KRAP regulates mitochondrial Ca2+ uptake by licensing IP3 receptor activity and stabilizing ER-mitochondrial junctions.

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.

Atakpa-Adaji P ，Ivanova A ，Kujawa K ，Taylor CW ... -  《-》

KRAP tethers IP(3) receptors to actin and licenses them to evoke cytosolic Ca(2+) signals.

Regulation of IP3 receptors (IP3Rs) by IP3 and Ca2+ allows regenerative Ca2+ signals, the smallest being Ca2+ puffs, which arise from coordinated openings of a few clustered IP3Rs. Cells express thousands of mostly mobile IP3Rs, yet Ca2+ puffs occur at a few immobile IP3R clusters. By imaging cells with endogenous IP3Rs tagged with EGFP, we show that KRas-induced actin-interacting protein (KRAP) tethers IP3Rs to actin beneath the plasma membrane. Loss of KRAP abolishes Ca2+ puffs and the global increases in cytosolic Ca2+ concentration evoked by more intense stimulation. Over-expressing KRAP immobilizes additional IP3R clusters and results in more Ca2+ puffs and larger global Ca2+ signals. Endogenous KRAP determines which IP3Rs will respond: it tethers IP3R clusters to actin alongside sites where store-operated Ca2+ entry occurs, licenses IP3Rs to evoke Ca2+ puffs and global cytosolic Ca2+ signals, implicates the actin cytoskeleton in IP3R regulation and may allow local activation of Ca2+ entry.

Thillaiappan NB ，Smith HA ，Atakpa-Adaji P ，Taylor CW ... -  《Nature Communications》

IP(3) receptors: An "elementary" journey from structure to signals.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are large tetrameric channels which sit mostly in the membrane of the endoplasmic reticulum (ER) and mediate Ca2+ release from intracellular stores in response to extracellular stimuli in almost all cells. Dual regulation of IP3Rs by IP3 and Ca2+ itself, upstream "licensing", and the arrangement of IP3Rs into small clusters in the ER membrane, allow IP3Rs to generate spatially and temporally diverse Ca2+ signals. The characteristic biphasic regulation of IP3Rs by cytosolic Ca2+ concentration underpins regenerative Ca2+ signals by Ca2+-induced Ca2+-release, while also preventing uncontrolled explosive Ca2+ release. In this way, cells can harness a simple ion such as Ca2+ as a near-universal intracellular messenger to regulate diverse cellular functions, including those with conflicting outcomes such as cell survival and cell death. High-resolution structures of the IP3R bound to IP3 and Ca2+ in different combinations have together started to unravel the workings of this giant channel. Here we discuss, in the context of recently published structures, how the tight regulation of IP3Rs and their cellular geography lead to generation of "elementary" local Ca2+ signals known as Ca2+ "puffs", which form the fundamental bottleneck through which all IP3-mediated cytosolic Ca2+ signals must first pass.

Smith HA ，Thillaiappan NB ，Rossi AM 《-》

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 (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.

Vorontsova I ，Lock JT ，Parker I 《-》

IP(3) Receptors Preferentially Associate with ER-Lysosome Contact Sites and Selectively Deliver Ca(2+) to Lysosomes.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) allow extracellular stimuli to redistribute Ca2+ from the ER to cytosol or other organelles. We show, using small interfering RNA (siRNA) and vacuolar H+-ATPase (V-ATPase) inhibitors, that lysosomes sequester Ca2+ released by all IP3R subtypes, but not Ca2+ entering cells through store-operated Ca2+ entry (SOCE). A low-affinity Ca2+ sensor targeted to lysosomal membranes reports large, local increases in cytosolic [Ca2+] during IP3-evoked Ca2+ release, but not during SOCE. Most lysosomes associate with endoplasmic reticulum (ER) and dwell at regions populated by IP3R clusters, but IP3Rs do not assemble ER-lysosome contacts. Increasing lysosomal pH does not immediately prevent Ca2+ uptake, but it causes lysosomes to slowly redistribute and enlarge, reduces their association with IP3Rs, and disrupts Ca2+ exchange with ER. In a "piston-like" fashion, ER concentrates cytosolic Ca2+ and delivers it, through large-conductance IP3Rs, to a low-affinity lysosomal uptake system. The involvement of IP3Rs allows extracellular stimuli to regulate Ca2+ exchange between the ER and lysosomes.

Atakpa P ，Thillaiappan NB ，Mataragka S ，Prole DL ，Taylor CW ... -  《Cell Reports》