Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca 2+-activated cl − channel responsible for essential physiological processes. each monomer harbours a pore and a ca 2+-binding pocket; the voltage-dependent binding of two intracellular ca 2+ ions to the pocket gates the pore. However, in the absence of intracellular ca 2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with ca 2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/ Asp residues in the ca 2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca 2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca 2+. Anoctamin-1 (ANO1 or TMEM16A) and Anoctamin-2 (ANO2 or TMEM16B) are the pore-forming subunits of Ca 2+-activated Cl − channels (CaCCs) 1-3. Several tissues express CaCCs that participate in vital physiological functions 4,5. Thus, a role for TMEM16A and TMEM16B in smooth muscle contraction, control of blood pressure, control of gastrointestinal movements, regulation of cardiac and neuronal excitability, fluid secretion in exocrine glands, secretion of melatonin, mucin and insulin, sperm capacitation and motility, inhibition of polyspermy, and sensory transduction was established using tissue-specific knockout mice 6-16. In addition, TMEM16A modulates the partitioning of membrane phosphoinositides and endocytic transport by controlling the [Cl − ] i 17. Overexpression of TMEM16A is associated with hypertension, increased cell proliferation and cancer progression 18-21. Activation of CaCCs is triggered by voltage-dependent binding of two Ca 2+ ions to the channel when the intracellular Ca 2+ concentration ([Ca 2+ ] i) increases 22-26. Structural and mutagenesis analysis show that Ca 2+ ions bind to an acidic Ca 2+ pocket formed by four Glu, one Asp and one Asn 25-27. The pocket is located near the cytosolic side facing the permeation pathway. However, other divalent cations and maybe trivalent cations too can support TMEM16A activation. Based on the cation concentrations to obtain the half-maximum response, the cation selectivity of TMEM16A gating machinery is Ca 2+ »Sr 2+ »Ba 2+ »Cd 2+ , 23,27-29. Gd 3+ may also activate TMEM16A since its application removed the inward rectification of the Gly644Pro TMEM16A mutant channel 30 .

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Numerous essential physiological processes depend on the TMEM16A-mediated Ca2+-activated chloride fluxes. Extensive structure–function studies have helped to elucidate the Ca2+ gating mechanism of TMEM16A, revealing a Ca2+-sensing element close to the anion pore that alters conduction. However, substrate selection and the substrate–gating relationship in TMEM16A remain less explored. Here, we study the gating–permeant anion relationship on mouse TMEM16A expressed in HEK 293 cells using electrophysiological recordings coupled with site-directed mutagenesis. We show that the apparent Ca2+ sensitivity of TMEM16A increased with highly permeant anions and SCN− mole fractions, likely by stabilizing bound Ca2+. Conversely, mutations at crucial gating elements, including the Ca2+-binding site 1, the transmembrane helix 6 (TM6), and the hydrophobic gate, impaired the anion permeability and selectivity of TMEM16A. Finally, we found that, unlike anion-selective wild-type channels, the voltage dependence of unselective TMEM16A mutant channels was less sensitive to SCN−. Therefore, our work identifies structural determinants of selectivity at the Ca2+ site, TM6, and hydrophobic gate and reveals a reciprocal regulation of gating and selectivity. We suggest that this regulation is essential to set ionic selectivity and the Ca2+ and voltage sensitivities in TMEM16A.

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New vaccine design approaches, platforms, and immunization strategies might foster antiviral mucosal effector and memory responses to reduce asymptomatic infection and transmission in vaccinated individuals. Here, we investigated a combined parenteral and mucosal immunization scheme to induce local and serum antibody responses, employing the epitope-based antigens 3BT and NG19m. These antigens target the important emerging and re-emerging viruses PRRSV-2 and SARS-CoV-2, respectively. We assessed two versions of the 3BT protein, which contains conserved epitopes from the GP5 envelope protein of PRRSV-2: soluble and expressed by the recombinant baculovirus BacDual-3BT. On the other hand, NG19m, comprising the receptor-binding motif of the S protein of SARS-CoV-2, was evaluated as a soluble recombinant protein only. Vietnamese mini-pigs were immunized employing different inoculation routes: subcutaneous, intranasal, or a combination of both (s.c.-i.n.). Animals produced antigen-binding and neut1ralizing antibodies in serum and mucosal fluids, with varying patterns of concentration and activity, depending on the antigen and the immunization schedule. Soluble 3BT was a potent immunogen to elicit binding and neutralizing antibodies in serum, nasal mucus, and vaginal swabs. The vectored immunogen BacDual-3BT induced binding antibodies in serum and mucosae, but PRRSV-2 neutralizing activity was found in nasal mucus exclusively when administered intranasally. NG19m promoted serum and mucosal binding antibodies, which showed differing neutralizing activity. Only serum samples from subcutaneously immunized animals inhibited RBD-ACE2 interaction, while mini-pigs inoculated intranasally or via the combined s.c.-i.n. scheme produced subtle neutralizing humoral responses in the upper and lower respiratory mucosae. Our results show that intranasal immunization, alone or combined with subcutaneous delivery of epitope-based antigens, generates local and systemic binding and neutralizing antibodies. Further investigation is needed to evaluate the capability of the induced responses to prevent infection and reduce transmission.