Probing the structure of the affinity-purified and lipid-reconstituted torpedo nicotinic acetylcholine receptor

Abstract

The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical and biophysical techniques, detergent solubilization, purification, and lipid reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) to compare the state-dependent photolabeling of the Torpedo nAChR before and after purification and reincorporation into lipid. For the purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9, and M2-13, the agonist-enhanced labeling of deltaThr274 (deltaM2-18) within the delta subunit helix bundle, and the labeling at the lipid-protein interface (alphaMu4) were the same as for the nAChR in native membranes. However, addition of agonist did not enhance [(125)I]TID photolabeling of deltaIle288 within the deltaM2-M3 loop. These results indicate that after purification and reconstitution of the Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the deltaM2-M3 loop. To further characterize the pharmacology of [(125)I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [(125)I]TID photolabeling. PCP inhibited [(125)I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6) while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the delta subunit helix bundle.

Figure 1. The effect of affinity-purification and lipid-reconstitution on [¹²⁵I]TID photoincorporation into δM2 in nAChRs in the resting and desensitized states

¹²⁵I (○, ●) and PTH-amino acids (□, ■) released during sequence analysis through δM2 from native (A and B) or purified (C and D) nAChRs. Native Torpedo nAChR-rich membranes or affinity-purified and lipid-reconstituted nAChR were photolabeled with [¹²⁵I]TID in the absence (○, □) or presence (●, ■) of Carb, and EndoLys-C digests of δ subunits were fractionated by Tricine SDS-PAGE and by rpHPLC (Supplemental Figure S1) to isolate fragments beginning at δMet257. The primary sequence began at δMet257 for native nAChR (A and B, Io, control (□), 31 pmol,+ Carb (■), 37 pmol) and for purified nAChR (C and D, Io, −Carb (□), 35 pmol, +Carb (■), 30 pmol), with a fragment beginning at δAsn437 present at ~ 1 pmol in all samples. For both the native and purified nAChRs, in the absence of Carb the peaks of ¹²⁵I release in cycles 9 and 13 reflected labeling of δLeu265 and δVal269, which was reduced by >95% for the +Carb samples which had the major peak of ¹²⁵I release in cycle 18, consistent with labeling of δThr-274, and in cycles 6 (δSer262). B and D, ¹²⁵I release profiles from panels A and C are replotted on an expanded scale to show clearly the peaks of release for the +Carb samples. The amino acid sequence quantified is shown above each panel, with the solid bar indicating the span of the M2.

Figure 2. The effect of affinity-purification and lipid-reconstitution on [¹²⁵I]TID photoincorporation in δM1 (A and C) and in the δM2-δM3 loop (B and D) in the absence or presence of agonist

¹²⁵I (○, ●) and PTH-amino acids (□, ■) released during sequencing of fragments isolated from EndoLys-C digests (A and C) and V8 protease digests (B and D) of δ subunits isolated from [¹²⁵I]TID-labeled native (A, B) or purified Torpedo nAChR (C, D) as described in “Experimental Procedures” and in Supplemental Figures S1 and S2. During sequencing the filters were treated with OPA at cycle 20 to chemically isolate δM1 (A & C) or in cycle 6 to chemically isolate M3 (B & D) by preventing further sequencing of fragments not containing a proline in those cycles. A & C, Before treatment with OPA the primary amino acid sequence began at δPhe206 for native (A, Io, 4 pmol, − and +Carb) and purified nAChRs (B, Io, 14 pmol, − and +Carb), and sequencing of that fragment continued after OPA treatment. For the native membranes, before OPA treatment the fragment beginning at δMet257 was present as a secondary sequence (+Carb (◆), Io, 2 pmol; −Carb, 1 pmol (not shown)), and it is this fragment that is the source of the peaks of ¹²⁵I release in cycles 9 and 13 (−Carb, ○) and in cycle 18 (●, +Carb). After treatment with OPA in cycle 20, for the native nAChR there were peaks of ¹²⁵I release in cycles 27 (δPhe232), and 31 (δCys236) +Carb, with no detectable ¹²⁵I release in the −Carb sample. For the purified nAChR, there were peaks of ¹²⁵I release in cycles 27 and 31 in the absence and presence of Carb. B & D, Before OPA treatment in cycle 6, fragments were present beginning at δIle192, δVal443, and δThr281, each at ~ 12 pmol (B), while after OPA treatment, sequencing continued only for the δThr281 fragment from native (B, Io, 12 pmol − or +Carb) and purified nAChRs (D, Io, 8 pmol − or +Carb). The amino acid sequence quantified is shown above each panel, with the solid bar indicating the span of the M1 (A &C) and M3 (B & D) segments.

