Extended Synaptotagmin-1 (ESYT1) Antibody

Cystic fibrosis  (CF) is the most common life-limiting (median age at death ~28 years) rare disease, affecting ~32,000 people in Europe (~85,000 worldwide). This inherited disorder results from mutations in the gene encoding the  CF transmembrane conductance regulator  (CFTR), an epithelial chloride (Cl  ^–  ) and bicarbonate (HCO  ₃  ^–  ) anion channel. Despite symptomatic therapies, quality of life and life expectancy are still limited. Therefore, correction of the basic CF defect, ie restoring epithelial Cl  ^–  /HCO  ₃  ^–  secretion by CFTR modulators, is the much sought-after alternative. 

Despite some success in introducing it to the clinic, CFTR modulators still cannot be used to pharmacologically rescue all CFTR mutations, e.g. B.

large deletions such as the dele2,3(21 kb) mutation, which is fairly common in Slavic countries. For CF patients with these ‚unsalvageable‘ mutations, recently classified as class (theratype) VII   , the alternative is to develop ‚mutation-independent‘ therapies. One such possibility is the activation of alternative (non-CFTR) anion channels .

• Anoctamins consist of a family of 10 different proteins, ANO1-10 (also known as TMEM16A-H, J, and K, respectively), some of whose members are reported to be (or regulate) Cl  ^–  channels. 
• Indeed, anoctamine 1 (ANO1) has been identified as a calcium (Ca  ²⁺  ) activated Cl  ^–  channel (CaCC). It has also been reported that ANO2 is the CaCC-mediated olfactory  signal transduction  [7]  and ANO6 has been identified as the major component of the outward rectifying chloride channel (ORCC).
•  However, ANO5, ANO7, ANO8, ANO9 and ANO10  do not appear to mediate  ion transport [9]  ,  [10] . Besides transepithelial ion transport, anoctamines also play a variety of physiological functions ranging from Ca  ²⁺  -dependent lipid scramble – ANO3– to regulation of other ion channels , to mediate  smooth muscle contraction  ,  olfaction, phototransduction  ,  nociception  , and control of neuronal excitability.
• ANO1/TMEM16A is of fundamental importance for the Ca  ²⁺  -dependent Cl  ^–  secretion in different epithelia, namely in the respiratory tract, the salivary gland, the pancreas and the hepatocytes.
• Indeed, siRNA knockdown of ANO1 in vivo resulted in reduced fluid secretion in the salivary glands. Importantly, native epithelia from mice with  genetic ablation  of ANO1/TMEM16A do not produce Ca  ²⁺  -activated Cl  ^{– transport}  , namely in the airways, and also show impaired  mucociliary clearance  with accumulation of mucus in the airways, in striking resemblance with respiratory CF disease .
•  In addition to Cl  ⁻  , ANO1 has also been shown to transport HCO  ₃  ^{−  following physiological stimulatio} , suggesting that this channel could also  ^{replace  CFTR-mediated HCO} ₃  − transport .
• Taken together, these data demonstrate the important role of ANO1 in fluid secretion, making it an attractive candidate for novel treatments of CF to compensate for defective CFTR-mediated  anion transport  .

Notwithstanding, we still lack basic understanding of how ANO1  ^{can be stimulated independently of Ca 2+}  , an essential requirement for its potential pharmacological applications. 

Although ANO1 has been reported to accumulate in the  apical membrane  of airway epithelial cells  [17]  ,  [20]  , it is present at the cell surface in low amounts  [17]  . Furthermore, little is known about the regulation of ANO1 trafficking, namely what factors promote its plasma membrane (PM) localization.  Interestingly, recent studies indicate that ANO1 may also play a tethering role of receptors, e.g. B. the  inositol trisphosphate  (IP  ₃  ) receptor – and endoplasmic reticulum (ER) stores Ca  ²⁺ to the PM  [21] . According to this hypothesis, intracellular ANO1 (and possibly other ANO proteins) may not be the channels themselves, but instead mediate activation through coupling of Ca  ²⁺  signals from other membrane-localized channels . Understanding how this regulation occurs is very relevant to human diseases, as ANO1 has been reported to play a role in other diseases as well, namely various types of cancer .

In any case, essential understanding of the molecular basis of Ca  ²⁺  -dependent Cl  ⁻  transport by anoctamines is lacking, as we still lack key information on the most fundamental aspects of how (and if) they are transported to the PM. However, this is a crucial aspect for a detailed molecular mechanism of ANO1 regulation or its regulation of other channels. An open question is therefore under which conditions ANO1 is localized intracellularly and which factors favor its PM localization.

Early studies on TMEM16A suggested the alternative name anoctamine 1 as it showed selectivity for  an  ions and presumably possesses eight (  oct-  )  transmembrane domains  [4]  . Indeed, a high-resolution structural analysis of the fungal homologue from the fungus  Nectria haematococca  nh TMEM16 revealed 10 instead of eight transmembrane domains  [24]  , which was subsequently confirmed by a topological model proposed for TMEM16A. According to initial biochemical analyzes and structural studies, TMEM16 acts as a dimer  [25].

Against this background, we report here the establishment of a microscopic trafficking assay on the physiologically relevant CFBE (CF Bronchial Epithelial) cell line stably transduced to express a reporter of ANO1 trafficking under an inducible promoter. This ANO1 traffic reporter contains enhanced  green fluorescent protein  (eGFP) fused to the C-tail of ANO1 and a triple  hemagglutinin  (3HA) tag at its first extracellular loop to target PM-localized ANO1 through the use of anti-HA -Detect antibodies without cell  permeabilization  . 

Similar to that previously described for CFTR, this dual-labeled reporter allows for ratiometric readouts of traffic efficiency on a per-cell basis, thus providing a reliable cell model to study ANO1 traffic. The results shown here for this cell model demonstrate the robustness and sensitivity of the assay. Application of this assay in systematic loss-of-function (siRNA knockdown) gene screens  [27]  will allow identification of ANO1 trafficking regulators and potential drug targets for CF.