidence for a centrally located gate that moves during channel activation. In the closed state, three cysteine substitutions, located on the extracellular side of M2, were modified by methanethiosulfonate (MTS) reagents. In contrast, 13 cysteine substitutions were modified in the open state with MTS reagents. The pattern of inhibition (every three to four substitutions) was consistent with an
helical structure for the middle and cytoplasmic segments of the M2 transmembrane domain. Unexpectedly, open-state modification of two amino acids in the center of M2 with three different MTS reagents prevented channels from fully closing in the absence of neurotransmitter. Our results are consistent with a model in which the central region of the M2 transmembrane domain is inaccessible in the closed state and moves during channel activation.
Key words: 5-HT3R; substituted cysteine accessibility method; serotonin; gating; ligand-gated ion channel; ion channel structure; ionotropic receptor
INTRODUCTION
5-HT3 channels are nonselective, cationic ion channels that are activated directly by serotonin. They are expressed in the enteric nervous system, the spinal cord, and various regions of the brain, including the hippocampus, striatum, neocortex, amygdala, and raphe nucleus (Tecott et al., 1993
). Activation of 5-HT3 receptors leads to membrane depolarization and Ca2+ influx (MacDermott et al., 1999
5-HT3 channels are members of a superfamily of ligand-gated ion channels in which the extracellular N-terminal domain encodes the entire ligand-binding site. The second putative transmembrane domain, M2, lines part of the channel pore and is involved in determining ion selectivity (see Fig. 1) (Corringer et al., 2000
The location of the gate in the nicotinic acetylcholine-gated receptor (nAChR) has been equivocal. In one set of studies, the location of the gate was inferred using the substituted cysteine accessibility method (SCAM) (Akabas et al., 1994
2'G and 2'T [this nomenclature allows the comparison of homologous amino acids in the M2 transmembrane domain from different types of neurotransmitter-gated ion channels (refer to Fig. 1A)]. Mutagenesis studies of a conserved leucine (9'L), on the other hand, implicated the middle of the M2 transmembrane domain in the gating of nAChR. Mutating the 9'L dramatically changed the EC50 for activation (Filatov and White, 1995
MATERIALS AND METHODS
Molecular biology. Mouse 5-HT3a cDNA (M74425; kindly provided by Dr. Julius, UCSF, San Francisco, CA) was subcloned into pGEMHE for high expression in Xenopus oocytes (Maricq et al., 1991
A304). Cysteine mutants were created by overlap PCR or whole-plasmid PCR. The numbering nomenclature for the mutants refers to the amino acid number in the corrected full-length sequence of 5-HT3a. All PCR-generated products were subjected to DNA sequencing (Salk Sequencing Facility) for potential errors generated by Taq polymerase. In vitro methyl-capped cRNA was made from linearized cDNA and T7 RNA polymerase (Stratagene, La Jolla, CA). The concentration and quality of cRNA were assessed using an ethidium-stained formaldehyde gel and RNA molecular weight marker. Xenopus oocytes were isolated as described previously (Slesinger et al., 1996
Electrophysiology. Macroscopic currents were recorded from oocytes with a two-electrode voltage-clamp amplifier (Geneclamp 500; Axon Instruments, Union City, CA), filtered at 0.05-2 kHz, digitized (0.1-2 kHz) with a Digidata 1200 analog-to-digital interface (Axon Instruments), and stored on a laboratory computer. Electrodes were filled with 3 M KCl and had resistances of 0.4-2 M
. Oocytes were perfused continuously with a solution containing 86 mM NaCl (Sigma-Aldrich, St. Louis, MO), 2 mM KCl, 4.7 mM MgCl2 (free Mg2+ of 3.3 mM), 0.5 mM EGTA, and 5 mM HEPES, pH 7.5 with ~5 mM NaOH. For experiments with zero divalents, MgCl2 was omitted. Programmable pinch valves (Warner Instruments, Hamden, CT) were used to change the extracellular solution flowing through a small chamber (0.3175 × 1.524 cm). The extracellular solution was connected to ground via a 3 M KCl agarose bridge. Because of instability of MTS (Toronto Research Chemicals, North York, Ontario, Canada), MTSET [(2-(trimethylammonium) ethyl)-methanethiosulfonate], MTSEA (2-aminoethyl-methanethiosulfonate-bromide), and MTSES [sodium (2-sulfonatoethyl)-methanethiosulfonate] stocks were made on the day of the experiment, stored on ice, and diluted immediately before perfusion. For experiments with DTT, 50 mM DTT was prepared in 0.5× of the extracellular solution to minimize osmotic shock. MDL-72222 (Tocris, Ballwin, MO) stock of 10 mM was diluted (1 µM) just before use. For MDL-72222 experiments, we compared the change in 5-HT-induced current after exposure to MDL-72222 (control) with that of MDL-72222 plus MTS, after 10 min of wash to allow for sufficient removal of MDL-72222.
