Light-flash photolysis of caged inositol 1,4,5-trisphosphate (InsP3) was used to generate reproducible transients of free InsP3 in Xenopus oocytes, and the resulting liberation of Ca2+ from intracellular stores was monitored by recording Ca2+-activated membrane currents and by use of the fluorescent Ca2+ indicator fluo-3. InsP3-mediated Ca2+ release was inhibited by elevating the intracellular free Ca2+ level, either by microinjecting Ca2+ into the cell or by applying conditioning light flashes to liberate Ca2+. This inhibition followed a slow time course, being maximal after about 2 s and subsequently declining over several seconds. Negative feedback of Ca2+ ions on InsP3-mediated Ca2+ liberation may explain the oscillatory release of Ca2+ seen during activation of inositol phospholipid signaling in the oocyte, and the time course of the inhibition is consistent with the period of the oscillations.

Laser confocal microscopy was used to monitor calcium ion (Ca ²⁺ ) liberation from highly localized (micrometer) regions of intact Xenopus oocytes in response to photo-released inositol 1,4,5-trisphosphate (InsP ₃ ). Local Ca ²⁺ release varied in an all-or-none manner with increasing amount of InsP ₃ , in contrast to signals recorded from larger areas, which grew progressively as the concentration of InsP ₃ was raised above a threshold. Liberation of Ca ²⁺ was restricted to within a few microns of the site of InsP ₃ release and, in response to agonist activation, localized regions of the oocyte showed asynchronous oscillations in cytoplasmic Ca ²⁺ release. Results obtained with this technique provided direct evidence that InsP ₃ -induced Ca ²⁺ liberation was quantized and suggest that the InsP ₃ -sensitive Ca ²⁺ pool may be a collection of independent, localized compartments that release Ca ²⁺ in an all-or-none manner.

Xenopus oocytes are a valuable aid for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Their use has recently been extended by the demonstration that oocytes can incorporate foreign membranes carrying preassembled receptors and channels. Here we show that when reconstituted in an artificial lipid matrix and injected into Xenopus oocytes, purified nicotinic acetylcholine receptors are efficiently inserted into the plasma membrane, where they form "clusters" of receptors that retain their native properties. This constitutes an innovative approach that, besides allowing the analyses of membrane fusion processes, is also a powerful technique for studying the characteristics and regulation of many membrane proteins (with their native stoichiometry and configuration) upon reinsertion into the membrane of a very convenient host cell system.

Injection of Ins(1,3,4,6)P4 into Xenopus oocytes evoked Ca2(+)-dependent membrane currents with a potency 5-10 times less than Ins(1,4,5)P3, whereas Ins(1,3,4)P3 and Ins(1,3,4,5,6)P5 were almost ineffective. Responses to Ins(1,3,4,6)P4 arose through liberation of intracellular Ca2+ and through entry of extracellular Ca2+. These results, together with the observation that Ins(1,3,4,6)P4 facilitated responses to Ins(1,4,5)P3, suggests that both of these compounds may act on the same intracellular receptors.