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

Abstract

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.