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Functional properties of human nicotinic AChRs expressed by IMR-32 neuroblastoma cells resemble those of alpha3beta4 AChRs expressed in permanently transfected HEK cells.

Nelson ME, Wang F, Kuryatov A, Choi CH, Gerzanich V, Lindstrom J - J. Gen. Physiol. (2001)

Bottom Line: Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells.Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells.The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1).

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, PA 19104, USA.

ABSTRACT
We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human alpha3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express alpha3, alpha5, alpha7, beta2, and beta4 AChR subunits. From these subunits, several types of alpha3 AChRs as well as homomeric alpha7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect alpha3beta4 AChRs. alpha7 AChR function was not detected, yet we estimate that there are 70% as many surface alpha7 AChRs in IMR-32 when compared with alpha3 AChRs. Agonist potencies (EC(50) values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16+/-1 microM) > nicotine (Nic; 48 +/- 7 microM) > or = cytisine (Cyt; 57 +/- 3 microM) = acetylcholine (ACh; 59 +/- 6 microM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s(-1) (300 microM ACh; -60 mV). Assays that used mAbs confirmed the predominance of alpha3- and beta4-containing AChRs in IMR-32 cells. Although 18% of total alpha3 AChRs contained beta2 subunits, no beta2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface alpha3beta2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells. Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC(50) values) for this line was DMPP (14 +/- 1 microM) = Cyt (18 +/- 1 microM) > Nic (56 +/- 15 microM > ACh (79 +/- 8 microM). The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1). These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to alpha3beta4 AChRs.

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Confocal microscopy of α3 AChRs expressed by IMR-32 neuroblastoma cells and HEK cells transfected with α3β4 and α3β2 AChRs. Cells were plated, fixed, permeabilized, and labeled as described in materials and methods. For each cell type, green reflects labeling of α3 subunits by Alexa 488-labeled mAb 210. For IMR-32 cells and α3β4 cells, red reflects secondary labeling by Alexa 568-labeled GAM antibody against mAb 337 which bound β4 subunits. For α3β2 cells, red reflects secondary labeling by Alexa 594-labeled GART antibody against mAb 295 which bound β2 subunits. Yellow is a result of overlap in the labeling of colocalized subunits. For all cells, ToPro-3 iodide (blue) was included as a nuclear counterstain. The scale bars in the α3β4 panel represent 1, 2, 5, and 10 μm, respectively.
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Figure 11: Confocal microscopy of α3 AChRs expressed by IMR-32 neuroblastoma cells and HEK cells transfected with α3β4 and α3β2 AChRs. Cells were plated, fixed, permeabilized, and labeled as described in materials and methods. For each cell type, green reflects labeling of α3 subunits by Alexa 488-labeled mAb 210. For IMR-32 cells and α3β4 cells, red reflects secondary labeling by Alexa 568-labeled GAM antibody against mAb 337 which bound β4 subunits. For α3β2 cells, red reflects secondary labeling by Alexa 594-labeled GART antibody against mAb 295 which bound β2 subunits. Yellow is a result of overlap in the labeling of colocalized subunits. For all cells, ToPro-3 iodide (blue) was included as a nuclear counterstain. The scale bars in the α3β4 panel represent 1, 2, 5, and 10 μm, respectively.

Mentions: Confocal microscopy was used to visualize the distribution of α3 AChRs in IMR-32 neuroblastoma cells and the α3β2 or the α3β4 transfected cells. For the transfected cells, intense, clustered labeling was observed with antibody directed against the α3 subunit. The labeling of α3 largely overlapped with the labeling observed with antibody directed against the β4 subunit in the α3β4 cells and the β2 subunit in the α3β2 cells (Fig. 11). In the case of IMR-32 cells, the labeling of α3 subunit was also extensive and appeared in clusters that overlapped with the labeling observed for antibody directed at the β4 subunit (Fig. 11). Since mAb 337 (to β4) targets a cytoplasmic epitope, all images are shown for permeabilized cells. The clustered appearance of the α3 label was also observed on nonpermeabilized cells, indicating that surface AChRs were expressed in concentrated densities (unpublished data). The α3β4 cells seem to have the largest size clusters, which probably reflects the fact that these cells express the greatest levels of AChR (Wang et al. 1998).


