Fast kinetics of calcium liberation induced in Xenopus oocytes by photoreleased inositol trisphosphate.
Inositol 1,4,5-trisphosphate (InsP3) acts on intracellular receptors to cause liberation of Ca2+ ions into the cytosol as repetitive spikes and propagating waves. We studied the processes underlying this regenerative release of Ca2+ by monitoring with high resolution the kinetics of Ca2+ flux evoked in Xenopus oocytes by flash photolysis of caged InsP3. Confocal microfluorimetry was used to monitor intracellular free [Ca2+] from femtoliter volumes within the cell, and the underlying Ca2+ flux was then derived from the rate of increase of the fluorescence signals. A threshold amount of InsP3 had to be photoreleased to evoke any appreciable Ca2+ signal, and the amount of liberated Ca2+ then increased only approximately fourfold with maximal stimulation, whereas the peak rate of increase of Ca2+ varied over a range of nearly 20-fold, reaching a maximum of approximately 150 microMs-1. Ca2+ flux increased as a first-order function of [InsP3]. Indicating a lack of cooperativity in channel opening, and was half-maximal with stimuli approximately 10 times threshold. After a brief photolysis flash, Ca2+ efflux began after a quiescent latent period that shortened from several hundred milliseconds with near-threshold stimuli to 25 ms with maximal flashes. This delay could not be explained by an initial "foot" of Ca2+ increasing toward a threshold at which regenerative release was triggered, and the onset of release seemed too abrupt to be accounted for by multiple sequential steps involved in channel opening. Ca2+ efflux increased to a maximum after the latent period in a time that reduced from > 100 ms to approximately 8 ms with increasing [InsP3] and subsequently declined along a two-exponential time course: a rapid fall with a time constant shortening from > 100 ms to approximately 25 ms with increasing [InsP3], followed by a much smaller fail persisting for several seconds. The results are discussed in terms of a model in which InsP3 receptors must undergo a slow transition after binding InsP3 before they can be activated by cytosolic Ca2+ acting as a co-agonist. Positive feedback by liberated Ca2+ ions then leads to a rapid increase in efflux to a maximal rate set by the proportion of receptors binding InsP3. Subsequently, Ca2+ efflux terminates because of a slower inhibitory action of cytosolic Ca2+ on gating of InsP3 receptor-channels.
Parker
I
I
Department of Psychobiology, University of California, Irvine 92717 USA. iparker@uci.edu
Yao
Y
Y
Ilyin
V
V
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005260:Female; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000528:radiation effects; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015640:Ion Channel Gating; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D010782:Photolysis; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism; D013269:Stochastic Processes; D014982:Xenopus laevis
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D011978:Receptors, Nicotinic; C061868:inositol 1,4,5-trisphosphate 1-(2-nitrophenyl)ethyl ester; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium