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J Neurochem. 1996 Jul;67(1):145-54.

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

Author information

1
Department of Pharmacology, McGill University, Montreal, Quebec, Canada.

Abstract

The present results demonstrate stable expression of alpha-bungarotoxin (alpha-BGT) binding sites by cells of the GH4C1 rat pituitary clonal line. Wild-type GH4C1 cells do not express alpha-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor alpha2, alpha3, alpha4, alpha5, alpha7, beta2, or beta3 subunits. However, GH4C1 cells stably transfected with rat nicotinic receptor alpha7 cDNA (alpha7/GH4C1 cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of teh predicted size. The alpha7/GH4C1 cells also express saturable, high-affinity binding sites for 125I-labeled alpha-BGT, with a KD of 0.4 nM and Bmax 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 alpha7/GH4C1 cells. Furthermore, KD and Ki values for 125I-alpha-BGT binding sites on intact alpha7/GH4C1 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 alpha7/GH4C1 cells was similar to that of the native brain alpha-BGT receptor. Chronic exposure of alpha7/GH4C1 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 alpha7/GH4C1 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 alpha7 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 alpha7 plus some other previously identified or novel subunit(s), the data would indicate that the alpha7 subunits play a major role in determining properties of the alpha-BGT receptor.

[Indexed for MEDLINE]

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