IP3R at ER-Mitochondrial Contact Sites: Beyond the IP3R-GRP75-VDAC1 Ca2+ Funnel Peace Atakpa-Adaji1 and Adelina Ivanova1 Abstract Membrane contact sites (MCS) circumvent the topological constraints of functional coupling between different membrane- bound organelles by providing a means of communication and exchange of materials. One of the most characterised contact sites in the cell is that between the endoplasmic reticulum and the mitochondrial (ERMCS) whose function is to couple cel- lular Ca2+ homeostasis and mitochondrial function. Inositol 1,4,5-trisphosphate receptors (IP3Rs) on the ER, glucose-regu- lated protein 75 (GRP 75) and voltage-dependent anion channel 1 (VDAC1) on the outer mitochondrial membrane are the canonical component of the Ca2+ transfer unit at ERMCS. These are often reported to form a Ca2+ funnel that fuels the mitochondrial low-affinity Ca2+ uptake system. We assess the available evidence on the IP3R subtype selectivity at the ERMCS and consider if IP3Rs have other roles at the ERMCS beyond providing Ca2+. Growing evidence suggests that all three IP3R subtypes can localise and regulate Ca2+ signalling at ERMCS. Furthermore, IP3Rs may be structurally important for assembly of the ERMCS in addition to their role in providing Ca2+ at these sites. Evidence that various binding partners regulate the assembly and Ca2+ transfer at ERMCS populated by IP3R-GRP75-VDAC1, suggesting that cells have evolved mechanisms that stabilise these junctions forming a Ca2+ microdomain that is required to fuel mitochondrial Ca2+ uptake. Keywords Ca2+, endoplasmic reticulum, mitochondria, membrane contact sites, IP3 receptor, VDAC1, GRP75 Highlight • IP3Rs may have a structural role in forming ERMCS • All IP3R subtypes localise to ER–mitochondrial contact sites • Various tethering complexes and binding partners ensure the stability of ERMCS where IP3R delivers Ca2+ to the mitochondria Introduction Membrane contact sites (MCS) provide a means of biochem- ical exchange of signalling mediators between distinct membran e-bound organelles. For cellular homeostasis, intra- cellular compartments are required to cooperate via func- tional junctions in order to regulate various processes including Ca2+ signalling, lipid synthesis and trafficking (Chang and Liou, 2016, Zaman et al., 2020), glucose homeo- stasis (Rieusset, 2018), mitochondrial nucleoid transporta- tion (Qin et al., 2020), autophagy (Kohler et al., 2020), organelle dynamics (Prinz, 2014) and various pathological processes (Kim et al., 2022). MCS are characterised as the regions of proximity between two organelles or cellular com- partments that do not completely fuse but are held apart at short distances of ∼30 nm by distinct tethering complexes (Fernandez-Busnadiego et al., 2015; Prinz, 2014). First reported over 60 years ago, the endoplasmic reticulum (ER)–mitochondrial junction is one of the most characterised MCS (Copeland and Dalton, 1959). Tethering complexes of various compositions have been identified between the mito- chondria and the ER, posing an important need to understand if these signify the occurrence of functionally distinct junc- tions, or if independent tethering complexes contribute to the maintenance of the same junctions, so facilitating the overall functions of these contact sites. The composition of the ER–mitochondrial MCS (ERMCS) has been thoroughly reviewed previously (de Brito and Scorrano, 2010, Prinz, 2014, Sassano et al., 2022, Xu et al., 2020). The morphology and composition of these junctions are not static but are dynamic and responsive to changing cellular metabolic demands. Cryogenic-electron microscopy (Cryo-EM) analysis has shown that the length of ERMCS changes in response to nutrient availability (Sood et al., 2014), while biochemical analysis reveals a change in the number of ERMCS during transitions in nutrient availability 1Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK Received February 28, 2023. Revised February 29, 2023; Accepted May 23, 2023. Corresponding Author: Peace Atakpa-Adaji, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK. Email: pa376@cam.ac.uk Creative Commons CC BY: This article is distributed under the terms of the Creative Commons Attribution 4.0 License (https:// creativecommons.org/licenses/by/4.0/) which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage). Membrane Contact sites as hubs for Ca2+ signaling – Review Contact Volume 6: 1–12 © The Author(s) 2023 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/25152564231181020 journals.sagepub.com/home/ctc https://orcid.org/0000-0002-9398-7750 mailto:pa376@cam.ac.uk https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://us.sagepub.com/en-us/nam/open-access-at-sage https://us.sagepub.com/en-us/nam/open-access-at-sage https://us.sagepub.com/en-us/journals-permissions https://journals.sagepub.com/home/ctc http://crossmark.crossref.org/dialog/?doi=10.1177%2F25152564231181020&domain=pdf&date_stamp=2023-06-22 (Theurey et al., 2016). The onset of ER stress is reportedly marked by an increase in ERMCS and subsequent changes in mitochondrial functions that drive the cellular adaptation to this signal (Bravo et al., 2011). Similarly, a change in ER–mitochondria coupling is also observed following chemically-induced mitochondrial stress (Lopez-Crisosto et al., 2021). Advanced imaging techniques have also shown the dynamism of these junctions, revealing highly motile VAMP-associated protein B (VAPB) positive contact sites that are altered in response to nutrient stress (Obara et al., 2022). In this review, we will discuss the role of ERMCS in shaping the delivery of Ca2+ to the mitochondria. Specifically, we will highlight the role and regulation of com- ponent proteins that drive the stability and delivery of Ca2+ at these junctions. ERMCS and Ca2+ Signalling The role of ERMCS in shaping Ca2+ signals is one of the most characterised functions of these contact sites. Ca2+ must permeate the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM) to reach the mitochondrial matrix. The OMM has a high Ca2+ permeabil- ity. This is possibly due to its abundant expression of voltage-dependent anion-selective channel proteins (VDACs) (of which there are 3 subtypes (VDAC1-3)); although VDAC1 is predominantly implicated in mitochon- drial Ca2+ dynamics, it was shown to selectively co-immunoprecipitate with inositol 1,4,5-trisphosphate receptors (IP3R; see later)(De Stefani et al., 2012; Gincel et al., 2001, Rapizzi et al., 2002). There may yet be uniden- tified Ca2+ permeable channels on the OMM that drive its high permeability to Ca2+ as the loss of all VDAC isoforms does not affect mitochondrial-dependent cell death, a process thought to occur following mitochondrial Ca2+ overload (Baines et al., 2007). Following many years of debate, the mitochondrial Ca2+ uniporter (MCU) was identified as the mediator of Ca2+ transport through the IMM into the mito- chondrial matrix (De Stefani et al., 2011; Kirichok et al., 2004). The Ca2+ uptake into mitochondria is now strongly encouraged to involve a tightly regulated multiprotein complex (reviewed in (Giorgi et al. 2018), (De Stefani et al., 2015)). The low affinity of the MCU (KD Ca2+ for MCU, ∼20 −30 µM) is evolutionary important to avoid unregulated Ca2+ overload of the mitochondria for triggering apoptosis (Bragadin et al., 1979). The accumulation of high concentrations of Ca2+ in the mitochondria occurs following the stimulation of Ca2+ channels on the ER. This is only made possible due to their close apposition to the ER (Csordas et al., 2010; Rizzuto et al., 1998). Between 5% and 20% of the surface of the mitochondrial network in HeLa cells is in contact with the ER under resting conditions (Rizzuto et al., 1998). The canonical view of the ERMCS protein complex driving Ca2+ exchange is the interaction of IP3R with VDAC1 via the cytosolic protein linker GRP75. GRP75 is part of