From the Department of Neurobiology, Harvard Medical
School, Boston, Massachusetts 02115 and ¶ Biomembrane Lab,
Department of Chemistry, Indian Institute of Technology Bombay,
Powai, Bombay 400076, India
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ABSTRACT |
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The uncharged photoactivable probe
2-[3H]diazofluorene ([3H]DAF) was
used to examine structural changes in the Torpedo
californica nicotinic acetylcholine receptor (AChR) ion channel
induced by agonists. Photoincorporation of [3H]DAF into
the AChR consisted of the following two components: a nonspecific
component consistent with incorporation into residues situated at the
lipid-protein interface, and a specific component, inhibitable by
noncompetitive antagonists and localized to the M2 hydrophobic segments
of AChR subunits. The nonspecific [3H]DAF incorporation
was characterized in the M4 segment of each AChR subunit. The observed
distribution and periodicity of labeled residues reinforce the
conclusion that the M4 segments are organized as transmembrane
-helices with a common "face" of each helix in contact with
lipid. Within the M2 segments, in the absence of agonist
[3H]DAF specifically labeled homologous residues
Val-261 and
Val-269, with incorporation into
Val-269 at a
5-fold greater efficiency than into
Val-261. This observation,
coupled with the lack of detectable incorporation into
-M2 including
the homologous
Val-255, indicates that within the resting channel
[3H]DAF is bound with its photoreactive diazo group
oriented toward
Val-269. In the presence of agonist, there is an
~90% reduction in the labeling of
Val-261 and
Val-269
accompanied by specific incorporation into residues (
Leu-257,
Ala-258,
Ser-262, and
Leu-265) situated 1 or 2 turns of an
-helix closer to the cytoplasmic end of the M2 segments. The results
provide a further characterization of agonist-induced rearrangements of
the M2 (ion channel) domain of the AChR.
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INTRODUCTION |
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The nicotinic acetylcholine receptor
(AChR)1 isolated from the
electric organ of the marine elasmobranch Torpedo
californica is the best characterized member of a family of
ligand-gated ion channels which includes the -aminobutyric acid,
glycine, and serotonin 5-HT3 receptors (for recent reviews, see
Refs. 1-3). The AChR is composed of four homologous subunits
(
2
) arranged quasi-symmetrically around a
central cation-selective ion channel (4). The subunits each have a
characteristic topology as follows: a large hydrophilic N-terminal
domain containing the agonist binding sites, followed in the primary
structure by three hydrophobic membrane-spanning segments (M1-M3), a
cytoplasmic domain, a fourth hydrophobic transmembrane segment (M4),
and a short extracellular C-terminal tail.
Noncompetitive antagonists (NCAs) are agents that block the AChR
permeability response without binding to the agonist site. These
compounds are structurally diverse and include many aromatic amines but
also general anesthetics, steroids, and even the neuropeptide substance
P (reviewed in Ref. 5). A number of NCAs have been instrumental in
identifying regions of the AChR which contribute to the formation of
the pore of the ion channel. Photoaffinity labeling studies with
[3H]chlorpromazine (6-9) and
[3H]triphenylmethylphosphonium (10) as well as reaction
with [3H]meproadifen mustard (11) have all identified
labeled residues within the M2 segments that would all lie on a common
side of an -helix. These results, in conjunction with the observed
functional properties of AChRs with mutations in the M2 segments (1,
2), provide the basis for a model of the ion channel comprised of M2
segments of each subunit arranged as transmembrane
-helices around
the central axis, a model consistent with studies of AChR three-dimensional structure derived from electron micrographic image
analysis (4, 12).
The sites of [3H]chlorpromazine,
[3H]triphenylmethylphosphonium, and
[3H]meproadifen mustard incorporation were all identified
in the presence of agonist under conditions where the AChR is expected to be in the desensitized state. More recently,
3-(trifluoromethyl)-3-(m-[125I]iodo-phenyl)diazirine
([125I]TID),a small, uncharged photoactivable probe, was
used to identify residues in the channel lining M2 region both in the
absence and presence of agonist, i.e. for AChRs
predominantly in the resting state or desensitized state, respectively
(13). [125I]TID is a potent NCA (14, 15) that reacts
nonspecifically with AChR amino acids in M3 and M4 hydrophobic segments
at the lipid-protein interface (14-17) and specifically with amino
acids in M2 segments of each AChR subunit (13). In the absence of agonist (resting state), [125I]TID labeled Leu-265 and
Val-269 and the homologous residues in the other subunits that are
located 9 and 13 amino acids to the C-terminal side of the conserved
lysine residue at the N terminus of the M2 region (positions 9 and 13).
