Activated Chloride Channel Stoichiometrically Interacts with an Ezrin–Radixin–Moesin Network

Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

Patricia Perez-Cornejoa,1, Avanti Gokhaleb,1, Charity Duranb,1, Yuanyuan Cuib, Qinghuan Xiaob, H. Criss Hartzellb,2, and Victor Faundezb,2

aPhysiology Department, School of Medicine, Universidad Autónoma de San Luis Potosí, San Luis Potosí, SLP 78210, Mexico; and bDepartment of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322

Edited by David E. Clapham, Howard Hughes Medical Institute, Children’s Hospital Boston, Boston, MA, and approved May 9, 2012 (received for review January 4, 2012)

The newly discovered Ca2+-activated Cl− channel (CaCC), Anocta- approach to identify Ano1-interacting proteins. We find that min 1 (Ano1 or TMEM16A), has been implicated in vital physiolog- Ano1 forms a complex with two high stochiometry interactomes. ical functions including epithelial fluid secretion, gut motility, and One protein network is centered on the signaling/scaffolding smooth muscle tone. Overexpression of Ano1 in HEK cells or Xen- actin-binding regulatory proteins ezrin, radixin, moesin, and opus oocytes is sufficient to generate Ca2+-activated Cl− currents, RhoA. The ezrin–radixin–moesin (ERM) proteins organize the but the details of channel composition and the regulatory factors cortical cytoskeleton by linking actin to the plasma membrane that control channel biology are incompletely understood. We and coordinate cell signaling events by scaffolding signaling used a highly sensitive quantitative SILAC proteomics approach molecules (19). The other major interactome is centered on the to obtain insights into stoichiometric protein networks associated SNARE and SM proteins VAMP3, syntaxins 2 and -4, and the with the Ano1 channel. These studies provide a comprehensive syntaxin-binding proteins munc18b and munc18c. This complex footprint of putative Ano1 regulatory networks. We find that is involved in docking and translocation of vesicles to the plasma Ano1 associates with the signaling/scaffolding proteins ezrin, rad- membrane (20). These studies provide a comprehensive foot- ixin, moesin, and RhoA, which link the plasma membrane to the print of putative Ano1 regulatory networks encompassing 2+ cytoskeleton with very high stoichiometry. Ano1, ezrin, and moe- a spectrum from Ca sensors to actin cytokeleton scaffolding sin/radixin colocalize apically in salivary gland epithelial cells, and networks and suggest mechanisms for the polarized localization overexpression of moesin and Ano1 in HEK cells alters the sub- of Ano1 in epithelial cells. cellular localization of both proteins. Moreover, interfering RNA Results for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes To characterize the Ano1 interactome, we developed a method the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syn- for high-level purification of Ano1 with specifically interacting taxin-binding proteins munc18b and munc18c, which are integral proteins while minimizing nonspecific protein associations. This to translocation of vesicles to the plasma membrane. A number of was accomplished using immunoaffinity chromatography of the other regulatory proteins, including GTPases, Ca2+-binding pro- Ano1 complex, which was stabilized with the cross-linker DSP teins, kinases, and lipid-interacting proteins are enriched in the [dithiobis(succinimidyl propionate)]. We have previously vali- Ano1 complex. These data provide stoichiometrically prioritized dated the robustness and specificity of this method using genetic information about mechanisms regulating Ano1 function and tools (21). We constructed a HEK-293 cell line stably expressing trafficking to polarized domains of the plasma membrane. Ano1 tagged on its C terminus with three FLAG epitopes – × 2+ (Ano1 FLAG3 ). The cell line− exhibited robust Ca -de- calcium | interactome | cross-linker | apical targeting pendent, outwardly rectifying Cl selective Ano1 currents (>200 pA/pF at +100 mV with 1 μM intracellular Ca2+). The cells were − a2+-activated Cl channels (CaCCs) play critical roles in treated with the cell-permeant, amino-reactive, homobifunctional cross-linker DSP to stabilize low-affinity protein–protein Cepithelial secretion, sensory transduction and adaptation, fi regulation of smooth muscle contraction, control of neuronal interactions. DSP has a 12-Å spacer arm and a disul de bond, and cardiac excitability, and nociception (1, 2). In 2008, after which is cleaved with reducing agents to release cross-linked many years of controversy about the molecular identity of proteins (22, 23). Effective cross-linking was demonstrated by – CaCCs, Ano1 and Ano2 (also called Tmem16A and Tmem16B) a moderate increase in the sedimentation velocity of Ano1 of the Anoctamin superfamily were identified as essential sub- FLAG3× protein complexes in sucrose gradients (Fig. S1). units of CaCCs (3–5). However, the regulatory networks that Cross-linking was limited to closely neighboring proteins and did control Ano1 function remain largely unexplored. not result in large protein aggregates, because the sedimentation The physiological significance of Ano1 cannot be understated. profile of total protein visualized by silver stain was not signifi- Ano1 is widely expressed in epithelia including salivary gland, cantly altered by the DSP treatment (Fig. S1) (22, 24). pancreas, gut, mammary gland, and airway. Disruption of the − Ano1 gene in mice eliminates Ca2+-dependent Cl secretion in several epithelial tissues including salivary gland (3, 5–9). Ano1 Author contributions: P.P.-C., A.G., C.D., Y.C., H.C.H., and V.F. designed research; P.P.-C., also performs important functions in nonepithelial tissues in- A.G., C.D., Y.C., Q.X., and V.F. performed research; P.P.-C., A.G., C.D., Y.C., Q.X., H.C.H., cluding pacemaker activity in the gut and regulation of vascular and V.F. analyzed data; and P.P.-C., A.G., C.D., H.C.H., and V.F. wrote the paper. and airway smooth muscle tone. Ano1 has also attracted the The authors declare no conflict of interest. interest of cancer biologists because the gene is amplified in oral, This article is a PNAS Direct Submission. head, and neck squamous cell carcinomas, and its expression 1P.P-C., A.G., and C.D. contributed equally to this work. level may correlate with cell proliferation (see recent reviews, 2To whom correspondence may be addressed. E-mail: [email protected] or refs. 10–18). [email protected]. To identify potential accessory subunits and/or regulatory pro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tein networks, we used a highly sensitive quantitative proteomic 1073/pnas.1200174109/-/DCSupplemental.

