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alpha-bungarotoxin receptors contain alpha7 subunits in two different disulfide-bonded conformations.

Rakhilin S, Drisdel RC, Sagher D, McGehee DS, Vallejo Y, Green WN - J. Cell Biol. (1999)

Bottom Line: Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells.Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, alpha-bungarotoxin-binding receptors with two alpha7 conformations.Our results suggest that alpha7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological and Physiological Sciences, Department of Anesthesia and Critical Care, University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells. Functional alpha-bungarotoxin receptors are expressed if the membrane-spanning and cytoplasmic domains of the alpha7 subunit are replaced by the homologous regions of the serotonin-3 receptor subunit. Bgt-binding surface receptors assembled from chimeric alpha7/serotonin-3 subunits contain subunits in two different conformations as shown by differences in redox state and other features of the subunits. In contrast, alpha7 subunit complexes in the same cell line contain subunits in a single conformation. The appearance of a second alpha7/serotonin-3 subunit conformation coincides with the formation of alpha-bungarotoxin-binding sites and intrasubunit disulfide bonding, apparently within the alpha7 domain of the alpha7/serotonin-3 chimera. In cell lines of neuronal origin that produce functional alpha7 receptors, alpha7 subunits undergo a conformational change similar to alpha7/serotonin-3 subunits. alpha7 subunits, thus, can fold and assemble by two different pathways. Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, alpha-bungarotoxin-binding receptors with two alpha7 conformations. Our results suggest that alpha7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.

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Differences in the folding of α7-HA and α7/5HT3-HA subunits. (A) A difference in α7 and α7/5HT3 subunit redox state. 6-cm cultures of tsA201 cells, transfected with α7-HA or α7/5HT3-HA cDNAs, were metabolically labeled for 1 h and chased for 1 h. The cells were solubilized in the absence of NEM and labeled subunits immunoprecipitated with anti-HA mAb. Samples, each from one 6-cm culture, were loaded on the gel with or without treatment with 10 mM DTT. α7-HA samples were loaded into lanes 1 and 3 and α7/5HT3-HA samples were loaded into lanes 2 and 4. Arrows on the left of the figure are the indicated molecular weight markers run on a separate lane. Arrows on the right of the figure indicate positions of monomer, dimer, trimer, tetramer, and pentamer subunit complexes. (B) α7 subunit alkylation prevents its aggregation. TsA201 cells were transfected with α7-HA cDNA, metabolically labeled, and precipitated as in A, except that NEM (0–100 μM) was included in the culture medium for the final 10 min of the chase. A sample from sham-transfected cells (no DNA) was run in lane 1. Arrows on the left and right of the figure are the same as in A. (C) SDS resistance of subunit multimers. TsA201 cells were transfected with α7-HA cDNA, metabolically labeled, and precipitated as in A. As indicated, NEM (2 mM) was added to the solubilization buffer and to the loading buffer after addition of DTT (1 mM). After alkylation by NEM (lane 3) and reduction by DTT before gel loading (lane 4), some subunits remained in complexes as well as migrating as monomers. Arrows on the left of the figure are the indicated molecular weight markers run on a separate lane. Arrows on the right of the figure indicate positions corresponding to monomer, dimer, trimer, tetramer, and pentamer subunit complexes. (D) Bgt-binding subunit multimers. TsA201 cells transfected with α7/5HT3-HA cDNA were metabolically labeled as in A, chased for 2 h, and subunits precipitated with Bgt-Sepharose. Subunit multimers were greatly decreased by NEM alkylation (2 mM; lane 2) and addition of DTT before gel loading (1 mM; lane 3). A sample from sham-transfected cells (no DNA) was run in lane 1. Molecular weight markers are on the left of the gel. (E and F) The truncated α7 subunit. TsA201 cells were transfected with the truncated α7 subunit cDNA (Tα7-HA; Fig. 1 A), metabolically labeled, and immunoprecipitated as in A. Labeled subunits were analyzed on 10% (E) or 4–8% gradient (F) gels. As indicated, NEM (2 mM) was added to the solubilization buffer and to the loading buffer after addition of DTT (1 mM). Truncated α7 subunits migrated as aggregates and multimers similar to full-length α7 subunits (A–C). Arrows on the left of the figures are the indicated molecular weight markers run on a separate lane. Arrows on the right of the figures indicate positions corresponding to monomer, dimer, trimer, and tetramer (E and F) plus pentamer (F) subunit complexes.
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Figure 3: Differences in the folding of α7-HA and α7/5HT3-HA subunits. (A) A difference in α7 and α7/5HT3 subunit redox state. 6-cm cultures of tsA201 cells, transfected with α7-HA or α7/5HT3-HA cDNAs, were metabolically labeled for 1 h and chased for 1 h. The cells were solubilized in the absence of NEM and labeled subunits immunoprecipitated with anti-HA mAb. Samples, each from one 6-cm culture, were loaded on the gel with or without treatment with 10 mM DTT. α7-HA samples were loaded into lanes 1 and 3 and α7/5HT3-HA samples were loaded into lanes 2 and 4. Arrows on the left of the figure are the indicated molecular weight markers run on a separate lane. Arrows on the right of the figure indicate positions of monomer, dimer, trimer, tetramer, and pentamer subunit complexes. (B) α7 subunit alkylation prevents its aggregation. TsA201 cells were transfected with α7-HA cDNA, metabolically labeled, and precipitated as in A, except that NEM (0–100 μM) was included in the culture medium for the final 10 min of the chase. A sample from sham-transfected cells (no DNA) was run in lane 1. Arrows on the left and right of the figure are the same as in A. (C) SDS resistance of subunit multimers. TsA201 cells were transfected with α7-HA cDNA, metabolically labeled, and precipitated as in A. As indicated, NEM (2 mM) was added to the solubilization buffer and to the loading buffer after addition of DTT (1 mM). After alkylation by NEM (lane 3) and reduction by DTT before gel loading (lane 4), some subunits remained in complexes as well as migrating as monomers. Arrows on the left of the figure are the indicated molecular weight markers run on a separate lane. Arrows on the right of the figure indicate positions corresponding to monomer, dimer, trimer, tetramer, and pentamer subunit complexes. (D) Bgt-binding subunit multimers. TsA201 cells transfected with α7/5HT3-HA cDNA were metabolically labeled as in A, chased for 2 h, and subunits precipitated with Bgt-Sepharose. Subunit multimers were greatly decreased by NEM alkylation (2 mM; lane 2) and addition of DTT before gel loading (1 mM; lane 3). A sample from sham-transfected cells (no DNA) was run in lane 1. Molecular weight markers are on the left of the gel. (E and F) The truncated α7 subunit. TsA201 cells were transfected with the truncated α7 subunit cDNA (Tα7-HA; Fig. 1 A), metabolically labeled, and immunoprecipitated as in A. Labeled subunits were analyzed on 10% (E) or 4–8% gradient (F) gels. As indicated, NEM (2 mM) was added to the solubilization buffer and to the loading buffer after addition of DTT (1 mM). Truncated α7 subunits migrated as aggregates and multimers similar to full-length α7 subunits (A–C). Arrows on the left of the figures are the indicated molecular weight markers run on a separate lane. Arrows on the right of the figures indicate positions corresponding to monomer, dimer, trimer, and tetramer (E and F) plus pentamer (F) subunit complexes.

