NICOTINE-INDUCED SUBUNIT STOICHIOMETRY AFFECTS THE STABILITY AND THE INTRACELLULAR TRAFFICKING OF THE ALPHA3BETA4 NICOTINIC RECEPTOR.

The α3β4 is a heteromeric nicotinic acetylcholine receptor (nAChR) implicated in the risk of nicotine dependence, smoking, and lung cancer. Little is known about the effect of nicotine on this subtype, so to study it, the α3β4 subunits were expressed in epithelial Hela and neuroblastoma SH-SY5Y cells: in both cells the receptor is very inefficiently exported from the Endoplasmic Reticulum (ER) to plasmamembrane, indicating that a stringent quality control is present at the level of ER to avoid the exit of unfolded/misassembled protein. In this project, It has been demonstrated that nicotine, in a dose dependent manner, is able to up-regulate the newly-synthesized receptor and to increase the percentage of receptor at the plasmamembrane. Furthermore, It has been revealed that an ER export motif (LFM) present in the M3-M4 intracellular loop of the β4 subunit is necessary for ER export of the receptor. The presence of nicotine during receptor synthesis facilitates the assembly of a pentameric receptor with a (α3)2(β4)3 stoichiometry. The effect of nicotine is double:1) on receptor degradation, that is reduced, and 2) on receptor trafficking,that it is increased, through favoring the assembly of (α3)2(β4)3 receptors. The same effect is obtained with other nicotinic agonists and/or antagonists providing that they are cell permeable and, like nicotine, bind the orthosteric binding site. The receptor with this stoichiometry is less prone to proteasome degradation and thanks to the presence of more export signals can more easily exit from the ER. To confirm the importance of the orthosteric binding site for the nicotine upregulation, the amino acid tryptophan, located in the binding site of the α3 subunit sequence, was mutated to alanine. This mutated α3*β4 nAChR is no more up-regulated by nicotine but is efficiently exported to plasmamembrane, suggesting a gain of function effect of the mutation.