10376–10381 | PNAS | June 26, 2012 | vol. 109 | no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1200174109 Downloaded by guest on September 27, 2021 The cross-linked Ano1 protein complex was captured by in- cubating the cell lysate with anti-FLAG antibody-coated magnetic beads. The material that bound to the anti-FLAG beads consisted of a spectrum of bands on silver-stained gels (Fig. 1, lane 3). The prominent 130-kDa and 260-kDa bands correspond to Ano1 monomers and dimers as shown by Western blot (lane 3′). The predicted molecular mass of Ano1–FLAG3× is 113 kDa, but the glycosylated monomers and dimers migrate as diffuse bands at 130 kDa and 260 kDa (25). Many of the other bound proteins were nonspecific, because they bound to magnetic beads lacking anti- FLAG (lane 1), to beads coated with an irrelevant antibody (lane 2), or to beads coated with anti-FLAG in the presence of excess competing FLAG3× peptide (lane 4). Under these control conditions, Ano1–FLAG3× did not bind (lanes 1′,2′,and4′). The bound Ano1–FLAG3× complex was separated from nonspecifically bound proteins by elution from the anti-FLAG beads with excess FLAG3× peptide. The eluted material was excep- tionally clean on silver-stained gels, consisting predominantly of the 130-kDa and 260-kDa Ano1 bands (lanes 6 and 6′). To analyze Ano1 interacting proteins quantitatively, we coupled the immunoaffinity purification just described with stable isotope labeling with amino acids in culture (SILAC) (Fig. 2). Untransfected HEK cells were grown in medium containing ar- 12 14 ginine and lysine with “light” C and N, (R0K0), whereas the Ano1–FLAG3× stable cells were grown in medium containing 13 15 “heavy” C- and N-labeled arginine and lysine (R10K8) for more than six passages to ensure equilibrium labeling of the proteome (97.5% R10K8 saturation). The cross-linked Ano1 Fig. 2. Summary of SILAC experiment. Untransfected HEK cells were in- “ ” complex purified by immunoaffinity chromatography (Fig. S2B) cubated in isotopically light (R0K0) DMEM. HEK cells stably transfected – × “ ” was analyzed by nano-LC MS/MS. A total of 509 proteins were with Ano1 FLAG3 were incubated in isotopically heavy (R10K8) medium. identified at a 1% false discovery rate. Of these, there was suf- Cells were substoichiometrically cross-linked using DSP. Lysates were ficient signal to quantify SILAC enrichment for 392 (Fig. 2). The immunoprecipitated using magnetic beads decorated with FLAG antibody. list was refined to 209 by curation against a database of poly- Ano1 supramolecular complexes were eluted with FLAG3× peptide. Samples peptides that nonselectively bind to anti-FLAG beads from were combined at a 1:1 ratio and analyzed by nano-LS MS/MS. Peptides – enriched more than twofold with the R10K8 amino acids were considered as untransfected DSP cross-linked cell extracts (26 28). A total of fi 93 of these proteins were enriched more than twofold and 73 potential Ano1 interactors. The list of proteins was re ned by curation fi against a list of peptides that nonspecifically bind to the immunomagnetic were enriched more than vefold in the Ano1-expressing cells beads. The Venn diagram shows the number of peptides identified in the CELL BIOLOGY (Fig. 2 and Dataset S1). A twofold SILAC enrichment is experiment. A total of 509 proteins were identified, 93 of which were a stringent cutoff criterion to reliably detect differences between enriched more than twofold in R10K8 and did not bind nonspecifically to two samples (26, 28, 29). immunomagnetic beads.

