TMEM16A alternative splicing isoforms in Xenopus tropicalis: Distribution and functional properties

Huanosta-Gutiérrez, A.; Espino-Saldaña, Ángeles Edith; Reyes, Juan Pablo; Pétriz, Adriana; Miledi, Ricardo; Martı́nez-Torres, Ataúlfo

doi:10.1016/j.bbrc.2014.03.057

Cited by 5 publications

(10 citation statements)

References 15 publications

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“…In comparison with other nonsteroidal anti-inflammatory drugs or nonopioid analgesics, it is not associated with side effects, particularly, reactions in children (Sturkenboom et al, 2005). NFA is also widely used for inhibition of some types of Cl − -selective channels, blocking primary voltage-gated CLC-1 channels (Sturkenboom et al, 2005) and Ca 2+ -activated Cl − channels (CaCCs) (White and Aylwin, 1990; Yang et al, 2008; Huanosta-Gutierrez et al, 2014). …”

Section: Discussionmentioning

confidence: 99%

“…However, mechanism of its action on these proteins remains unclear. NFA blocks voltage-gated chloride channels, CLC-1 (Liantonio et al, 2007), as well as Ca 2+ -activated Cl − - channels (CaCCs) (White and Aylwin, 1990; Yang et al, 2008; Huanosta-Gutierrez et al, 2014). The effect of NFA on CaCCs is voltage-independent and it blocks the channels when applied from either outside or inside the cell (Qu and Hartzell, 2001).…”

Section: Introductionmentioning

confidence: 99%

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Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid

Maleeva

Peiretti

Zhorov

et al. 2017

Front. Mol. Neurosci.

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Niflumic acid (NFA) is a member of the fenamate class of nonsteroidal anti-inflammatory drugs. This compound and its derivatives are used worldwide clinically for the relief of chronic and acute pain. NFA is also a commonly used blocker of voltage-gated chloride channels. Here we present evidence that NFA is an efficient blocker of chloride-permeable glycine receptors (GlyRs) with subunit heterogeneity of action. Using the whole-cell configuration of patch-clamp recordings and molecular modeling, we analyzed the action of NFA on homomeric α1ΔIns, α2B, α3L, and heteromeric α1β and α2β GlyRs expressed in CHO cells. NFA inhibited glycine-induced currents in a voltage-dependent manner and its blocking potency in α2 and α3 GlyRs was higher than that in α1 GlyR. The Woodhull analysis suggests that NFA blocks α1 and α2 GlyRs at the fractional electrical distances of 0.16 and 0.65 from the external membrane surface, respectively. Thus, NFA binding site in α1 GlyR is closer to the external part of the membrane, while in α2 GlyR it is significantly deeper in the pore. Mutation G254A at the cytoplasmic part of the α1 GlyR pore-lining TM2 helix (level 2′) increased the NFA blocking potency, while incorporation of the β subunit did not have a significant effect. The Hill plot analysis suggests that α1 and α2 GlyRs are preferably blocked by two and one NFA molecules, respectively. Molecular modeling using Monte Carlo energy minimizations provides the structural rationale for the experimental data and proposes more than one interaction site along the pore where NFA can suppress the ion permeation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid

Maleeva

Peiretti

Zhorov

et al. 2017

Front. Mol. Neurosci.

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“…tropicalis tmem16a is expressed in mucin-producing small secretory cells in tadpole skin Functional studies of Tmem16a in Xenopus are limited to its role in the oocyte and the prevention of polyspermy [39,40,41]. RT-PCR expression analysis in adults shows expression in various tissues [42,43], while bulk RNA sequencing in developing embryos shows expression at Nieuwkoop-Faber (NF) stages NF1, NF9 and NF24-NF42 [42]. We therefore sought tissue-speci c expression during skin development.…”

Section: Resultsmentioning

confidence: 99%

The Tmem16a chloride channel is required for mucin maturation after secretion from goblet-like cells in the Xenopus tropicalis tadpole skin.

Dubaissi,

Emma,

Lilley

et al. 2024

Preprint

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The TMEM16A chloride channel is proposed as a therapeutic target in cystic fibrosis, where activation of this ion channel might restore airway surface hydration and mitigate respiratory symptoms. While TMEM16A is associated with increased mucin production under stimulated or pro-inflammatory conditions, its role in baseline mucin production, secretion and/or maturation is less well understood. Here, we use the Xenopus tadpole skin mucociliary surface as a model of human upper airway epithelium to study Tmem16a function in mucus production. We found that Xenopus tropicalis Tmem16a is present at the apical membrane surface of tadpole skin small secretory cells that express canonical markers of mammalian “goblet cells” such as Foxa1 and Spdef. X. tropicalis Tmem16a functions as a voltage-gated, calcium-activated chloride channel when transfected into mammalian cells in culture. Depletion of Tmem16a from the tadpole skin results in dysregulated mucin maturation post-secretion, with secreted mucins having a disrupted molecular size distribution and altered morphology assessed by sucrose gradient centrifugation and electron microscopy, respectively. Our results show that in the Xenopus tadpole skin Tmem16a is necessary for normal mucus barrier formation and demonstrate the utility of this model system to discover new biology relevant to human mucosal biology in health and disease.

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“…16 The Goldman-Hodgkin-Katz equation was used to fit data points in Figure 2, where P Na /P Cl was left as a free parameter. With regard to the data in Figure 3, a Three-Barrier, TwoSite permeation model was used to fit the experimental data for the reversal potential as a function of Iodide mole fraction (Er vs. MF), as described in Reyes et al 6 Voltage dependence of the blockage (Figures 4-6) was assessed using voltage ramps by calculating the following ratio: I BDx /I BD0 , where I BDx is the current with settled effect of the blocker tested at any given x concentration, and I BD0 is the control maximum current (i.e., the concentration of blocker equals zero).…”

Section: Discussionmentioning

confidence: 99%

Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

Reyes¹,

Huanosta-Gutiérrez²,

Lopez-Rodriguez

et al. 2015

Channels

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We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na C permeation, whereas K613E, R646E and the double mutant K613E C R646E affect anion selectivity and permeability to Na C . Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.

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TMEM16A alternative splicing isoforms in Xenopus tropicalis: Distribution and functional properties

Cited by 5 publications

References 15 publications

Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid

Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid

The Tmem16a chloride channel is required for mucin maturation after secretion from goblet-like cells in the Xenopus tropicalis tadpole skin.

Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

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