the heat shock protein 70 family of proteins and is reported to stabilise the IP3R-VDAC1 inter- action and therefore promote the formation of ERMCS enab- ling Ca2+ transfer into the mitochondria (Xu et al., 2018). It is important to note that other than at ERMCS, mitochondrial Ca2+ uptake dynamics have also been implicated in regulat- ing Ca2+ signals at ER–PM membrane contact sites by regu- lating STIM1 oligomerisation at these junctions (Deak et al., 2014, Nan et al., 2021). The transfer of Ca2+ at ERMCS not only shapes the spatiotemporal properties of global Ca2+ signals but also dir- ectly affects mitochondria functions. For example, the activ- ity of three of the important enzymes in the electron transport chain pyruvate dehydrogenase, isocitrate dehydrogenase, oxoglutarate dehydrogenase (OGDH) and α-ketoglutarate dehydrogenase is Ca2+-dependent (Denton, 2009). Ca2+ sig- nalling at ERMCS is also an important regulator of cellular apoptosis (Kroemer and Reed, 2000). Ca2+ overload into the mitochondria is an important trigger for apoptosis leading to mitochondrial swelling, the opening of the mito- chondrial permeability transition pore (mPTP) and release into the cytosol of apoptotic factors such as cytochrome c, procaspase 9 and apoptosis-inducing factor 9 (Giorgi et al. 2018, Sukumaran et al., 2021). Furthermore, B cell lymph- oma 2 (Bcl-2), mainly resident on the OMM, is an anti- apoptotic protein which reduces the steady-state ER Ca2+ concentration and subsequent Ca2+ transfer to the mitochon- dria so enhancing the apoptotic signals (Pinton and Rizzuto, 2006, Sukumaran et al., 2021). Conversely, the pro-apoptotic protein BAX promotes apoptosis by increasing Ca2+ transfer to the mitochondria from the ER (Sukumaran et al., 2021). VDAC1 oligomerisation is also implicated in regulating apoptosis (Shoshan-Barmatz et al., 2018). Moreover, a reduction in mitochondrial Ca2+ uptake, such as following down-regulation of IP3R on the ER, leads to the impairment of cellular bioenergetics and the activation of autophagy, a process where obsolete cellular components are degraded or recycled in lysosomes (Sukumaran et al., 2021). It is there- fore not unexpected that disruptions of ERMCS are impli- cated in the pathophysiology of multiple diseases such as amyotrophic lateral sclerosis (Sakai et al., 2021), Alzheimer’s disease (Area-Gomez et al., 2009, Yu et al., 2021), Parkinson’s disease (Guardia-Laguarta et al., 2014), Huntington’s disease (Kim et al., 2010; Milakovic et al., 2006) and cancer (Simoes et al., 2020) (Figure 1). Localisation and Functioning of IP3Rs at ERMCS IP3Rs are high-conductance intracellular Ca 2+ channels, ubi- quitously expressed in eukaryotic cells, which open in response to extracellular stimuli. Ca2+ signals elicited by IP3Rs regulate a vast plethora of biological processes such as motility, neurotransmitter release, apoptosis and gene 2 Contact transcription (Berridge, 2016; Clapham, 2007). IP3Rs are predominantly located on the ER but have also been reported to be in the nuclear envelope, plasma membrane, Golgi and secretory vesicles (Dellis et al., 2006; Malviya et al., 1990; Pinton et al., 1998, Yoo and Albanesi, 1990). There are 3 subtypes of IP3R, which have a molecular mass of ∼300 kDa and share 60-80% sequence homology, although they are encoded by different genes and expressed differentially in tissues (Foskett et al., 2007; Taylor et al., 1999). The exist- ence of alternative splice variants confers further diversity to IP3R expression in cells (Foskett et al., 2007). Functionally, IP3R exists as tetrameric channels which can be homomeric or heteromeric (Alzayady et al., 2013). IP3R are co-regulated by binding to IP3 and Ca2+, with the latter regulating the channel in a biphasic manner such that at low cytosolic concentrations of Ca2+ in the presence of IP3 activates the channel (Mak and Foskett, 1998) while high Ca2+ concentrations conversely inhibit the IP3 receptor (Kaftan et al., 1997, Mak and Foskett, 1998). Recent insights into the structure of IP3Rs continue to improve our under- standing of the structural basis for IP3R activation and channel opening (Schmitz et al., 2022). Whilst most cells express more than one IP3R subtype, preferential expression of the subtypes has been reported, with IP3R1 reported as the predominant subtype in Purkinje cell neurons (Nakanishi et al., 1991), IP3R2 in cardiac myocytes (Vervloessem et al., 2015) but also in the liver and epithelium (Klar et al., 2014), and IP3R3 in pancreatic β cells (Blondel et al., 1994), testis and endothelial cells (De Smedt et al., 1997). While all three IP3R subtypes are regulated by IP3 and Ca2+, they have different IP3 affinities in the order IP3R2 > IP3R1 > IP3R3 (Iwai et al., 2007, Miyakawa et al., 1999; Newton et al., 1994; Tu et al. 2005b, Zhang et al., 2011) and are distinctly modulated by additional signals such as Ca2+ and ATP (Mak et al., 2001; Prole and Taylor, 2016, Tu et al. 2005a, Wagner and Yule, 2012; Yoneshima et al., 1997). In systems containing all three IP3R subtypes, the composition of IP3R channels that participate in Ca2+ release remains a question, as minor subtypes could be select- ively contributing to the Ca2+ signals while the major subtype remains silent (Lock et al., 2018). The Ca2+ signals evoked by IP3R occur in a hierarchical manner, with increasing IP3 stimulus corresponding to a graded response from local Ca2+ events, such as Ca2+ puffs involving the opening of a few IP3R to global Ca2+ waves that spread across the entire cell (Berridge et al., 2000). All IP3R subtypes can evoke Ca2+ puffs with a similar frequency and amplitude, but IP3R2’s high affinity for IP3 is reflected in the slower kinetics of individual puffs (Lock et al., 2018, Mataragka and Taylor, 2018). The original notion that Ca2+ puffs are the building blocks of all modes of Ca2+ signalling (Berridge, 1997, Bootman and Berridge, 1996, Bootman et al., 1997, Marchant et al., 1999, Parker et al., 1996) has recently been challenged; it has been sug- gested that, sustained global signals are evoked by a diffuse mode of Ca2+ release distinct from Ca2+ puffs which terminate during the early stages of global Ca2+ waves (Lock and Parker, 2020). Another layer of complexity in the strict regulation of IP3R activity is that not all IP3Rs are licensed to release Ca2+. The majority of IP3Rs in a cell are mobile. However, Ca2+ signals originate from a subset of IP3R immobilised on actin via KRas-induced actin-binding protein (KRAP). The loss of KRAP reduces the number of immobile IP3R clusters in a cell, abrogates Ca2+ puffs and global Ca2+ signals (Thillaiappan et al., 2021). There are thought to be approximately 8 IP3Rs in a licensed IP3R cluster from which elementary Ca2+ events occur (Thillaiappan et al., 2017, 2021). However, further analysis has suggested that KRAP may license individual IP3R chan- nels rather than the entire cluster as a signalling unit (Vorontsova et al., 2022). Furthermore, cyclic AMP response element-binding protein (CREB) may affect IP3R1 licensing Figure 1. Ca2+ Transfer at ER–Mitochondria Contact Sites Is Important for Cellular Homeostasis. IP3R forms a tightly regulated complex with GRP75 in the cytosol and VDAC1 in the outer mitochondrial membrane to allow preferential access of high Ca2+ concentrations to fuel the low-affinity MCU. Ca2+ taken up into the mitochondria is important for mitochondrial function and dynamics. Dysregulation of ERMCS abrogates mitochondrial Ca2+ uptake with consequences for cellular Ca2+ dynamics and various pathophysiological processes. Atakpa-Adaji and Ivanova 3 by regulating the expression of KRAP in HEK293 cells (Arige et al., 2021). Even with our increased knowledge of the structure and function of IP3R, there continues to be active research on the fine-tuning of its licensing, activity and spatial localisation. All IP3R Subtypes Localise to ER–Mitochondrial