Direct measurement of calcium transport across chloroplast inner-envelope vesicles

The initial rate of Ca2+ movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spec trofluorometry using membrane vesicles loaded with the Ca2+-sensitive fluor ophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca2+-selective minielectrodes to determine pCa v alues. The initial rate of Ca2+ influx in predominantly right-side-out inne r-envelope membrane vesicles was greater than that in largely inside-out ve sicles. Ca2+ movement was stimulated by an inwardly directed electrochemica l proton gradient across the membrane vesicles, an effect that was diminish ed by the addition of valinomycin in the presence of K+. In addition, Ca2was shown to move across the membrane vesicles in the presence of a K+ diff usion potential gradient. The potential-stimulated rate of Ca2+ transport w as slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl3, verapamil, and nifedipine had l ittle or no effect. These results indicate that Ca2+ transport across the c hloroplast inner envelope can occur by a potential-stimulated uniport mecha nism.