Transient and Persistent Depolarization‐Induced Changes of Protein Phosphorylation in a Molluscan Nervous System

Phosphoproteins in the CNS of the nudibranch mollusc, Hermissenda crassicornis, were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography. After preincubation in artificial sea‐water containing 32P, nervous systems were exposed to elevation of external K+ (100 or 300 mM) for a period (e.g., 30 min) approximating a period of depolarization which occurs during classical conditioning. Elevated external K+ was found to change the state of phosphorylation of three distinct proteins (Mr 56,000, 25,000, and 20,000) in three distinct ways without consistently changing that of any other proteins. Phosphorylation of an Mr 56,000 protein was increased by high K+ about twofold only in the presence of external Ca2+ ([Ca2+]o). Phosphorylation of Mr 25,000 protein, on the other hand, was decreased up to 10‐fold by high K+, irrespective of the level of [Ca2+]o. The effect of depolarization on Mr 25,000 protein phosphorylation most likely represents dephosphorylation rather than proteolysis. This interpretation is consistent with the observations that (a) reappearance of the Mr 25,000 protein occurred in the presence of the protein synthesis inhibitors cycloheximide, puromycin, or anisomycin, and (b) the Hermissenda nervous system apparently contains a NaF and EDTA‐sensitive protein phosphatase capable of de‐phosphorylating Mr 25,000 protein. High K+ also reduced Mr 20,000 protein phosphorylation which was dependent on [Ca2+]o even in normal low K+ (10 mM) medium. Removal of [Ca2+]o enhanced reduction of Mr 20,000 phosphorylation due to the high K+ treatment. Interestingly, reduction of the Mr 25,000 protein phosphorylation was long‐lasting, i.e., its phosphorylation did not fully recover to a control level for at least 30 min after the high K+ conditions had been removed. On the other hand, changes of Mr 56,000 and Mr 20,000 phosphorylation did not persist after depolarization had been terminated. A23187, a Ca2+ ionophore, partially reduced Mr 25,000 phosphorylation. These results suggested that depolarization‐induced modification of protein phosphatase and/or protein kinase activity which regulates Mr 25,000 phosphorylation may be involved in biochemical pathways important for memory storage.