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 n icotinic receptors expressed in Xenopus oocytes are substrates for cyc lic AMP-dependent protein kinase (PKA) and whether nicotine affects re ceptor phosphorylation. The cRNAs for the subunits were coinjected int o oocytes, and cells were incubated for 24 h in the absence or presenc e 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 recep tors. When receptors isolated from oocytes expressing alpha 4 beta 2 r eceptors were incubated with [gamma-P-32]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiograp hy, a labeled phosphoprotein with the predicted molecular size of the alpha 4 subunit was present. Phosphorylation of alpha 4 subunits of al pha 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 lab eled phosphoprotein corresponding to the size of the alpha 3 subunit. Results suggest that the PKA-mediated phosphorylation of alpha 4 and n ot alpha 3 subunits may explain the differential inactivation by nicot ine of these receptor subtypes expressed in oocytes.