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Similarity Between Rat Brain Nicotinic α‐Bungarotoxin Receptors and Stably Expressed α‐Bungarotoxin Binding Sites
Author(s) -
Quik M.,
Choremis J.,
Komourian J.,
Lukas R. J.,
Puchacz E.
Publication year - 1996
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1996.67010145.x
Subject(s) - bungarotoxin , nicotinic agonist , ganglion type nicotinic receptor , receptor , alpha 4 beta 2 nicotinic receptor , chemistry , neuroscience , biology , biochemistry , nicotinic acetylcholine receptor
The present results demonstrate stable expression of α‐bungarotoxin (α‐BGT) binding sites by cells of the GH 4 C 1 rat pituitary clonal line. Wild‐type GH 4 C 1 cells do not express α‐BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor α2, α3, α4, α5, α7, β2, or β3 subunits. However, GH 4 C 1 cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH 4 C 1 cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH 4 C 1 cells also express saturable, high‐affinity binding sites for 125 I‐labeled α‐BGT, with a K D of 0.4 n M and B max of 3.2 fmol/10 6 intact cells. 125 I‐α‐BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH 4 C 1 cells. Furthermore, K D and K i values for 125 I‐α‐BGT binding sites on intact α7/GH 4 C 1 cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the α‐BGT binding sites expressed in α7/GH 4 C 1 cells was similar to that of the native brain α‐BGT receptor. Chronic exposure of α7/GH 4 C 1 cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of α‐BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of α‐BGT binding sites in transfected α7/GH 4 C 1 cells resemble those for brain nicotinic α‐BGT receptors. If the heterologously expressed α‐BGT binding sites in the present study are composed solely of α7 subunits, the results could suggest that the rat brain α‐BGT receptor has a similar homooligomeric structure. Alternatively, if α‐BGT binding sites exist as heterooligomers of α7 plus some other previously identified or novel subunit(s), the data would indicate that the α7 subunits play a major role in determining properties of the α‐BGT receptor.