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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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Bgt-binding site formation and the change in redox state. (A) Formation of Bgt-binding site correlates with the change in redox state. TsA201 cells transfected with α7/5HT3-HA cDNA were metabolically labeled for 10 min and chased for the times specified in the figure. The cells were solubilized in the absence of NEM and labeled subunits precipitated with anti-HA mAb (lanes 2–7 on the left) or Bgt-Sepharose (lanes 2–7 on the right). Bgt-Sepharose appeared not to precipitate all of the α7/5HT3 subunit monomers precipitated by anti-HA mAb. It is possible that not all α7/5HT3 subunit monomers assemble into Bgt-binding complexes or, alternatively, Bgt-Sepharose fails to quantitatively precipitate all Bgt-binding sites. All samples were loaded on the gels without DTT, and a sample from sham-transfected cells (no DNA) was run in lane 1 (left and right). Arrows on the right of the figure indicate positions of putative monomer, dimer, trimer, tetramer, and pentamer α7/5HT3-HA subunit complexes. (B) Addition of 5 mM DTT to the cell medium blocks Bgt-binding site formation. The experiment was performed as in Fig. 4 C except that labeled subunits were precipitated with Bgt-Sepharose.
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Figure 5: Bgt-binding site formation and the change in redox state. (A) Formation of Bgt-binding site correlates with the change in redox state. TsA201 cells transfected with α7/5HT3-HA cDNA were metabolically labeled for 10 min and chased for the times specified in the figure. The cells were solubilized in the absence of NEM and labeled subunits precipitated with anti-HA mAb (lanes 2–7 on the left) or Bgt-Sepharose (lanes 2–7 on the right). Bgt-Sepharose appeared not to precipitate all of the α7/5HT3 subunit monomers precipitated by anti-HA mAb. It is possible that not all α7/5HT3 subunit monomers assemble into Bgt-binding complexes or, alternatively, Bgt-Sepharose fails to quantitatively precipitate all Bgt-binding sites. All samples were loaded on the gels without DTT, and a sample from sham-transfected cells (no DNA) was run in lane 1 (left and right). Arrows on the right of the figure indicate positions of putative monomer, dimer, trimer, tetramer, and pentamer α7/5HT3-HA subunit complexes. (B) Addition of 5 mM DTT to the cell medium blocks Bgt-binding site formation. The experiment was performed as in Fig. 4 C except that labeled subunits were precipitated with Bgt-Sepharose.

Mentions: In addition to aggregates and subunit monomers on the gels, α7 and α7/5HT3 subunits ran as a ladder of higher molecular weight bands where each rung was a multiple of the monomer (Fig. 3A, Fig. C, and Fig. D). Bands corresponding to subunit dimers, trimers, tetramers, and pentamers were observed, but there were no complexes larger than pentamers (see Fig. 4Fig. 5Fig. 6). These data are consistent with α7 subunits complexing into pentamers, similar to α7/5HT3 subunit receptors (Rangwala et al. 1997) and other characterized AChRs (Karlin and Akabas 1995). For α7 subunits, a large number of the multimers was present even on reducing gels or alkylated with NEM, though alkylation did disperse some of the larger multimers (Fig. 3 C). Since most of the α7 multimers remained tightly associated even in the presence of SDS, they must be held together by SDS-resistant associations in addition to any disulfide bonds. α7/5HT3 subunit multimers were also observed and most remained intact on reducing gels or after NEM alkylation (Fig. 3 A and 4 B). These SDS-resistant associations between subunits are similar to those observed for other oligomeric proteins such as K+ channel subunits (Cortes and Perozo 1997). α7/5HT3 subunit multimers, precipitated with Bgt-affinity resin, were dispersed after NEM alkylation and reduction (Fig. 3 D). Thus, there was a loss of the SDS-resistant subunit associations after Bgt-binding sites appeared on α7/5HT3 subunits.


