Alpha-Bungarotoxin-sensitive nicotinic receptors on bovine chromaffin cells: molecular cloning, functional expression and alternative splicing of the alpha 7 subunit.

Abstract

Chromaffin cells from the bovine adrenal medulla express alpha-bungarotoxin-sensitive acetylcholine receptors whose subunit composition is unknown. Northern blot analysis showed that the alpha 7 subunit, a main component of these alpha-bungarotoxin-sensitive acetylcholine receptors in avian and rat brain, is expressed in chromaffin cells. The cDNA of this bovine alpha 7 subunit was cloned by polymerase chain reaction amplification of adrenal medulla RNA for detailed characterization of structure and function. The protein-coding region revealed 92% amino acid sequence identity to rat alpha 7 and 89% to chicken alpha 7 subunits. The alpha-bungarotoxin affinity of alpha 7 homomers expressed in Xenopus oocytes was similar to that observed previously with native chromaffin alpha-bungarotoxin-sensitive acetylcholine receptors. Cross-linking and sucrose gradient experiments suggested that, like the muscular and neuronal acetylcholine receptors; the alpha 7 receptor has a pentameric structure. Upon activation with nicotinic agonists the alpha 7 receptor exhibited rapidly desensitizing cation currents that were blocked by nicotinic antagonists and showed inward rectification. The amplification of adrenal medulla RNA by reverse transcription-polymerase chain reaction methods revealed an alternatively spliced isoform of the bovine alpha 7 subunit, where the exon that codes for the M2 transmembrane segment was skipped during mRNA processing. Oocyte expression of this isoform does not yield functional channels. However, this alternative mRNA exhibits dose-dependent inhibition of alpha 7 homomer expression when coinjected with the undeleted isoform.