SIMILARITY BETWEEN RAT-BRAIN NICOTINIC ALPHA-BUNGAROTOXIN RECEPTORS AND STABLY EXPRESSED ALPHA-BUNGAROTOXIN BINDING-SITES

The present results demonstrate stable expression of alpha-bungarotoxi n (alpha-BGT) binding sites by cells of the GH(4)C(1) rat pituitary cl onal line. Wild-type GH(4)C(1) cells do not express alpha-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor alph a 2, alpha 3, alpha 4, alpha 5, alpha 7, beta 2, or beta 3 subunits. H owever, GH(4)C(1) cells stably transfected with rat nicotinic receptor alpha 7 cDNA (alpha 7/GH(4)C(1) cells) express the transgene abundant ly as mRNA, and northern analysis showed that the message is of the pr edicted size, The alpha 7/GH(4)C(1) cells also express saturable, high -affinity binding sites for I-125-labeled alpha-BGT, with a K-D of 0.4 nM and B-max of 3.2 fmol/10(6) intact cells. I-125-alpha-BGT binding affinities and pharmacological profiles are not significantly differen t for sites in membranes prepared either from rat brain or alpha 7/GH( 4)C(1) cells, Furthermore, K-D and K-i values for I-125-alpha-BGT bind ing sites on intact alpha 7/GH(4)C(1) cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient an alysis showed that the size of the alpha-BGT binding sites expressed i n alpha 7/GH(4)C(1) cells was similar to that of the native brain alph a-BGT receptor. Chronic exposure of alpha 7/GH(4)C(1) cells in culture to nicotine or an elevated extracellular potassium concentration indu ces changes in the number of alpha-BGT binding sites comparable to tho se observed in cultured neurons. Collectively, the present results sho w that the properties of alpha-BGT binding sites in transfected alpha 7/GH(4)C(1) cells resemble those for brain nicotinic alpha-BGT recepto rs, If the heterologously expressed alpha-BGT binding sites in the pre sent study are composed solely of alpha 7 subunits, the results could suggest that the rat brain alpha-BGT receptor has a similar homooligom eric structure. Alternatively, if alpha-BGT binding sites exist as het erooligomers 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.