In the desensitized state, the pattern of
[125I]TID-labeled residues broadened to include
homologous serine residues at positions 2 and 6 (i.e.
Ser-258 and
Ser-262). These results provided the first direct
evidence of an agonist-induced structural rearrangement of the channel
lining M2 helices. They further suggested that in the closed ion
channel the aliphatic residues at positions 9 and 13 form a
permeability barrier to the passage of ions.
To examine further the structure of the channel in different functional
states, we examined the incorporation into the AChR of another
lipophilic photoactivable probe, [3H]diazofluorene
([3H]DAF), both in the absence and presence of agonist.
[3H]DAF has been used to probe the hydrophobic core of
erythrocyte membranes (18) as well as the sites of lipid exposure of
Staphylococcus aureus -toxin (19). Whereas incorporation
of [125I]TID and [3H]DAF both proceed
through a UV-induced reactive carbene, the two compounds are
structurally distinct, and TID produces a singlet carbene (20) whereas
DAF produces a carbene with substantial triplet character (21, 22). We
report here that like [125I]TID, [3H]DAF
not only incorporates into residues situated at the lipid-protein interface but also into residues in the channel lining M2 segments. We
identify the amino acids within the M4 segments that are labeled nonspecifically as well as the pattern of specific photoincorporation within the M2 segments. The subunit selectivity of photolabeling within
the M2 domain in conjunction with the agonist-dependent redistribution of labeling provide further information about the change
in structure of the AChR ion channel domain between resting and
desensitized states.
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EXPERIMENTAL PROCEDURES |
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Materials--
AChR-rich membranes were isolated from T. californica electric organ (17). 2-[3H]Diazofluorene
([3H]DAF) of specific activities ranging from 0.67 to 1.4 Ci/mmol was prepared from 2-[3H]fluorenone according to
the procedure described by Pradhan and Lala (18), repurified, and
stored at 20 °C in ethanol. [3H]Phencyclidine (43 Ci/mmol) was from NEN Life Science Products, and
[3H]tetracaine (47 Ci/mmol) was prepared at NEN Life
Science Products by catalytic tritiation of 3,3-dibromotetracaine.
1-Azidopyrene was purchased from Molecular Probes. Carbamylcholine and
tetracaine were from Sigma, and phencyclidine (PCP) was from Alltech
Associates. L-1-Tosylamido-2-phenylethyl chloromethyl
ketone-treated trypsin was purchased from Worthington and
endoproteinase Lys-C from Boehringer Mannheim. Genapol C-100 (10%) was
purchased from Calbiochem. Prestained low molecular weight gel
standards were purchased from Life Technologies, Inc.
Photolabeling AchR-rich Membranes with [3H]DAF-- For analytical labeling experiments, Torpedo membranes (2 mg/ml) in Torpedo physiological saline (TPS, 250 mM NaCl, 5 mM KCl, 3 mM CaCl2, 2 mM MgCl2, 5 mM sodium phosphate, pH 7.0) were incubated with [3H]DAF at a final concentration of 5 µM in the absence or presence of 100 µM carbamylcholine and in the absence or presence of additional ligands. After a 30-min incubation, suspensions were irradiated for 5 min at a distance of less than 1 cm with a 365-nm lamp (EN-Spectroline). Following irradiation, each sample was pelleted (15,000 × g), the pellet solubilized in sample loading buffer (23), and then submitted to SDS-PAGE. Preparative photolabelings (12-15 mg per condition) were carried out at 10 µM [3H]DAF (±100 µM tetracaine) and in the presence of carbamylcholine (±100 µM phencyclidine). After 1 h incubation, sequential photoincorporation of [3H]DAF and then 1-azidopyrene (1-AP) was carried out as described previously for [125I]TID (17), except that irradiation of suspensions with 1-AP was limited to 5 min. Samples were then pelleted and solubilized in electrophoresis sample loading buffer and submitted to SDS-PAGE.