In contrast, in the absence of cross-linking, only six proteins copurifying with Ano1–FLAG3× were enriched more than twofold (Fig. 3C and Dataset S2). Most of these proteins are not present in the proteome of DSP-treated cells and some are located in subcellular compartments where Ano1–FLAG3× is unlikely to reside, such as the inner mitochondrial membrane. These data emphasize the importance of cross-linking for stabilizing weak and transient interactions. The finding that many of the cross-linked interacting proteins are found in plasma-membrane–associated compartments supports the contention that the cross-linking procedure only cross-links proteins that are physiologically relevant. Fig. 3 A and B quantifies the SILAC enrichment in cross-linked cells. Two additional non-SILAC nano-liquid chromatography (LC) MS/MS experiments independently confirmed the presence of most of the proteins identified in the SILAC run (Dataset S1). The five proteins that were most highly enriched were radixin, Ano1, syntaxin binding protein 3 (munc18c), ezrin, and moesin fi fi Fig. 1. Immunoaf nity puri cation of Ano1 supramolecular complexes (∼30-fold enrichment). Examples of spectra of several high-rank- from Ano1–FLAG3× cell line. (A) Silver-stained SDS/PAGE gel. (B) Immuno- ∼ ∼ ing proteins in the SILAC enrichment are shown in Fig. S3. blot. Ano1 migrates as 130-kDa monomer and a 260-kDa dimer (25). HEK The experimentally verified functions of the 93 proteins were cells were cross-linked with DSP. Triton X-100 soluble lysates were incubated – × evaluated with Ingenuity pathways analysis (Fig. S4). The func- with anti-FLAG magnetic beads. Input: total lysate from Ano1 FLAG3 HEK P cells. Lanes 1–4: Bound proteins were eluted with SDS/PAGE sample buffer. tions with the smallest values were: organization of cytoplasm and cytoskeleton, followed by other functions that encompassed Lane 1: No antibody control, beads lacking antibody. Lane 2: Irrelevant an- fi tibody control, beads coated with SV2 antibody. Lane 3: beads coated with cell motility, protein traf cking, secretion, and signaling. To anti-FLAG antibody. Note the band corresponding to Ano1 at ∼130 kDa. better understand the Ano1 interactome, the 93 proteins were Lane 4: Excess antigen control, beads coated with anti-FLAG antibody in the subjected to analysis using DAPPLE (30). DAPPLE looks for presence of 340 μM FLAG3× peptide. Lane 5: Proteins eluted from anti-FLAG significant physical connectivity among proteins according to coated beads (as in lane 3) with wash buffer A. Lane 6: Proteins eluted from protein–protein interactions from MINT, BIND, IntAct, PPrel, anti-FLAG coated beads (as in lane 3) with 340 μM FLAG3× peptide. ECrel, Reactome, and other databases. The dataset contains

