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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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Disulfide bonding is required to effect a change in α7/5HT3 subunit redox state. (A and B) Time-dependent change in α7/5HT3-HA subunit redox state. TsA201 cells transfected with α7-HA (A) or α7/5HT3-HA (B) cDNAs were metabolically labeled for 10 min and chased for the times specified in the figure. The cells were solubilized in the absence of NEM, labeled subunits immunoprecipitated with anti-HA mAb, and samples loaded on the gels without (nonreducing) or with (reducing) 10 mM DTT. A sample from sham-transfected cells (no DNA) was run in lane 1 of each of the four gels. Arrows on the right of the figure indicate positions of putative monomer, dimer, trimer, tetramer, and pentamer α7/5HT3-HA subunit complexes. (C) Addition of 5 mM DTT to the cell medium blocks the redox state change. TsA201 cells transfected with α7/5HT3-HA cDNA were metabolically labeled for 10 min and chased for the times specified in the figure in the absence (left) or presence (right) of 5 mM DTT in the medium. Cells were solubilized in the absence of NEM and labeled subunits precipitated with anti-HA mAb. 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.
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Figure 4: Disulfide bonding is required to effect a change in α7/5HT3 subunit redox state. (A and B) Time-dependent change in α7/5HT3-HA subunit redox state. TsA201 cells transfected with α7-HA (A) or α7/5HT3-HA (B) cDNAs were metabolically labeled for 10 min and chased for the times specified in the figure. The cells were solubilized in the absence of NEM, labeled subunits immunoprecipitated with anti-HA mAb, and samples loaded on the gels without (nonreducing) or with (reducing) 10 mM DTT. A sample from sham-transfected cells (no DNA) was run in lane 1 of each of the four gels. Arrows on the right of the figure indicate positions of putative monomer, dimer, trimer, tetramer, and pentamer α7/5HT3-HA subunit complexes. (C) Addition of 5 mM DTT to the cell medium blocks the redox state change. TsA201 cells transfected with α7/5HT3-HA cDNA were metabolically labeled for 10 min and chased for the times specified in the figure in the absence (left) or presence (right) of 5 mM DTT in the medium. Cells were solubilized in the absence of NEM and labeled subunits precipitated with anti-HA mAb. 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.

Mentions: In light of earlier findings that expression of α7 subunits does not produce Bgt-binding sites, it was surprising that large numbers of the cells transfected with the α7-HA subunit stained positive with the anti-HA mAb (Fig. 1 B). As expected, no cells stained positive with TMR-Bgt. Thus, α7-HA subunits on the surface of these cells lack Bgt-binding sites. The relative amounts of cell-surface α7-HA and α7/5HT3-HA subunits were determined using the anti-HA mAb and 125I-protein A. Fig. 1 C illustrates that α7/5HT3-HA subunit expression was sixfold higher than α7-HA subunit expression. The reason for the difference in the levels of surface expression is unclear, but it is not caused by differences in rates of α7-HA and α7/5HT3-HA subunit synthesis (see Fig. 3 and Fig. 4).


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)

Disulfide bonding is required to effect a change in α7/5HT3 subunit redox state. (A and B) Time-dependent change in α7/5HT3-HA subunit redox state. TsA201 cells transfected with α7-HA (A) or α7/5HT3-HA (B) cDNAs were metabolically labeled for 10 min and chased for the times specified in the figure. The cells were solubilized in the absence of NEM, labeled subunits immunoprecipitated with anti-HA mAb, and samples loaded on the gels without (nonreducing) or with (reducing) 10 mM DTT. A sample from sham-transfected cells (no DNA) was run in lane 1 of each of the four gels. Arrows on the right of the figure indicate positions of putative monomer, dimer, trimer, tetramer, and pentamer α7/5HT3-HA subunit complexes. (C) Addition of 5 mM DTT to the cell medium blocks the redox state change. TsA201 cells transfected with α7/5HT3-HA cDNA were metabolically labeled for 10 min and chased for the times specified in the figure in the absence (left) or presence (right) of 5 mM DTT in the medium. Cells were solubilized in the absence of NEM and labeled subunits precipitated with anti-HA mAb. 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.
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Figure 4: Disulfide bonding is required to effect a change in α7/5HT3 subunit redox state. (A and B) Time-dependent change in α7/5HT3-HA subunit redox state. TsA201 cells transfected with α7-HA (A) or α7/5HT3-HA (B) cDNAs were metabolically labeled for 10 min and chased for the times specified in the figure. The cells were solubilized in the absence of NEM, labeled subunits immunoprecipitated with anti-HA mAb, and samples loaded on the gels without (nonreducing) or with (reducing) 10 mM DTT. A sample from sham-transfected cells (no DNA) was run in lane 1 of each of the four gels. Arrows on the right of the figure indicate positions of putative monomer, dimer, trimer, tetramer, and pentamer α7/5HT3-HA subunit complexes. (C) Addition of 5 mM DTT to the cell medium blocks the redox state change. TsA201 cells transfected with α7/5HT3-HA cDNA were metabolically labeled for 10 min and chased for the times specified in the figure in the absence (left) or presence (right) of 5 mM DTT in the medium. Cells were solubilized in the absence of NEM and labeled subunits precipitated with anti-HA mAb. 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.
Mentions: In light of earlier findings that expression of α7 subunits does not produce Bgt-binding sites, it was surprising that large numbers of the cells transfected with the α7-HA subunit stained positive with the anti-HA mAb (Fig. 1 B). As expected, no cells stained positive with TMR-Bgt. Thus, α7-HA subunits on the surface of these cells lack Bgt-binding sites. The relative amounts of cell-surface α7-HA and α7/5HT3-HA subunits were determined using the anti-HA mAb and 125I-protein A. Fig. 1 C illustrates that α7/5HT3-HA subunit expression was sixfold higher than α7-HA subunit expression. The reason for the difference in the levels of surface expression is unclear, but it is not caused by differences in rates of α7-HA and α7/5HT3-HA subunit synthesis (see Fig. 3 and Fig. 4).

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