SDS-Polyacrylamide Gel Electrophoresis--
SDS-PAGE was
performed as described by Laemmli (23) using either 1.0-mm (analytical)
or 1.5-mm (preparative scale) thick 8% polyacrylamide gels with 0.33%
bis(acrylamide). For analytical gels, polypeptides were visualized by
staining with Coomassie Blue R-250 (0.25% w/v in 45% methanol and
10% acetic acid) and destaining in 25% methanol, 10% acetic acid.
The gels were then impregnated with fluor (Amplify, Amersham Pharmacia
Biotech) for 20 min with rapid shaking, dried, and exposed at
80 °C to Kodak X-OMAT LS film for various times (3-12 weeks).
Incorporation of 3H into individual polypeptides was
quantified by scintillation counting of excised gel pieces as described
(24). For preparative scale gels, polypeptides incorporating 1-AP were
visualized from their associated fluorescence when the gels were
illuminated at 365 nm on a UV-light box. Bands corresponding to AChR
subunits were excised, and in some cases the gel pieces were
transferred to the wells of individual 15% mapping gels (25, 26).
Mapping gels were composed of a 4.5% acrylamide stacking gel and a
15% acrylamide separating gel. The gel pieces were overlaid with 350 µl of buffer (5% sucrose, 125 mM Tris-HCl, 0.1% SDS, pH
6.8) containing 250 µg of S. aureus V8 protease (500 µg
V8 protease for gel pieces containing the
-subunit). Electrophoresis
was carried out overnight at 25 mA constant current. In the course of
this work it was determined that for membranes labeled with 1-AP and
[3H]DAF, the fluorescent- and 3H-labeled-AChR
subunits comigrated, as did their large proteolytic fragments.
Purification of Proteolytic Digests of
[3H]DAF/1-AP-labeled AChR Subunits to Isolate Fragments
Containing the M2 Segment--
For trypsin digestion,
acetone-precipitated subunits ( and
) were resuspended in a small
volume (~50 µl) of buffer (100 mM
NH4HCO3, 0.1% SDS, pH 7.8). The SDS
concentration was then reduced by diluting with buffer without SDS, and
Genapol C-100 was added, resulting in final concentrations of 0.02%
SDS, 0.5% Genapol C-100, and 1-2 mg/ml protein. Trypsin was added to
a 1:5 (w/w) enzyme to substrate ratio and incubated at room temperature for 4 days. For endoproteinase Lys-C (EKC) digestion, subunits (
)
were resuspended in 15 mM Tris-HCl, 0.1% SDS, pH 8.1, at
1-2 mg/ml protein. Approximately 1.5 units of EKC was added and
incubated at room temperature for 6 days. Both trypsin and EKC digests
were separated on Tricine/SDS-polyacrylamide gels prepared as described (17, 28). Aliquots of each of the digests (~5%) were routinely resolved on analytical scale Tricine/SDS-polyacrylamide gels (1.0-mm thick) along with prestained molecular weight standards (Life Technologies, Inc.) as follows: ovalbumin (43,000), carbonic anhydrase (29,000),
-lactoglobulin (18, 400), lysozyme (14, 300), bovine trypsin inhibitor (6, 200), the A chain of insulin (3, 400), and the B
chain of insulin (2, 300). Analytical gels were soaked in 25%
methanol, 10% acetic acid for 30 min, and then prepared for
fluorography.
Generation and Isolation of Fragments of AChR Subunits Containing
[3H]DAF/1-AP-labeled M4 Segments--
Fragments
beginning near the N terminus of the M4 segment of each AChR subunit
were isolated as described (17) for
[125I]TID/1-AP-labeled subunits. Briefly, in gel
digestion of each isolated subunit with S. aureus V8
protease was used to generate 10-14-kDa subunit fragments as follows:
V8-10 (
Asn-339 to
Gly-437);
V8-12 (
Met-384/
Ser-417
to
Ala-469);
V8-14 (
Leu-373/
Ile-413 to
Pro-489;
V8-11 (Lys-
436 to
Ala-501). Trypsin digests of these
fragments were fractionated by Tricine/SDS-PAGE yielding fluorescent
and 3H containing bands of 3-4 kDa for
-subunit
(
T-4K), 5 kDa for
- and
-subunits (
T-5K and
T-5K), and 6 kDa for
-subunit (
T-6K). Material eluted from these bands was
further purified by reversed-phase HPLC. With the exception of the
-subunit digest, each digest yielded a single major peak of
3H which coeluted with 1-AP fluorescence, and these peaks
eluted at the same concentrations of organic solvent as had been seen for the [125I]TID/1-AP-labeled M4 segments. Material in
these fractions were pooled, dried, and resuspended for protein
microsequence analysis. The tryptic digest of
T-6K yielded a broader
distribution of 3H without significant fluorescence, and
material was pooled from the concentrations of organic eluent that had
been found to contain [125I]TID/1-AP-labeled
M4.
