The Tether Function of the Anoctamins

Abstract

The core functions of the anoctamins are Cl⁻ channel activity and phosphatidylserine (and perhaps other lipids) scrambling. These functions have been extensively studied in various tissues and cells. However, another function of the anoctamins that is less recognized and minimally explored is as tethers at membrane contact sites. This short review aims to examine evidence supporting the localization of the anoctamins at membrane contact sites, their tether properties, and their functions as tethers.

Graphical Abstract

Introduction:

The endoplasmic reticulum (ER) serves as the communication network of the cell, facilitating the conveyance and distribution of messages from the cell exterior to the interior and throughout the cell. It mediates communication between cellular organelles and transfer of material both within and between organelles [1–3]. These functions are achieved through the formation of membrane contact sites (MCS) with all cellular membranes, including those of the mitochondria [4, 5], lysosomes and phagosomes [6], endosomes [7], lipid droplets [8] and the plasma membrane leading to the formation of contact junctions that in the case of the ER and PM are the ER/PM junctions [1]. MCS are formed by tether proteins that are typically anchored in the ER, have cytoplasmic domain spanning the distance between the ER and target membrane, and a domain that attaches them to the target membrane Some tether proteins are specific to certain MCS, as observed in the ER-mitochondria MCS (MAM) [4, 5]. Others, such as the ER-localized VAMP-associated proteins (VAPs) VAPA and VAPB,MOSPD2, and MOSPD1, are ubiquitous in virtually all MCS [9]. These proteins feature an MSP (Major Sperm Protein) domain that binds a two phenylalanine in acidic tract (FFAT) motif, recruiting the proteins to various MCS [10]. MOSPD2 and MOSPD1 bind an FFAT-related FFNT motif (two phenylalanines [FF] in a neutral tract) [11]. Similarly, the lipid transfer protein (LTP) families of the extended synaptotagmins (E-Syts) [12] and the Oxysterol-binding protein (OSBP)-related proteins (ORPs) [13] localize to various MCS.

A prominent MCS established between the ER and plasma membrane (PM) are the ER/PM junctions [1]. Key tethers at the ER/PM junctions are the STIM proteins, namely STIM1 and STIM2 [14]. The STIMs are dynamic tethers that feature a luminal ER Ca²⁺ sensor, a single transmembrane domain, a folded cytoplasmic domain encompassing the SOAR domain responsible for activating the Ca²⁺ influx channel Orai1 and a C-terminal polybasic domain [15, 16]. The cytoplasmic domain unfolds in response to Ca²⁺ release from the ER, while the polybasic domain anchors the STIMs to the plasma membrane though interaction with PI(4)P and PI(4,5)P₂ [17–19]. The SOAR domain’s binding to Orai1 [16] and its interaction with PI(4,5)P2 and PI(4)P [20]contribute to further stabilizing the STIM proteins at the plasma membrane. This interaction leads to the formation and enlargement of ER/PM junctions [20, 21]. Among the several LTP localized at the ER/PM junctions, all three E-Syts [22] are included. Notably, E-Syt1 influences the integration and function of STIM1-Orai1 [19], while E-Syt2 affects the function of the phosphoinositides phosphatase Sac1 to control junctional PI(4,5)P₂ [23]. Other LTPs impacting junctional PI(4,5)P₂ dynamics are the Nir proteins [24] which are targeted to ER/PM junctions by VAP proteins [25]. Lastly, the ORPs, ORP5 and ORP8 function as PI(4)P/phosphatidylserine (PtdSer) exchangers at the ER/PM junctions [26]. ORP5 and ORP8 mediate reciprocal PI(4)P/PtdSer exchange ratio, selectively regulating junctional PI(4)P/PtdSer ratio to inhibit (ORP5) or facilitate (ORP8) STIM1 clustering. This modulation impacts Orai1-mediated Ca²⁺ influx and the receptor-evoked Ca²⁺ signal [27].

Tether proteins mediating formation of the ER/PM junctions in yeast includes the homologues of mammalian VAPA and the E-Syts [28]. Early studies in yeast also identified the Ist2 protein at the ER/PM contact site [29], demonstrating its essential role in maintaining the integrity of these ER/PM contact sites [28]. Ist2, an ER protein, possesses multiple N-terminus transmembrane domains embedded in the ER, a long unstructured cytoplasmic domain, and a polybasic C-terminus that binds to PI(4,5)P_2, and likely interact with other phosphoinositides at the PM [30]. Ist2 shares sequence and domain homology with members of the anoctamins family [31–34], the topic of this Special Issue. Below we describe features and functions of the tether functions of the anoctamins.

