Received for publication December 1, 2004.
Revised February 17, 2005.
Accepted for publication March 16, 2005.

Expression of Native 34* Neuronal Nicotinic Receptors: Binding and Functional Studies Investigating Turnover of Surface and Intracellular Receptor Populations

Abstract

Several pathological conditions involve alterations in expression of neuronal nicotinic acetylcholine receptors (nAChRs). While some studies have addressed processes involved with muscle nAChR expression, knowledge of the regulation of neuronal nAChRs is particularly sparse. The following studies were designed to investigate cellular mechanisms involved with expression of neuronal 34* nAChRs. Catecholamine secretion assays and receptor binding studies coupled with receptor alkylation were used to study the nAChR regulation and turnover. Alkylation of adrenal nAChRs results in a rapid and complete loss of receptor-mediated neurosecretion and surface [³H]epibatidine binding sites. Following alkylation both neurosecretory function and nAChR binding slowly (24-48 hrs) return to pre-alkylation levels. When cells are treated with the protein synthesis inhibitor puromycin, after alkylation, receptor-mediated neurosecretion does not recover. Chronic (24-48 hrs) puromycin treatment, in the absence of alkylation, results in a slow, time-dependent shift to the right, followed by a downward shift, in the nicotine concentration-response curve, documenting a disappearance of surface nAChRs. Puromycin treatment alone also results in a loss to both surface and intracellular [³H]epibatidine binding sites. nAChR 4 subunit levels are significantly decreased following treatment with puromycin. These data support a constitutive turnover of adrenal 34* nAChRs, requiring continual de novo synthesis of new receptor protein.

Key words: Nicotinic cholinergic, Ion channel regulation, Receptor binding studies, Protein synthesis inhibitors