Sequence Analysis-- N-terminal sequence analysis was performed on an Applied Biosystems model 477A protein sequencer using gas phase cycles. Pooled HPLC samples were dried by vacuum centrifugation, resuspended in a small volume of 0.05% SDS (~20 µl), and immobilized on chemically modified glass fiber disks (Beckman Instruments). Approximately 30% of the released PTH-derivatives were separated by an on-line Model 120A PTH-derivative analyzer, and approximately 60% was collected for determination of released 3H by scintillation counting of each sample for three 5-min intervals. Initial yield (I0) and repetitive yield (R) were calculated by nonlinear least squares regression of the observed release (M) for each cycle (n): M = I0Rn (PTH-derivatives of Ser, Thr, Cys, and His were omitted from the fit).
Radioligand Binding Assays-- The equilibrium binding of [3H]PCP (6 nM), [3H]tetracaine (2 nM), and [3H]histrionicotoxin (10 nM) to Torpedo membranes was assayed by centrifugation. 500-µl aliquots of membrane suspensions (0.5 mg of protein/ml in TPS, ~0.6 µM AChR) were equilibrated with the radioligand and the nonradioactive cholinergic ligands for 2-3 h in 10 × 75-mm Pyrex disposable culture tubes (Corning) and then transferred to 1.5-ml plastic microcentrifuge tubes and pelleted for 45 min at 15,000 rpm in a Sorvall SA-600 rotor. After removal of the supernatants, the membrane pellets were solubilized in 100 µl of 10% SDS, and the pellet 3H was determined by liquid scintillation counting.
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RESULTS |
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In initial experiments, nonradioactive DAF (Fig. 1) was tested as an inhibitor of the equilibrium binding of radiolabeled, positively charged AChR NCAs. [3H]Tetracaine binds with high affinity (Keq = 0.3 µM) in the absence of agonist to one site per AChR monomer, whereas it binds ~100-fold more weakly to desensitized AChRs (29). The sites of specific photoincorporation of [3H]tetracaine are restricted to amino acids within each M2 segment (30). In the absence of agonist, DAF produced a dose-dependent inhibition of [3H]tetracaine binding (IC50 = 6 µM), with high concentrations inhibiting ~60% of specific binding2 (Fig. 1A). For desensitized AChRs, [3H]phencyclidine binds with high affinity (Keq = 1 µM) to a single site per AChR (31), and DAF also acted as an allosteric inhibitor of [3H]phencyclidine binding (Fig. 1B, IC50 = 10 µM, 60% maximal inhibition). DAF also acted as an allosteric inhibitor of [3H]histrionicotoxin binding, with IC50 = 2 µM and maximal inhibition of 50% in the presence of agonist (data not shown).
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Initial photolabeling experiments were designed to characterize the
general pattern of [3H]DAF photoincorporation into
Torpedo AChR-rich membranes as well as to test the
sensitivity of the photoincorporation to cholinergic ligands. Membranes
(2 mg/ml) were equilibrated with 5 µM
[3H]DAF in the absence and in the presence of 100 µM carbamylcholine. After irradiation, membrane
suspensions were pelleted and resuspended in electrophoresis sample
buffer, and the pattern of incorporation was monitored by SDS-PAGE
followed by fluorography. As is evident in the fluorograph of an 8%
polyacrylamide gel (Fig. 2, lanes 3 and 4), there was appreciable incorporation of
[3H]DAF into each of the AChR subunits. Neither the
overall labeling pattern nor the relative incorporation into individual
AChR subunits was affected by the inclusion of 100 µM
carbamylcholine (Fig. 2, lane 4). Based on liquid
scintillation counting of excised gel bands, ~1% of subunits
incorporated 3H, with approximately equal incorporation in
each subunit (/
/
/
: 1/(0.8 ± 0.2)/(0.93 ± 0.3)/(1.2 ± 0.3)). The presence of carbamylcholine resulted in a
<10% change of subunit labeling. Incorporation of [3H]DAF into the AChR subunits accounted for
approximately 60% of the total labeling in polypeptides present in
Torpedo AChR-rich membranes.
