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.

Keywords: Anoctamins; Tethers.

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].