Neuronal alpha-bungarotoxin receptors are alpha7 subunit homomers

Abstract

Nicotinic acetylcholine receptors in the nervous system are heterogeneous with distinct pharmacological and functional properties resulting from differences in post-translational processing and subunit composition. Because of nicotinic receptor diversity, receptor purification and biochemical characterization have been difficult, and the precise subunit composition of each receptor subtype is poorly characterized. Evidence is presented that alpha-bungarotoxin (Bgt)-binding nicotinic receptors found in pheochromocytoma 12 (PC12) cells are pentamers composed solely of alpha7 subunits. Metabolically labeled, affinity-purified Bgt receptors (BgtRs) consisted of a single 55 kDa band on SDS gels, which was recognized by anti-alpha7 antibodies on immunoblots. Isoelectric focusing separated the 55 kDa band into multiple spots, all recognized by anti-alpha7 antibodies and, therefore, each a differentially processed alpha7 subunit. Cell-surface BgtR subunits, cross-linked to each other and (125)I-Bgt, migrated on gels as a ladder of five bands with each band a multiple of an alpha7 subunit monomer. Similar characteristics of BgtRs from rat brain suggest that they, like PC12 BgtRs, are alpha7 pentamers containing differentially processed alpha7 subunits.

Fig. 1.

Affinity-purified BgtRs are composed of α7 subunits. BgtRs were affinity-purified from PC12 cells using Bgt-Sepharose and were analyzed on 4–8% gradient SDS-PAGE. BgtRs were metabolically labeled and processed for fluorography (left) or subjected to immunoblot analysis with α7-specific antibodies (right). Metabolically labeled BgtR subunits migrated as a single band at 55 kDa (lane 2), identical to the position of α7 subunits identified on immunoblots (lane 5). A mean value of 55 ± 1 kDa (±SD) was obtained for the labeledbandin the figure together with the results from three other experiments. Alkylation of the subunits with the sulfhydryl-specific agent NEM produced two closely spaced bands centered at 55 kDa for both labeled subunits (lane 3) and α7 subunits on immunoblots (lane 6). No additional bands were observed in the 20–40 kDa range when affinity-purified BgtRs were analyzed on 7.5% gels, and no bands were observed when 100 μmnicotine was present during the Bgt-Sepharose precipitation (lanes 1, 4). The positions of molecular markers are shown on the left: myosin, 200 kDa; β-galactosidase, 116 kDa; phosphorylase b, 97.4 kDa; BSA, 66 kDa; and actin, 45 kDa.

Fig. 2.

Affinity-purified BgtRs are fully assembled.A, Metabolically labeled BgtRs were solubilized in the absence of NEM and size-fractionated on 5–20% linear sucrose gradients before affinity purification. Labeled subunits were precipitated from each gradient fraction, 1 (top) to 16 (bottom), and analyzed on SDS-PAGE as in Figure 1. The majority of the labeled subunits migrated at 10 S (fractions 12, 13). Arrows at thetop mark the peak fractions of the standards: alkaline phosphatase, 5.4 S; ¹²⁵I-Bgt-bound α7/5HT₃receptors, 9 S; and catalase, 11 S. Arrows on theright indicate the positions of molecular weight standards: actin, 45 kDa, and BSA, 66 kDa. The arrowheadindicates the position of α7 subunits. B, Immunoblot analysis performed on BgtRs size-fractionated on 5–20% linear sucrose gradients is shown. BgtRs, precipitated from each gradient fraction with Bgt-Sepharose, were prepared on SDS-PAGE as described inA, followed by immunoblot analysis with anti-α7-specific antibodies. Standards used are identical to those inA. C,¹²⁵I-Bgt-binding sites were size-fractionated on 5–20% linear sucrose gradients. After sedimentation on sucrose gradients, BgtRs in each gradient fraction were bound with ¹²⁵I-Bgt and precipitated using conconavalin A-Sepharose. The majority of the sites (92%) sedimented at 10 S (fractions 12, 13), whereas a smaller number (8%) sedimented at ∼6 S (fractions 5, 6).

Fig. 3.

Affinity-purified BgtRs contain five subunits of equal molecular weight. A, B, Cross-linking of surface¹²⁵I-Bgt-bound BgtRs is shown. BgtRs on the surface of PC12 cells (A) or from the surface of cells expressing α7/5HT₃ chimeras (B) were bound with ¹²⁵I-Bgt and cross-linked with the indicated concentrations of DTSSP. Cross-linked complexes were solubilized in the presence of NEM, immunoprecipitated with anti-Bgt antibodies conjugated to protein A-Sepharose, and run on nonreducing SDS-PAGE.Arrowheads on the right indicate the positions of subunit monomers, dimers, trimers, tetramers, and pentamers, whereas arrows on theleft in A indicate positions of the molecular weight markers (see Fig. 1). C, Molecular weights of the cross-linked complexes were estimated and plotted as a function of the number of subunits in the cross-linked PC12 (▪) and α7/5HT₃ (○) complexes. Molecular weight values are the mean ± SD from four experiments. The linesrepresent a least-squares linear regression fit to each data set.D, Cross-linking of surface ¹²⁵I-Bgt-bound BgtRs with the shorter-arm reagent sDST is shown. BgtRs on the surface of PC12 cells (leftlane) or from cells expressing α7/5HT₃ chimeras (rightlane) were bound with ¹²⁵I-Bgt and cross-linked with sDST. Samples were treated as described inA except they were solubilized without NEM.Arrowheads on the left andright represent the positions of oligomers for PC12 BgtR and α7/5HT₃ subunits, respectively. Arrowsshow the migration of molecular weight markers.

Fig. 4.

Two-dimensional gel analysis of affinity-purified BgtRs. A, PC12 BgtRs were metabolically labeled, solubilized without NEM, precipitated with Bgt-Sepharose in the presence (top) or absence (bottom) of 100 μm nicotine, and run on two-dimensional gels. The 55 kDa band observed on SDS-PAGE in Figure 1 separated into six different spots with pIs between 5.5 and 5.7 (labeled 1–6) and a seventh spot at 5.9 (labeled 7). Arrows on the bottommark the positions of the standards for the IEF dimension (left to right): actin, pI 5.0–5.1; BSA, pI 5.4–5.6; carbonic anhydrase, pI 5.9–6.0; and conalbumin, pI 6.0–6.6. Each arrowhead marks the position of 55 kDa in the SDS-PAGE dimension, and a marks the position of actin. B, PC12 BgtRs were precipitated with Bgt-Sepharose in the presence (top) or absence (bottom) of 100 μm nicotine and analyzed by immunoblot after being run on two-dimensional gels. All seven spots observed on two-dimensional gels for metabolically labeled BgtR subunits were stained by anti-α7 antibodies. Standards used are identical to those in A.

Fig. 5.

Rat brain BgtRs. BgtRs were affinity-purified from solubilized rat brain membranes and compared with PC12 BgtRs using immunoblot analysis with anti-α7 antibodies. Rat brain (left) and PC12 (right) BgtRs were solubilized in the absence or presence of NEM as indicated and run on reducing (+DTT) or nonreducing (−DTT) SDS-PAGE. The positions of oligomers (arrowheads) and molecular weights of the standards (arrows) are shown (Fig. 3). Results are representative of three separate experiments.