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. 2011 Jan 21;286(3):2365-74.
doi: 10.1074/jbc.M110.175109. Epub 2010 Nov 17.

TMEM16A inhibitors reveal TMEM16A as a minor component of calcium-activated chloride channel conductance in airway and intestinal epithelial cells

Wan Namkung  1 , Puay-Wah Phuan, A S Verkman
Affiliations

TMEM16A inhibitors reveal TMEM16A as a minor component of calcium-activated chloride channel conductance in airway and intestinal epithelial cells

Wan Namkung et al. J Biol Chem. .

Abstract (VSports)

TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of ∼110,000 compounds revealed four novel chemical classes of small molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with an IC(50) < 10 μM, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor, an aminophenylthiazole (T16A(inh)-A01), had an IC(50) of ∼1 μM. Two distinct types of inhibitors were identified. Some compounds, such as tannic acid and the arylaminothiophene CaCC(inh)-A01, fully inhibited CaCC current in human bronchial and intestinal cells VSports手机版. Other compounds, including T16A(inh)-A01 and digallic acid, inhibited total CaCC current in these cells poorly, but blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia. The small molecule inhibitors identified here permit pharmacological dissection of TMEM16A/CaCC function and are potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production. .

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Figures

FIGURE 1.
FIGURE 1.
Identification of small molecule inhibitors of human TMEM16A. A, TMEM16A-transfected cells showing green cytoplasmic YFP-H148Q/I152L/F46L fluorescence (left) and TMEM16A protein by immunoblotting (right). B, short circuit current analysis of TMEM16A-expressing FRT cells. Representative current traces showing ionomycin- and ATP-stimulated TMEM16A chloride current are shown. C, screening protocol. FRT cells stably expressing TMEM16A and the YFP halide sensor were incubated for 10 min with test compound. Fluorescence was monitored in response to the addition of iodide and ATP. D, fluorescence measured in single wells of 96-well plates, showing vehicle control and examples of active and inactive compounds.
FIGURE 2.
FIGURE 2.
Chemical structures of TMEM16A inhibitors. A, structures shown of the most potent TMEM16A inhibitor of each of four classes (T16Ainh-X01, where X = A, B, C, or D), along with structure of digallic acid and the previously identified CaCC inhibitors CaCCinh-A01 (16) and tannic acid (19). B, summary of SAR analysis for class A TMEM16A inhibitors. Inhibition data for most potent class A compounds are summarized in Table 1. Functional data and SAR analysis for class B–D compounds provided in supplementary Figs. S1–S3, respectively.
FIGURE 3.
FIGURE 3.
Characterization of small molecule TMEM16A inhibitors. A, left, short circuit (apical membrane) current measured in TMEM16A-expressing FRT cells in the presence of a transepithelial chloride gradient (see “Experimental Procedures”). Inhibitors were added 5 min prior to TMEM16A activation by 100 μm ATP. A, right, summary of dose-response data (mean ± S.E., n = 4). B, cytoplasmic calcium measured by Fluo-4 fluorescence. Cells were pretreated for 5 min with 10 μm T16Ainh-A01 or 100 μm digallic acid (or vehicle control), with 100 μm ATP or 1 μm ionomycin added as a calcium agonist as indicated. C, CFTR activated by 10 μm forskolin in primary cultured human bronchial epithelial cells and 10 μm T16Ainh-A01 (black line) or 100 μm digallic acid (gray line) added to apical bath as indicated. D, CaCC current measured in TMEM16B-transfected FRT cells expressing YFP-H148Q/I152L/F46L, showing inhibition by 10 μm T16Ainh-A01, 100 μm digallic acid, 30 μm CaCCinh-A01, and 100 μm tannic acid. E, whole cell TMEM16A current recorded at a holding potential at 0 mV and pulsing to voltages between ±100 mV (in steps of 20 mV) in the absence and presence of 10 μm T16Ainh-A01 (left) or 100 μm digallic acid (center). TMEM16A was stimulated by 275 nm free calcium in pipette solution. E, right, current/voltage (I/V) plot of mean currents at the middle of each voltage pulse.
