CALCIUM-DEPENDENT PROTEIN PHOSPHORYLATION AND THE REGULATION OF ELECTRICAL EXCITABILITY IN THE BAG CELL NEURONS OF APLYSIA (PHORBOL, CALMODULIN, PROTEIN KINASE C)

Calcium-dependent protein phosphorylation is one of the major means by which intracellular calcium can modulate the activity of cells. In order to determine whether the calcium-dependent protein kinases found in the nervous system regulate electrical excitability, I have characterized these enzymes and the effects of altering their activity in the bag cell neurons of Aplysia californica.;Trifluoperazine, calmidazolium (R24571), and W7 inhibited the activity of both the calcium/calmodulin-independent protein kinase and protein kinase C. Pretreatment of intact bag cell clusters with these drugs prevented the onset of electrically evoked afterdischarges. Addition of the drug after the initiation of an afterdischarge however did not affect the ongoing activity or the duration of discharge.;Exposure of bag cell neurons maintained in primary culture to TPA enhanced the height of action potentials elicited by depolarizing current pulses. This effect was not seen with the non-tumorogenic phorbols 4-alpha-phorbol, 4-alpha-phorbol-12,13-didecanoate or 4-O-methyl-TPA. It was not observed when calcium was removed from the extracellular solution, and voltage clamp experiments indicate that it is due to an increase in inward current. Intracellular injection of protein kinase C purified from bovine brain produced changes in the bag cell action potential similar to those produced by exposure of the cells to TPA. Injection of heat-inactivated enzyme or the carrier medium (20 mM potassium-phosphate buffer, pH 7.2; 0.9 M KCl) did not produce these effects. Injection of calmodulin kinase II purified from rat brain also had no consistent effect on the action potentials of cultured bag cell neurons.;Incubation of homogenates of the bag cell neurons with calcium leads to an increase in phosphate incorporation into a number of proteins. This phosphorylation was shown to be due to the activity of at least three distinct protein kinases. The activity of one of these, Protein kinase C, was stimulated by phosphatidylserine plus diolein or by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA,PMA). The two other enzymes were stimulated by the calcium-binding protein calmodulin. One of these was shown to be homologous to an enzyme present in the vertebrate brain, calmodulin kinase II.