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Two NCAs, tetracaine and proadifen, were tested at concentrations of 3, 30, and 250 µM for their effects on [3H]DAF
photoincorporation into AChR-rich membranes in the absence and in the
presence of 100 µM carbamylcholine, respectively. The effects of these ligands on [3H]DAF (5 µM)
photoincorporation was examined by fluorography and by liquid
scintillation counting of excised gel pieces. The pattern of
incorporation was qualitatively very similar in each of the different
conditions, with a small dose-dependent decrease
(~15-25%) in the labeling of the subunits evident for both
tetracaine and proadifen that was not seen for other labeled non-AChR
polypeptides (data not shown). The relative distribution of
3H incorporation within the -subunit was examined by
determining 3H incorporation within the four large,
non-overlapping
-subunit fragments that can be generated by
digestion with S. aureus V8 protease (14, 26). Inspection of
the fluorograph of the dried mapping gel indicated that all the visible
labeling was contained within a 20-kDa fragment (
V8-20,
Ser-173-Glu-338) containing hydrophobic segments M1-M3 and a 10-kDa
fragment (
V8-10, Asn-339-Gly-437) containing the M4 segment. Based
on liquid scintillation counting of the excised gel pieces, 75% of
3H cpm was incorporated in
V8-10 and 25% was in
V8-20, with the relative incorporation of [3H]DAF
into
V8-10 similar for labelings carried out in the absence (77%)
and in the presence (74%) of 100 µM carbamylcholine.
Sites of [3H]DAF Incorporation in M4 Segments of Each
AChR Subunit--
The M4 segments were isolated from tryptic digests
of large V8-protease fragments of each of the receptor subunits as
described under "Experimental Procedures." For -subunit, tryptic
digestion of
V8-10 produced a fluorescent and radioactive band of
3-4 kDa (
T-4K), which was further purified by reversed-phase HPLC.
N-terminal sequence analysis (Fig.
3A) revealed the presence of a
single sequence beginning at
Tyr-401 (490 pmol) that was present in a 10-20-fold greater abundance than any secondary sequence. The largest release of 3H occurred in cycle 12, with additional
release in cycles 8, 15, and 18. The same pattern of 3H
release was seen for the M4 segment isolated from membranes labeled in
the presence of 100 µM carbamylcholine (data not shown). 3H release in cycle 12 indicated that
Cys-412 was the
primary site of incorporation of [3H]DAF in
M4, as it
was for [125I]TID (17), with lower level reaction with
His-408,
Met-415, and
Cys-418.
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[3H]DAF Labeling in the Ion Channel-- To determine the sites, the agonist sensitivity, and the specificity of [3H]DAF photoincorporation in the M2 regions of AChR subunits, T. californica AChR-rich membranes (~1.4 µM AChR) were photolabeled with 10 µM [3H]DAF under four different conditions as follows: 1) in the absence of agonist; 2) in the absence of agonist and in the presence of 100 µM tetracaine; 3) in the presence of 100 µM carbamylcholine; 4) in the presence of 100 µM carbamylcholine and in the presence of 100 µM phencyclidine (PCP).
Identification of the Sites of [3H]DAF Incorporation
in -M2--
The
-subunits isolated from AChRs labeled with
[3H]DAF under the four different conditions (~300
µg/condition) were digested with 20% (w/w) trypsin for 4 days. The
digests were resolved by Tricine/SDS-PAGE, and a 5.1-kDa fragment
(
T-5.1K; Fig. 4), known to contain the
M2-M3 region (13), was isolated from the gel as described under
"Experimental Procedures." The material eluted from the
T-5.1K
band was further purified by reversed-phase HPLC (Fig.
5), and for each labeling condition, the
majority of 3H counts eluted in a peak centered at 74%
solvent B. HPLC fractions 29-31 were pooled and sequenced (Fig.
