KINETICS OF CALCIUM-CHANNEL OPENING BY INOSITOL 1,4,5-TRISPHOSPHATE

The subsecond mobilization of intracellular Ca2+ by IP3 was measured with rapid mixing techniques to determine how cells achieve rapid rises in cytosolic [Ca2+] during receptor-triggered calcium spiking. In permeabilized rat basophilic leukemia cells at 11 °C, more than 80% of the 0.7 fmol of Ca2+/cell sequestered by the ATP-driven pump could be released by IP3. Half of the stored Ca2+ was released within 200 ms after addition of saturating (1 μM) IP3. The flux rate was half-maximal at 120 nM IP3. Ca2+ release from fully loaded stores was highly cooperative; the Hill coefficient over the 2–40 nM range was greater than 3. The delay time of channel opening was inversely proportional to [IP3], increasing from 150 ms at 100 nM IP3 to 1s at 15 nM, indicating that the rate-limiting step in channel opening is IP3 binding. Multiple binding steps are required to account for the observed delay and nonexponential character of channel opening. A simple model is proposed in which the binding of four IP3 molecules to identical and independent sites leads to channel opening. The model agrees well with the data for KD = 18 nM, kon = 1.2 X 108 M−1 s−1, and koff = 2.2 s−1. The ~l-s exchange time of bound IP3 indicates that the channel gating sites are distinct from binding sites having ~100-s exchange times that were previously found with radiolabeled IP3. The ~l-s response time of [Ca2+] to a rapid increase in IP3 level can account for observed rise times of calcium spikes. © 1990, American Chemical Society. All rights reserved.