DETECTION OF FUNCTIONAL NICOTINIC RECEPTORS BLOCKED BY BUNGAROTOXIN ON PC12 CELLS AND DEPENDENCE OF THEIR EXPRESSION ON POSTTRANSLATIONAL EVENTS

A major class of nicotinic receptors in the nervous system is one that binds alpha-bungarotoxin and contains the alpha 7 gene product. PC12 cells, frequently used to study nicotinic receptors, express the alpha 7 gene and have binding sites for the toxin, but previous attempts to elicit currents from the putative receptors have failed. Using whole- cell patch-clamp recording techniques and rapid application of agonist , we find a rapidly desensitizing acetylcholine-induced current in the cells that can be blocked by alpha-bungarotoxin. The current amplitud e varies dramatically among three populations of PC12 cells but correl ates well with the number of toxin-binding receptors. In contrast, the current shows no correlation with alpha 7 transcript; cells with high levels of alpha 7 mRNA can be negative for toxin binding and yet have other functional nicotinic receptors. Northern blot analysis and reve rse transcription-PCR reveal no defects in alpha 7 RNA from the negati ve cells, and immunoblot analysis demonstrates that they contain full- length alpha 7 protein, although at reduced levels. Affinity purificat ion of toxin-binding receptors from cells expressing them confirms tha t the receptors contain alpha 7 protein. Transfection experiments demo nstrate that PC12 cells lacking native toxin-binding receptors are def icient at producing receptors from alpha 7 gene constructs, although t he same cells can produce receptors from other transfected gene constr ucts. The results indicate that nicotinic receptors that bind alpha-bu ngarotoxin and contain alpha 7 subunits require additional gene produc ts to facilitate assembly and stabilization of the receptors. PC12 cel ls offer a model system for identifying those gene products.