Perez-Cornejo et al. PNAS | June 26, 2012 | vol. 109 | no. 26 | 10377 Downloaded by guest on September 27, 2021 networks were represented in the 93-protein Ano1 interactome. One network is involved in vesicle trafficking and centers on the SNARE proteins syntaxin 4, syntaxin 7, VAMP3, and munc18. The other pathway is centered on the ezrin–radixin–moesin signaling/scaffolding complex and rhoA, which is involved in linking cell surface proteins to the cytoskeleton. Thus, the SILAC enrichment experiment, the accumulated peptide counts in three independent mass spectrometry experiments, and two independent in silico analyses support the association of two important signaling networks with Ano1. To verify the significance of the interactions identified by our proteomic analysis, we selected the ERM proteins for further analysis. We first examined the ability to coimmunoprecipitate ezrin, radixin, and moesin with Ano1. All three endogenous ERM proteins were robustly detected in the affinity-purified Ano1–FLAG3× complex (Fig. 5A). Radixin (68.5 kDa) and moesin (67.8 kDa) were not well separated in routine gels, but they were clearly separated when the gels were run for longer times (Figs. 5A(i) and see Fig. 8A). We independently confirmed these associations by coimmunoprecipitation of CFP-tagged ezrin or radixin coexpressed with Ano1–FLAG3× (Fig. 5B). We then asked whether native Ano1 and ERM proteins associate endogenously in adult mouse salivary gland in the absence of cross-linking. Subcellular fractionation of salivary gland homoge- nates demonstrated that a large proportion of Ano1 cosedimented with ERM proteins and actin in a fraction (P2) that was Triton fi Fig. 3. Fold-enrichment and spectral counts of peptides identified in SILAC X-100 insoluble. This nding supports the conclusion that Ano1 experiment. (A) Fold enrichment of heavy:light of the top 209 proteins vs. and ERM proteins exist in a stable macromolecular complex in the spectral counts. (B) Data in A replotted as fold enrichment vs. protein salivary gland cells (Fig. S5). To determine whether Ano1 and rank. The top five enriched proteins are labeled. (C) Number of proteins ERM proteins are molecularly associated, we tested whether Ano1 enriched more than twofold from cells treated with vehicle (DMSO) or DSP. and ERM proteins could be coimmunoprecipitated from the de- tergent-soluble P2 fraction using a polyclonal antibody to mAno1.

428,430 reported interactions, 169,810 of which are deemed high-conﬁdence nonself interactions across 12,793 proteins. This analysis revealed 80 interactions between the 93 proteins with an average direct connectivity per protein of 2.7 (Fig. 4). Two major

Fig. 5. Coimmunoprecipitation of ezrin, radixin, and moesin with Ano1. (A) Ano1–FLAG3× stable cell line was cross-linked and Ano1 complexes were purified by immunoaffinity chromatography (lane 1). Control sample (lane 2) was applied to the anti-FLAG affinity column in the presence of excess FLAG3× peptide. Eluted proteins were run on Western blot and probed with antibodies against moesin (67.8 kDa) and radixin (67.8 kDa), ezrin, or FLAG (Ano1). Insert Fig. 4. DAPPLE analysis of interactions among the proteins in the Ano1 A(i) shows a better separation of radixin and moesin than in Fig. 8A.(B)HEK interactome. Proteins from Dataset S1 were submitted for analysis using cells transiently coexpressing Ano1–FLAG3× and radixin–CFP or moesin–cyan parameters: iterations = 10,000, common interactor binding degree cutoff = fluorescent protein were cross-linked and Ano1 complexes purified and probed 6, iteration = on. Red labels are nodes that achieved a P value <0.05, in- as in A.(C) Moesin/radixin were immunoprecipiated from salivary gland P2 dicating the probability that the connections at these nodes were observed Triton X-100 soluble fraction (Fig. S5) with a polyclonal antibody against Ano1 by chance. (lane 2) or an irrelevant antibody against hemaglutinin (control, lane 1).

