Similarity Between Rat Brain Nicotinic α‐Bungarotoxin Receptors and Stably Expressed α‐Bungarotoxin Binding Sites

: The present results demonstrate stable expression of α‐bungarotoxin (α‐BGT) binding sites by cells of the GH4C1 rat pituitary clonal line. Wild‐type GH4C1 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, GH4C1 cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH4C1 cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH4C1 cells also express saturable, high‐affinity binding sites for 125I‐labeled α‐BGT, with a KD of 0.4 nM and Bmax of 3.2 fmol/106 intact cells. 125I‐α‐BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH4C1 cells. Furthermore, KD and Ki values for 125I‐α‐BGT binding sites on intact α7/GH4C1 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/GH4C1 cells was similar to that of the native brain α‐BGT receptor. Chronic exposure of α7/GH4C1 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/GH4C1 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.