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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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α7 subunit complexes are nonfunctional. (A) Whole-cell response to nicotine for cells expressing α7/5HT3-HA subunits. α7-5HT3-HA transfected cells were identified by positive staining with anti-HA mAb, and whole cell patch clamp recordings showed functional nicotine-gated currents at a potential of −70 mV (100 μM nicotine; similar responses were seen in 15 of 15 cells). No responses were seen in cells that were not positively stained with the anti-HA mAb . (B) Whole-cell response to nicotine for cells expressing α7-HA subunits. Transfection with α7-HA results in positive staining of live cells, but no inward currents were seen at holding potentials of −70 mV. In total, 11 cells were exposed to a 10-fold higher nicotine concentration (1 mM). Except for their response to nicotine, cells expressing α7-HA subunits appeared to be identical to cells expressing α7/5HT3-HA subunits.
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Figure 2: α7 subunit complexes are nonfunctional. (A) Whole-cell response to nicotine for cells expressing α7/5HT3-HA subunits. α7-5HT3-HA transfected cells were identified by positive staining with anti-HA mAb, and whole cell patch clamp recordings showed functional nicotine-gated currents at a potential of −70 mV (100 μM nicotine; similar responses were seen in 15 of 15 cells). No responses were seen in cells that were not positively stained with the anti-HA mAb . (B) Whole-cell response to nicotine for cells expressing α7-HA subunits. Transfection with α7-HA results in positive staining of live cells, but no inward currents were seen at holding potentials of −70 mV. In total, 11 cells were exposed to a 10-fold higher nicotine concentration (1 mM). Except for their response to nicotine, cells expressing α7-HA subunits appeared to be identical to cells expressing α7/5HT3-HA subunits.

Mentions: Since significant numbers of α7 subunits are transported to the surface of transfected cells, we examined whether these subunits form functional receptors. We tested the nicotine sensitivity of tsA201 cells transfected with the α7/5HT3-HA construct and stained with the anti-HA antibody. In whole-cell voltage clamp recordings, 15 of 15 cells responded to nicotine with robust inward currents that desensitized in the continued presence of the agonist, as expected for an AChR-mediated response (Fig. 2 A). In the same cultures, cells that did not stain positive with the anti-HA antibody did not respond to nicotine application (; not shown). In contrast to the cells expressing α7/5HT3-HA, the application of nicotine to α7-HA–transfected cells that stained with anti-HA antibody, did not elicit any response (; Fig. 2 B). These findings indicate that the α7-HA subunit complexes expressed on the surface of these cells do not form functional receptors.


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)

α7 subunit complexes are nonfunctional. (A) Whole-cell response to nicotine for cells expressing α7/5HT3-HA subunits. α7-5HT3-HA transfected cells were identified by positive staining with anti-HA mAb, and whole cell patch clamp recordings showed functional nicotine-gated currents at a potential of −70 mV (100 μM nicotine; similar responses were seen in 15 of 15 cells). No responses were seen in cells that were not positively stained with the anti-HA mAb . (B) Whole-cell response to nicotine for cells expressing α7-HA subunits. Transfection with α7-HA results in positive staining of live cells, but no inward currents were seen at holding potentials of −70 mV. In total, 11 cells were exposed to a 10-fold higher nicotine concentration (1 mM). Except for their response to nicotine, cells expressing α7-HA subunits appeared to be identical to cells expressing α7/5HT3-HA subunits.
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Related In: Results  -  Collection

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Figure 2: α7 subunit complexes are nonfunctional. (A) Whole-cell response to nicotine for cells expressing α7/5HT3-HA subunits. α7-5HT3-HA transfected cells were identified by positive staining with anti-HA mAb, and whole cell patch clamp recordings showed functional nicotine-gated currents at a potential of −70 mV (100 μM nicotine; similar responses were seen in 15 of 15 cells). No responses were seen in cells that were not positively stained with the anti-HA mAb . (B) Whole-cell response to nicotine for cells expressing α7-HA subunits. Transfection with α7-HA results in positive staining of live cells, but no inward currents were seen at holding potentials of −70 mV. In total, 11 cells were exposed to a 10-fold higher nicotine concentration (1 mM). Except for their response to nicotine, cells expressing α7-HA subunits appeared to be identical to cells expressing α7/5HT3-HA subunits.
Mentions: Since significant numbers of α7 subunits are transported to the surface of transfected cells, we examined whether these subunits form functional receptors. We tested the nicotine sensitivity of tsA201 cells transfected with the α7/5HT3-HA construct and stained with the anti-HA antibody. In whole-cell voltage clamp recordings, 15 of 15 cells responded to nicotine with robust inward currents that desensitized in the continued presence of the agonist, as expected for an AChR-mediated response (Fig. 2 A). In the same cultures, cells that did not stain positive with the anti-HA antibody did not respond to nicotine application (; not shown). In contrast to the cells expressing α7/5HT3-HA, the application of nicotine to α7-HA–transfected cells that stained with anti-HA antibody, did not elicit any response (; Fig. 2 B). These findings indicate that the α7-HA subunit complexes expressed on the surface of these cells do not form functional receptors.

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