The majority of cysteine-substituted mutants expressed in oocytes yielded small (<300 nA) agonist-independent currents and 5-HT-induced currents between 0.5 and 15 µA. L286C, G288C, Y289C, and F292C were coinjected with wild-type 5-HT3a cRNA (e.g., F292Cwt) in ~1:1 ratio because injection of the mutant cRNA alone resulted in little or no 5-HT-induced currents. Injection of the cRNA for I295C sometimes gave rise to larger agonist-independent currents and/or unstable agonist-independent currents. Only recordings that were stable and displayed reproducible 5-HT-induced currents were included in the analyses.
Analysis. The dose-response curve for 5-HT was determined by recording the current (voltage clamped at
h), where the EC50 is the concentration at which there is half-activation, and h is the Hill coefficient. The deactivation time (T50) was determined by measuring the time taken for the current to decrease to 50% of its level before removing the 5-HT.
The irreversible effect of MTS on 5-HT-induced currents was examined using one of two protocols. Protocol 1 consisted of two 5-10 sec pulses of 10 µM 5-HT (I1, I2), 2-3 min of wash, 1 min of MTS (1 mM) reagent in the absence or presence of 10 µM 5-HT, 3.5 min of wash, and two 5-10 sec pulses of 5-HT (I3, I4). The percentage inhibition-potentiation was calculated using the equation (I4/I2
t/
for potentiation or y = y0 + a(1
* [MTS]). The change in agonist-independent current was quantified by taking the difference between the basal current just before I3 (after the 3.5 min wash) and the initial basal current (after I2) and then dividing by I3 (this represents the total amount of current that can pass through modified channels in the open state).
All values were reported as mean ± SEM. Data were analyzed for statistical significance (SigmaStat 2.0; SPSS, Chicago, IL) using one-way ANOVA, followed by Bonferroni's post hoc test with wild-type as control or using a paired t test for Mg2+-dependent inhibition. Values of p < 0.05 were considered significant.
RESULTS
Effect of cysteine substitutions on 5-HT3a
Neurotransmitter stimulation of ligand-gated channels produces a mixed population of closed, open, and desensitized states. For example, in the presence of a saturating dose of 5-HT (10 µM), 5-HT3a channels opened rapidly and then quickly entered a nonconducting, desensitized state (Fig. 1B). Wilson et al. (2001)
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Figure 1. Properties of wild-type and cysteine-substituted 5-HT3a channels expressed in Xenopus oocytes. A, Alignment of the M2 transmembrane domain sequences from mouse 5HT3a (M74425), mouse 5-HT3b (AF155045), mouse nAChR
To localize the position of the gate in 5-HT3a, we examined the accessibility of cysteine substitutions in the M2 domain of open and closed mutant channels. If treatment with MTS leads to an irreversible reduction in the amplitude of agonist-induced current, then the cysteine side chain is likely positioned in or near the pore of the channel. If a cysteine-substituted channel is modified equally well in the open and closed states with extracellularly applied MTS reagents, then the channel gate is located on the cytoplasmic side of the engineered cysteine.
