Similarity Between Rat Brain Nicotinic Î±-Bungarotoxin Receptors And Stably Expressed Î±-Bungarotoxin Binding Sites
The present results demonstrate stable expression of Î±-bungarotoxin (Î±- BGT) binding sites by cells of the GH4C1 rat pituitary clonal line. Wild- type GH4C1 cells do not express Î±-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor Î±2, Î±3, Î±4, Î±5, Î±7, Î²2, or Î²3 subunits. However, GH4C1 cells stably transfected with rat nicotinic receptor Î±7 cDNA (Î±7/GH4C1 cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The Î±7/GH4C1 cells also express saturable, high-affinity binding sites for 125I-labeled Î±-BGT, with a K(D) of 0.4 nM and B(max) of 3.2 fmol/106 intact cells. 125I-Î±-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or Î±7/GH4C1 cells. Furthermore, K(D) and K(i) values for 125I-Î±-BGT binding sites on intact Î±7/GH4C1 cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the Î±-BGT binding sites expressed in Î±7/GH4C1 cells was similar to that of the native brain Î±-BGT receptor. Chronic exposure of Î±7/GH4C1 cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of Î±- BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of Î±-BGT binding sites in transfected Î±7/GH4C1 cells resemble those for brain nicotinic Î±- BGT receptors. If the heterologously expressed Î±-BGT binding sites in the present study are composed solely of Î±7 subunits, the results could suggest that the rat brain Î±-BGT receptor has a similar homooligomeric structure. Alternatively, if Î±-BGT binding sites exist as heterooligomers of Î±7 plus some other previously identified or novel subunit(s), the data would indicate that the Î±7 subunits play a major role in determining properties of the Î±- BGT receptor.
Journal of Neurochemistry
Quik, M.; Choremis, J.; Komourian, J.; Lukas, R. J.; and Puchacz, E., "Similarity Between Rat Brain Nicotinic Î±-Bungarotoxin Receptors And Stably Expressed Î±-Bungarotoxin Binding Sites" (1996). Neurobiology. 275.