Similarity between rat brain nicotinic alpha-bungarotoxin receptors and stably expressed alpha-bungarotoxin binding sites

Quik, M.; Choremis, J.; Komourian, J.; Lukas, R.J.; Puchacz, E.

Journal of Neurochemistry 67(1): 145-154


ISSN/ISBN: 0022-3042
PMID: 8666985
DOI: 10.1046/j.1471-4159.1996.67010145.x
Accession: 009417214

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The present results demonstrate stable expression of alpha-bungarotoxin (alpha-BGT) binding sites by cells of the GH-4C-1 rat pituitary clonal line. Wild-type GH-4C-1 cells do not express alpha-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor alpha-2, alpha-3, alpha-4, alpha-5, alpha-7, beta-2, or beta-3 subunits. However, GH-4C-1 cells stably transfected with rat nicotinic receptor alpha-7 cDNA (alpha-7/GH-4C-1 cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The alpha-7/GH-4C-1 cells also express saturable, high-affinity binding sites for 125I-labeled alpha-BGT, with a K-D of 0.4 nM and B-max of 3.2 fmol/10-6 intact cells. 125I-alpha-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or alpha-7/GH-4C-1 cells. Furthermore, K-D and K-i values for 125I-alpha-BGT binding sites on intact alpha-7/GH-4C-1 cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the alpha-BGT binding sites expressed in alpha-7/GH-4C-1 cells was similar to that of the native brain alpha-BGT receptor. Chronic exposure of alpha-7/GH-4C-1 cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of alpha-BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of alpha-BGT binding sites in transfected alpha-7/GH-4C-1 cells resemble those for brain nicotinic alpha-BGT receptors. If the heterologously expressed alpha-BGT binding sites in the present study are composed solely of alpha-7 subunits, the results could suggest that the rat brain alpha-BGT receptor has a similar homooligomeric structure. Alternatively, if alpha-BGT binding sites exist as heterooligomers of alpha-7 plus some other previously identified or novel subunit(s), the data would indicate that the alpha-7 subunits play a major role in determining properties of the alpha-BGT receptor.