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)

Bgt-binding site formation and the change in redox state. (A) Formation of Bgt-binding site correlates with the change in redox state. TsA201 cells transfected with α7/5HT3-HA cDNA were metabolically labeled for 10 min and chased for the times specified in the figure. The cells were solubilized in the absence of NEM and labeled subunits precipitated with anti-HA mAb (lanes 2–7 on the left) or Bgt-Sepharose (lanes 2–7 on the right). Bgt-Sepharose appeared not to precipitate all of the α7/5HT3 subunit monomers precipitated by anti-HA mAb. It is possible that not all α7/5HT3 subunit monomers assemble into Bgt-binding complexes or, alternatively, Bgt-Sepharose fails to quantitatively precipitate all Bgt-binding sites. All samples were loaded on the gels without DTT, and a sample from sham-transfected cells (no DNA) was run in lane 1 (left and right). Arrows on the right of the figure indicate positions of putative monomer, dimer, trimer, tetramer, and pentamer α7/5HT3-HA subunit complexes. (B) Addition of 5 mM DTT to the cell medium blocks Bgt-binding site formation. The experiment was performed as in Fig. 4 C except that labeled subunits were precipitated with Bgt-Sepharose.
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Related In: Results  -  Collection

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Figure 5: Bgt-binding site formation and the change in redox state. (A) Formation of Bgt-binding site correlates with the change in redox state. TsA201 cells transfected with α7/5HT3-HA cDNA were metabolically labeled for 10 min and chased for the times specified in the figure. The cells were solubilized in the absence of NEM and labeled subunits precipitated with anti-HA mAb (lanes 2–7 on the left) or Bgt-Sepharose (lanes 2–7 on the right). Bgt-Sepharose appeared not to precipitate all of the α7/5HT3 subunit monomers precipitated by anti-HA mAb. It is possible that not all α7/5HT3 subunit monomers assemble into Bgt-binding complexes or, alternatively, Bgt-Sepharose fails to quantitatively precipitate all Bgt-binding sites. All samples were loaded on the gels without DTT, and a sample from sham-transfected cells (no DNA) was run in lane 1 (left and right). Arrows on the right of the figure indicate positions of putative monomer, dimer, trimer, tetramer, and pentamer α7/5HT3-HA subunit complexes. (B) Addition of 5 mM DTT to the cell medium blocks Bgt-binding site formation. The experiment was performed as in Fig. 4 C except that labeled subunits were precipitated with Bgt-Sepharose.
Mentions: In addition to aggregates and subunit monomers on the gels, α7 and α7/5HT3 subunits ran as a ladder of higher molecular weight bands where each rung was a multiple of the monomer (Fig. 3A, Fig. C, and Fig. D). Bands corresponding to subunit dimers, trimers, tetramers, and pentamers were observed, but there were no complexes larger than pentamers (see Fig. 4Fig. 5Fig. 6). These data are consistent with α7 subunits complexing into pentamers, similar to α7/5HT3 subunit receptors (Rangwala et al. 1997) and other characterized AChRs (Karlin and Akabas 1995). For α7 subunits, a large number of the multimers was present even on reducing gels or alkylated with NEM, though alkylation did disperse some of the larger multimers (Fig. 3 C). Since most of the α7 multimers remained tightly associated even in the presence of SDS, they must be held together by SDS-resistant associations in addition to any disulfide bonds. α7/5HT3 subunit multimers were also observed and most remained intact on reducing gels or after NEM alkylation (Fig. 3 A and 4 B). These SDS-resistant associations between subunits are similar to those observed for other oligomeric proteins such as K+ channel subunits (Cortes and Perozo 1997). α7/5HT3 subunit multimers, precipitated with Bgt-affinity resin, were dispersed after NEM alkylation and reduction (Fig. 3 D). Thus, there was a loss of the SDS-resistant subunit associations after Bgt-binding sites appeared on α7/5HT3 subunits.

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