10378 | www.pnas.org/cgi/doi/10.1073/pnas.1200174109 Perez-Cornejo et al. Downloaded by guest on September 27, 2021 The Ano1 antibody coimmunoprecipitated moesin/radixin in the conclude that Ano1 colocalizes with moesin/radixin and ezrin at absence of cross-linker, showing that these proteins interact in the apical membrane of salivary acinar and duct cells. native salivary gland (Fig. 5C). To investigate the functional interaction between Ano1 and Additional approaches were taken to explore the association moesin, HEK cells were treated with shRNA directed against of Ano1 and moesin. HEK-293 cells transfected with Ano1– moesin. Four different shRNA plasmids were tested. Hairpin 5 mCherry and moesin–EGFP were analyzed by confocal micros- was most effective in knocking down moesin expression, copy. Ano1 and moesin are both intensely localized at the whereas hairpin 3 was ineffective (Fig. 8A). Control cells were plasma membrane (Fig. 6 A–C). The plasma membrane locali- treated identically but with empty vector. On average, the zation of both Ano1 and moesin were augmented when the two amplitude of the Ano1 currents was reduced by >50% after were expressed together compared with either expressed alone treatment with shRNA 5, but not by treatment with shRNA 3 B (Fig. 6 D–F). Bimolecular fluorescence complementation was compared with control cells (Fig. 8 ). The decrease in Ano1 then used to establish whether morphological colocalization of current amplitude is not due to decreased levels of Ano1 at the cell surface, as determined by biotinylation of the Ano1 cell Ano1 with moesin represented molecular binding. Ano1 tagged A with the N-terminal half of the fluorescent protein Venus surface pool (Fig. 8 ). (Ano1–N–Venus) was coexpressed with moesin tagged with the Discussion – – C-terminal half of Venus (moesin C Venus). Coexpression of fi the two proteins produced intense plasma membrane fluores- We have developed a procedure to af nity purify Ano1 with its cence (Fig. 6 G and H), showing that Ano1 and moesin interact. interacting proteins. Three independent proteomic analyses identified protein networks associated with Ano1 at high stoi- No fluorescence was seen when Ano1–N–Venus was expressed chiometry: the actin-binding regulatory ERM proteins and alone or with either of two irrelevant proteins, the trans- a SNARE protein complex. In addition, Ano1 interacts with both membrane Kv1.2 channel or the SMN complex protein unrip, – I J B large and small GTPases, calcium binding proteins, kinases, and tagged with C Venus (Fig. 6 and and Fig. S6 ). lipid-modifying enzymes (Dataset S1). Because HEK cells do not To determine whether colocalization of moesin and Ano1 also naturally express Ano1, it is possible that we have missed some occurs in native tissue, we examined the distribution of Ano1 and interacting proteins that might be present in cell types that na- moesin/radixin by immunostaining mouse salivary gland. Salivary tively express Ano1. However, because the currents that we re- gland was chosen because we have shown that Ano1 is responsible − cord in transfected HEK cells are virtually identical to those that for Ca2+-mediated Cl secretion in this tissue, and because this 2+ − we have recorded in native cells, such as salivary gland acinar tissue is a model system for studying Ca -activated Cl channels cells (8), Xenopus oocytes (32), and inner medullary collecting (8, 31). Ano1 was highly concentrated at the apical membrane of duct (IMCD) cells (33), it seems unlikely that essential binding acinar cells and intercalated excretory ducts and colocalized with partners are missing. However, our investigation was limited to moesin/radixin, ezrin, and actin (Fig. 7). Some staining for moesin/ nominally “basal” intracellular Ca2+ conditions. Future experi- radixin and ezrin was sometimes observed on the basolateral ments will be directed at examining the interactome when Ca2+ membranes, but this staining was also present in cells in which is elevated before cross-linking as well as in cells that endoge- primary antibody was omitted (Fig. S6A). This background stain- nously express Ano1. The converse question is whether cross- ing is probably due to the polymeric IgA receptor, which is widely linking could produce false positives that are not actually in-