6). For each of the labeling conditions,
a single sequence was evident beginning at
Met-257 at the N terminus
of
-M2. In addition, each of the samples sequenced with similar
efficiencies and mass levels (see legend to Fig. 6). For
T-5.1K
labeled in the absence of agonist, 3H release occurred
primarily in cycle 13 (Fig. 6A (
)), a result that
indicates that the labeled amino acid is
Val-269 (26 cpm/pmol) in
-M2. For the 3H release profile of
T-5.1K labeled in
the absence of agonist but in the presence of 100 µM
tetracaine (
, Fig. 6A), there was an approximately 90%
reduction in [3H]DAF incorporation into
Val-269 (3 cpm/pmol). The presence of agonist alone (Fig. 6B,
)
caused a similar reduction in 3H incorporation into
Val-269 (2 cpm/pmol), and there was also a small but significant
release of 3H in cycles 6 and 9 that corresponds to
[3H]DAF incorporation into
Ser-262 (1.9 cpm/pmol) and
Leu-265 (1.3 cpm/pmol) with similar efficiency as in
Val-269.
Finally, in the presence of both agonist and 100 µM PCP,
there was no detectable 3H release in cycles 6 and 9, whereas release in cycle 13 was unaffected (Fig. 6B,
).
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Identification of the Sites of [3H]DAF Incorporation
in -M2--
In a manner similar to that for
-subunit, the sites
of [3H]DAF photoincorporation in the M2 region of
-subunit were determined by digesting the subunit (~250
µg/labeling condition) with 20% (w/w) trypsin for 4 days. The
digests from each of the four labeling conditions were then
fractionated by Tricine/SDS-PAGE and a 7.2-kDa band (
T-7.2K), known
to contain the M2-M3 region (13), was isolated (see under
"Experimental Procedures"). When the gels were illuminated at 365 nm on a UV-light box, the
T-7.2K fragment, which migrates as a sharp
band of 3H in the fluorograph of an analytical
Tricine/SDS-PAGE gel, migrated in an area of weak fluorescence between
two strongly fluorescent bands of 10 and 5.5 kDas (data not shown). The
material eluted from the
T-7.2K fragment was further purified by
reversed-phase HPLC (Fig. 7), and for
each labeling condition the majority of 3H counts eluted in
a peak centered at 84% solvent B. HPLC fractions 29-33 were pooled
and sequenced (Fig. 8). In each of the
four labeling conditions, sequence analysis revealed the presence of a
single peptide beginning at
Met-249 at the N terminus of
M2 that
was sequenced at similar efficiency and mass level for each sample (see
legend to Fig. 8). For the sample labeled in the absence of any ligands
other than [3H]DAF itself, the major release of
3H release was in cycle 13, with lower release in cycle 9 (
, Fig. 8A). This pattern of release corresponds to
[3H]DAF incorporation into
Leu-257 (0.7 cpm/pmol) and
Val-261 (5.3 cpm/pmol) within
-M2. The presence of tetracaine
(
, Fig. 8A) resulted in an approximately 95% reduction
in the incorporation into
Val-261 (0.2 cpm/pmol). The addition of
agonist (
, Fig. 8B) resulted in a substantial reduction
in the amount of [3H]DAF incorporated into
Val-261,
although the extent of inhibition was difficult to quantify because
that reduced release in cycle 13 was associated with a dramatic
increase in 3H release in cycles 9 and 10 that corresponds
to increased incorporation of [3H]DAF into
Leu-257 (2 cpm/pmol) and
Ala-258 (~3 cpm/pmol). Interestingly, the presence
of excess PCP (
, Fig. 8B) resulted in an 80% reduction in the amount of incorporation into
Leu-257 (0.4 cpm/pmol) but only
a 50% reduction in the labeling of
Ala-258 (1.5 cpm/pmol).
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Identification of the Sites of [3H]DAF Incorporation
in -M2--
The
-subunits isolated from AChRs labeled under each
of the four different conditions (~500 µg of
-subunit/labeling
condition) were each digested with 1.5 units of endoproteinase Lys-C
for 6 days. The digests were then resolved by Tricine/SDS-PAGE, and an
approximately 10-kDa band (
K-10K), previously shown to contain the
M2-M3 region (11), was excised (see "Experimental Procedures"). Inspection of the fluorogram of the gel of the analytical digests (Fig.
