Expression of α3β2β4 nicotinic acetylcholine receptors by rat adrenal chromaffin cells determined using novel conopeptide antagonists

Adrenal chromaffin cells release neurotransmitters in response to stress and may be involved in conditions such as post-traumatic stress and anxiety disorders. Neurotransmitter release is triggered, in part, by activation of nicotinic acetylcholine receptors (nAChRs). However, despite decades of use as a model system for studying exocytosis, the nAChR subtypes involved have not been pharmacologically identified. Quantitative real-time PCR of rat adrenal medulla revealed an abundance of mRNAs for alpha 3, alpha 7, beta 2, and beta 4 subunits. Whole-cell patch-clamp electrophysiology of chromaffin cells and subtype-selective ligands were used to probe for nAChRs derived from the mRNAs found in adrenal medulla. A novel conopeptide antagonist, PeIA-5469, was created that is highly selective for alpha 3 beta 2 over other nAChR subtypes heterologously expressed in Xenopus laevis oocytes. Experiments using PeIA-5469 and the alpha 3 beta 4-selective alpha-conotoxin TxID revealed that rat adrenal medulla contain two populations of chromaffin cells that express either alpha 3 beta 4 nAChRs alone or alpha 3 beta 4 together with the alpha 3 beta 2 beta 4 subtype. Conclusions were derived from observations that acetylcholine-gated currents in some cells were sensitive to inhibition by PeIA-5469 and TxID, while in other cells, currents were sensitive only to TxID. Expression of functional alpha 7 nAChRs was determined using three alpha 7-selective ligands: the agonist PNU282987, the positive allosteric modulator PNU120596, and the antagonist alpha-conotoxin [V11L,V16D]ArIB. The results of these studies identify for the first time the expression of alpha 3 beta 2 beta 4 nAChRs as well as functional alpha 7 nAChRs by rat adrenal chromaffin cells.