Contact Sites With different biophysical properties of the IP3R subtypes, there has been a long interest in the subtype-specific regula- tion of ER–mitochondrial contact sites. Initial analysis into the importance of subtype selectively in the role of IP3R at ER–mitochondria MCS in CHO-KI and HEK293 cells has suggested a preferentiality for IP3R3 in delivering Ca2+ to mitochondria and subsequent regulation of apoptosis (Mendes et al., 2005). Furthermore, Mendes et al. using con- focal microscopy suggested that IP3R3 colocalises more extensively with mitochondria compared to the other 2 sub- types (Mendes et al., 2005). For many years it was thus con- sidered that IP3R3 selectively interacts with mitochondria at ERMCS and is the required mediator of apoptotic Ca2+ over- load. Nonetheless, there have been isolated studies showing the roles of the other IP3R subtypes at ERMCS in different biological settings. Recombinant expression of IP3R1 was shown to enhance Ca2+ accumulation in mitochondria in the rat liver and HeLa cells (Szabadkai et al., 2006). Furthermore, IP3R1 forms a tripartite complex with GRP75 and VDAC1 modulating diabetic atrial remodelling (Yuan et al., 2022). In astrocytes, IP3R2 was found to be accumu- lated together with mitochondria at the sites of regenerative Ca2+ wave initiation (Simpson et al., 1998). However, in a systematic study, Bartok et al. showed that all IP3R subtypes are competent to restore contacts between the ER and mito- chondria in a null IP3R background with compromised ERMCS (Bartok et al., 2019). Super-resolution analysis shows that all IP3R subtypes can form clusters near to the mitochondria, but IP3R2 may be more enriched at ER– mitochondrial junctions in DT40 cells. Whilst all IP3R sub- types were found to be competent in transferring Ca2+ to mitochondria, IP3R2 displayed higher efficacy in mediating Ca2+ transfer from the ER to the mitochondria in DT40 cells (Bartok et al., 2019). This work further suggests that IP3R1-mediated Ca2+ transfer to the mitochondria may be predominantly sustained by Ca2+ entry across the plasma membrane in DT40 cells (Bartok et al., 2019). Therefore, there is growing evidence of the localisation of all IP3R sub- types at ERMCS (Table 1). Further assessment of the context-specific contribution of IP3R subtypes to ERMCS formation and function is required to address the potential involvement of all three subtypes in junction stability and Ca2+ transfer from the ER to the mitochondria. IP3Rs Have a Structural Role in Forming ERMCS Recent evidence suggests that IP3Rs may play a structural role in ERMCS formation which is independent of their ability to deliver Ca2+ from the ER for mitochondrial uptake via the MCU. HEK293 cells are reported to show limited ER–mitochondrial Ca2+ transfer, but tight IP3R-dependent ERMCS (Katona et al., 2022). HEK293 cells lacking IP3R showed fewer regions of close apposition (<20 nm) between the ER and mitochondria and re-expression of IP3Rs including a non-Ca2+ conducting mutant in HEK293 cells rescued the contacts between the ER and mitochondria (Katona et al., 2022). Recruitment of mobile IP3R at ER–mitochondrial contacts has been shown to license the IP3Rs to rapidly deliver Ca2+ to the mitochon- dria and increase the activity of mitochondrial Ca2+-sensitive dehydrogenases (Katona et al., 2022). Analysis using drug-inducible ER–mitochondrial linkers showed that ERMCS junctions formed more quickly in WT DT40 cells compared to IP3R knock-out cells where IP3R-mediated Ca2+ release was completely attenuated (Bartok et al., 2019). This further suggests that IP3R are not situated at junc- tions with mitochondria solely for the provision of Ca2+, but Table 1. Summary of IP3R subtype identification at ERMCS in different cell types. Subtype Cell line Comment Reference IP3R1 DT40 IP3R1 may increase [Ca2+]mito, fuelled by Ca2+ influx from PM (Bartok et al., 2019) Rat liver IP3R1 forms a complex with GRP75 and VDAC1 (Szabadkai et al., 2006) HeLa IP3R1 forms a complex with GRP75 and VDAC1 (Szabadkai et al., 2006) IP3R2 Astrocytes IP3R2 sits with mitochondria at the sites of regenerative Ca2+ signals (Simpson et al., 1998) DT40 Suggests all IP3R can exist at ERMCS but IP3R2 most effective in Ca2+ transfer to mitochondria (Bartok et al., 2019) IP3R3 HeLa IP3R3 forms a complex with GRP75 and VDAC1 (Gomez-Suaga et al., 2017) HEK siRNA to IP3R3 selectively reduces [Ca2+]mito (Mendes et al., 2005) CHO siRNA to IP3R3 selectively reduces [Ca2+]mito (Mendes et al., 2005) C57B6/J mice brain slices IP3R3 at MAM fractions with VDAC1, Tom70 and VAPB (Filadi et al., 2018) 4 Contact are required for junction assembly. IP3R-mediated regulation of the gap length at specialised junctions is independent of their Ca2+ flux property as pore-dead IP3Rs are equally as competent in restoring tight junctions. Close ER–mitochon- dria proximity nonetheless remains key for Ca2+ transfer to the mitochondria as forcing the mitochondria to the PM and away from the ER disrupts IP3R-mediated Ca2+ transfer into the mitochondria. Such proximity is likely maintained by several tethering complexes acting synergistically. Co-Regulators of the IP3R-GRP75-VDAC1 complex at ERMCS Although the IP3R–VDAC–GRP75 interaction in the canon- ical tethering complex is often reported at the ERMCS Ca2+ microdomain, these junctions can be modulated by other membrane-anchored or cytosolic binding partners (Figure 2). Tespa1. Thymocyte-expressed positive selection-associated gene 1 (Tespa1) is an important regulator of T cell develop- ment in the thymus which shares sequence homology (including the double phenylalanine motif that interacts with IP3Rs) with KRAP (Matsuzaki et al., 2012, Thillaiappan et al., 2021). Although it lacks the actin-binding domain of KRAP, Tespa1 has been shown to interact with IP3Rs as well as GRP75 in T and B lymphocytes (Matsuzaki et al., 2012, Matsuzaki et al., 2013). The loss of Tespa1 leads to a reduction in both cytosolic and mito- chondrial Ca2+ signals following T-cell receptor stimulation (Matsuzaki et al., 2013). This would suggest that Tespa1 functions in these cells to regulate IP3R activity at ERMCS (Matsuzaki et al., 2012). Sigma-1 Receptor. The ER chaperone protein sigma-1 recep- tor is an important regulator of Ca2+ signalling at ERMCS. The sigma-1 receptor is thought to remain bound to another chaperone protein binding immunoglobulin protein (BiP) under physiological conditions. However, upon ER stress, such as during ER Ca2+ depletion, sigma-1 dissociates from BiP and translocates to ERMCS, stabilising IP3R3 at these junctions and promoting the prolonged release of Ca2+ from the ER into the mitochondria (Hayashi and Su, 2007). Other functions of the sigma-1 receptor at ERMCS include stabilising the ER stress sensor inositol-requiring enzyme 1 (IRE-1) and promoting its ability to respond to mitochondria-derived reactive oxygen species during ER stress (Mori et al., 2013). Furthermore, the sigma-1 receptor may regulate dendritic spine formation by modulating the free radical formation around ERMCS to dampen caspase-3 activation and consequent inactivation of Rac GTPase via the degradation of guanine nucleotide exchange factor (Tsai et al., 2009). Sigma-1 receptors functioning at ERMCS are implicated in the pathophysiology of multiple neurodegenerative diseases (Nguyen et al., 2017). BOK. B cell lymphoma 2 (BCL-2) ovarian killer (BOK) is an ER-resident pro-apoptotic protein. It has been shown to bind IP3Rs, regulating both Ca2+ dynamics at ERMCS under physiological conditions, and following a stimulus (Carpio et al., 2021). BOK is also shown to regulate the stability and composition of proteins at ERMCS (Carpio et al., 2021). The loss of BOK leads to a decreased localisation of IP3R1, IP3R3 and the sigma-1 receptor at ERMCS as assessed by microscopy (Carpio et al., 2021). Furthermore, whilst the amount of VDAC1 is unchanged