Mentions: 15 h after transfection, 6 cm cultures were starved in methionine-free DMEM for 10–15 min and labeled in methionine-free DMEM containing 100 μCi/ml of an [35S]methionine [35S]cysteine mixture (NEN EXPE35S35S) for the specified times. To follow the subsequent changes in the labeled subunits, the cells were chased by incubation for the indicated times in medium at 37°C. Cells that were chased were washed twice with DMEM supplemented with 5 mM cold methionine and incubated at 37°C in complete medium for the duration of the chase. Cells were then transferred from the plates into Eppendorf tubes, washed twice with PBS, and solubilized in lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 0.02% NaN3) containing 2 mM phenylmethylsulfonyl fluoride, 10 μg/ml each of chymostatin, leupeptin, pepstatin and tosyl-lysine chloromethyl ketone, and 1% Triton X-100. Lysates were clarified by centrifugation at 10,000 g for 30 min at 4°C, and the supernatants precleared by incubation with Sepharose 4B (Pharmacia) overnight at 4°C. The resin was removed by centrifugation and the supernatant was rotated overnight at 4°C with either saturating amounts of the anti-HA antibody, mAb 12CA5, or Bgt-Sepharose. Bgt-Sepharose was prepared by coupling Bgt to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer's directions. The receptor-antibody complex was precipitated with protein G–Sepharose. The resin was washed twice in lysis buffer similar to the one used above, but with 500 mM NaCl instead of 150 mM and addition of 0.1% SDS, and twice with regular lysis buffer. Precipitates were eluted from the beads with gel loading buffer and separated on 4–8% gradient SDS-PAGE with the exception of the truncated α7 construct where 10% SDS-PAGE was used (see Fig. 3 D). Gels were stained, fixed, treated with Amplify (Amersham) for 30 min, dried, and exposed to Kodak XRP film at −70°C with intensifying screens.


alpha-bungarotoxin receptors contain alpha7 subunits in two different disulfide-bonded conformations.

