KRAP is required for diffuse and punctate IP 3 -mediated Ca 2+ liberation and determines the number of functional IP 3 R 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 (IP ₃ Rs) that mediate Ca ²⁺ 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 ₃ -mediated local Ca ²⁺ puffs and global, cell-wide Ca ²⁺ signals. High resolution Ca ²⁺ imaging revealed that the mean amplitude of puffs was strongly reduced by KRAP knockdown, whereas the Ca ²⁺ flux during openings of individual IP ₃ R 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 IP ₃ R 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 ²⁺ release), global, cell-wide Ca ²⁺ signals evoked by higher levels of IP ₃ are further composed from a discrete 'diffuse' mode of Ca ²⁺ release. By applying fluctuation analysis to isolate the punctate component during global Ca ²⁺ signals, we find that KRAP knockdown suppresses to similar extents punctate and diffuse Ca ²⁺ 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 IP ₃ Rs involved in diffuse Ca ²⁺ release as well as the clustered IP ₃ Rs that generate local Ca ²⁺ puffs.
Competing Interests: Declaration of Competing Interest None of the authors have any conflict of interest to declare.
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