9) revealed that the
K-10K fragment
migrated with lower mobility than the major radiolabeled band of 7.8 kDa that was also intensely fluorescent (not shown). When the material
eluted from the
K-10K band was further purified by reversed-phase
HPLC, for each labeling condition the majority of 3H counts
were in a peak centered at 78% solvent B. HPLC fractions 31-34 were
pooled and sequenced (Fig. 10). For
each sample the primary sequence began at
Met-243 before
-M2
(~25 pmol), and a secondary sequence beginning at
Tyr-401 before
M4 was present at ~8 pmol.4
For all four of the samples, which correspond to each of the different
labeling conditions, low level 3H release was seen in
cycles 12, 15, and 18. This pattern of release corresponds to that
observed for nonspecific [3H]DAF incorporation into
-M4 region, i.e. labeling of
Cys-412,
Met-415, and
Cys-418 (Fig. 3A).4 For both of the
-K-10K
samples labeled in the absence of agonist (with and without
tetracaine), no other sites of 3H release were evident
(Fig. 10), a result that indicates that [3H]DAF does not
incorporate into
-M2. Had [3H]DAF incorporated into
Val-255 in
-M2 at the same efficiency as it did into
Val-269,
release of 180 cpm would be observed in cycle 13. For the
K-10K
sample isolated from AChRs labeled in the presence of agonist, there
was also 3H release in cycles 6 and 9 at levels similar to
cycle 12 (data not shown) that was eliminated (cycle 6) by the presence
of PCP. Thus, in the presence of agonist there was low level labeling (~1 cpm/pmol) of
Ser-248 and
Leu-251 as was seen for the
equivalent amino acids in
-M2 (Fig. 6B).
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DISCUSSION |
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The results presented here demonstrate that the uncharged
hydrophobic compound 2-[3H]diazofluorene
([3H]DAF) photoincorporates in a specific and
agonist-sensitive fashion into residues in the channel lining M2 region
of the nicotinic AChR. In addition, [3H]DAF
photoincorporates in a agonist-insensitive manner into the M4 segment
of each receptor subunit. Therefore, there exist two components to the
labeling of the AChR by [3H]DAF as follows: a nonspecific
component consistent with incorporation into residues situated at the
lipid-protein interface of the AChR, and a specific component,
inhibitable by noncompetitive antagonists, sensitive to
state-dependent transitions, and localized to residues within the ion channel. It is significant that even in the absence of
agonist the specific photoincorporation of [3H]DAF within
- or
-M2 can be clearly revealed only during N-terminal sequence
analyses of isolated peptides, since that component of the
photolabeling is
20% of the total labeling at the level of isolated
subunits or even of proteolytic fragments after fractionation by
SDS-PAGE and reversed-phase HPLC (Figs. 5 and 7). Unlike
[125I]TID at micromolar concentrations for which the
reaction with residues within M2 segments comprises 70% of the total
labeling at the subunit level, for [3H]DAF at micromolar
concentrations labeling of
-M2 does not predominate over labeling of
residues in the M4 segments. For example, for M2 and M4 segments
isolated from the same labeling experiment, the inhibitable
3H incorporation in
Val-269 (7-8 cpm/pmol) is ~twice
the level of nonspecific labeling in
Cys-412 (3-4 cpm/pmol), the
most highly labeled residue in M4.
Characterization of [3H]DAF Labeling at the
Lipid-Protein Interface--
The majority of AChR labeling by
[3H]DAF appears similar to the component of
[125I]TID labeling which is inhibitable neither by
agonist nor by an excess of non-radioactive TID and which is consistent
with photoincorporation into lipid-exposed regions of the AChR
(14-17). [3H]DAF incorporation into each AChR subunit M4
segment was determined under labeling conditions done in the absence
and in the presence of 100 µM carbamylcholine. In either
condition [3H]DAF reacted in -M4 with His-408,
Cys-412, Met-415, and Cys-418 (Fig. 3A); in
-M4 with
Tyr-441, Phe-443, Phe-444, and Cys-447 (Fig. 3B); in
-M4
with Cys-451 and Trp-453 (Fig. 3C). The low level of
3H release within
-M4 allows only a tentative assignment
for Met-458 (Fig. 3D). Although the isolation of M4
fragments by alternative digestion strategies would provide unambiguous
proof that the observed 3H release originates from the
observed M4 peptides, this is the expected result based upon previous
studies with [125I]TID that establish that the labeled
and unlabeled peptides coelute from the reversed-phase column (16, 17).
It is also important to point out that due to the lags resulting from
the ~90% repetitive yields inherent in the Edman degradation
reaction, cycles that follow a labeled amino acid also contain prior
PTH-derivatives and associated 3H. It is therefore
difficult to detect the presence of labeled amino acids which
immediately follow another labeled amino acid, particularly if the
efficiency of incorporation into those residues is significantly lower.
