Adenosine-mediated modulation of muscular nAChRs both transplanted into Xenopus laevis oocytes and expressed naturally in skeletal muscle cells

Adenosine is an important nucleotide widely distributed in all living organisms that contributes to the regulation of highly heterogeneous cellular function acting via P1-purinergic receptors (P1-receptors). The P1-receptors belong to the superfamily of G-protein coupled receptor and are divided in 4 subtypes according both to their affinity for adenosine (high affinity: A1R and A2AR; low affinity: A2BR and A3R) and their ability to activate or inhibit adenylyl cyclase (A1R and A3 subtypes mainly inhibit adenylyl cyclase activity via Gi proteins, whereas A2A and A2B receptors stimulate adenylyl cyclase via Gs proteins). Mouse skeletal myotubes differentiated in vitro exhibited spontaneous contraction and the open probability and the mean open time of single embryonic nicotinic acetylcholine channels (nAChRs) were modulated through the activation of P1-receptors, specifically the A2BR subtype (Bernareggi et al., 2015). This cross-talk is shown to be very important for the maintenance of the muscle tropism before the nerve arrival. The microtransplantation of membranes in Xenopus oocytes is a useful approach to study the activity of channels and receptors still embedded in their native lipid envoronment and transplanted into the foreign oocyte membrane. In oocytes the biophysical activity of exogenous nAChRs was studied using the two-electrode voltage clamp technique. For comparison, experiments were also performed on the mouse myotubes in vitro with the patch-clamp technique in whole cell configuration. The activation of AChR-channels was evoked by high ACh concentration and the current desensitization was measured as a current decay T0.1 and T0.5, time necessary for the current to decay by 10% and 50% from its peak value. These experiments were performed before and after a pre-treatment with nonselective and selective ligands for P1-receptors. I found that the interplay between P1Rs and nAChRs is altered when the receptors are transplanted into the oocyte and more precisely the activity of nAChRs is modulated by A1 subtype P1 adenosine receptor. This could be due to different reasons: altered colocalization of the nAChRs in the oolemma, some key proteins normally involved in the cross-talk modulation were lose during the isolation protocol and the hypothesis of endogenous expression of P1-receptors from the same oocytes. In the second part of this study, the ADO-mediated activity was studied in adult skeletal muscle fiber where the embryonic nAChR is replaced by the adult isoform, and the receptors are clustered at the endplate region. At the endplate region the presence of P1R, in particular the low affinity P1R subtypes, it has been recently reported (Garcia et al., 2014), but their role is still unknown. Here, it has been reported the role of the low affinity P1 subtypes in mediating the nAChR-channel activity at the adult endplate.