COOPERATIVE ACTIVATION OF IP3 RECEPTORS BY SEQUENTIAL BINDING OF IP3 AND CA2+ SAFEGUARDS AGAINST SPONTANEOUS ACTIVITY

Background: Ca2+ waves allow effective delivery of intracellular Ca2signals to cytosolic targets. Propagation of these regenerative Ca2+ s ignals probably results from the activation of intracellular Ca2+ chan nels by the increase in cytosolic [Ca2+] that follows the opening of t hese channels. Such positive feedback is potentially explosive. Mechan isms that limit the spontaneous opening of intracellular Ca2+ channels are therefore likely to have evolved in parallel with the mechanism o f Ca2+-induced Ca2+ release. Results: Maximal rates of Ca-45(2+) efflu x from permeabilised hepatocytes superfused with medium in which the [ Ca2+] was clamped were cooperatively stimulated by inositol 1,4,5-tris phosphate (lP(3)). A minimal interval of similar to 400 msec between l P(3) addition and the peak rate of Ca2+ release as well as an absolute latency of 30 msec before initiation of Ca2+ mohilisation indicate th at channel opening does not immediately follow binding of IP3. Althoug h the absolute latency of Ca2+ release was unaffected by further incre asing the IP3 concentration. it was reduced by increased [Ca2+]. Concl usions: We propose that the closed conformation of the IP3 receptor is very stable and therefore minimally susceptible to spontaneous activa tion; at least three (probably four) IP3 molecules may be required to provide enough binding energy to drive the receptor into a stable open conformation. We suggest that a further defence from noise is provide d by an extreme form of coincidence detection. Binding of IP3 to each of its four receptor subunits unmasks a site to which Ca2+ must bind b efore the channel can open. As IP3 binding may also initiate receptor inactivation, there may be only a narrow temporal window during which each receptor subunit must bind both of its agonists if the channel is to open rather than inactivate.