ALPHA-BUNGAROTOXIN-SENSITIVE NICOTINIC RECEPTORS ON BOVINE CHROMAFFINCELLS - MOLECULAR-CLONING, FUNCTIONAL EXPRESSION AND ALTERNATIVE SPLICING OF THE ALPHA-7 SUBUNIT

Chromaffin cells from the bovine adrenal medulla express alpha-bungaro toxin-sensitive acetylcholine receptors whose subunit composition is u nknown. Northern blot analysis showed that the alpha 7 subunit, a main component of these alpha-bungarotoxin-sensitive acetylcholine recepto rs 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 subun its. The alpha-bungarotoxin affinity of alpha 7 homomers expressed in Xenopus oocytes was similar to that observed previously with native ch romaffin alpha-bungarotoxin-sensitive acetylcholine receptors. Cross-l inking and sucrose gradient experiments suggested that, like the muscu lar and neuronal acetylcholine receptors, the alpha 7 receptor has a p entameric 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-polymera se chain reaction methods revealed an alternatively spliced isoform of the bovine alpha 7 subunit, where the exon that codes for the M2 tran smembrane segment was skipped during mRNA processing. Oocyte expressio n of this isoform does not yield functional channels. However, this al ternative mRNA exhibits dose-dependent inhibition of alpha 7 homomer e xpression when coinjected with the undeleted isoform.