Anoctamins features compatible with tether function:

To serve as tethers the anoctamins must localize at the ER/PM junctions. Each member of the anoctamin family has 10 transmembrane domains with both the C and N terminus localized in the cytoplasm [35–37]. Several anoctamins such as ANO1, ANO2, and ANO6, have their transmembrane domains embedded in the plasma membrane, while other like ANO5, ANO8 and ANO10 are embedded in the ER [38, 39]. Numerous studies support the notion that anoctamins are localized at MCS. Many of the anoctamins affect receptor-stimulated Ca²⁺ signaling [40–45]. Given that all components of the Ca²⁺ signaling pathway are localized at the ER/PM junctions [1, 41, 46], it is plausible that the anoctamins impact on Ca²⁺ signaling is a result of their shared localization at MCS, including the ER/PM junctions.

The anoctamins interact with several PM Ca²⁺ permeable channels at the ER/PM junctions, effectively activating them. ANO1 colocalizes and interacts with several TRP channels to mediate key physiological functions. ANO1 has a significant role in TRPV1-dependent pain sensation [47]. ANO1 is prominently involved in primary sensory neurons. Here, ANO1 is activated by Ca2+ influx through TRPV1, leading to neuronal depolarization and the inhibition of action potentials evoked by capsaicin Activation of ANO1 by TRPV1 occurs within the same nanodomain since it was inhibited by the fast Ca²⁺ buffer BAPTA, but not by the slow Ca²⁺ buffer EGTA [47]. Further examples are colocalization and functional interaction of ANO1 with the Ca²⁺ permeable TRPV4 channel [48]. In choroid plexus receptor activation and Ca²⁺ increase by TRPV4 activates ANO1 current. Notably, Ca²⁺ influx by TRPV4 markedly augmented activation of ANO1 by warm temperature and cell swelling [49]. Direct pharmacologic activation of TRPV4 or receptor-mediated activation of TRPV4 activated ANO1 and fluid secretion in salivary and lacrimal glands [48]. In endothelial cells, single-molecule localization imaging was used to show localization of ANO1 with TRPV4 within a few nanometers. Receptor stimulation of TRPV4 mediated the Ca²⁺ influx to activate ANO1 and control vasodilation [50]. In epidermal keratinocytes ANO1 was activated by Ca²⁺ influx through TRPV3 to regulate the cell cycle [51]. In cerebral arteries, co-immunoprecipitation and FRET microscopy suggested localization of ANO1 with TRPC6 [52]. Activation of ANO1 by TRPC6-mediated Ca²⁺ influx was blocked by BAPTA, but not by EGTA, indicating functional proximity and coupling [52]. In placental trophoblast, Cl⁻ current and PtdSer externalization by ANO6 are activated by Ca²⁺ influx through TRPV4, mediating trophoblast cell-cell fusion essential for trophoblast syncytialization [53]. In addition, it was reported that ANO6 can be activated by Ca²⁺ influx mediated by TRPC1 [54].

Additional compelling evidence supporting the presence of anoctamins at the ER/PM junctions comes from the observation that ANO1 is localized in caveolae. Deletion of caveolin 1 disrupted localization of native ANO1 in interstitial cells of Cajal [55] and ANO1 expression and current are increased in portal vein smooth muscle cells with deleted caveolin 1 [56]. Furthermore, expressed ANO1 physically and functionally interacts with the volume-regulated Cl⁻ channel LRRCA in caveolae [57]. ANO1 is most likely localized at the STIM1 domain of the ER/PM junctions. An elegant study in sweat glands showed that ANO1 was specifically activated by Ca²⁺ influx through Orai1 that was activated by the unfolded STIM1 [58]. Interestingly, ANO1 could be transiently activated by the limited IP₃-evoked Ca²⁺ release from the ER, but the subsequent sustained activity of ANO1 was strictly dependent on Ca²⁺ influx through Orai1 to maintain fluid secretion by glandular serosal acinar cells [58].