We first examined the effect of 1 mM MTSET (extracellular) on the 5-HT-induced current through wild-type channels (Fig. 1C). Wild-type 5-HT3a channels exposed to MTSET in either the closed or open state showed no decrement in 5-HT-induced current. We therefore used the wild-type 5-HT3a channel for the introduction of new cysteine substitutions and assumed that the eight native cysteines remained silent. Twenty-three amino acids in the M2 transmembrane domain (D274-V296) were systematically replaced with a cysteine (one at a time) (Fig. 1A). Nineteen 5-HT3a cysteine mutants gave rise to measurable 5-HT-induced currents after injection of the cRNA into Xenopus oocytes. In contrast to our results with S290C, Reeves et al. (2001)
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Table 1. Properties of cysteine-substituted 5-HT3a channels To assess whether the cysteine mutation grossly altered the channel structure, we compared the dose-response curves (EC50) for serotonin activation of the cysteine-substituted mutants that formed homomultimers. The EC50 changed less than twofold for 13 cysteine substitutions and twofold to eightfold for the remaining six cysteine substitutions. The largest shifts in EC50 occurred with cysteine substitutions near the extracellular half of the M2 transmembrane domain. These changes in EC50 are relatively small considering that each homopentamer contained five cysteine mutations. For comparison, some single cysteine mutations in two of five nAChR subunits produced 50-fold changes in the EC50 (Akabas et al., 1994
State-dependent MTS modification of 5-HT3a mutants
We first examined the MTS sensitivity of all 5-HT3a cysteine mutant channels with MTSET in either the absence (closed state) or presence (open state) of 10 µM 5-HT (this dose was 4.5-45 times the EC50). We chose MTSET because it has a permanent positive charge and is not membrane permeant (Holmgren et al., 1996
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Figure 2. Reactivity of cysteine-substituted 5-HT3a channels in open and closed states with MTSET. A, Continuous current recorded from oocyte injected with cRNA for wild-type, I295C, S290C, S280C, or R278C. Solid bar indicates 10 µM 5-HT, hatched bar indicates 1 mM MTSET, and + and
The accessibility of cysteine mutants in the open state, on the other hand, was strikingly different from that of closed channels. MTSET irreversibly reduced the 5-HT-induced current by 40-80% of control for six cysteine 5-HT3a mutants and potentiated the 5-HT-induced current for four mutants (Fig. 2A,B). Although G276C and V296C fell out of statistical significance using one-way ANOVA, they both appeared to be inhibited after MTSET treatment in the open state. Our results with MTSET are similar but not identical to those of Reeves et al. (2001)
To test this idea, we studied the effect of MTSEA, which is smaller than MTSET [3.6 vs 5.8 Å for the head group (see Fig. 5)], on cysteine substitutions L286C through V296C. In the closed state, V296C and L293C were both modified by 1 mM MTSEA (Fig. 3A,C). Interestingly, MTSEA treatment increased the 5-HT-induced current by 350% for L293C in the closed state, whereas MTSET had little effect (Fig. 3A,C). Although L287C exhibited some potentiation of 5-HT-induced current with MTSEA treatment in the closed state, this increase was not statistically significant. In the open state, MTSEA treatment modified six cysteine substitutions [G288Cwt, S290C, V291C, F292Cwt, L293C, and V296C (Fig. 3C)]. Of these, F292Cwt and L293C were not significantly modified with MTSET, suggesting that they lie within a region of the channel that is too small for MTSET to access but large enough for MTSEA to enter. Additionally, MTSET modification of residues S290C and V291C inhibited 5-HT-induced current, whereas modification with MTSEA did not. One possibility is that the pore is relatively wide at this level in the channel, and attachment of a smaller MTS moiety, such as MTSEA, may have negligible effects on current flow. Combining the results from experiments with MTSEA and MTSET, we conclude that amino acids L293 through V296 are accessible in the closed and open state, suggesting that the gate is on the cytoplasmic side of L293.
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Figure 3. Reactivity of cysteine-substituted 5-HT3a channels in open and closed states with MTSEA. A, B, Continuous current recorded from oocyte injected with the cRNA for L293C or G288Cwt. The 5-HT-induced current through L293C was potentiated with MTSEA (1 mM) in the closed and open states. G288Cwt was modified only in the open state. C, Summary of the percentage of inhibition or potentiation of current for mutants L286Cwt through V296C after 1 min exposure to MTSEA (1 mM) in the closed (open bars) or open (solid bars) states (n = 4-10). V296C and L293C were modified in both the closed and open states. The inhibition or potentiation for S290C, V291C, I294C, I295C, and V296C were adjusted as described in Figure 2. *p < 0.05 indicates statistical difference from wild-type channels using one-way ANOVA.