Functional properties of human nicotinic AChRs expressed by IMR-32 neuroblastoma cells resemble those of alpha3beta4 AChRs expressed in permanently transfected HEK cells.

Nelson ME, Wang F, Kuryatov A, Choi CH, Gerzanich V, Lindstrom J - J. Gen. Physiol. (2001)

Confocal microscopy of α3 AChRs expressed by IMR-32 neuroblastoma cells and HEK cells transfected with α3β4 and α3β2 AChRs. Cells were plated, fixed, permeabilized, and labeled as described in materials and methods. For each cell type, green reflects labeling of α3 subunits by Alexa 488-labeled mAb 210. For IMR-32 cells and α3β4 cells, red reflects secondary labeling by Alexa 568-labeled GAM antibody against mAb 337 which bound β4 subunits. For α3β2 cells, red reflects secondary labeling by Alexa 594-labeled GART antibody against mAb 295 which bound β2 subunits. Yellow is a result of overlap in the labeling of colocalized subunits. For all cells, ToPro-3 iodide (blue) was included as a nuclear counterstain. The scale bars in the α3β4 panel represent 1, 2, 5, and 10 μm, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233843&req=5

Figure 11: Confocal microscopy of α3 AChRs expressed by IMR-32 neuroblastoma cells and HEK cells transfected with α3β4 and α3β2 AChRs. Cells were plated, fixed, permeabilized, and labeled as described in materials and methods. For each cell type, green reflects labeling of α3 subunits by Alexa 488-labeled mAb 210. For IMR-32 cells and α3β4 cells, red reflects secondary labeling by Alexa 568-labeled GAM antibody against mAb 337 which bound β4 subunits. For α3β2 cells, red reflects secondary labeling by Alexa 594-labeled GART antibody against mAb 295 which bound β2 subunits. Yellow is a result of overlap in the labeling of colocalized subunits. For all cells, ToPro-3 iodide (blue) was included as a nuclear counterstain. The scale bars in the α3β4 panel represent 1, 2, 5, and 10 μm, respectively.
Mentions: Confocal microscopy was used to visualize the distribution of α3 AChRs in IMR-32 neuroblastoma cells and the α3β2 or the α3β4 transfected cells. For the transfected cells, intense, clustered labeling was observed with antibody directed against the α3 subunit. The labeling of α3 largely overlapped with the labeling observed with antibody directed against the β4 subunit in the α3β4 cells and the β2 subunit in the α3β2 cells (Fig. 11). In the case of IMR-32 cells, the labeling of α3 subunit was also extensive and appeared in clusters that overlapped with the labeling observed for antibody directed at the β4 subunit (Fig. 11). Since mAb 337 (to β4) targets a cytoplasmic epitope, all images are shown for permeabilized cells. The clustered appearance of the α3 label was also observed on nonpermeabilized cells, indicating that surface AChRs were expressed in concentrated densities (unpublished data). The α3β4 cells seem to have the largest size clusters, which probably reflects the fact that these cells express the greatest levels of AChR (Wang et al. 1998).

Bottom Line: Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells.Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells.The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1).

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, PA 19104, USA.

ABSTRACT
We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human alpha3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express alpha3, alpha5, alpha7, beta2, and beta4 AChR subunits. From these subunits, several types of alpha3 AChRs as well as homomeric alpha7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect alpha3beta4 AChRs. alpha7 AChR function was not detected, yet we estimate that there are 70% as many surface alpha7 AChRs in IMR-32 when compared with alpha3 AChRs. Agonist potencies (EC(50) values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16+/-1 microM) > nicotine (Nic; 48 +/- 7 microM) > or = cytisine (Cyt; 57 +/- 3 microM) = acetylcholine (ACh; 59 +/- 6 microM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s(-1) (300 microM ACh; -60 mV). Assays that used mAbs confirmed the predominance of alpha3- and beta4-containing AChRs in IMR-32 cells. Although 18% of total alpha3 AChRs contained beta2 subunits, no beta2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface alpha3beta2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells. Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC(50) values) for this line was DMPP (14 +/- 1 microM) = Cyt (18 +/- 1 microM) > Nic (56 +/- 15 microM > ACh (79 +/- 8 microM). The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1). These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to alpha3beta4 AChRs.

Show MeSH
Related in: MedlinePlus