An important lipid enriched in MCS, in particular the ER/PM junctions, is phosphatidylinositol (4,5) bisphosphate (PI(4,5)P₂). In addition to its role in Ca²⁺ signaling, PI(4,5)P₂ activates many ion transporters and channels and other transport proteins [59, 60]. Moreover, PI(4,5)P₂ provides a platform for interaction of many proteins with the plasma membrane by their polybasic domain, [61], C2 domains [62] and PH domains [63]. Several anoctamins are regulated by PI(4,5)P₂ [41, 64–68] which affects their conductive and kinetic properties. The anoctamins have additional PI(4,5)P₂ binding sites that may serve to localize them at MCS. Such an effect was reported for ANO8 where localization at the ER/PM junctions was altered by PI(4,5)P₂ depletion and mutation of a potential PI(4,5)P₂ binding sequence [41].

Several anoctamins have protein-protein interacting motifs that mediate formation of protein complexes in specific cellular domains. Proteome analysis of ANO1 identified several PDZ domain-containing proteins including Ezrin, MAGUK family Membrane protein MPP6, MPP2, MPP1 and Disks large homolog 1 (DLG1) [69].In the luminal membrane of the mouse intestine, ANO1 interacts with the PDZ containing protein NHERF1, and this interaction relies on the ANO1 PDZ binding motif [70]. Additionally, receptor stimulated ANO1 shows reduced activity in conditions where NHERF1 is knocked down [70]. Table 1 shows that only ANO1, ANO2, ANO5, ANO8 and ANO9 have identifiable PDZ motifs in their C terminus. The PDZ-domains binding specificity analysis of the various anoctamins predicted by https://pow.baderlab.org/ [71], suggest that these PDZ motifs exhibit significant affinity for multiple PDZ binding domains (the full list of the PDZ domains is given in supplementary table 1), raising the possibility that they can form multiple complexes mediated by various PDZ domains containing scaffolding proteins.

Table 1:

The anoctamins PDZ ligands and the number of the various PDZ domains that they can interact with.

Anoctamin	PDZ motif	# of Interacting PDZ Domains
ANO1	HGDAL	7
ANO2	QHTNV	21
ANO5	AKSTL	7
ANO8	PSGWH	2
ANO9	RSTV	18

Among the crucial motifs are the two phenylalanine in acidic tract (FFAT) and the two phenylalanine in neutral tract (FFNT). These motifs bind to the VAMP-associated proteins VAPA and VAPB [9, 72, 73]. VAPA/B feature an ER transmembrane domain and a cytoplasmic MSP (Major Sperm Protein) domain that recognizes the FFAT and FFNT motifs. The VAPs are found in virtually all MCS, acting as tethers that connect the ER to various cellular membranes, including the plasma membrane [10, 74]. VAPA was found in the ANO1 interactome [69] and ANO10 was detected in the VAPA interactome [9]. We therefore searched for FFAT and FFNT motifs in the anoctamins. Table 2 shows that with the exception of ANO9, all other anoctamins have conserved two phenylalanines, although not all are predicted as strong or even weak FFAT sites. As indicated by the FFAT scores, ANO1 has strong FFAT site, ANO2, ANO7 and ANO8 have modest FFAT site, while the two phenylalanines of ANO3–6 and ANO10 do not form classical FFAT sites as defined in [10]. However, the ANO10 two phenylalanines form a good FFNT motif. It is plausible that VAPA/B utilize these motifs to recruit the respective anoctamins to various MCS. In the case of the PM localized ANO1 and ANO2, interaction with the VAPA/B is straightforward, where their cytoplasmic FFAT motifs interact with the ER localized VAPs to localize them in the junctions where they can then form VAPA/B-ANO1/2 tethers. It is not evident how the FFAT and FFNT motifs of the ER or organellar localized anoctamins are used. One possibility is that the VAPs recruit these anoctamins to the ER domain of the MCS to aid and facilitate their interaction with the PM.

Table 2:

The conserved anoctamins FFAT motifs and their FFAT scores (red-strong; Green-week; Blue-not FFAT site. ANO10 is not FFAT motif but is an FFNT motif.

Anoctamins as tethers:

ANO1 engages in the formation of complexes with several cytoskeletal and junctional proteins. Quantitative proteomics revealed association of ANO1 with the cytoskeletal complex of ezrin-radixin-moesin that showed restricted co-localization at the luminal pole of salivary gland acinar cells [69]. Notably, when overexpressed in HEK cells, moesin and ANO1 showed punctate localization at the plasma membrane, typical of ER/PM junctional appearance. Moreover, localization of moesin to the puncta required ANO1 [69], suggesting a tether function of ANO1.