Without direct characterization of the 3H-labeled amino
acids released in each cycle, one cannot determine whether some of the
3H release in cycles immediately following these labeled
residues is due to amino acids which have also reacted with
[3H]DAF.
|
Characterization of [3H] DAF Labeling in the
Channel--
In the absence of agonist, [3H]DAF labels
within M2 segments two homologous aliphatic residues, Val-261 and
Val-269. Incorporation into these residues is reduced greater than
90% by the addition of an excess of tetracaine, which binds within the
channel in the absence of agonist (30). Surprisingly, in the absence of agonist, no [3H]DAF incorporation was detected in
-M2.
It is also striking that the incorporation of [3H]DAF
into
Val-269 occurred five times more efficiently than into
Val-261. The preferential incorporation into
Val-269 cannot be
easily explained by differences in amino acid side chain reactivities, since a valine is present at that position in all three subunits (
,
,
). Thus [3H]DAF bound within the ion channel must
be oriented with its photoreactive diazo group facing the side chain of
Val-269. This selective orientation is then a consequence of the
asymmetry of the [3H]DAF molecule and of the pore of the
ion channel itself. The lack of incorporation into
-M2 and the less
efficient incorporation into
-M2 compared with
-M2 reflects the
arrangement of subunits in the pentameric receptor:
-
-
-
-
(11, 36, 38). Only by determining if [3H]DAF also reacts
with
-M2 can it be determined whether [3H]DAF can
exist in the channel in more than one possible orientation.
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ACKNOWLEDGEMENT |
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We thank Martin Gallagher for helpful comments and suggestions.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Grants NS 19522 and GM 15904 (to J. B. C.) and by an award in Structural Neurobiology from the Keck Foundation.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.
§ Present address: Dept. of Pharmacology, Texas Tech Univ. Health Sciences Center, Lubbock, TX 79430.
To whom correspondence should be addressed: Dept. of
Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA
02115. Tel.: 617-432-1728; Fax: 617-734-7557;
E-mail:jbcohen{at}warren.med.harvard.edu.
1 The abbreviations used are: AChR, nicotinic acetylcholine receptor; 1-AP, 1-azidopyrene; [3H]DAF, 2-[3H]diazofluorene; [125I]TID, 3-trifluoromethyl-3-(-m-[125I]iodophenyl) diazirine; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; EKC, endoproteinase Lys-C; PCP, phencyclidine; PTH, phenylthiohydantoin; NCAs, noncompetitive antagonists; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
2 Although high concentrations of DAF were apparently unable to fully displace [3H]PCP or [3H]tetracaine, further studies are required to determine whether this reflects a true allosteric inhibition or the limited aqueous solubility of DAF.
3
The presence of a contaminating -subunit
fragment in
-subunit fractions occurred uniquely in the particular
experiment in Fig. 3B. Since
-subunit does not
contaminate
-subunit preparations in detectable quantities, the
contamination in this sample most likely resulted when the
reversed-phase HPLC column was washed insufficiently between the
purification of the
-subunit T-4K fragment and the purification of
the
-subunit T-5K band. Based upon an estimated 8.6 pmol of
Cys-412 in cycle 12 of the
-subunit sample (Fig. 3B,
calculated from the mass of the
Tyr-401 peptide) and the amount of
[3H]DAF incorporation into
Cys-412 (~4 cpm/pmol,
Fig. 3A (cycle 12)), the 8 cpm release in cycle 12 of Fig.
3B must result from the
-subunit fragment. In contrast,
the 3H release in cycles 15, 17, 18, and 21 cannot result
from the
Tyr-401 peptide.
4
Fragments containing -M4 beginning at
Tyr-401 and
Ser-388 were found to be present at higher mass
levels (40 pmol) in the material purified from the intensely
fluorescent, radiolabeled 7.8-kDa fragment (
K-7.8K). Based upon the
mass level of the
Tyr-401 peptide in the
K-10K band (Fig.
10A), the 3H release in cycle 12 would
correspond to labeling of
Cys-412 in M4 at a level (9 cpm/pmol)
similar to that seen for labeling of
Cys-412 (8 cpm/pmol) in the
sequence analysis of the
K-7.8K band.
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REFERENCES |
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