KRAP is required for diffuse and punctate IP₃-mediated Ca²⁺ liberation and determines the number of functional IP₃R...

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.