@article {Kemp356, author = {G Kemp and M Edge}, title = {Cholinergic function and alpha-bungarotoxin binding in PC12 cells.}, volume = {32}, number = {3}, pages = {356--363}, year = {1987}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {The cell line PC12, derived from an adrenal chromaffin cell tumor, expresses both ganglionic (C6) acetylcholine receptors (nAChR) and an alpha-bungarotoxin (BGT) binding protein of unknown function. We measured nicotinic Na+ fluxes of 180-260 nmol/mg protein X min and 0.35-0.8 pmol [125I]BGT binding sites/mg protein; 45-65\% of the [125I]BGT binding was to intracellular sites. We blocked ganglionic Na+ fluxes with reversible and irreversible inhibitors and tested whether a residual BGT-sensitive flux could be identified. No such flux was detected. These experiments place an upper limit on the amount of an undetected Na+ flux such that we question whether the BGT binding protein could act as a functional nAChR X Na+ flux and [125I]BGT binding were irreversibly inactivated by the affinity-directed antagonist 4-(N-maleimido)benzyltrimethylammonium bromide (MBTA), and the appearance of new nAChRs and BGT binding proteins was monitored. New ganglionic nAChRs appeared at a rate of 0.029 hr-1, corresponding to a steady state turnover t1/2 of 24 hr. BGT binding protein was synthesized more rapidly (K = 0.11 hr-1, t1/2 = 6.5 hr). When protein synthesis was simultaneously blocked with cycloheximide, insertion of BGT binding protein into the plasma membrane decreased to 11\% of control values. Cycloheximide also induced a biphasic decline in intracellular BGT binding sites. Incubation of PC12 cells in 5 mM carbamylcholine for varying intervals resulted in a rapid 30\% loss of Na+ flux activity. In contrast, the concentration of BGT binding protein did not change.}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/32/3/356}, eprint = {https://molpharm.aspetjournals.org/content/32/3/356.full.pdf}, journal = {Molecular Pharmacology} }