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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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Mentions: PC12 and SH-SY5Y cell lines were used to look at the processing of α7 subunits in cells that produce functional, Bgt-binding α7 receptors. The PC12 cells have endogenous Bgt-binding receptors that contain α7 subunits (Blumenthal et al. 1997; Rangwala et al. 1997) whereas the SH-SY5Y cells stably express rat α7 subunits in addition to endogenous α7 subunits (Peng et al. 1994; Puchacz et al. 1994). The α7 subunits in these cell lines lack the HA epitope and the level of subunit synthesis is not as high as that achieved by transient transfection. These differences prevented us from characterizing α7 subunits in these cell lines to the same degree as the α7-HA and α7/5HT3-HA subunits in tsA201 cells. Nonetheless, we were able to precipitate α7 subunits from SH-SY5Y and PC12 cells using Bgt-Sepharose and performed Western blot analysis on the α7 subunits from both cell lines (Fig. 6b and Fig. c). The processing of α7 subunits in SH-SY5Y and PC12 cells was different from that of α7 subunits in tsA201 cells. In the absence of NEM alkylation, many α7 subunits in these cells migrated as monomers on a nonreducing gel (Fig. 6b and Fig. c, lane 1). Such monomers were never observed on nonreducing gels with the expression of the α7-HA subunits in tsA201 cells. In addition to the monomers, there were subunit aggregates and multimers. Like Bgt-binding α7/5HT3 subunits in tsA201 cells (Fig. 3 D), the α7 subunit multimers, precipitated with Bgt-affinity resin, were dispersed by NEM alkylation and reduction (Fig. 6b and Fig. c, lane 2). Identical results were found for cell-surface PC12 BgtRs specifically isolated by binding Bgt to intact cells and immunoprecipitating the Bgt-bound receptors with anti-Bgt antibodies (Fig. 6 C, lanes 4 and 5).


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)

© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199736&req=5

Mentions: PC12 and SH-SY5Y cell lines were used to look at the processing of α7 subunits in cells that produce functional, Bgt-binding α7 receptors. The PC12 cells have endogenous Bgt-binding receptors that contain α7 subunits (Blumenthal et al. 1997; Rangwala et al. 1997) whereas the SH-SY5Y cells stably express rat α7 subunits in addition to endogenous α7 subunits (Peng et al. 1994; Puchacz et al. 1994). The α7 subunits in these cell lines lack the HA epitope and the level of subunit synthesis is not as high as that achieved by transient transfection. These differences prevented us from characterizing α7 subunits in these cell lines to the same degree as the α7-HA and α7/5HT3-HA subunits in tsA201 cells. Nonetheless, we were able to precipitate α7 subunits from SH-SY5Y and PC12 cells using Bgt-Sepharose and performed Western blot analysis on the α7 subunits from both cell lines (Fig. 6b and Fig. c). The processing of α7 subunits in SH-SY5Y and PC12 cells was different from that of α7 subunits in tsA201 cells. In the absence of NEM alkylation, many α7 subunits in these cells migrated as monomers on a nonreducing gel (Fig. 6b and Fig. c, lane 1). Such monomers were never observed on nonreducing gels with the expression of the α7-HA subunits in tsA201 cells. In addition to the monomers, there were subunit aggregates and multimers. Like Bgt-binding α7/5HT3 subunits in tsA201 cells (Fig. 3 D), the α7 subunit multimers, precipitated with Bgt-affinity resin, were dispersed by NEM alkylation and reduction (Fig. 6b and Fig. c, lane 2). Identical results were found for cell-surface PC12 BgtRs specifically isolated by binding Bgt to intact cells and immunoprecipitating the Bgt-bound receptors with anti-Bgt antibodies (Fig. 6 C, lanes 4 and 5).

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