expressed in salivary gland and is very difﬁcult to eliminate when volved in regulating Ano1. We previously validated the experi- CELL BIOLOGY using mouse monoclonal secondary antibodies. Therefore, we mental approach used here genetically and have shown that it

Fig. 6. Colocalization of moesin and Ano1 in HEK cells. (A–C) HEK cells were transfected with Ano1–mCherry (red, A) and moesin–EGFP (green, B). (C) A and B superimposed. (D and E) Cells transfected with Ano1–mCherry alone (D) or moesin–EGFP alone (E). (F) Profiles of transcellular fluorescence in cells expressing only moesin–EGFP or moesin–EGFP plus Ano1–mCherry. Profiles were obtained by drawing a line across the cell avoiding the nucleus where moesin is excluded. Profiles are representative of >10 cells selected at random. (G–J) Bimolecular fluorescence complementation (BiFC). (G) Cells were cotransfected with Ano1 tagged with Venus(1–155) and Moesin tagged with Venus(156–239). (H) High power showing membrane localization of the BiFC fluorescence. (I) Control for BiFC. Cells were transfected with Ano1–Venus(1–155) and Kv1.2–Venus(156–239).(J) Quantification of BiFC. Histograms of pixel intensity for fields transfected with Ano1–Venus(1–155) plus moesin–Venus(156–239), the potassium channel Kv1.2–Venus(156–239), or the survival of motor neurons (SMN) complex subunit unrip–Venus(156–239).

Perez-Cornejo et al. PNAS | June 26, 2012 | vol. 109 | no. 26 | 10379 Downloaded by guest on September 27, 2021 Fig. 7. Localization of Ano1, ezrin, moesin, and actin in salivary gland. Col- oring is as follows: moesin and ezrin are red, Ano1 is green, and actin (stained with Alexa 647 phalloidin) is yellow. (A–F). Colocalization of Ano1, ezrin, and actin. (G–L). Colocalization of Ano1, moesin, and actin. (A and G) DIC images. (B and H) Actin. (C and I) Ano1. (D) Ezrin. (E) Ano1 and ezrin overlay. (J) Moesin. (K) Ano1 and moesin overlay. (F and L) Ano1 and DIC overlay.

