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

Neutral carrier–based Ca²⁺ –selective microelectrodeshave been examined for application in concentrated multi–ionsolutions. Calculations with data from the literature and ourcalibration series with Ca²⁺ –EGTA buffers (a convenientalgorithm for theircalculation is given) provide the physico–chemicalconditions for determination of submicromolar concentrationsof free Ca²⁺ in the cytoplasm (with about 400 mM K⁺ and 70 mMNa⁺) of the marine alga Acetabularia acetabulum. The experimentalresults give a cytoplasmic concentration of 560 nM free Ca²⁺corresponding to 140 nM activity. Recordings of cytoplasmicCa²⁺ uponremoval and re-addition of external (10 mM) Ca²⁺ showsteady–state changes by about 50 nM (following the directionof external Ca²⁺) which are preceded by transient over shoots.These kinetics are better described by damped oscillations ofa feedback control system than by two superimposed exponentials.Using the maximum rate of decrease of cytoplasmic Ca²⁺ uponremovalof external Ca²⁺, a unidirectional Ca²⁺ efflux of 0.3µmol m^–2 s^–1 is determined which is consideredto mark the steady–state turnover of Ca²⁺ at the plasmalemma.This high rate and the high electrochemical driving force forCa²⁺ (about – 580 mV)across the plasmalemma at a restingvoltage of about – 170 mV, point to a powerful Ca²⁺ transportsystem which cannot sufficiently be fuelled by ATP–hydrolysisbut requires additional energy Key words: Acetabularia, Ca²⁺–selective microelectrode, cytoplasmic free calcium, EGTA–buffer, homeostasis, plasmalemma