Figure 3. Affinity-purification and lipid-reconstitution has no effect on [¹²⁵I]TID photolabeling within αM4

¹²⁵I (○, ●) and PTH-amino acids (□, ■) released during sequence analysis of the fragment beginning at αTyr401, which was isolated by rpHPLC from trypsin digests of αV8-10 from [¹²⁵I]TID-labeled native (A) or purified nAChR (B) photolabeled with [¹²⁵I]TID in the absence (□, ○) or presence of Carb (■, ●). In each condition, the primary amino acid sequence began at αTyr401 (A, Io, 15 pmol, − or +Carb; B, 3 pmol − or +Carb) and the major peak of ¹²⁵I release was in cycle 12 (αCys412). The amino acid sequence detected is shown above the panel, with the solid bar indicating the span of the M4 segment.

Figure 4. For nAChRs in the desensitized state, PCP inhibits [¹²⁵I]TID photoincorporation into amino acids only at the cytoplasmic end of the M2 segments

¹²⁵I (●, △) and PTH-amino acids (■, ◇) released during sequence analysis of the fragments beginning at the N-termini of αM2 (A), βM2 (B), and δM2 (C) that were isolated from native Torpedo nAChR labeled with [¹²⁵I]TID in the presence of Carb (●, ■) or Carb and PCP (△, ◇). A, The primary amino acid sequence began at αMet243 (Io, 4 pmol each condition). The major peak of ¹²⁵I release in cycle 6 +Carb (αSer248, 90 cpm) was reduced to 10 cpm +Carb +PCP. B, The primary amino acid sequence began at βMet249 (I_o, +Carb, 20 pmol; +Carb+PCP, 16 pmol) with a secondary sequence beginning at βLys216 (I_o, < 1 pmol). In the presence of PCP, the peaks of ¹²⁵I release in cycles 2 (βSer250) and 6 (βSer254) were reduced by >90%, the release in cycle 9 (βLeu257) was reduced by <15%, and the release in cycle 13 (βVal261) was increased by 200%. C, The primary amino acid sequence began at δMet257 (Io, 16 pmol, both conditions). In the presence of Carb, the major peak of release in cycle 18 indicated labeling of δThr274 at 22 cpm/pmol which was reduced by 30% in the presence of PCP. The peaks of ¹²⁵I release in cycles 2 and 6 indicated labeling of δSer258 and δSer262 at 0.4 and 1.7 cpm/pmol which was reduced by 90% +PCP. δLeu265 (cycle 9) and δVal269 (cycle 13) were labeled at 2.5 and 3.3 cpm/pmol (+Carb), and that labeling was increased by 100% and 20%, respectively, in the presence of PCP.

Figure 5. [¹²⁵I]TID photoincorporation within δM1 and the δM2-M3 loop in the presence of PCP

¹²⁵I (●, △) and PTH-amino acids (■, ◇) released during sequence analysis of the fragments beginning at δPhe206 before δM1 (A) and δThr281 in the δM2-M3 loop (B) that were isolated from native Torpedo nAChR labeled with [¹²⁵I]TID in the presence of Carb (●, ■) or Carb and PCP (△, ◇) as described under the Experimental Procedures and the legend of Figure 2. During sequencing the filters treated with OPA before cycle 20 (A) or 6 (B) to prevent sequencing of fragments not containing a proline in those cycles. A, The primary amino acid sequence began at δPhe206 (+Carb (■), I₀, 7 pmol; +Carb + PCP (◇), 10 pmol). In the presence of Carb, the peaks of ¹²⁵I release in cycles 27 and 31 indicated labeling of δPhe232 and δCys236 at 8 and 38 cpm/pmol, respectively, which was reduced by 50% in the presence of PCP. B, The fragment beginning at δThr281 was present at 20 pmol (both conditions). In the presence of Carb, the major peak of ¹²⁵I release in cycle 8 indicated labeling of δIle288 at 11 cpm/pmol, which was reduced to 6 cpm/pmol in the presence of PCP. The amino acid sequences of the fragments are shown above each panel, with solid bars indicating the span of the δM1 (A) and δM3 (B) segments.

Figure 6. Residues photolabeled by [¹²⁵I]TID within the transmembrane domain of native and reconstituted nAChRs

Views of the membrane-spanning helices (shown as cylinders) of the Torpedo nAChR structure (PDB # 2BG9): A, looking down the channel from the base of the extracellular domain; and B, (A) looking parallel to the membrane with 2 subunits removed for clarity, rotated 90° from (A). Subunits are color-coded: α, gold; β blue; γ, green; and δ, magenta. Residues photolabeled by TID are included in stick format, color coded by domain and conformation: ion channel, resting state (red); ion channel, desensitized state (PCP inhibitable) (yellow); δ subunit helix bundle, desensitized state (cyan); lipid-protein interface (green). A Connolly surface model of TID is included in A for scale.