minimizes false positive interactors between a membrane protein and a cytosolic adapter (34) and in multicomponent DSP interactomes (21). DSP is a homobifunctional reagent with a spacer arm of 12 Å that interacts only with N-terminal α-amino and ε-amino groups of lysine. Thus, only amino groups within this distance will be cross-linked. We used DSP in substoichiometric and precisely defined conditions that avoid the generation of Fig. 8. Effect of knockdown of moesin on Ano1 currents. (A) Western blot of large protein aggregates (21, 22, 24, 34). In the case of Ano1, total cell extracts (lanes 1–4) and surface biotinylated proteins (lanes 5–8). Cells evidence for restricted cross-linking is shown by the small effect were transfected with Ano1–FLAG3× and with empty shRNA vector or two of DSP on the sedimentation profile of total proteins in sucrose different shRNA constructs (nos. 3 and 5). Cells transfected with shRNA vectors gradients. Importantly, the Ano1 interactome identified in DSP were selected with puromycin. (Upper) Ano1 expression detected with Ano1 cross-linked HEK cells has led to the identification of associa- antibody. (Lower) Radixin and moesin expression. Control shRNA: cells were tions between Ano1 and ERM proteins in salivary gland in the transfected with the empty shRNA vector and selected with puromycin as with absence of cross-linker. The isotopic enrichment of the top 10 the other shRNA constructs. shRNA 5 almost completely eliminated moesin proteins in the SILAC experiment (Dataset S1)is>20, com- expression. (B) IV curves of whole-cell patch clamped Ano1 currents in a cell pared with 30 for Ano1 itself, suggesting that the top-ranking line stably expressing Ano1 tagged with EGFP on the C terminus treated with control vector, shRNA 3 or shRNA 5. Intracellular (pipet) Ca2+ concentration proteins are likely interacting at high stoichiometry with Ano1. was 600 nM. (C) Average current densities at +100 mV. *P < 0.05. One notable observation is that there are no known ion channels or Ano-like membrane proteins in addition to Ano1 in the fi curated interactome. Even among the un ltered list of the highest The physiological significance of the ERM proteins is high- 200 SILAC enriched proteins, there were no known ion channels lighted by the colocalization of moesin and Ano1 in native and and only three ion transporters. Although it is widely thought that fi transfected cells and by the inhibitory effect of moesin knock- Ano1 is a pore-forming channel subunit, the molecular identi - down on Ano1 current amplitude. An interesting observation is cation of Cl channels has been fraught with problems (1). Because that the surface localization of moesin is increased when the two unambiguous proof that Ano1 forms the pore has not yet been proteins are coexpressed. This suggests the possibility that Ano1 achieved (for example, by incorporation into bilayers) and HEK may play a role in organization of the actin cytoskeleton. The cells endogenously express several Ano orthologs, it is reassuring actin cytoskeleton and its associated regulatory proteins could be that our proteomic data provide no evidence for an additional implicated in Ano1 function by modulating Ano1 channel gating, potential pore-forming subunit that associates with Ano1. fi The mechanism of regulation of Ano1 by Ca2+ remains un- directing the traf cking Ano1 to the apical membrane, or as- resolved (35, 36). Some data support the idea that Ca2+ binds di- sembling Ano1 into signaling networks. It has been reported that rectly to the Ano1 protein, but other data implicate a role for CaM both depolymerization of the actin cytoskeleton with cytochala- (review in ref. 18). It is notable that CaM was not found in the Ano1 sin-D and stabilization of the actin network with phalloidin interactome. The interactome does include a member of the S100 suppresses Ano1 currents (42). A number of ion channels have – been shown to interact with the actin cytoskeleton (43). It is Ca-binding protein superfamily, S100 A10. However, this member − of the family does not appear to bind Ca2+. Other proteins that are particularly interesting that another epithelial Cl channel, regulated by Ca2+ or may bind Ca include annexin A1 (ANXA1), CFTR, interacts with some of the same proteins that Ano1 calnexin (CANX), calmodulin-activated serine kinase (CASK), interacts with, notably ezrin and NHERF1. The actin cytoskel- coiled-coil and C2-domain interacting protein (CC2D1A), phos- eton modulates CFTR by multiple mechanisms including traf- pholipid scramblase (PLSCR1), and extended synaptotagmin ficking, gating, and assembly into signaling complexes. CFTR, (FAM62A). Annexin A1 is intriguing because various annexins like Ano1, also interacts with SNARES (syntaxins 1A, -3, -6, -7, − have been shown to regulate native Ca2+-activated Cl channels. -8, -16, and VAMP8) (44), which play a role in CFTR trafficking. − For example, annexin IV inhibits calmodulin-sensitive Cl chan- In summary, these data provide a rich store of leads about 2+ − 2+ nels (37, 38) as well as Ca -activated Cl channels (39). In en- potential− regulatory mechanisms impinging on the Ca -acti- dothelial cells the annexin II–S100A10 complex might be in- vated Cl channel Ano1. Experiments to elucidate the roles that − volved in regulation of the swelling-activated Cl channel (40) the SNARE complex and ERM proteins play in Ano1 physiology and cystic fibrosis transmembrane conductance regulator (41). and cell biology are likely to yield exciting new information.

