Adenosine A2A receptor induces protein kinase A-dependent functional modulation of human alpha 3 beta 4 nicotinic receptor

Non-technical summary In many brain regions, adenosine regulates the function of the nicotinic acetylcholine receptor-channel, which allows the movement of ions across neuronal membrane if nicotine or acetylcholine is present. In this work we use a very controlled experimental system to study the details of adenosine action. We show that adenosine enhances the function of the receptor-channel when it is repeatedly stimulated. The intracellular signalling molecule protein kinase A mediates the effects of adenosine on the nicotinic receptor-channel. Our results contribute to understanding the function of receptor proteins involved in the normal function of the brain, but also in pathologies such as nicotine addiction.Adenosine modulates the function of nicotinic ACh receptors (nAChRs) in a variety of preparations, possibly through pathways involving protein kinase A (PKA), but these phenomena have not yet been investigated in detail. In this work we studied, using the patch clamp technique, the functional modulation of recombinant human alpha 3 beta 4 nAChR by the A2A adenosine receptor, co-expressed in HEK cells. Tonic activation of A2A receptor slowed current decay during prolonged applications of nicotine and accelerated receptor recovery from desensitization. Together, these changes resulted into a more sustained current response upon multiple nicotine or ACh applications. These findings were confirmed in cultured mouse superior cervical ganglion neurones, which express nAChR containing the alpha 3 subunit together with beta 2 and/or beta 4 and A2A receptor. Expression of the A2A receptor in HEK cells also increased the apparent potency of nAChR for nicotine, further supporting a general A2A-induced gain of function for nAChR. These effects were dependent on PKA since the direct activation of PKA mimicked, and its inhibition prevented almost completely, the effects of the A2A receptor. Mutations of R-385 and S-388 in the cytoplasmic loop of the alpha 3 subunit abolished the functional modulation of nAChR induced by activation of A2A receptor, PKA and other Ser/Thr kinases, suggesting that this region constitutes a putative consensus site for these kinases. These data provide conclusive evidence that activation of the A2A receptor determines functional changes of human alpha 3 beta 4 nAChR, through pathways involving PKA.