Intracellular Factors for the Maintenance of Calcium Currents in Perfused Neurones from the Snail, Lymnaea Stagnalis

Isolated nerve cell bodies from Lymnaea stagnalis were internally perfused and voltage-clamped. The magnitude of the Ca2+ current was monitored while perfusing with various intracellular solutions. When the intracellular perfusate was unenriched (containing only inorganic ions, 100 mM-HEPES and 5 mM-EGTA), the Ca2+ current was found to 'wash out', falling to half of its maximum value approximately 30-40 min from the beginning of perfusion. Stopping the flow of the perfusing solution increased this half-time to more than 50 min. The current-voltage relationship changed only slightly during wash-out. The addition of 2 mM-ATP and 1 mM-Mg2+ to the internal perfusate prevented, and even reversed, wash-out of the Ca2+ current. Both ATP and Mg2+ were necessary for maximal effect. Such current loss as occurred in the presence of ATP and Mg2+ was associated with a decrease in the capacitance of the cell and probably resulted from membrane being pulled into the pipette. The rate of inactivation of the Ca2+ current increased during perfusion with an unenriched internal solution, but decreased to initial values when ATP and Mg2+ were added to the internal perfusate. Although intracellular Mg2+ was necessary for the prevention of wash-out, levels higher than 1 mM had a blocking effect on the Ca2+ current. Certain factors that promote cyclic AMP-dependent protein phosphorylation (internal: cyclic AMP, theophylline and catalytic subunit of cyclic AMP-dependent protein kinase; external: dibutyryl cyclic AMP, 8-bromo cyclic AMP and forskolin) had no effect on the magnitude of the Ca2+ current in cells perfused with ATP and Mg2+. Externally applied theophylline blocked the Ca2+ current. The mechanism through which ATP and Mg2+ act to prevent wash-out of the Ca2+ current may be to enhance the ability of the cell to lower the Ca2+ concentration near the inner surface of the plasma membrane. This would prevent both the reversible block of Ca2+ current by intracellular Ca2+ and an irreversible loss of current due to high levels of intracellular Ca2+.