M. Quik et al., SIMILARITY BETWEEN RAT-BRAIN NICOTINIC ALPHA-BUNGAROTOXIN RECEPTORS AND STABLY EXPRESSED ALPHA-BUNGAROTOXIN BINDING-SITES, Journal of neurochemistry, 67(1), 1996, pp. 145-154
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.