Nicotine enhances the cyclic AMP-dependent protein kinase-mediated phosphorylation of alpha 4 subunits of neuronal nicotinic receptors

Studies determined whether alpha 4 beta 2 or alpha 3 beta 2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 nM for alpha 4 beta 2 and 500 nM for alpha 3 beta 2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing alpha 4 beta 2 receptors were incubated with [gamma-P-32]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the alpha 4 subunit was present. Phosphorylation of alpha 4 subunits of alpha 3 beta 2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. in contrast, receptors isolated from oocytes expressing alpha 3 beta 2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the alpha 3 subunit. Results suggest that the PKA-mediated phosphorylation of alpha 4 and not alpha 3 subunits may explain the differential inactivation by nicotine of these receptor subtypes expressed in oocytes.