Rakhilin S, Drisdel RC, Sagher D, McGehee DS, Vallejo Y, Green WN - J. Cell Biol. (1999)

Differences in the folding of α7-HA and α7/5HT3-HA subunits. (A) A difference in α7 and α7/5HT3 subunit redox state. 6-cm cultures of tsA201 cells, transfected with α7-HA or α7/5HT3-HA cDNAs, were metabolically labeled for 1 h and chased for 1 h. The cells were solubilized in the absence of NEM and labeled subunits immunoprecipitated with anti-HA mAb. Samples, each from one 6-cm culture, were loaded on the gel with or without treatment with 10 mM DTT. α7-HA samples were loaded into lanes 1 and 3 and α7/5HT3-HA samples were loaded into lanes 2 and 4. Arrows on the left of the figure are the indicated molecular weight markers run on a separate lane. Arrows on the right of the figure indicate positions of monomer, dimer, trimer, tetramer, and pentamer subunit complexes. (B) α7 subunit alkylation prevents its aggregation. TsA201 cells were transfected with α7-HA cDNA, metabolically labeled, and precipitated as in A, except that NEM (0–100 μM) was included in the culture medium for the final 10 min of the chase. A sample from sham-transfected cells (no DNA) was run in lane 1. Arrows on the left and right of the figure are the same as in A. (C) SDS resistance of subunit multimers. TsA201 cells were transfected with α7-HA cDNA, metabolically labeled, and precipitated as in A. As indicated, NEM (2 mM) was added to the solubilization buffer and to the loading buffer after addition of DTT (1 mM). After alkylation by NEM (lane 3) and reduction by DTT before gel loading (lane 4), some subunits remained in complexes as well as migrating as monomers. Arrows on the left of the figure are the indicated molecular weight markers run on a separate lane. Arrows on the right of the figure indicate positions corresponding to monomer, dimer, trimer, tetramer, and pentamer subunit complexes. (D) Bgt-binding subunit multimers. TsA201 cells transfected with α7/5HT3-HA cDNA were metabolically labeled as in A, chased for 2 h, and subunits precipitated with Bgt-Sepharose. Subunit multimers were greatly decreased by NEM alkylation (2 mM; lane 2) and addition of DTT before gel loading (1 mM; lane 3). A sample from sham-transfected cells (no DNA) was run in lane 1. Molecular weight markers are on the left of the gel. (E and F) The truncated α7 subunit. TsA201 cells were transfected with the truncated α7 subunit cDNA (Tα7-HA; Fig. 1 A), metabolically labeled, and immunoprecipitated as in A. Labeled subunits were analyzed on 10% (E) or 4–8% gradient (F) gels. As indicated, NEM (2 mM) was added to the solubilization buffer and to the loading buffer after addition of DTT (1 mM). Truncated α7 subunits migrated as aggregates and multimers similar to full-length α7 subunits (A–C). Arrows on the left of the figures are the indicated molecular weight markers run on a separate lane. Arrows on the right of the figures indicate positions corresponding to monomer, dimer, trimer, and tetramer (E and F) plus pentamer (F) subunit complexes.
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Figure 3: Differences in the folding of α7-HA and α7/5HT3-HA subunits. (A) A difference in α7 and α7/5HT3 subunit redox state. 6-cm cultures of tsA201 cells, transfected with α7-HA or α7/5HT3-HA cDNAs, were metabolically labeled for 1 h and chased for 1 h. The cells were solubilized in the absence of NEM and labeled subunits immunoprecipitated with anti-HA mAb. Samples, each from one 6-cm culture, were loaded on the gel with or without treatment with 10 mM DTT. α7-HA samples were loaded into lanes 1 and 3 and α7/5HT3-HA samples were loaded into lanes 2 and 4. Arrows on the left of the figure are the indicated molecular weight markers run on a separate lane. Arrows on the right of the figure indicate positions of monomer, dimer, trimer, tetramer, and pentamer subunit complexes. (B) α7 subunit alkylation prevents its aggregation. TsA201 cells were transfected with α7-HA cDNA, metabolically labeled, and precipitated as in A, except that NEM (0–100 μM) was included in the culture medium for the final 10 min of the chase. A sample from sham-transfected cells (no DNA) was run in lane 1. Arrows on the left and right of the figure are the same as in A. (C) SDS resistance of subunit multimers. TsA201 cells were transfected with α7-HA cDNA, metabolically labeled, and precipitated as in A. As indicated, NEM (2 mM) was added to the solubilization buffer and to the loading buffer after addition of DTT (1 mM). After alkylation by NEM (lane 3) and reduction by DTT before gel loading (lane 4), some subunits remained in complexes as well as migrating as monomers. Arrows on the left of the figure are the indicated molecular weight markers run on a separate lane. Arrows on the right of the figure indicate