following the loss of BOK in pure mitochondria-associated membrane (MAM) fractions, the amount of IP3R1 and IP3R3 is signifi- cantly attenuated (Carpio et al., 2021). Conversely, overex- pression of MCL-1 and BOK transmembrane domains in HeLa cells increases ERMCS populated by IP3R and VDAC as assessed via proximity ligation assay (Lucendo et al., 2020). The BOK-IP3R interaction at ERMCS is impli- cated in the regulation of apoptosis in these cells (Carpio Figure 2. Composition of ER–Mitochondrial Contact Sites Where Ca2+ Exchange Occurs. Various binding partners and regulators of the IP3R-GRP75-VDAC1 complex have been described. These act either by binding directly to one or more of the components (top) or by stabilising the ERMCS (bottom) thereby allowing IP3R to remain localised preferentially to provide Ca2+ for the MCU. Atakpa-Adaji and Ivanova 5 et al., 2021). Altogether, these suggest BOK plays a role in the recruitment and localisation of IP3R at ERMCS. Tom70. The translocase of the outer membrane (TOM) family of proteins accounts for a significant proportion of the OMM protein content. TOM proteins, together with the translocase of the inner membrane (TIM), are key regulators of the trafficking of mitochondrial proteins encoded in the nucleus (Chacinska et al., 2009). Unlike TOM20 which has a uniform distribution across the OMM, TOM70 appears more punctate, coinciding with ERMCS contact sites iden- tified with an engineered split-GFP construct (Filadi et al., 2018). TOM70 was also found to be present in pure MAM fractions physically bind to IP3R2 (as assessed by co-immunoprecipitation analysis) and regulate IP3R- mediated Ca2+ transfer and cellular bioenergetics (Filadi et al., 2018). Of note, the depletion of TOM70 did not affect the number of ERMCS junctions detected (Filadi et al., 2018). This suggests that TOM70 is not structurally required in the assembly of the junction but may be involved in ‘trapping’ IP3R at these junctions. Transglutaminase Type 2 (TG2). Transglutaminase type 2 (TG2) is an important mediator of post-translational modifi- cations of proteins and localises to various cellular compart- ments including the cytosol, mitochondria and nucleus (Szondy et al., 2017). Mass spectrometry reveals that TG2 interacts with multiple proteins on the ER and mitochondria including BiP, TOM70 and GRP75 (D’Eletto et al., 2018). TG2 was found to immunoprecipitate with GRP75 and was also enriched in pure MAM fractions (D’Eletto et al., 2018). Overexpression of TG2 reduces the IP3R3–GRP75 interaction in MEFs, suggesting that TG2 is a negative regu- lator of IP3R-GRP75 (D’Eletto et al., 2018). TG2 also mod- ulates the composition of ERMCS and mitochondrial Ca2+ uptake following agonist stimulation (D’Eletto et al., 2018). Although the IP3R3–GRP75 association is increased in the absence of TG2, the overall ERMCS number is reduced (D’Eletto et al., 2018). One argument is that the increase in the IP3R3–GRP75 interaction occurs as a com- pensatory mechanism for the reduction in physical coupling between the ER and mitochondria. It remains to be elucidated if TG2 is a physical tether or only a regulatory partner at these ERMCS. iRhom. iRhom, a catalytically-dead member of the rhomboid family of serine proteases is best characterised as a cofactor of ADAM metallopeptidase domain 17 (ADAM17) (Dulloo et al., 2019). Although not exclusively, iRhoms which has 2 subtypes (iRhom1/2) can be found on the ER and have been implicated in regulating the transfer of Ca2+ to the mitochondria via IP3Rs in ER stress (Dulloo et al., 2022). Depletion of iRhom1/2 under ER stress leads to defective mitochondrial membrane potential and abrogated IP3R-mediated cytosolic and mitochondrial Ca2+ signals (Dulloo et al., 2022). Although iRhoms were shown to inter- act with IP3Rs, it is not clear if they reside within ERMCS, or if they indirectly affect mitochondrial Ca2+ uptake by glo- bally attenuating IP3R activity under ER stress. Seipin. Seipin is an ER resident protein implicated in lipody- strophy (Combot et al., 2022). The observation that patient- derived lymphoblast showed perturbed mitochondrial morphology prompted further investigation into the possible roles of this protein at ERMCS (Combot et al., 2022). Seipin is reported to be enriched at ERMCS populated by IP3R, VDAC and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) during starvation (Combot et al., 2022). Seipin deficiency leads to attenuation of the uptake of mitochondrial Ca2+ following IP3R stimulation and impaired mitochondrial metabolism and dynamics in A431 cells (Combot et al., 2022). Pyruvate Dehydrogenase Kinases (PDKs). Pyruvate dehydro- genase kinases (PDKs) are serine/threonine kinases mainly located in the mitochondria where they regulate the activity of pyruvate dehydrogenase (PDH) in glycolysis (Wang et al., 2021). There are four isoforms of PDK (PDK 1-4), showing broad tissue expression. Using proximity ligation assays and cellular fractionation analysis, PDK4 alone was shown to localise and interact with IP3R1–VDAC–GRP75 at ERMCS in skeletal muscle cells (Thoudam et al., 2018). Overexpression of PDK4 enhances the IP3R1–VDAC1– GRP75 interaction at ERMCS (Thoudam et al., 2018). The kinase activity of PDK4 is required for this enhancement as the pharmacological inhibition of its kinase activity, or the use of a kinase-dead mutant, does not show this enhancement (Thoudam et al., 2018). Inhibition of PDK4 in these cells also attenuates IP3R-mediated Ca2+ transfer to the mitochondria without affecting the ER store content (Thoudam et al., 2018). Using mice models, PDK4 has been shown to be increased in obesity, leading to the regulation of ERMCS for- mation and Ca2+ transfer to the mitochondria (Thoudam et al., 2018). This suggests PDK4 as a structural tether and stabiliser of IP3R-positive ERMCS in these specialised cells. PDKs are traditionally known to reside in the mito- chondrial matrix (Wang et al., 2021), therefore the mechan- ism by which they interact and couple with IP3R1, GRP75 and VDAC in MAM fractions remains unclear. Furthermore, PDK4 can be found in other cell types such as the liver, kidneys and pancreatic cells (Moon et al., 2012), but it remains to be determined if it also interacts at ERMCS in these other cells. Other Co-Regulators of ERMCS Where Ca2+ Exchange Occurs. Other regulators of ERMCS important for Ca2+ exchange have been described in metabolic regulation, neurodegenera- tive disease models and in cancer. In metabolic regulation, abrogation of the tethers VAPB on the ER membrane and protein tyrosine phosphatase interacting protein 51 6 Contact (PTPIP51) on the mitochondrial membrane at ERMCS that regulate autophagy attenuates the IP3R3–VDAC1 interaction and subsequent IP3R mediated Ca2+ signals to the mitochon- dria (Gomez-Suaga et al., 2017). Furthermore, the deletion of PTP1P51 coiled-coil domain which affects its localisation at ERMCS, abrogates IP3R-mediated Ca2+ delivery to the mito- chondria (Mórotz et al., 2022). In cardiomyocytes, the mito- chondrial resident protein FUN14 domain containing 1 (FUNDC1) binds to IP3R2 to regulate ERMCS and modulate Ca2+ exchange at these junctions (Wu et al., 2017). In neurodegenerative disease models, the loss of function mutation in Niemann-Pick type C1 (NPC1) changes the spatial distribution of IP3R1, potentiates GPCR-mediated Ca2+ signals, and promotes IP3R-mediated mitochondrial Ca2+ uptake causing cytotoxicity (Tiscione et al., 2021). Furthermore, DJ-1 encoded for by PARK7 is a cytosolic and nuclear protein implicated in early onset Parkinson’s disease. PARK7 which translocates to ERMCS to interact with the IP3R–GRP75–VDAC1 complex to regulate both the integrity of the ERMCS and Ca2+ transfer at these junc- tions (Liu et al., 2019). DJ1 regulates the spatial localisation of IP3R, with the loss of DJ1, leading to the aggregation of IP3R via an unknown mechanism (Liu et al., 2019). Actin polymerisation via the ER-anchored inverted formin 2 (INF2) results in an increase in mitochondrial Ca2+ and further change in the mitochondrial morphology via dynamin-related protein 1 (Drp1) recruitment (Chakrabarti et al., 2018). In adrenocortical carcinoma, fetal and adult testis-expressed 1 (FATE1), which encodes a cancer-testis antigen has been shown to localise to MAMs (Doghman-Bouguerra et al., 2016). There it decreases ERMCS and negatively modulates the transfer of IP3R-mediated Ca2+ to the mitochondria. In addition, Furthermore, FATE1 has also been suggested to attenuate apoptosis in these adrenocortical carcinoma cells (Doghman-Bouguerra et al., 2016). Upregulation of FATE1 in cancers may occur to drive uncoupling of the ER and mito- chondria, attenuate Ca2+ delivery and increase the resistance to cell death in these cancer cells. Perspectives and Concluding Remarks As highlighted, multiple regulators of the IP3R-GRP75-VDAC complex at ERMCS mediate Ca2+ exchange in various cell types. This poses the question of how ERMCS are regulated? Furthermore, the dogma that the IP3R–GRP75–VDAC tripartite complex forms a funnel through which Ca2+ is exchanged from the ER to the mitochondria, requires closer scrutiny. Firstly, there is evidence that other binding partners can interact with IP3R to form ERMCS where Ca2+ exchange occurs, as is the case for IP3R interacting with the AKAP1 transmembrane domain or TOM70 on the mitochondrial membrane (Katona et al., 2022). Although it is important to note that TOM70 has been shown to interact directly with the IP3R–GRP75–VDAC complex (SECTION 3.3); therefore, TOM70 may well be a coincident reporter of the same IP3R–GRP75–VDAC junctions. Secondly, disruption of other tethering complexes such as between VAPB and PTPIP51 on the OMM, also disrupts Ca2+ uptake into the mitochondria (De Vos et al., 2012). This suggests a model where tethering complexes work syn- ergistically to promote ERMCS where Ca2+ exchange can occur. Indeed, overexpression of VAPB and PTP1P51 was shown to increase the IP3R3–VDAC1 interaction in HeLa cells and Ca2+ exchange at these junctions (Gomez-Suaga et al., 2017). Furthermore, the knockdown of mitofusin-2 which tethers at ERMCS via interaction with either mitofu- sin 1 or 2 on the OMM also regulates the stability of the junctions, with its ablation leading to attenuated mitochon- drial Ca2+ uptake (de Brito and Scorrano, 2008, Naon et al., 2016). This is not without controversy, since MFN2 has been postulated to act in the opposite direction where it reduces the stability of ERMCS and its reduction increases Ca2+ transfer to the mitochondria (Filadi et al., 2015). Thirdly, local Ca2+ transfer at ERMCS can be circumvented using a sufficiently high concentration of a rapid Ca2+ che- lating agent such as ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (Csordas et al., 1999). A tight Ca2+ microdomain synonymous with a funnel may be impenetrable by buffering agents as Ca2+ can be channelled through to the mitochondria regardless of the buffering activity in the cytosol. We suggest that the ER–mitochondrial Ca2+microdomain is maintained by a complex of different tethers and binding proteins working synergistically to promote ERMCS contact sites where IP3R sits to power the low-affinity mito- chondrial Ca2+ uptake mechanism. Further work is required to elucidate the interdependence of various tethering com- plexes on mitochondrial Ca2+ uptake and their role in health and disease. Whilst current technologies are limited in their capabilities, it is also important to assess how the individual tethering complexes at ERMCS change simultan- eously in response to metabolic demands, as it will help answer the longstanding questions around the role of the mul- tiple complexes found at these junctions. Abbreviations BAX Bcl-2-associated X protein Bcl-2 B cell lymphoma 2 BOK Bcl-2 related ovarian killer Ca2+ calcium CHO Chinese hamster ovary cell CREB cyclic AMP-response element-binding protein Cryo-EM cryogenic electron microscopy EGTA tetra(acetoxymethyl Ester) ER endoplasmic reticulum ERMCS ER-mitochondrial membrane contact sites FUNDC1 FUN14 domain containing 1 green fluorescent protein GPCR G protein-coupled receptor Atakpa-Adaji and Ivanova 7 GRP 75 glucose-regulated protein 75 HEK human embryonic kidney IMM inner mitochondrial membrane INF2 inverted formin 2 IP3R inositol 1,4,5-trisphosphate KRAP KRas-induced actin binding protein MAM mitochondria-associated membrane MCS membrane contact sites MCU mitochondrial Ca2+ uniporter mPTP mitochondrial permeability transition pore NPC1 Niemann-Pick type C1 OGDH oxoglutarate dehydrogenase OMM outer mitochondrial membrane PDK pyruvate dehydrogenase kinases PM plasma membrane PTPIP51 protein tyrosine phosphatase interacting protein 51 SERCA sarco/endoplasmic reticulum Ca2+-ATPase STIM1 stromal interaction molecule 1 Tespa1 thymocyte-expressed positive selection-associated gene 1 TG2 transglutaminase type 2 TIM translocase of the inner membrane TOM70 translocase of the outer membrane 70 VAPB VAMP associated protein B VDAC voltage-dependent anion-selective channel proteins WT wild type Acknowledgement This work was supported by the Biotechnology and Biological Sciences Research Council UK (BB/T012986/1). P.A-A is a fellow of Emmanuel College, Cambridge. A.I is supported by a Cambridge European, Department of Pharmacology & Wolfson Medical Research Scholarship. We would like to thank Prof Colin Taylor for helpful discussions during the write-up of this review. We thank Prof Graham Ladds for providing guidance and for reviewing the manuscript to enhance its clarity. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. ORCID iDs Peace Atakpa-Adaji https://orcid.org/0000-0002-9398-7750 References Alzayady KJ, Chandrasekhar R, Yule DI (2013). Fragmented inosi- tol 1,4,5-trisphosphate receptors retain tetrameric architecture and form functional Ca2+ release channels. Journal of Biological Chemistry 288(16), 11122–11134. https://doi.org/ 10.1074/jbc.M113.453241 Area-Gomez E, de Groof AJ, Boldogh I, Bird TD, Gibson GE, Koehler CM, Yu WH, Duff KE, Yaffe MP, Pon LA et al. (2009). Presenilins are enriched in endoplasmic reticulum membranes associated with mitochondria. The American Journal of Pathology 175(5), 1810–1816. Arige V, Terry LE, Malik S, Knebel TR, Wagner Ii LE, Yule DI (2021). CREB Regulates the expression of type 1 inositol 1,4,5-trisphosphate receptors. Journal of Cell Science 134(20), jcs258875. Baines CP, Kaiser RA, Sheiko T, Craigen WJ, Molkentin JD (2007). Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death. Nature Cell Biology 9(5), 550–555. Bartok A, Weaver D, Golenar T, Nichtova Z, Katona M, Bansaghi S, Alzayady KJ, Thomas VK, Ando H, Mikoshiba K et al. (2019). IP3 Receptor isoforms differently regulate ER-mitochondrial contacts and local calcium transfer. Nature Communications 10(1), 3726. Berridge MJ (1997) Elementary and global aspects of calcium sig- nalling. Journal of Physiology 499, 291–306. Berridge MJ (2016). The inositol trisphosphate/calcium signaling pathway in health and disease. Physiological Reviews 96, 1261–1296. Berridge MJ, Lipp P, Bootman MD (2000). The versatility and uni- versality of calcium signalling. Nature Reviews: Molecular Cell Biology 1, 11–21. Blondel O, Bell GI, Moody M, Miller RJ, Gibbons SJ (1994). Creation of an inositol 1,4,5-trisphosphate-sensitive Ca2+ store in secretory granules of insulin-producing cells. Journal of Biological Chemistry 269, 27167–27170. Bootman MD, Berridge MJ (1996). Subcellular Ca2+ signals under- lying waves and graded responses in HeLa cells. Current Biology 6, 855–865. Bootman MD, Berridge MJ, Lipp P (1997). Cooking with calcium: The recipes for composing global signals from elementary events. Cell 91, 367–373. Bragadin M, Pozzan T, Azzone GF (1979). Kinetics of Ca2+ carrier in rat liver