Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase enhances calcium action potentials of bag cell neurons in cell culture

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Abstract
We have found that the calcium action potentials of bag cell neurons from the abdominal ganglion of Aplysia may be enhanced by intracellular microinjection of the catalytic subunit of cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The catalytic subunit was purified from bovine heart and shown to be effective in stimulating the phosphorylation of bag cell proteins in homogenates at concentrations of 10-50 nM. Intracellular injection into isolated bag cell neurons maintained in primary culture was through pressure applied to microelectrodes filled at the tip with catalytic subunit (5-22 μM). In 11 of 16 injected cells, both the slope of the rising phase and the height of the action potentials evoked by a constant depolarizing current were markedly enhanced relative to the pre-injection control (mean increases, 73% and 35%, respectively). This effect could occur with no change in resting potential or in the latency of the action potential from the onset of the depolarizing pulse. The effect was observed with enzyme dissolved in three different salt solutions (Na phosphate, K phosphate, or KCl). In two experiments, tetrodotoxin (50 μM) added to the extracellular medium had no effect on the enhanced action potentials. Subsequent addition of the calcium antagonist Co2+, however, diminished or abolished the spikes. In more than half of the experiments, the injection of catalytic subunit was accompanied by an increase in the input resistance of the cells as measured by applying small hyperpolarizing current pulses. In three experiments, subthreshold oscillations in membrane potential resulted from the injections. Control injections (24 cells), carried out either with carrier medium alone or with heat-inactivated enzyme preparations, did not produce spike enhancement, increased input resistance, or oscillations. Our data suggest that the stimulation of intracellular protein phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase enhances the excitability of bag cell neurons by modifying calcium and potassium channels or currents.