CYTOPLASMIC FREE CA2+ IN THE MARINE ALGA ACETABULARIA - MEASUREMENT WITH CA2+-SELECTIVE MICROELECTRODES AND KINETIC-ANALYSIS

Neutral carrier-based Ca2+-selective microelectrodes have been examined for application in concentrated multi-ion solutions. Calculations with data from the literature and our calibration series with Ca2+-EGTA buffers (a convenient algorithm for their calculation is given) provide the physico-chemical conditions for determination of submicromolar concentrations of free Ca2+ in the cytoplasm (with about 400 mM K+ and 70 mM Na+) of the marine alga Acetabularia acetabulum. The experimental results give a cytoplasmic concentration of 560 nM free Ca2+ corresponding to 140 nM activity. Recordings of cytoplasmic Ca2+ upon removal and re-addition of external (10 mM) Ca2+ show steady-state changes by about 50 nM (following the direction of external Ca2+) which are preceded by transient overshoots. These kinetics are better described by damped oscillations of a feedback control system than by two superimposed exponentials. Using the maximum rate of decrease of cytoplasmic Ca2+ upon removal of external Ca2+, a unidirectional Ca2+ efflux of greater-than-or-equal-to 0.3-mu-mol m-2 s-1 is determined which is considered to mark the steady-state turnover of Ca2+ at the plasmalemma. This high rate and the high electrochemical driving force for Ca2+ (about -580 mV) across the plasmalemma at a resting voltage of about -170 mV, point to a powerful Ca2+ transport system which cannot sufficiently be fuelled by ATP-hydrolysis but requires additional energy.