mitochondria. Biochemistry 18(26), 5972–5978. Bravo R, Vicencio JM, Parra V, Troncoso R, Munoz JP, Bui M, Quiroga C, Rodriguez AE, Verdejo HE, Ferreira J et al. (2011). Increased ER-mitochondrial coupling promotes mitochondrial respiration and bioenergetics during early phases of ER stress. Journal of Cell Science 124(Pt 13), 2143–2152. Carpio MA, Means RE, Brill AL, Sainz A, Ehrlich BE, Katz SG (2021). BOK Controls apoptosis by Ca2+ transfer through ER-mitochondrial contact sites. Cell Reports 34(10), 108827. Chacinska A, Koehler CM, Milenkovic D, Lithgow T, Pfanner N (2009). Importing mitochondrial proteins: machineries and mechanisms. Cell 138(4), 628–644. Chakrabarti R, Ji WK, Stan RV, de Juan Sanz J, Ryan TA, Higgs HN (2018). INF2-mediated Actin polymerization at the ER sti- mulates mitochondrial calcium uptake, inner membrane con- striction, and division. Journal of Cell Biology 217, 251–268. Chang CL, Liou J (2016). Homeostatic regulation of the PI(4,5) P2-Ca 2+ signaling system at ER-PM junctions. Biochimica et Biophysica Acta 1861, 862–873. Clapham DE (2007). Calcium signaling. Cell 131, 1047–1058. Combot Y, Salo VT, Chadeuf G, Hölttä M, Ven K, Pulli I, Ducheix S, Pecqueur C, Renoult O, Lak B et al. (2022). Seipin localizes at endoplasmic-reticulum-mitochondria contact sites to control mitochondrial calcium import and metabolism in adipocytes. Cell Reports 38(2), 110213. 8 Contact https://orcid.org/0000-0002-9398-7750 https://orcid.org/0000-0002-9398-7750 https://orcid.org/0000-0002-9398-7750 https://orcid.org/0000-0002-9398-7750 https://orcid.org/0000-0002-9398-7750 https://doi.org/10.1074/jbc.M113.453241 https://doi.org/10.1074/jbc.M113.453241 https://doi.org/10.1074/jbc.M113.453241 Copeland DE, Dalton AJ (1959). An association between mitochon- dria and the endoplasmic reticulum in cells of the pseudobranch gland of a teleost. The Journal of Biophysical and Biochemical Cytology 5(3), 393–396. Csordas G, Thomas AP, Hajnóczky G (1999). Quasi-synaptic calcium signal transmission between endoplasmic reticulum and mitochondria. European Molecular Biology Organisation Journal 18, 96–108. Csordas G, Varnai P, Golenar T, Roy S, Purkins G, Schneider TG, Balla T, Hajnoczky G (2010). Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface. Molecular Cell 39, 121–132. Deak AT, Blass S, Khan MJ, Groschner LN, Waldeck-Weiermair M, Hallström S, Graier WF, Malli R (2014). IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake. Journal of Cell Science 127(Pt 13), 2944–2955. de Brito OM, Scorrano L (2008). Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456(7222), 605–610. de Brito OM, Scorrano L (2010). An intimate liaison: Spatial organ- ization of the endoplasmic reticulum-mitochondria relationship. European Molecular Biology Organisation Journal 29(16), 2715–2723. D’Eletto M, Rossin F, Occhigrossi L, Farrace MG, Faccenda D, Desai R, Marchi S, Refolo G, Falasca L, Antonioli M et al. (2018). Transglutaminase type 2 regulates ER-mitochondria contact sites by interacting with GRP75. Cell Reports 25(13), 3573–3581.e3574. Dellis O, Dedos S, Tovey SC, Rahman T-U-, Dubel SJ, Taylor CW (2006). Ca2+ entry through plasma membrane IP3 receptors. Science 313, 229–233. Denton RM (2009). Regulation of mitochondrial dehydrogenases by calcium ions. Biochimica et Biophysica Acta 1787(11), 1309–1316. De Smedt H, Missiaen L, Parys JB, Henning RH, Sienaert I, Vanlingen S, Gijsens A, Himpens B, Caseels R (1997). Isoform diversity of the inositol trisphosphate receptor in cell types of mouse origin. Biochemical Journal 322, 575–583. De Stefani D, Bononi A, Romagnoli A, Messina A, De Pinto V, Pinton P, Rizzuto R (2012). VDAC1 Selectively transfers apop- totic Ca2+ signals to mitochondria. Cell Death & Differentiation 19(2), 267–273. De Stefani D, Patron M, Rizzuto R (2015). Structure and function of the mitochondrial calcium uniporter complex. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1853(9), 2006–2011. De Stefani D, Raffaello A, Teardo E, Szabo I, Rizzuto R (2011). A forty-kilodalton protein of the inner membrane is the mitochon- drial calcium uniporter. Nature 476, 336–340. De Vos KJ, Mórotz GM, Stoica R, Tudor EL, Lau KF, Ackerley S, Warley A, Shaw CE, Miller CC (2012). VAPB Interacts with the mitochondrial protein PTPIP51 to regulate calcium homeostasis. Human Molecular Genetics 21(6), 1299–1311. Doghman-Bouguerra M, Granatiero V, Sbiera S, Sbiera I, Lacas-Gervais S, Brau F, Fassnacht M, Rizzuto R, Lalli E (2016). FATE1 Antagonizes calcium- and drug-induced apop- tosis by uncoupling ER and mitochondria. EMBO Reports 17(9), 1264–1280. Dulloo I, Atakpa-Adaji P, Yeh YC, Levet C, Muliyil S, Lu F, Taylor CW, Freeman M (2022). Irhom pseudoproteases regulate ER stress-induced cell death through IP3 receptors and BCL-2. Nature Communications 13(1), 1257. Dulloo I, Muliyil S, Freeman M (2019). The molecular, cellular and pathophysiological roles of iRhom pseudoproteases. Open Biology 9(3), 190003. Fernandez-Busnadiego R, Saheki Y, De Camilli P (2015). Three-dimensional architecture of extended synaptotagmin- mediated endoplasmic reticulum-plasma membrane contact sites. Proceedings of the National Academy of Sciences 112, E2004–E2013. Filadi R, Greotti E, Turacchio G, Luini A, Pozzan T, Pizzo P (2015). Mitofusin 2 ablation increases endoplasmic reticulum- mitochondria coupling. Proceedings of the National Academy of Sciences 112(17), E2174–E2181. Filadi R, Leal NS, Schreiner B, Rossi A, Dentoni G, Pinho CM, Wiehager B, Cieri D, Calì T, Pizzo P et al. (2018). TOM70 Sustains cell bioenergetics by promoting IP3R3-mediated ER to mitochondria Ca2+ transfer. Current Biology 28(3), 369–382.e366. Foskett JK, White C, Cheung KH, Mak DO (2007). Inositol trispho- sphate receptor Ca2+ release channels. Physiological Reviews 87, 593–658. Gincel D, Zaid H, Shoshan-Barmatz V (2001). Calcium binding and translocation by the voltage-dependent anion channel: A pos- sible regulatory mechanism in mitochondrial function. Biochemical Journal 358(Pt 1), 147–155. Giorgi C, Marchi S, Pinton P (2018). The machineries, regulation and cellular functions of mitochondrial calcium. Nature Reviews Molecular Cell Biology 19(11), 713–730. Gomez-Suaga P, Paillusson S, Stoica R, Noble W, Hanger DP, Miller CCJ (2017). The ER-mitochondria tethering Complex VAPB-PTPIP51 regulates autophagy. Current Biology 27(3), 371–385. Guardia-Laguarta C, Area-Gomez E, Rüb C, Liu Y, Magrané J, Becker D, Voos W, Schon EA, Przedborski S (2014). α-Synuclein is localized to mitochondria-associated ER membranes. The Journal of Neuroscience 34(1), 249–259. Hayashi T, Su TP (2007). Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca2+ signaling and cell sur- vival. Cell 131(giot), 596–610. Iwai M, Michikawa T, Bosanac I, Ikura M, Mikoshiba K (2007). Molecular basis of the isoform-specific ligand-binding affinity of inositol 1,4,5-trisphosphate receptors. Journal of Biological Chemistry 282, 12755–12764. Kaftan EJ, Ehrlich BE, Watras J (1997). Inositol 1,4,5-trisphosphate (InsP3) and calcium interact to increase the dynamic range of InsP3 receptor-dependent calcium signaling. Journal of General Physiology 110, 529–538. Katona M, Bartók Á, Nichtova Z, Csordás G, Berezhnaya E, Weaver D, Ghosh A, Várnai P, Yule DI, Hajnóczky G (2022). Capture at the ER-mitochondrial contacts licenses IP3 receptors to stimulate local Ca2+ transfer and oxidative metabolism. Nature Communications 13(1), 6779. Kim J, Moody JP, Edgerly CK, Bordiuk OL, Cormier K, Smith K, Beal MF, Ferrante RJ (2010). Mitochondrial loss, dysfunction and altered dynamics in Huntington’s disease. Human Molecular Genetics 19(20), 3919–3935. Kim S, Coukos R, Gao F, Krainc D (2022). Dysregulation of organ- elle membrane contact sites in neurological diseases. Neuron 110(15), 2386–2408. Kirichok Y, Krapavinsky G, Clapham DE (2004). The mitochon- drial calcium uniporter is a highly selective ion channel. Nature 427, 360–364. Atakpa-Adaji