KRAP is required for diffuse and punctate IP-mediated Ca liberation and determines the number of functional IPR channels within clusters.

KRas-induced actin-interacting protein (KRAP) has been identified as crucial for the appropriate localization and functioning of the inositol trisphosphate receptors (IPRs) that mediate Ca 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 IP-mediated local Ca puffs and global, cell-wide Ca signals. High resolution Ca imaging revealed that the mean amplitude of puffs was strongly reduced by KRAP knockdown, whereas the Ca flux during openings of individual IPR 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 IPR 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' Ca release), global, cell-wide Ca signals evoked by higher levels of IP are further composed from a discrete 'diffuse' mode of Ca release. By applying fluctuation analysis to isolate the punctate component during global Ca signals, we find that KRAP knockdown suppresses to similar extents punctate and diffuse Ca 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 IPRs involved in diffuse Ca release as well as the clustered IPRs that generate local Ca puffs.