Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles

The initial rate of Ca(2+) movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca(2+)-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca(2+)-selective minielectrodes to determine pCa values. The initial rate of Ca(2+) influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca(2+) movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K(+). In addition, Ca(2+) was shown to move across the membrane vesicles in the presence of a K(+) diffusion potential gradient. The potential-stimulated rate of Ca(2+) transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl(3), verapamil, and nifedipine had little or no effect. These results indicate that Ca(2+) transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.