IP₃ receptors are ligand-operated intracellular Ca²⁺ channels, which are involved in the generation of cellular Ca²⁺ signals in many different physiological and pathological conditions. Control of IP₃-induced Ca²⁺ release (IICR) is possible either by regulation of the ligand binding, thus affecting the affinity for IP₃, or by changing the coupling between ligand binding and channel opening. The latter mechanism is much less understood but many data indicate that malfunction of this coupling mechanism may either produce an inactive channel or inversely may produce an hyperactive or even a leaky channel (Szlufcik et al. 2006). A functional element, which is involved in both types of regulation is the ‘suppressor/coupling’ domain; this is the N-terminal 225 AA of the IP₃R. This suppressor/coupling domain attenuates the interaction between IP₃ and the IP₃-binding core, but it is also essential for activation of IICR. By using chimeric IP₃Rs, in which the suppressor domains of IP₃R1 and IP₃R3 were exchanged, we have shown that the suppressor was an important determinant of the affinity of these different isoforms. Moreover, deletion of 11 AA in IP₃R1 eliminated the suppression effect and yielded a high affinity [Δ76-86]-IP₃R1 mutant. Among the proteins that directly control IICR, we have particularly focused on calmodulin (CaM). We have demonstrated that CaM and CaM-like proteins such as CaBP1 shared a Ca²⁺-independent binding site on the suppressor domain (AA 49-81) in a position which is critical for determining the IP₃ affinity. We showed that CaM did not function as a Ca²⁺ sensor, though inhibition of IICR by CaM only occurred in the presence of Ca²⁺. CaM also binds to a less conserved Ca²⁺-dependent binding site in middle portion of IP₃R1, but no regulatory role on IICR was as yet demonstrated. In addition we have found that endogenously bound CaM is essential for IICR. Depletion of CaM by high-affinity CaM-binding peptides reversibly inhibited IICR. This effect was on the coupling function as IP₃ binding was not affected. The data suggested that CaM is constitutively associated with the IP₃R and is essential for proper coupling between IP₃ binding and channel activation. There is increasing evidence that IP₃Rs play an essential role in apoptosis and that multiple mechanisms may be involved (Hanson et al. 2004). We observed that increasing the affinity for IP₃ in the [Δ76-86]-IP₃R1 mutant directly increased staurosporin (STS)–induced apoptosis. In addition however IP₃R1 is a substrate for caspase-3 cleavage, which yields a C-terminal 95kDa fragment containing the domains responsible for tetramerisation and for formation of a constitutively active channel. We have demonstrated that caspase-3 cleavage occurred during STS-induced apoptosis and that it may represent a positive feedback mechanism accelerating cell death (Assefa et al. 2004). Although the caspase-3 site is only present in IP₃R1 we have evidence that a similar truncation may occur for IP₃R3 as a consequence of calpain activity. Truncation of IP₃R1 by caspase-3 is an extreme case illustrating the possibility that IP₃Rs may operate as IP₃-independent or leaky channels. Such behavior was also observed as a consequence of IP₃R1 hyper-phosphorylation (Oakes et al. 2005). The Ca²⁺ leak may affect the luminal Ca²⁺ content which is a critical parameter controlling cell death pathways. It is conceivable that such leak pathways may occur particularly in some pathological conditions such as Alzheimer’s disease. We have observed a specific, five-fold up-regulation of IP₃R1 in mouse embryonic fibroblast deficient in presenilin-1 and -2 (MEF_DKO). MEF_DKO cells showed a decrease in the [Ca²⁺]_ER as measured by ER-targeted aequorin luminescence and the lower [Ca²⁺]_ER was associated with an increase in a Ca²⁺ leak from the ER. Using RNA-interference-mediated reduction of IP₃R1 we could demonstrate that the up-regulation of this isoform was responsible for the increased Ca²⁺ leak and the lowered [Ca²⁺]_ER in PS_DKO cells. We also showed that the decreased [Ca²⁺]_ER in PS_DKO cells was protective against apoptosis. In conclusion, our data indicate that IP₃Rs may be directly regulated at the level of the apparent affinity for IP₃. In addition, parameters that affect the structural requirements for proper channel opening may result in inactivation or in the formation of hyperactive channels or even in the generation of an IP₃-independent Ca²⁺ leak. Whereas an increased Ca²⁺ mobilization can contribute to pro-apoptotic mechanisms, Ca²⁺ leak pathways may also affect the Ca²⁺ store content. Such a decrease of the ER Ca²⁺ content may then be operative as an anti-apoptotic mechanism.