Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are high-conductance channels that allow the regulated redistribution of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and, at specialized membrane contact sites (MCSs), to other organelles. Only a subset of IP3Rs release Ca2+ to the cytosol in response to IP3. These \'licensed\' IP3Rs are associated with Kras-induced actin-interacting protein (KRAP, also known as ITPRID2) beneath the plasma membrane. It is unclear whether KRAP regulates IP3Rs at MCSs. We show, using simultaneous measurements of Ca2+ concentration in the cytosol and mitochondrial matrix, that KRAP also licenses IP3Rs to release Ca2+ to mitochondria. Loss of KRAP abolishes cytosolic and mitochondrial Ca2+ signals evoked by stimulation of IP3Rs via endogenous receptors. KRAP is located at ER-mitochondrial membrane contact sites (ERMCSs) populated by IP3R clusters. Using a proximity ligation assay between IP3R and voltage-dependent anion channel 1 (VDAC1), we show that loss of KRAP reduces the number of ERMCSs. We conclude that KRAP regulates Ca2+ transfer from IP3Rs to mitochondria by both licensing IP3R activity and stabilizing ERMCSs.

KRas-induced actin-interacting protein (KRAP) has been identified as crucial for the appropriate localization and functioning of the inositol trisphosphate receptors (IP3Rs) that mediate Ca2+ release from the endoplasmic reticulum. Here, we used siRNA knockdown of KRAP expression in HeLa and HEK293 cells to examine the roles of KRAP in the generation of IP3-mediated local Ca2+ puffs and global, cell-wide Ca2+ signals. High resolution Ca2+ imaging revealed that the mean amplitude of puffs was strongly reduced by KRAP knockdown, whereas the Ca2+ flux during openings of individual IP3R channels was little affected. In both control and KRAP knockdown cells the numbers of functional channels in the clusters underlying puff sites were stochastically distributed following a Poisson relationship, but the mean number of functional channels per site was reduced by about two thirds by KRAP knockdown. We conclude that KRAP is required for activity of IP3R channels at puff sites and stochastically \'licenses\' the function of individual channels on a one-to-one basis, rather than determining the functioning of the puff site as a whole. In addition to puff activity (\'punctate\' Ca2+ release), global, cell-wide Ca2+ signals evoked by higher levels of IP3 are further composed from a discrete \'diffuse\' mode of Ca2+ release. By applying fluctuation analysis to isolate the punctate component during global Ca2+ signals, we find that KRAP knockdown suppresses to similar extents punctate and diffuse Ca2+ release in wild-type cells and in HEK293 cells exclusively expressing type 1 and type 3 IP3Rs. Thus, KRAP appears essential for the functioning of the IP3Rs involved in diffuse Ca2+ release as well as the clustered IP3Rs that generate local Ca2+ puffs.