α‐Bungarotoxin‐sensitive Nicotinic Receptors on Bovine Chromaffin Cells: Molecular Cloning, Functional Expression and Alternative Splicing of the α7 Subunit

Abstract

Chromaffin cells from the bovine adrenal medulla express α-bungarotoxin-sensitive acetylcholine receptors whose subunit composition is unknown. Northern blot analysis showed that the α7 subunit, a main component of these α-bungarotoxin-sensitive acetylcholine receptors in avian and rat brain, is expressed in chromaffin cells. The cDNA of this bovine α7 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 α7 and 89% to chicken α7 subunits. The α-bungarotoxin affinity of α7 homomers expressed in Xenopus oocytes was similar to that observed previously with native chromaffin α-bungarotoxin sensitive acetylcholine receptors. Cross-linking and sucrose gradient experiments suggested that, like the muscular and neuronal acetylcholine receptors, the α7 receptor has a pentameric structure. Upon activation with nicotinic agonists the α7 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 α7 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 α7 homomer expression when coinjected with the undeleted isoform.