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 degrees 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 microM) 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 1 s 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 10(8) M-1 s-1, and koff = 2.2 s-1. The approximately 1-s exchange time of bound IP3 indicates that the channel gating sites are distinct from binding sites having approximately 100-s exchange times that were previously found with radiolabeled IP3. The approximately 1-1s response time of [Ca2+] to a rapid increase in IP3 level can account for observed rise times of calcium spikes.
Department of Cell Biology, Sherman Fairchild Center, Stanford University School of Medicine, California 94305.
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D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000502:physiology*; D054815:Cyclic Nucleotide-Gated Cation Channels; D004305:Dose-Response Relationship, Drug; D005136:Eye Proteins / Q000502:physiology; D006706:Homeostasis; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007473:Ion Channels / Q000502:physiology; D007700:Kinetics; D008954:Models, Biological; D051381:Rats; D011950:Receptors, Cholinergic / Q000502:physiology; D011978:Receptors, Nicotinic / Q000502:physiology; D012374:Rod Cell Outer Segment / Q000648:ultrastructure; D013997:Time Factors
D015220:Calcium Channels; D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins; D007473:Ion Channels; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium