Characterization of the Oligomeric Structure of the Ca2+-activated Cl− Channel Ano1/TMEM16A

Transmembrane proteins with unknown function 16 (TMEM16A) is a calcium-activated chloride channel (CaCC) important for neuronal, exocrine, and smooth muscle functions. TMEM16A belongs to a family of integral membrane proteins that includes another CaCC, TMEM16B, responsible for controlling action potential waveform and synaptic efficacy, and a small-conductance calcium-activated nonselective cation channel, TMEM16F, linked to Scott syndrome. We find that these channels in the TMEM16 family share a homodimeric architecture facilitated by their cytoplasmic N termini. This dimerization domain is important for channel assembly in eukaryotic cells, and the in vitro association of peptides containing the dimerization domain is consistent with a homotypic protein–protein interaction. Amino acid substitutions in the dimerization domain affect functional TMEM16A-CaCC channel expression, as expected from its critical role in channel subunit assembly.

Section: Tmem16 Family Proteins Form Dimeric Proteinmentioning

confidence: 99%

“…Several groups have recently used biochemical methods to characterize the quaternary structure of TMEM16A channels as homodimers (13,14). These previous studies on channel stoichiometry have raised the following questions: Is dimerization necessary for channel function?…”

mentioning

confidence: 99%

Identification of a dimerization domain in the TMEM16A calcium-activated chloride channel (CaCC)

Tien

Lee

Minor

et al. 2013

“…However, the halide permeability sequence of ORCC in the different cell lines is comparable and is SCN − > I − > Br − = Cl − >> gluconate (18). Homodimerization has been shown meanwhile for Ano1 (24,25). Although heterooligomerization has not yet been detected, we speculate that Ano6 may form variable oligomeric protein complexes in different cell types, thereby shaping ORCC in different cell types (15).…”

Section: Resultsmentioning

confidence: 94%

Anoctamin 6 is an essential component of the outwardly rectifying chloride channel

Martins

Faria

Kongsuphol

et al. 2011

145

161

Outwardly rectifying chloride channels (ORCC, ICOR) of intermediate single-channel conductance of around 50 pS, are ubiquitously expressed, but have remained a mystery since their description more than 25 y ago. These channels have been shown to be activated on membrane excision and depolarization of the membrane voltage and by cAMP in the presence of the cystic fibrosis transmembrane conductance regulator. We show that anoctamin 6 (Ano6), a member of the recently identified family of putative Cl − channels, is the crucial component of ORCC single-channel and whole-cell currents in airway epithelial cells and Jurkat T lymphocytes. Cystic fibrosis transmembrane conductance regulator augmented ORCC produced by Ano6 in A549 airway epithelial cells. Ano6 is activated during membrane depolarization or apoptosis of Jurkat T lymphocytes and epithelial cells, and is inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid, 4,4′-diisothio-cyanostilbene-2,2′-disulfonic acid, or AO1. Ano6 belongs to the basic equipment of any cell type, including colonic surface epithelial cells. It forms the essential component of ORCC and seems to have a role for cell shrinkage and programmed cell death.

“…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).…”

Section: Resultsmentioning

confidence: 99%

Anoctamin 1 (Tmem16A) Ca ² ⁺ -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

Pérez‐Cornejo

Gokhale

Duran

et al. 2012

Self Cite

114

139

The newly discovered Ca 2+ -activated Cl − channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca 2+ -activated Cl − currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca 2+ -binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane.calcium | interactome | cross-linker | apical targeting