In pulmonary arterial smooth muscle cells ANO1 is efficiently activated by receptor-evoked Ca²⁺ release from the ER/SR and subsequent Ca²⁺ influx through Orai1 but not by comparable Ca²⁺ increase mediated by the voltage-gated Ca²⁺ channel CaV1.2, although both IP₃Rs and CaV1.2 coimmunoprecipitated and colocalized with ANO1 as ermined by superresolution nano-microscopy [42]. Interestingly, deletion of ANO1 strongly inhibited the receptor stimulated Ca²⁺ oscillations and contraction, but not contraction induced by membrane depolarization and Ca²⁺ influx through CaV1.2. Most notably, deletion of ANO1 depleted the ER/SR Ca²⁺ store [42], suggesting that ANO1 functions as a tether in the assembly of the Ca²⁺ signaling complex in these cells. Communication between ANO1 and the GPCRs Ca²⁺ signaling pathway was also reported in Nociceptive Sensory Neurons in which Ca²⁺ influx by Orai1, but not by CaV1.2 activated ANO1 [45]. The privileged activation of ANO1 by receptor-activated Orai1-mediated Ca²⁺ influx required tethering of ANO1 C-terminus and first intracellular loop to the type 1 IP₃ receptor (IP₃R1) at an ER/PM nanodomain [45], providing strong evidence for a tethering function of ANO1.

ANO5 may also function as a tether. In myotubes, ANO5 interacts with caveolin 3 and associates with the RyR/L-type Ca²⁺ channel complex, the muscle ER/PM junction [75]. Interestingly, muscle damage in dysferlin-deficient myotubes results in enhanced ANO5 association with the RyR-L-type Ca²⁺ channel complex as part of the muscle repair process [75]. ANO5 is largely expressed in an intracellular compartment [75–77]. However, upon Ca²⁺ release from the ER/SR, part of ANO5 translocates to the plasma membrane and functions as a PtdSer scramblase to externalize PtdSer and promote cell-cell fusion [77, 78]. The combined findings raise the possibility that ANO5 functions as a mobile and transient tether at the ER/PM junctions.

Direct and specific evidence supporting tether function was examined for ANO8. The tether function of Ist2 and its homology to the anoctamins [28, 79] prompted examination of the anoctamins as tethers in the STIM1-Orai1 system [41]. Among all anoctamins, ANO8 exhibited the most prominent effect on the current mediated by the STIM1-Orai1 complex. ANO8 noticeably increased the number and density of the ER/PM junctions, thereby facilitating STIM1-STIM1 interaction at the ER, clustering at the junctions, and interaction of the STIM1 clusters with Orai1 effectively doubling the Orai1 Ca²⁺ current. Notably, these effects required the PI(4,5)P₂ binding sites of ANO8 and a PI(4,5)P₂-rich domain at the plasma membrane [41]. This is illustrated in Figure 1, which shows that depletion of PI(4,5)P₂ (A, B) and mutation of the PI(4,5)P₂ binding site (C-E) eliminate the increased current caused by expression of ANO8. An additional consequence of the ANO8-mediated increased ER/PM junction numbers and density was enhanced assembly of all core Ca²⁺ signaling proteins, PMCA, Orai1, STIM1, IP₃ receptors and SERCA2, into complexes. This resulted in a reshaping of receptor-evoked Ca²⁺ signaling. Moreover, the increased proximity of SERCA2 to Orai1 facilitated Ca²⁺ uptake into the ER even when the cytosol was buffered by 10 mM of the fast Ca²⁺ buffer BAPTA [41], as depicted in Figure 2. ANO8 affects assembly of the native (A, B) and the expressed (C) Ca²⁺ signaling proteins into complexes. Inhibition of SERCA2 Ca²⁺ pump with CPA (D), maintain activation of the IP₃Rs with cytoplasmic IP₃ and loading the ER with 10 mM of the ER Ca²⁺ buffer TEPN, all markedly reduced the rate and extent of current inactivation (Figure 2).

Figure 1: The PI(4,5)P₂ dependence of ANO8 function.