To quantitate the difference in accessibility of cysteines in the open and closed states, we measured the bimolecular rate constant for MTS modification (Fig. 4). We used a protocol in which the MTS reagent was applied repeatedly in the absence or presence of 5-HT for 5-10 sec (see legend of Fig. 4). The rates of closed-state modification were not measured for E277C, R278C, S280C, T284C, G288Cwt, F292Cwt, and I294C because 1 mM MTS treatment did not significantly affect the 5-HT-induced current (Fig. 2B). The rate of modification for S290C, V291C, L293C, I295C, and V296C in the closed state was <50 M/sec. However, the rates for S290C, V291C, I295C, and V296C are overestimates because these mutants were directly opened by MTSET (Figs. 1D, 4C, a) (MDL-72222 washed out too slowly for using in rate measurements). Because there was no significant modification of S290C and V291C when MTS was coapplied with MDL-72222 (Fig. 2B), the actual rate of modification in the closed state was likely <10 M/sec (Fig. 4C, dotted lines). In contrast to the closed state, the rate of modification for open channels varied from ~500 to ~5000 M/sec, with a majority of the faster rates occurring on the cytoplasmic side of M2 transmembrane domain. For comparison, these rate constants are comparable with those reported for modification of nAChR channels (Pascual and Karlin, 1998
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Figure 4. Rate constants for gated access to cysteine substitutions in the M2 transmembrane domain of 5-HT3a. A-C, Determination of the rate constant for modification with MTSET or MTSEA. A, Continuous current recorded from oocyte injected with the cRNA for E277C. Alternating pulses of 5-HT alone and 5-HT with MTSET (0.1 mM) were delivered. B, The amplitude of 5-HT-induced current was plotted as a function of cumulative exposure time to MTSET and fit with a single exponential having a time constant of 3.8 sec. C, Average rate constants for modification with MTSET (white circles, black circles) or MTSEA (circles with horizontal stripes, circles with diagonal stripes) in the closed and open states, respectively (n = 4-6). Circles with crosses indicate that closed-channel modification was not statistically different from wild type and was likely 10 M/sec (see Fig. 2B). Note that closed rates are overestimates for those mutants in which MTSET opened the channel directly, denoted by a. Dashed line for S290C and V291C indicates there was no detectable effect of MTSET or MTSEA when coapplied with MDL-72222.
We next searched for patterns of inhibition or potentiation with MTS reagents in the open state. In an
). Interestingly, those cysteine substitutions that were potentiated by MTS cluster on the opposite side of the helix (see Fig. 7A,
). If we define pore-facing amino acids as those that were irreversibly inhibited with MTS reagents, then the M2 transmembrane domain appears to be
Could the introduction of the cysteine in the mutant increase the MTS reactivity of one or more of the native cysteines? To test this idea, the S280C mutation was generated in the background of a 5HT3a subunit in which two native cysteines positioned close to the M2 transmembrane domain, C270 (in M1) and C316 (in M3), were mutated to alanine. In this triple mutant, MTSET irreversibly reduced the 5-HT-induced current by ~70%, similar to the reduction observed with S280C alone. Removing all of the native cysteines in 5-HT3a is impractical, because a canonical pair of cysteines is located in the ligand-binding domain of most ligand-gated channels and is essential for function (Corringer et al., 2000
MTS-dependent potentiation of 5-HT-induced current
The potentiation of 5-HT-induced current with MTSET or MTSEA for six cysteine substitutions (S275C, R278C, G288Cwt, F292Cwt, L293C, and I295C) was unexpected. We anticipated that covalent attachment of a positively charged moiety to a cysteine in the channel would reduce the 5-HT-induced current, because 5-HT3a is a cationic channel. One possible explanation for potentiation of current is that the MTS treatment shifted the EC50 to lower concentrations of 5-HT. This possibility was unlikely because a maximal dose of 5-HT (10 µM) was used to activate wild-type and mutant channels. Alternatively, MTSET modification might have altered divalent inhibition; 5-HT3 channels are inhibited by extracellular divalent cations (Lovinger, 1991
A change in Mg2+-dependent inhibition, on the other hand, did not appear to explain the potentiation of 5-HT-induced current for G288Cwt, F292Cwt, L293C, and I295C mutants (Table 1). An alternative explanation is that MTS modification increased the single-channel conductance or open-channel probability, which would produce a potentiation of 5-HT-induced current. The small conductance of wild-type 5-HT3a channels (<1 pS), however, precluded measuring the mean open-channel lifetime or conductance directly (Hussy et al., 1994
Middle of M2 transmembrane domain implicated in conformational change
