Intracellular calcium ions and calcium currents in perfused neurones of the snail, Lymnaea stagnalis.

Neuronal somata of Lymnaea stagnalis were internally perfused and voltage clamped using the suction pipette method. The cells were exposed to internal solutions buffered to various concentrations of Ca2+ while the cytoplasmic Ca2+ activity ( Ca2+i) was monitored with a Ca2+ -sensitive micro-electrode. Ca2+i was usually about 10(-7) M when the cell was perfused with a solution buffered to any level of Ca2+ from 9 X 10(-7) to below 10(-8) M. With internal solutions buffered to 10(-6) M-Ca2+ or greater, Ca2+i increased rapidly and overshot the perfusate Ca2+ activity by up to two orders of magnitude. It was thus virtually impossible to hold Ca2+i steady at any levels other than about 10(-7) M or 10(-4) M using internal perfusion of simple ionic internal solutions. The excess Ca2+ which caused the overshoot of Ca2+i entered the cell from the external solution through Cd2+ -sensitive channels. Cd2+ in the external solution prevented or reversed the overshoot of Ca2+i and brought Ca2+i to near the perfusate level. ATP added to the internal solution also prevented Ca2+i from overshooting the perfusate level during perfusion with high-Ca2+ buffers. By monitoring Ca2+i with a Ca2+ -sensitive micro-electrode, we were able to estimate the relationship between Ca2+i and the Ca2+ current (ICa) measured under voltage clamp. ICa was completely blocked as Ca2+i was raised to 10(-6) M. We believe that the discrepancy between our data and other estimates of the ICa vs. Ca2+i relationship using internal perfusion of molluscan nerve cells results from the incorrect assumption that Ca2+i is controlled adequately during internal perfusion.