In Figures 1 and 2 current was measured in HEK cells transfected with STIM1 and Orai1 and with patch pipette solution containing 10 mM BAPTA to deplete the ER Ca²⁺ store and activate the current. (A, B): To deplete the PI(4,5)P₂ cells were transfected with FRB and PI5 phosphatase tagged with FKBP. The cells were transfected with Orai1, STIM1 and the FRD-FKBP system and (blue, red) or without (black) ANO8. The controls were left untreated (blue trace and control in B) and PI(4,5)P₂ depletion was achieved by treating the cells with 0.5 μM rapamycin for 3 min (red and PI(4,5)P₂ depleted in B). Current measurement was initiated by exposing the cells to 10 mM Ca²⁺ in (A) or cells were imaged by confocal microscopy (B). Depletion of PI(4,5)P₂ eliminated the increased STIM1-Orai1 current caused by ANO8 (A) and translocation of ANO8 to the STIM1 ER/PM junctions (B). (C): putative structure of ANO8 with the PI(4,5)P₂ site in red spheres as predicted by Robetta (https://robetta.bakerlab.org/login.php). (D, E): Curremt was measured as in (A) with the ANO8 putative PI(4,5)P₂ binding site mutant that eliminated the increased STIM1-Orai1 current caused by ANO8 (D) and translocation of ANO8 to the STIM1 ER/PM junctions (E). The results are reproduced from [41].

Figure 2: Assembly of Ca²⁺ signaling complexes and stimulation of SERCA2 activity by ANO8.

(A-C): Interaction between the Ca²⁺ signaling proteins was measured by Co-IP in cells depleted of ANO8 or overexpressing ANO8. Cell were under resting conditions (R) or stimulated by store depletion with 25 μM of the SERCA inhibitor CPA (S). Knockdown of ANO8 (A) reduced the formation of the native STIM1-Orai1 and STIM1-ANO8 complexes, while overexpression of ANO8 (B) increased formation of the native STIM1-IP₃Rs and STIM1-SERCA2 complexes. (C) ANO8 also increased assembly of the expressed STIM1-PMCA4, STIM1-SERCA2 and Orai1-SERCA2 complexes. (D): Current was measured as in Figure 1A. Inhibition of SERCA activity with 25 μM CPA markedly reduced Orai1 slow Ca²⁺-dependent inactivation induced by ANO8, indicating that most of the inactivation was mediated by SERCA-mediated Ca²⁺ uptake into the ER. (E): Increasing ER Ca²⁺ permeability by including high concentration of 100 μM cytoplasmic IP₃ in the pipette solution (red traces) and by increasing ER Ca²⁺ buffering capacity by including in the patch pipette 10 mM of the ER Ca²⁺ puffer TPEN (green), reduced and slowed Orai1 Ca²⁺-dependent inactivation induced by ANO8. The results are reproduced from [41].

It is of note that the classical PtdSer scrambler ANO6 does not appear to have either PDZ ligand or FFAT motif (tables 1, 2) and thus may not have tether functions. However, as a ubiquitous scrambler ANO6 affects PtdSer that is involved in many cellular functions that take place at MCS. For example, ANO6-dependent PtdSer externalization is required for phagocytosis, formation of osteoclasts and myocytes syncytia, fertilization, synaptic pruning and (reviewed in [80]) Moreover, PtdSer scrambling by ANO6 is required for formation of the immunological synapse [81] and T cell receptor signaling [82]. For these functions ANO6 may be recruited to the ER/PM junctions by specific tethers to shape the PtdSer and lipid environment at the junctions and other MCS and thus their shape, stability and functions.

Our understanding of the tether functions of the anoctamins remains rudimentary, marked by numerous open questions. One key area requiring extensive exploration is the mechanisms and domains utilized by the anoctamins to function as tethers. The PDZ and VAPA motifs very likely serve both to assemble protein complexes by the anoctamins and to localize them and their partners at MCS. In addition all anoctamins bind PI(4,5)P₂ and other lipids that are enriched at MCS. Binding to PI(4)P and PI(4,5)P₂ by polybasic sequences are commonly found in proteins that localize at the ER/PM junctions and may serve the same function in the anoctamins. Many of the anoctamins pave lipid scramblase activity [37, 83, 84] which may affect the lipid environment at the MCS either directly or indirectly by interacting with and recruiting other lipid transfer proteins, as was reported for Ist2 [85, 86]. Understanding the role of lipids at MCS and how the anoctamins affect the lipids at MCS deserve attention. Future studies are likely to address the role of this remarkable family of dual functional proteins in cell physiology and in disease.

Supplementary Material

1

Supplementary Table 1: A list of the anoctamins PDZ domains binding partners

Highlights:

• The anoctamins can function as tethers at multiple cellular sites.

• ANO1 and ANO8 and likely other anoctamins function as tethers at the ER/PM junctions.

• ANO1 and ANO8 communicate with and regulate the Ca²⁺ signaling pathway.

• The anoctamin motifs FFAT, FFNT and PDZ ligands may facilitate their function as tethers+