Cytoplasmic Free Ca2+in the Marine Alga Acetabularia: Measurement with Ca2+–selective Microelectrodes and Kinetic Analysis

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Abstract
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 theircalculation 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+ uponremoval 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 over shoots. 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 removalof external Ca2+, a unidirectional Ca2+ efflux of ≥0.3 μmol 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

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