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.