positions corresponding to monomer, dimer, trimer, tetramer, and pentamer subunit complexes. (D) Bgt-binding subunit multimers. TsA201 cells transfected with α7/5HT3-HA cDNA were metabolically labeled as in A, chased for 2 h, and subunits precipitated with Bgt-Sepharose. Subunit multimers were greatly decreased by NEM alkylation (2 mM; lane 2) and addition of DTT before gel loading (1 mM; lane 3). A sample from sham-transfected cells (no DNA) was run in lane 1. Molecular weight markers are on the left of the gel. (E and F) The truncated α7 subunit. TsA201 cells were transfected with the truncated α7 subunit cDNA (Tα7-HA; Fig. 1 A), metabolically labeled, and immunoprecipitated as in A. Labeled subunits were analyzed on 10% (E) or 4–8% gradient (F) gels. As indicated, NEM (2 mM) was added to the solubilization buffer and to the loading buffer after addition of DTT (1 mM). Truncated α7 subunits migrated as aggregates and multimers similar to full-length α7 subunits (A–C). Arrows on the left of the figures are the indicated molecular weight markers run on a separate lane. Arrows on the right of the figures indicate positions corresponding to monomer, dimer, trimer, and tetramer (E and F) plus pentamer (F) subunit complexes.
Mentions: 15 h after transfection, 6 cm cultures were starved in methionine-free DMEM for 10–15 min and labeled in methionine-free DMEM containing 100 μCi/ml of an [35S]methionine [35S]cysteine mixture (NEN EXPE35S35S) for the specified times. To follow the subsequent changes in the labeled subunits, the cells were chased by incubation for the indicated times in medium at 37°C. Cells that were chased were washed twice with DMEM supplemented with 5 mM cold methionine and incubated at 37°C in complete medium for the duration of the chase. Cells were then transferred from the plates into Eppendorf tubes, washed twice with PBS, and solubilized in lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 0.02% NaN3) containing 2 mM phenylmethylsulfonyl fluoride, 10 μg/ml each of chymostatin, leupeptin, pepstatin and tosyl-lysine chloromethyl ketone, and 1% Triton X-100. Lysates were clarified by centrifugation at 10,000 g for 30 min at 4°C, and the supernatants precleared by incubation with Sepharose 4B (Pharmacia) overnight at 4°C. The resin was removed by centrifugation and the supernatant was rotated overnight at 4°C with either saturating amounts of the anti-HA antibody, mAb 12CA5, or Bgt-Sepharose. Bgt-Sepharose was prepared by coupling Bgt to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer's directions. The receptor-antibody complex was precipitated with protein G–Sepharose. The resin was washed twice in lysis buffer similar to the one used above, but with 500 mM NaCl instead of 150 mM and addition of 0.1% SDS, and twice with regular lysis buffer. Precipitates were eluted from the beads with gel loading buffer and separated on 4–8% gradient SDS-PAGE with the exception of the truncated α7 construct where 10% SDS-PAGE was used (see Fig. 3 D). Gels were stained, fixed, treated with Amplify (Amersham) for 30 min, dried, and exposed to Kodak XRP film at −70°C with intensifying screens.

Bottom Line: Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells.Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, alpha-bungarotoxin-binding receptors with two alpha7 conformations.Our results suggest that alpha7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological and Physiological Sciences, Department of Anesthesia and Critical Care, University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells. Functional alpha-bungarotoxin receptors are expressed if the membrane-spanning and cytoplasmic domains of the alpha7 subunit are replaced by the homologous regions of the serotonin-3 receptor subunit. Bgt-binding surface receptors assembled from chimeric alpha7/serotonin-3 subunits contain subunits in two different conformations as shown by differences in redox state and other features of the subunits. In contrast, alpha7 subunit complexes in the same cell line contain subunits in a single conformation. The appearance of a second alpha7/serotonin-3 subunit conformation coincides with the formation of alpha-bungarotoxin-binding sites and intrasubunit disulfide bonding, apparently within the alpha7 domain of the alpha7/serotonin-3 chimera. In cell lines of neuronal origin that produce functional alpha7 receptors, alpha7 subunits undergo a conformational change similar to alpha7/serotonin-3 subunits. alpha7 subunits, thus, can fold and assemble by two different pathways. Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, alpha-bungarotoxin-binding receptors with two alpha7 conformations. Our results suggest that alpha7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.

Show MeSH
Related in: MedlinePlus