Cholinergic function and alpha-bungarotoxin binding in PC12 cells.
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.
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D004079:Dihydro-beta-Erythroidine / Q000494:pharmacology; D018738:Hexamethonium; D006584:Hexamethonium Compounds / Q000494:pharmacology; D008024:Ligands; D008464:Mecamylamine / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D011950:Receptors, Cholinergic / Q000502:physiology*; D011978:Receptors, Nicotinic; D012964:Sodium / Q000378:metabolism; D014403:Tubocurarine / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002038:Bungarotoxins; D006584:Hexamethonium Compounds; D008024:Ligands; D000644:Quaternary Ammonium Compounds; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D004079:Dihydro-beta-Erythroidine; D018738:Hexamethonium; C008313:4-(N-maleimido)benzyltrimethylammonium; D008464:Mecamylamine; D002217:Carbachol; D012964:Sodium; D014403:Tubocurarine