FIGURE 4.
FIGURE 4.
TMEM16A inhibitors block CaCC chloride current in human salivary gland epithelial cells. A, left, immunoblot of TMEM16A protein in A253 salivary gland cells, T84 intestinal cells, and primary cultured human bronchial epithelial cells. Right, TMEM16A immunoblot at 24 h after incubation with 10 ng/ml IL-4 in human bronchial epithelial cells. Center, TMEM16A immunoblot at 48 h after the two transfections with nontargeting control siRNA and siRNA against TMEM16A in A253 cells. Representatives of three sets of studies are shown. B, whole cell patch clamp recordings of A253 cells. 10 μm T16Ainh-A01 (left) and 100 μm digallic acid (right) inhibition of TMEM16A chloride current induced by 275 nm free calcium in the pipette solution is shown. The bar graphs summarize current density data measured at +80 mV (mean ± S.E., n = 4).
FIGURE 5.
FIGURE 5.
TMEM16A inhibitors block the initial transient CaCC current in human intestinal epithelial cells following agonist stimulation. A, short circuit current measured in T84 cells. At 3 min after pretreatment with 10 μm T16Ainh-A01 (left) or 100 μm digallic acid (right), CaCC was activated by 100 μm ATP. Bar graphs summarize ATP-induced initial peak (#1, mean ± S.E., n = 7; *, p < 0.05). Remaining UTP-induced current was completely blocked by 100 μm tannic acid as indicated. CFTR was inhibited by pretreatment of 20 μm CFTRinh-172. B, left, short circuit current in T84 cells measured at 48 h after the two transfections with nontargeting control siRNA and siRNA against TMEM16A. Bar graph summarizes ATP-induced peak current (mean ± S.E., n = 4; *, p < 0.05). Right, immunoblot (IB) of TMEM16A in T84 cells transfected with control and TMEM16A siRNA (mean ± S.E., n = 3; *, p < 0.05). C, representative short circuit current data for pretreatment with 30 μm CaCCinh-A01 or 100 μm tannic acid. D, effect of 100 μm tannic acid, 30 μm CaCCinh-A01, 100 μm digallic acid, and 10 μm T16Ainh-A01 on CaCC activity in YFP-expressing HT29 cells (mean ± S.E., n = 4).
FIGURE 6.
FIGURE 6.
TMEM16A inhibitors poorly block CaCC chloride current in human bronchial epithelial cells. A, short circuit current measured in primary cultures of CF human bronchial epithelial cells. CaCC was activated by 100 μm UTP after 3-min pretreatment with 10 μm T16Ainh-A01 (left) or 100 μm digallic acid (right). Bar graphs summarize inhibition of UTP-induced initial peak current (0 min) and current at 5 min (mean ± S.E., n = 7–9). Remaining UTP-induced current was completely blocked by 100 μm tannic acid as indicated. Epithelial sodium channel was inhibited by 10 μm amiloride. B, short circuit current showing UTP-induced CaCC current in CF human bronchial epithelial cells with 10 μm T16Ainh-A01 added at four different time points (−2 min, 1 min, 3 min, 5 min) as indicated. Bar graph summarizes inhibition of CaCC at each time point (mean ± S.E., n = 7–9). C, apical chloride conductance measured after basolateral membrane permeabilization by nystatin (360 μg/ml) and with indicated apical and basolateral solution [Cl−]. Cells were pretreated with 10 μm T16Ainh-A01 (red curve) or 100 μm digallic acid (blue curve), and CaCC was activated by 100 μm UTP. Bar graph summarizes inhibition of UTP-induced initial peak current (mean ± S.E. (error bars), n = 3).
FIGURE 7.
FIGURE 7.
IL-4 induced CaCC up-regulation in CF human bronchial epithelial cells. A, short circuit current. IL-4 was applied at 10 ng/ml for 24 h. CaCC was activated by 100 μm UTP after 3-min pretreatment with 10 μm T16Ainh-A01. CF-HBE, CF human bronchial epithelial cells. B, short circuit current measured in Calu-3 cells. IL-4 was applied at 10 ng/ml for 24 h. CaCC was activated by 100 μm ATP after 3-min pretreatment with 10 μm T16Ainh-A01. CFTR was inhibited by pretreatment of 10 μm CFTRinh-172. C, bar graph summarizes inhibition of UTP-induced initial peak current (mean ± S.E. (error bars), n = 3–5; *, p < 0.05).
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