Surprisingly, MTSET treatment of four cysteine mutants (L287C, S290C, V291C, and I295C) in the open state increased the basal current, which persisted in the absence of 5-HT ("agonist-independent current") (Figs. 2A, 5). The amplitude of this agonist-independent current increased to 15-50% of the modified 5-HT-induced current, which reflects the total amount of current that can flow through the modified channels (Fig. 6). For comparison, MTS modification of a cysteine-substituted voltage-gated K+ channel also produced constitutively open channels (Liu et al., 1997
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Figure 5. MTS modification in the open state produces agonist-independent currents for cysteine substitutions in the middle of the M2 transmembrane domain. Continuous current recorded from oocyte injected with the cRNA for V291C (A), S290C (B), or L287C (C). One minute exposure to MTS reagent (1 mM) in the presence of 5-HT (10 µM) produced an agonist-independent current for L287C, S290C, V291C, and I295C (data not shown) with MTSET and for S290C and V291C with MTSEA and MTSES. The structure of MTS moiety that would covalently attach to the cysteine sulfhydryl is shown above. D, Continuous current recorded from oocyte injected with cRNA for V291C. In the absence of 5-HT, 50 mM DTT decreased the agonist-independent current to the level before MTSET modification. Note that 5-HT (10 µM) fully activated the V291C channel after DTT treatment.
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Figure 6. Summary of the change in agonist-independent current produced by MTSET, MTSEA, and MTSES. Bar graphs show the average amplitude of agonist-independent current after MTS treatment, expressed as a percentage of the 5-HT-induced current after MTS modification (see Materials and Methods) (n = 3-13). $ indicates that the pre-modified 5-HT-induced current was used to calculate the percentage change in agonist-independent current because MTSEA treatment eliminated >95% of the 5-HT-induced current. * indicates statistical difference from wild type using one-way ANOVA. nt, Not tested.
One explanation for the agonist-independent current is that modified channels become locked open after removal of 5-HT (see Fig. 8). To test this idea, we examined the effect of DTT (50 mM) in the absence of 5-HT. If MTSET-modified channels were locked open, then DTT would be expected to have access to the pore and, during reduction of the newly formed disulfide bond, enable the mutant channel to close normally. Indeed, the agonist-independent current declined to within ~5% of the premodified level when V291C was exposed to DTT in the absence of 5-HT (Fig. 5E). DTT treatment also restored the agonist-independent current to within 5% of the premodified level for L287C, S290C, and I295C channels modified with MTSET. In addition to the recovery of agonist-independent current, the amplitude of 5-HT-induced current returned to 80-130% of the original level after DTT treatment.
DISCUSSION
Neurotransmitter-gated ion channels are important for mediating the response of "fast" neurotransmitters and have evolved a transduction mechanism that tightly couples the binding of neurotransmitter to a conformational change in the channel pore. Our analysis of 5-HT3a channels has revealed a cluster of amino acids in the middle of the M2 transmembrane domain that appears to move during channel activation. In addition, our results suggest that the middle and cytoplasmic segments of the M2 transmembrane domain are inaccessible to MTS reagents in the closed state and, during activation with 5-HT, become readily accessible to MTS reagents. Finally, the pattern of inhibition is consistent with an
SCAM of the M2 in 5-HT3a and nAChR
5-HT3a and nAChR share 29% homology in the M1-M3 transmembrane domains and likely conserve the molecular mechanism underlying channel activation. Consistent with this idea, replacing the N-terminal domain of 5-HT3a with the homologous sequence from
The number of cysteine substitutions that were modified in the open state with MTS reagents for both 5-HT3a and nAChR channels is remarkably large. In our study, seven cysteine substitutions modified in 5-HT3a overlap with those modified in cysteine-substituted muscle nAChR channels (Akabas et al., 1994
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Figure 7. A, Helical wheel representation of the MTS-dependent changes in current. Note the majority of cysteine substitutions that are inhibited with MTS reagents (bold and italicized,
In contrast to the open state, the accessibility of cysteine substitutions in the closed state appeared to be very different between nAChR and 5-HT3a. With cysteine substitutions S275C through F292Cwt, MTS exposure in the closed state produced no irreversible change in 5-HT-induced current. In contrast, MTS treatment of six cysteine-substituted nAChR channels in the closed state inhibited ~40-70% of the acetylcholine-induced current (Akabas et al., 1994
Working model for the gate in 5-HT3a channel
We define the "gate" as the region of the channel that prevents the inappropriate flow of ions through the channel pore. The activator (i.e., voltage, neurotransmitter, and pH) leads to a conformational change in the protein that moves the gate and allows ions to permeate the open pore. The gate therefore serves two functions: a barrier to ion permeation and a link to the transduction machinery.
