Inositol trisphosphate-mediated Ca2+ influx into Xenopus oocytes triggers Ca2+ liberation from intracellular stores.
1. Inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger by liberating Ca2+ from intracellular stores and by promoting influx of extracellular Ca2+. We examined whether Ca2+ influx modulates intracellular Ca2+ liberation in Xenopus oocytes by fluorescence monitoring of cytosolic free Ca2+ together with voltage clamp recording of Ca(2+)-activated Cl- membrane currents. Sustained activation of membrane Ca2+ permeability was induced by intracellular injections of a non-metabolizable InsP3 analogue, 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate (3-F-InsP3), and Ca2+ influx was controlled by applying step changes in membrane potential to alter the driving force for Ca2+ entry. 2. Negative-going potential steps evoked intracellular Ca2+ signals comprising two components; an initial transient peak followed by a slower rise. The initial transient grew steeply over a narrow (ca 40 mV) voltage range but then increased little with further polarization, whereas the second component showed a nearly linear voltage dependence. 3. The transient Ca2+ signal continued to rise almost unchanged when Ca2+ influx was interrupted by stepping the potential to more positive values after brief hyperpolarization. In contrast, Ca2+ levels declined monotonically when positive-going steps were applied after longer intervals during the second component of the Ca2+ signal. 4. Large Ca(2+)-dependent transient inward (T(in)) membrane currents were evoked during the rising phase of the initial Ca2+ signal, but little current was associated with the second component of the Ca2+ signal. 5. The T(in) currents evoked by hyperpolarization were mimicked at fixed clamp potential by re-admitting Ca2+ to the bathing solution, and by flash photolysis of caged Ca2+ loaded into the oocyte. 6. T(in) currents were strongly inhibited by prior release of Ca2+ from InsP3-sensitive intracellular stores, and vice versa. Experiments with paired hyperpolarizing pulses and paired photorelease of InsP3 showed that responses to both stimuli recovered with similar time courses. 7. We conclude that the transient Ca2+ signal and associated T(in) current evoked by hyperpolarization arise because Ca2+ entering the oocyte triggers regenerative release of Ca2+ from InsP3-sensitive intracellular stores. Since membrane currents evoked by liberated Ca2+ were much greater than those evoked by Ca2+ entry per se, a major function of InsP3-mediated Ca2+ entry may be to modulate the activity of intracellular Ca2+ stores.
Yao
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Y
Department of Psychobiology, University of California Irvine 92717.
Parker
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I
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000528:radiation effects; D005071:Evoked Potentials / Q000187:drug effects; D005260:Female; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D008564:Membrane Potentials; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D010782:Photolysis; D015290:Second Messenger Systems; D015398:Signal Transduction / Q000187:drug effects; D014982:Xenopus laevis
C084782:3-deoxy-3-fluoroinositol 1,4,5-trisphosphate; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium