Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18.
(Ca2+)-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] measured during stimulation in intact cells. This paradox might be explained if the local [Ca2+] at the cell membrane is very different from that in the rest of the cell. Soluble Ca2+ indicators, which indicate spatially averaged cytoplasmic [Ca2+], cannot resolve these localized, near-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 Ca2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Ca2+] transients recorded using FFP18 during membrane depolarization-induced Ca2+ influx show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fura-2, has risen to only a few hundred nanomolar. High-speed series of digital images of [Ca2+] show that near-membrane [Ca2+], reported by FFP18, rises within 20 msec, peaks at 50-100 msec, and then declines. [Ca2+] reported by fura-2 rose slowly and continuously throughout the time images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numerous (Ca2+)-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.
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Department of Physiology and Biomedical Imaging Group, University of Massachusetts Medical Center, Worcester, 01605, USA.
Minta
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Poenie
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Fay
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D000818:Animals; D002024:Bufo marinus; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology; D002614:Chelating Agents; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D066298:In Vitro Techniques; D007700:Kinetics; D008433:Mathematics; D008564:Membrane Potentials; D008954:Models, Biological; D009130:Muscle, Smooth / Q000166:cytology / Q000502:physiology*; D018408:Patch-Clamp Techniques; D013270:Stomach / Q000166:cytology / Q000502:physiology
D002614:Chelating Agents; D005456:Fluorescent Dyes; C098105:fura-FFP18; D002118:Calcium; D016257:Fura-2