A role for kinase/phosphatase action in the regulation of brain amino acid receptors

1. A rat neocortical slice preparation was used to examine the effects of protein kinase or alkaline phosphatase on two populations of amino acid receptors. 2. These receptors, the inhibitory GABA(A) and the excitatory α-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA) receptor populations, have been assayed using the selective antagonists [³H]-SR95531 and [³H]-CNQX (6-cyano-2,3-dihydroxy-7-nitroquinoxaline), respectively. To examine the effects of phosphorylation on receptor regulation, cortical slices were frozen and then thawed to eliminate living cortical circuitry before exposure to varying concentrations of alkaline phosphatase (2.5 x 10^-2 to 2.5 x 10^-9 mg/ml) or protein kinase (catalytic subunit, 5 x 10^-2 to 5 x 10^-9 pM/μg protein/ml). 3. For both receptor populations the addition of alkaline phosphatase (AP) led to a concentration-dependent increase in binding of radioligand with a significant 20% increase at 2.5 x 10^-7 mg/ml AP. In contrast, protein kinase (PK) led to a significant concentration-dependent decrease of 30% binding for both receptors at 5 x 10^-6 pM/μg protein/ml PK. Eadie-Hofstee analysis of saturation binding data revealed that these changes reflected changes in receptor numbers rather than affinities. 4. Inhibition experiments were performed in which alkaline phosphatase activity was blocked by phenylarsine oxide or sodium orthovanadate or in which protein kinase activity was blocked with protein kinase inhibitor. For both alkaline phosphatase and protein kinase, the regulation normally induced in GABA(A) and AMPA receptors was prevented by the appropriate inhibitor. 5. These data suggest a role for phosphorylation/dephosphorylation reactions in the events leading to the regulation of at least one type of inhibitory and one type of excitatory amino acid receptor in the cortex.