NEAR-MEMBRANE [CA2+] TRANSIENTS RESOLVED USING THE CA2+ INDICATOR FFP18

Ca2+-sensitive processes at cell membranes involved in contraction, se cretion, and neurotransmitter release are activated in situ or in vitr o by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] meas ured during stimulation in intact cells. This paradox might be explain ed if the local [Ca2+] at the cell membrane is very different from tha t in the rest of the cell. Soluble Ca2+ indicators, which indicate spa tially averaged cytoplasmic [Ca2+], cannot resolve these localized, ne ar-membrane [Ca2+] signals. FFP18, the newest Ca2+ indicator designed to selectively monitor near-membrane [Ca2+], has a lower Ca2+ affinity and is more water soluble than previously used membrane-associating C a2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Ca2+] transient s recorded using FFP18 during membrane depolarization-induced Ca2+ inf lux show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fur a-2, has risen to only a few hundred nanomolar. High-speed series of d igital images of [Ca2+] show that near-membrane [Ca2+], reported by FF P18, rises within 20 msec, peaks at 50-100 msec, and then declines. [C a2+] reported by fura-2 rose slowly and continuously throughout the ti me images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numer ous Ca2+-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.