There are two important findings with SCAM of 5-HT3a that help pinpoint the location of the gate. First, the middle and cytoplasmic segments of the M2 transmembrane domain (S275 through F292) were accessible in open but not closed states. In contrast, three amino acids on the extracellular side of F292 (L293C, I295C, and V296C) were accessible in both open and closed states. Surprisingly, I294C was only modified in the open state, whereas the more cytoplasmic L293C was modified in both open and closed states. Similarly, residues above the putative gate were also found to exhibit state-dependent modification in the nAChR (Akabas et al., 1994
The second finding localizing the gate, however, is that exposure of two cysteine mutants in the middle of M2, S290C and V291C, to all three MTS reagents generated agonist-independent currents. Notably, this change in current occurred only when MTS was applied in the open state. Similarly, Reeves et al. (2001)
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Figure 8. Two working models that explain the agonist-independent current. In both models, 5-HT produces a rotation of M2 transmembrane domain, thereby exposing an ionized sulfhydryl on the cysteine (black arrow), such as at position V291 (only two cysteines are shown for clarity). MTS (black sphere) covalently attaches to the sulfhydryl in the open state (open). A, After removal of 5-HT, the channel fails to close completely in Model 1 because of steric hindrance (locked open-MTS). B, In Model 2, modified channel closes fully in the absence of 5-HT. This closed-MTS state is unstable, however, allowing the channel to spontaneously open. In both models, DTT can reduce the disulfide to restore the channel to its original unmodified closed state.
Together, our data are consistent with a model in which the extracellular barrier to ion permeation exists in the middle of the M2 domain of 5-HT3a (Fig. 7B). During ligand binding, the M2 helix likely rotates (Figs. 7, 8), as has been proposed for the nAChR (Unwin, 1995
Studies on other ligand-gated channels also support a model for a centrally located gate that includes amino acids at the 12' and 13' positions, which are homologous to S290 and V291. First, amino acids in the 12' and 13' positions (and 9') are extremely well conserved (85%-100%) in cationic ligand-gated channels (Le Novere and Changeux, 1999
Our conclusions concerning the structure of the M2 are inferred after a systematic analysis of cysteine-substituted channels exposed to extracellular MTS reagents in closed and open states. Is there good concordance between the structure of a protein determined by SCAM and that by x-ray crystallography? In the handful of cases in which there is a three-dimensional structure, there appears to be good agreement. For example, the narrowest region of the
FOOTNOTES Received Oct. 29, 2001; revised Dec. 10, 2001; accepted Dec. 18, 2001.
* S.P. and H.C. contributed equally to this work.
This work was supported in part by the Sloan Foundation, the McKnight Endowment for Neuroscience, and the Fritz-Burns Foundation. S.P. is a Merck Fellow in the University of California, San Diego Neurosciences Graduate Program. P.A.S. is a McKnight Scholar and an Alfred P. Sloan Research Fellow. We thank D. Julius for providing the 5-HT3a cDNA, Drs. D. Berg, S. Heinemann, N. Unwin, and members of the Slesinger lab for providing discussion and comments on this and previous versions of this manuscript.
Correspondence should be addressed to Paul A. Slesinger, Peptide Biology Laboratory, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, CA 92037. E-mail: slesinger{at}salk.edu.
H. Cruz's present address: University of Geneva, Department of Biochemistry, 30, Quai Ernest-Ansermet, 1211 Geneva 4, Switzerland.
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