Detection of Functional Nicotinic Receptors Blocked by α-Bungarotoxin on PC12 Cells and Dependence of Their Expression on Post-Translational Events

Abstract

A major class of nicotinic receptors in the nervous system is one that binds ␣-bungarotoxin and contains the ␣7 gene product. PC12 cells, frequently used to study nicotinic receptors, express the ␣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 ␣-bungarotoxin. The current amplitude varies dramatically among three populations of PC12 cells but correlates well with the number of toxin-binding receptors. In contrast, the current shows no correlation with ␣7 transcript; cells with high levels of ␣7 mRNA can be negative for toxin binding and yet have other functional nicotinic receptors. Northern blot analysis and reverse transcription-PCR reveal no defects in ␣7 RNA from the negative cells, and immunoblot analysis demonstrates that they contain full-length ␣7 protein, although at reduced levels. Affinity purification of toxin-binding receptors from cells expressing them confirms that the receptors contain ␣7 protein. Transfection experiments demonstrate that PC12 cells lacking native toxin-binding receptors are deficient at producing receptors from ␣7 gene constructs, although the same cells can produce receptors from other transfected gene constructs. The results indicate that nicotinic receptors that bind ␣-bungarotoxin and contain ␣7 subunits require additional gene products to facilitate assembly and stabilization of the receptors. PC12 cells offer a model system for identifying those gene products.

T Cells Express α7-Nicotinic Acetylcholine Receptor Subunits That Require a Functional TCR and Leukocyte-Specific Protein Tyrosine Kinase for Nicotine-Induced Ca2+ Response

“…Given that T cells express ␣7-nAChR subunits essentially identical to the neuronal receptor, failure to detect a functional Ca 2ϩ -permeating channel is surprising. However, even in some PC12 cells and TsA201 cells transfected with ␣7-nAChR cDNA, the presence of full-length ␣7-nAChR mRNA transcripts and membrane-localized ␣7-nAChRs was not sufficient to generate a functional nicotine-sensitive ligand-gated Ca 2ϩ response (31,34). RIC-3, a chaperon protein, has recently been implicated in the functional association of ␣7-nAChRs (54).…”

“…It is generally assumed that nAChRs on all cell types, including those on nonneuronal cells, are cation channels (28,30), but no electrophysiological evidence supports the presence of nicotine-sensitive, ligand-gated cation channels on T cells. Recent evidence suggests that ␣7-nAChRs on some neuronal cells fail to raise nicotine-induced [Ca 2ϩ ] i (31), and in astrocytes the nicotine-induced rise in [Ca 2ϩ ] i is amplified by Ca 2ϩ -induced Ca 2ϩ influx (32,33).…”

Regulation by cycloheximide and lowered temperature of cell‐surface α7‐nicotinic acetylcholine receptor expression on transfected SH‐EP1 cells

“…Surface expression of homomeric nAChR has been shown to be host-cell and even cell-isolate dependent (Blumenthal et al 1997;Cooper and Millar 1997;Cooper and Millar 1998;Aztiria et al 2000;Sweileh et al 2000). An interesting question not addressed here is whether surface expression could be achieved at all using lowered temperature or CHX in transfected cell lines or isolates that exhibit no a7-nAChR surface expression.…”

“…This is consistent with studies using Xenopus oocytes, but not with studies employing a7-nAChR-expressing mammalian or avian cells. In ciliary ganglia and PC12 cells, which express a7-nAChRs endogenously, and in exogenously a7-nAChR-expressing chick fibroblasts, CsA had no effect on receptor surface expression (Blumenthal et al 1997;Kassner and Berg 1997). One possibility to explain this discrepancy is cell-type dependent redundancy in the chaperones or other regulatory molecules needed to achieve surface expression.…”

Desensitization of Catecholamine Release

“…Because homomeric ␣7 receptors also desensitize, it is likely that the ␤ subunit plays a modulatory rather than a permissive role in desensitization (28). ␣3, ␣5, ␣7, ␤2, and ␤4 nicotinic subunit expression is detectable in PC12 cells (4,5,31), although the precise subunit stoichiometry of any given nicotinic heteropentamer may be difficult to establish. Lack of effect of ␣-bungarotoxin on nicotinic-stimulated PC12 cell catecholamine release weighs against a role for ␣7 subunits in the secretion we observed (23).…”