and Ivanova 9 Klar J, Hisatsune C, Baig SM, Tariq M, Johansson AC, Rasool M, Malik NA, Ameur A, Sugiura K, Feuk L, Mikoshiba K, Dahl N (2014). Abolished InsP3R2 function inhibits sweat secretion in both humans and mice. Journal of Clinical Investigation 124, 4773–4780. Kohler V, Aufschnaiter A, Büttner S (2020). Closing the Gap, Membrane Contact Sites in the Regulation of Autophagy. Cells 9(5). Kroemer G, Reed JC (2000). Mitochondrial control of cell death. Nature Medicine 6(5), 513–519. Liu Y, Ma X, Fujioka H, Liu J, Chen S, Zhu X (2019). DJ-1 regu- lates the integrity and function of ER-mitochondria association through interaction with IP3R3-Grp75-VDAC1. Proceedings of the National Academy of Sciences 116(50), 25322–25328. Lock JT, Alzayady KJ, Yule DI, Parker I (2018). All three IP3 recep- tor isoforms generate Ca2+ puffs that display similar characteris- tics. Science Signaling 11, eaau0344. Lock JT, Parker I (2020). IP3 Mediated global Ca2+ signals arise through two temporally and spatially distinct modes of Ca2+ release. Elife 9, e55008. Lopez-Crisosto C, Díaz-Vegas A, Castro PF, Rothermel BA, Bravo-Sagua R, Lavandero S (2021). Endoplasmic reticulum- mitochondria coupling increases during doxycycline-induced mitochondrial stress in HeLa cells. Cell Death Discovery 12(7), 657. Lucendo E, Sancho M, Lolicato F, Javanainen M, Kulig W, Leiva D, Duart G, Andreu-Fernández V, Mingarro I, Orzáez M (2020). Mcl-1 and Bok transmembrane domains: Unexpected players in the modulation of apoptosis. Proceedings of the National Academy of Sciences 117(45), 27980–27988. Mak DO, Foskett JK (1998). Effects of divalent cations on single- channel conduction properties of Xenopus IP3 receptor. American Journal of Physiology 275, C179–C188. Mak D-O, McBride S, Foskett JK (2001). Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels: Ca 2+ activation uniquely distinguishes types 1 and 3 InsP3 receptors. Journal of General Physiology 117, 435–446. Malviya AN, Rogue P, Vincendon G (1990). Stereospecific inositol 1,4,5-[32P]trisphosphate binding to isolated rat liver nuclei: Evidence for inositol trisphosphate receptor-mediated calcium release from the nucleus. Proceedings of the National Academy of Sciences 87, 9270–9274. Marchant J, Callamaras N, Parker I (1999). Initiation of IP3-mediated Ca2+ waves in Xenopus oocytes. European Molecular Biology Organisation Journal 18, 5285–5299. Mataragka S, Taylor CW (2018). All three IP3 receptor subtypes generate Ca2+ puffs, the universal building blocks of IP3-evoked Ca2+ signals. Journal of Cell Science 131, jcs220848. Matsuzaki H, Fujimoto T, Ota T, Ogawa M, Tsunoda T, Doi K, Hamabashiri M, Tanaka M, Shirasawa S (2012). Tespa1 is a novel inositol 1,4,5-trisphosphate receptor binding protein in T and B lymphocytes. FEBS Open Biology 2, 255–259. Matsuzaki H, Fujimoto T, Tanaka M, Shirasawa S (2013). Tespa1 is a novel component of mitochondria-associated endoplasmic reticulum membranes and affects mitochondrial calcium flux. Biochemical and Biophysical Research Communications 433, 322–326. Mendes CC, Gomes DA, Thompson M, Souto NC, Goes TS, Goes AM, Rodrigues MA, Gomez MV, Nathanson MH, Leite MF (2005). The type III inositol 1,4,5-trisphosphate receptor prefer- entially transmits apoptotic Ca2+ signals into mitochondria. Journal of Biological Chemistry 280, 40892–40900. Milakovic T, Quintanilla RA, Johnson GV (2006). Mutant hunting- tin expression induces mitochondrial calcium handling defects in clonal striatal cells: Functional consequences. Journal of Biological Chemistry 281(46), 34785–34795. Miyakawa T, Maeda A, Yamazawa T, Hirose K, Kurosaki T, Iino M (1999). Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes. European Molecular Biology Organisation Journal 18, 1303–1308. Moon SS, Lee JE, Lee YS, Kim SW, Jeoung NH, Lee IK, Kim JG (2012). Association of pyruvate dehydrogenase kinase 4 gene polymorphisms with type 2 diabetes and metabolic syndrome. Diabetes Research and Clinical Practice 95(2), 230–236. Mori T, Hayashi T, Hayashi E, Su TP (2013). Sigma-1 receptor chaperone at the ER-mitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival. PLoS One 8(10), e76941. Mórotz GM, Martín-Guerrero SM, Markovinovic A, Paillusson S, Russell MRG, Machado PMP, Fleck RA, Noble W, Miller CCJ (2022). The PTPIP51 coiled-coil domain is important in VAPB binding, formation of ER-mitochondria contacts and IP3 receptor delivery of Ca2 + to mitochondria. Frontiers in Cell and Developmental Biology 10. Nakanishi S, Maeda N, Mikoshiba K (1991). Immunohistochemical localization of an inositol 1,4,5-trisphosphate receptor, P400, in neural tissue: Studies in developing and adult mouse brain. The Journal of Neuroscience 11(7), 2075. Nan J, Li J, Lin Y, Saif Ur Rahman M, Li Z, Zhu L (2021). The interplay between mitochondria and store-operated Ca2+ entry: Emerging insights into cardiac diseases. Journal of Cellular and Molecular Medicine 25(20), 9496–9512. Naon D, Zaninello M, Giacomello M, Varanita T, Grespi F, Lakshminaranayan S, Serafini A, Semenzato M, Herkenne S, Hernández-Alvarez MI et al. (2016). Critical reappraisal con- firms that Mitofusin 2 is an endoplasmic reticulum-mitochondria tether. Proceedings of the National Academy of Sciences 113(40), 11249–11254. Newton CL, Mignery GA, Südhof TC (1994). Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3. Journal of Biological Chemistry 269, 28613–28619. Nguyen L, Lucke-Wold BP, Mookerjee S, Kaushal N, Matsumoto RR (2017). Sigma-1 receptors and neurodegenerative diseases: Towards a hypothesis of sigma-1 receptors as amplifiers of neu- rodegeneration and neuroprotection. Advances in Experimental Medicine and Biology 964, 133–152. Obara CJ, Nixon-Abell J, Moore AS, Riccio F, Hoffman DP, Shtengel G, Xu CS, Schaefer K, Pasolli HA, Masson J-B et al. (2022). Motion of single molecular tethers reveals dynamic sub- domains at ER-mitochondria contact sites. bioRxiv: 2022.2009.2003.505525. Parker I, Choi J, Yao Y (1996). Elementary events of InsP3-induced Ca2+ liberation in Xenopus oocytes: Hot spots, puffs and blips. Cell Calcium 20, 105–121. Pinton P, Pozzan T, Rizzuto R (1998). The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, with functional 10 Contact properties distinct from those of the endoplasmic reticulum. European Molecular Biology Organisation Journal 17, 5298– 5308. Pinton P, Rizzuto R (2006). Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum. Cell Death & Differentiation 13(8), 1409–1418. Prinz WA (2014). Bridging the gap: Membrane contact sites in sig- naling, metabolism, and organelle dynamics. Journal of Cell Biology 205, 759–769. Prole DL, Taylor CW (2016). Inositol 1,4,5-trisphosphate receptors and their protein partners as signalling hubs. Journal of Physiology 594, 2849–2866. Qin J, Guo Y, Xue B, Shi P, Chen Y, Su QP, Hao H, Zhao S, Wu C, Yu L, Li D, Sun Y (2020). ER-mitochondria contacts promote mtDNA nucleoids active transportation via mitochondrial dynamic tubulation. Nature Communications 11(1), 4471. Rapizzi E, Pinton P, Szabadkai G, Wieckowski MR, Vandecasteele G, Baird G, Tuft RA, Fogarty KE, Rizzuto R (2002). Recombinant expression of the voltage-dependent anion channel enhances the transfer of Ca2+ microdomains to mito- chondria. Journal of Cell Biology 159(4), 613–624. Rieusset J (2018). The role of endoplasmic reticulum-mitochondria contact sites in the control of glucose homeostasis: An update. Cell Death & Disease 9(3), 388. Rizzuto R, Pinton P, Carrington W, Fay FS, Fogarty KE, Lifshitz LM, Tuft RA, Pozzan T (1998). Close contacts with the endo- plasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280, 1763–1766. Sakai S, Watanabe S, Komine O, Sobue A, Yamanaka K (2021). Novel reporters of mitochondria-associated membranes (MAM), MAMtrackers, demonstrate MAM disruption as a common pathological feature in amyotrophic lateral sclerosis. The FASEB Journal 35(7), e21688. Sassano ML, Felipe-Abrio B, Agostinis P (2022). ER-mitochondria contact sites; a multifaceted factory for Ca2+ signaling and lipid transport. Frontiers in Cell and Developmental Biology 10. Schmitz EA, Takahashi H, Karakas E (2022). Structural basis for activation and gating of IP3 receptors. Nature Communications 13(1), 1408. Shoshan-Barmatz V, Krelin Y, Shteinfer-Kuzmine A (2018). VDAC1 Functions in Ca2+ homeostasis and cell life and death in health and disease. Cell Calcium 69, 81–100. Simoes ICM, Morciano G, Lebiedzinska-Arciszewska M, Aguiari G, Pinton P, Potes Y, Wieckowski MR (2020). The mystery of mitochondria-ER contact sites in physiology and pathology: A cancer perspective. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1866(10), 165834. Simpson PB, Mehotra S, Langley D, Sheppard CA (1998). Specialized distributions of mitochondria and endoplasmic reticulum proteins define Ca2+ wave amplification sites in cultured astrocytes. Journal of Neuroscience Research 52(6), 672–683. Sood A, Jeyaraju DV, Prudent J, Caron A, Lemieux P, McBride HM, Laplante M, Tóth K, Pellegrini L (2014). A Mitofusin-2 dependent inactivating cleavage of Opa1 links changes in mito- chondria cristae and ER contacts in the postprandial liver. Proceedings of the National Academy of Sciences 111(45), 16017–16022. Sukumaran P, Nascimento Da Conceicao V, Sun Y, Ahamad N, Saraiva LR, Selvaraj S, Singh BB (2021). Calcium Signaling Regulates Autophagy and Apoptosis. Cells 10. Szabadkai G, Bianchi K, Varnai P, De Stefani D, Wieckowski MR, Cavagna D, Nagy AI, Balla T, Rizzuto R (2006). Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. Journal of Cell Biology 175, 901–911. Szondy Z, Korponay-Szabó I, Király R, Sarang Z, Tsay GJ (2017). Transglutaminase 2 in human diseases. Biomedicine (Taipei) 7(3), 15. Taylor CW, Genazzani AA, Morris SA (1999). Expression of inosi- tol trisphosphate receptors. Cell Calcium 26, 237–251. Theurey P, Tubbs E, Vial G, Jacquemetton J, Bendridi N, Chauvin MA, Alam MR, Le Romancer M, Vidal H, Rieusset J (2016). Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver. Journal of Molecular Cell Biology 8, 129–143. Thillaiappan NB, Chavda AP, Tovey SC, Prole DL, Taylor CW (2017). Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions. Nature Communications 8, 1505. Thillaiappan NB, Smith HA, Atakpa-Adaji P, Taylor CW (2021). KRAP Tethers IP3 receptors to actin and licenses them to evoke cytosolic Ca2+ signals. Nature Communications 12(1), 4514. Thoudam T, Ha C-M, Leem J, Chanda D, Park J-S, Kim H-J, Jeon J-H, Choi Y-K, Liangpunsakul S, Huh YH et al. (2018). PDK4 Augments ER–mitochondria contact to dampen skeletal muscle insulin signaling during obesity. Diabetes 68(3), 571–586. Tiscione SA, Casas M, Horvath JD, Lam V, Hino K, Ory DS, Santana LF, Simó S, Dixon RE, Dickson EJ (2021). IP3R-driven increases in mitochondrial Ca2+ promote neuronal death in NPC disease. Proceedings of the National Academy of Sciences 118(40). Tsai SY, Hayashi T, Harvey BK, Wang Y, Wu WW, Shen RF, Zhang Y, Becker KG, Hoffer BJ, Su TP (2009). Sigma-1 recep- tors regulate hippocampal dendritic spine formation via a free radical-sensitive mechanism involving Rac1xGTP pathway. Proceedings of the National Academy of Sciences 106(52), 22468–22473. Tu H, Wang Z, Bezprozvanny I (2005a). Modulation of mammalian inositol 1,4,5-trisphosphate receptor isoforms by calcium: A role of calcium sensor region. Biophysical Journal 88, 1056–1069. Tu H, Wang Z, Nosyreva E, De Smedt H, Bezprozvanny I (2005b). Functional characterization of mammalian inositol 1,4,5-trisphosphate receptor isoforms. Biophysical Journal 88, 1046–1055. Vervloessem T, Yule DI, Bultynck G, Parys JB (2015). The type 2 inositol 1,4,5-trisphosphate receptor, emerging functions for an intriguing Ca2+-release channel. Biochimica et Biophysica Acta 1853, 1992–2005. Vorontsova I, Lock JT, Parker I (2022). KRAP Is required for diffuse and punctate IP3-mediated Ca2+ liberation and deter- mines the number of functional IP3R channels within clusters. Cell Calcium 107, 102638. Wagner LE 2nd, Yule DI (2012). Differential regulation of the InsP3 receptor type-1 and −2 single channel properties by InsP3, Ca 2+ and ATP. Journal of Physiology 590, 3245–3259. Wang X, Shen X, Yan Y, Li H (2021). Pyruvate dehydrogenase kinases (PDKs): an overview toward clinical applications. Bioscience Reports 41(4). Atakpa-Adaji and Ivanova 11 Wu S, Lu Q, Wang Q, Ding Y, Ma Z, Mao X, Huang K, Xie Z, Zou M-H (2017). Binding of FUN14 domain containing 1 with inosi- tol 1,4,5-trisphosphate receptor in mitochondria-associated endoplasmic Reticulum membranes maintains mitochondrial dynamics and function in hearts in vivo. Circulation 136(23), 2248–2266. Xu H, Guan N, Ren YL, Wei QJ, Tao YH, Yang GS, Liu XY, Bu DF, Zhang Y, Zhu SN (2018). IP3R-Grp75-VDAC1-MCU Calcium regulation axis antagonists protect podocytes from apoptosis and decrease proteinuria in an Adriamycin nephropa- thy rat model. BMC Nephrology 19(1), 140. Xu L, Wang X, Tong C (2020). Endoplasmic Reticulum- mitochondria contact sites and neurodegeneration. Frontiers in Cell and Developmental Biology 8, 428. Yoneshima H, Miyawaki A, Michikawa T, Furuichi T, Mikoshiba K (1997). Ca2+ differentially regulates ligand-affinity states of type 1 and 3 inositol 1,4,5-trisphosphate receptors. Biochemical Journal 322, 591–596. Yoo SH, Albanesi JP (1990). Inositol 1,4,5-trisphosphate-triggered Ca2+ release from bovine adrenal medullary secretory vesicles. Journal of Biological Chemistry 265, 13446–13448. YuW, Jin H, Huang Y (2021). Mitochondria-associated membranes (MAMs): A potential therapeutic target for treating Alzheimer’s disease. Clinical Science 135(1), 109–126. Yuan M, Gong M, He J, Xie B, Zhang Z, Meng L, Tse G, Zhao Y, Bao Q, Zhang Y et al.(2022). IP3R1/GRP75/VDAC1 complex mediates endoplasmic reticulum stress-mitochondrial oxida- tive stress in diabetic atrial remodeling. Redox Biology 52, 102289. Zaman MF, Nenadic A, Radojičić A, Rosado A, Beh CT (2020). Sticking With It: ER-PM Membrane Contact Sites as a Coordinating Nexus for Regulating Lipids and Proteins at the Cell Cortex. Frontiers in Cell and Developmental Biology 8. Zhang S, Fritz N, Ibarra C, Uhlén P (2011). Inositol 1,4,5-trisphosphate receptor subtype-specific regulation of calcium oscillations. Neurochemical Research 36(7), 1175–1185. 12 Contact Highlight Introduction ERMCS and Ca2+ Signalling Localisation and Functioning of IP3Rs at ERMCS All IP3R Subtypes Localise to ER–Mitochondrial Contact Sites IP3Rs Have a Structural Role in Forming ERMCS Co-Regulators of the IP3R-GRP75-VDAC1 complex at ERMCS Tespa1 Sigma-1 Receptor BOK Tom70 Transglutaminase Type 2 (TG2) iRhom Seipin Pyruvate Dehydrogenase Kinases (PDKs) Other Co-Regulators of ERMCS Where Ca2+ Exchange Occurs Perspectives and Concluding Remarks References << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile () /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 5 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness false /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages false /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Average /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages false /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Average /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages false /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Average /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /PDFX1a:2003 ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError false /PDFXTrimBoxToMediaBoxOffset [ 33.84000 33.84000 33.84000 33.84000 ] /PDFXSetBleedBoxToMediaBox false /PDFXBleedBoxToTrimBoxOffset [ 9.00000 9.00000 9.00000 9.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV /HUN /ITA /JPN /KOR /LTH /LVI /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken voor kwaliteitsafdrukken op desktopprinters en proofers. 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