10380 | www.pnas.org/cgi/doi/10.1073/pnas.1200174109 Perez-Cornejo et al. Downloaded by guest on September 27, 2021 Materials and Methods on glass slides using ProLong Gold (Invitrogen). Salivary gland was obtained Methods are described in detail in SI Materials and Methods. from wild-type mice immediately after euthanasia induced by inhalation of isoﬂurane. Animals were bred and maintained in accordance with National ’ Proteomic Analysis. A stable cell line was generated by transfecting HEK cells Institutes of Health s institutional guidelines. All procedures were approved with a neomycin-selectable plasmid encoding Ano1 tagged with a concatamer by Emory University Institutional Animal Care and Use Committee. Tissue was ﬁxed in 4% paraformaldehyde buffered with 0.1 M phosphate buffer pH 7.4, of three FLAG epitopes [(DYKDDDDK)3]. SILAC was performed by growing cells in DMEM media containing either isotopically light arginine and lysine frozen, and embedded in OCT compound for frozen sectioning. Sections

(“R0K0”) or isotopically heavy arginine and lysine (“R10K8”). The incorporation were then processed as essentially as described for transfected cells. of labeled amino acids was 97.5%. To stabilize low-affinity interactions, intact cells were cross-linked with DSP as previously described (22, 24, 34). Cells were Patch Clamp. Whole-cell patch clamp recordings were performed as pre- lysed and the clarified supernatant was applied to 30 μL Dynal magnetic beads viously described (32). Intracellular pipette solution contained (in millimoles): 2+ coated with anti-FLAG antibody. Specifically bound proteins were eluted by 2- 146 CsCl, 2 MgCl2, 5 EGTA, 10 sucrose, and 8 Hepes, pH 7.3. Ca was ad- h incubation with 340 μM3× FLAG peptide. Samples were analyzed by SDS/ justed to 600 nM free Ca2+ (32). The extracellular solution contained (in

PAGE followed by immunoblot or silver staining. Mass spectrometry analysis millimoles): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 15 glucose, and 10 Hepes, pH was performed by MS Bioworks. Samples for proteomic analysis were sepa- 7.4. shRNA in pLKO.1 vector for moesin was purchased from Open Bio- rated on a 4–12% (wt/vol) Bis-Tris Novex minigel, digested with trypsin, and systems (RHS4533_NM_002444). Transfected cells were selected in the analyzed by nano-LC MS/MS with a Waters NanoAcquity HPLC system inter- presence of 1 μg/mL puromycin for 3 d. faced to a ThermoFisher LTQ Orbitrap Velos. ACKNOWLEDGMENTS. We thank Alexa Mattheyses for help with microscopy, Microscopy. HEK cells were plated on coverslips in 35-mm dishes and tran- Claudia Fallini with bimolecular ﬂuorescence complementation (BiFC), and siently transfected using Fugene 6 with 1 μg of DNA. Live cells were examined Richard Jones of MS Bioworks for excellent mass spectrometry analysis and by confocal microscopy or were ﬁxed for 20 min in 4% paraformaldehyde advice on sample preparation. This work was supported by National Institutes and permeabilized with paraformaldehyde before blocking and staining of Health (NIH) Grants GM60448 (to H.C.H.), EY014852 (to H.C.H.), NS42599 (to with antibodies. Primary antibodies were used against the following anti- V.F.), and GM077569 (to V.F.); the Emory University Research Committee (H.C.H.); National Eye Institute (NEI) Training Grant 5T32EY007092-25 (to gens: Ano1 (amino acids 878–960, SDIX Custom Genomic Antibody), anti- C.D.); and NIH Fellowships in Research and Science Teaching (FIRST) Program moesin (Abcam; A50007), and antiezrin. The cells were then washed and Fellowship K12 GM000608 (to A.G.). Additional support was provided by the incubated with a mixture of Dylight-488 or Dylight-568 conjugated (1:1,000) Microscopy Core of the Emory Neuroscience National Institute of Neurological secondary antibodies for 1 hr at 4 °C (Jackson Immunochemicals). Sometimes Disorders and Stroke Core Facilities Grant P30NS055077 and NEI Core Alexa-647–phalloidin was included to stain actin. Coverslips were mounted Grant P30EY006360.

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