Dataset Information

Studies on expression and function of the TMEM16A calcium-activated chloride channel.

ABSTRACT: Calcium-activated chloride channels (CaCC) with similar hallmark features are present in many cell types and mediate important physiological functions including epithelial secretion, sensory signal transduction, and smooth muscle contraction. Having identified TMEM16A of the transmembrane proteins with unknown function (TMEM) 16 family as a CaCC subunit, we have developed antibodies specific for mouse TMEM16A, as evidenced by the absence of immunoreactivity in TMEM16A knockout mice. Here, we show that TMEM16A is located in the apical membranes of epithelial cells in exocrine glands and trachea. In addition, TMEM16A is expressed in airway smooth muscle cells and the smooth muscle cells of reproductive tracts, the oviduct and ductus epididymis. In the gastrointestinal (GI) tract, TMEM16A is absent from smooth muscle cells, but present in the interstitial cells of Cajal (ICC), the pacemaker cells that control smooth muscle contraction. The physiological importance of TMEM16A is underscored by the diminished rhythmic contraction of gastric smooth muscle from TMEM16A knockout mice. The TMEM16A expression pattern established in this study thus provides a roadmap for the analyses of physiological functions of calcium-activated chloride channels that contain TMEM16A subunits.

SUBMITTER: Huang F

PROVIDER: S-EPMC2781737 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Studies on expression and function of the TMEM16A calcium-activated chloride channel.

Huang Fen F Rock Jason R JR Harfe Brian D BD Cheng Tong T Huang Xiaozhu X Jan Yuh Nung YN Jan Lily Yeh LY

Proceedings of the National Academy of Sciences of the United States of America 20091124 50

Calcium-activated chloride channels (CaCC) with similar hallmark features are present in many cell types and mediate important physiological functions including epithelial secretion, sensory signal transduction, and smooth muscle contraction. Having identified TMEM16A of the transmembrane proteins with unknown function (TMEM) 16 family as a CaCC subunit, we have developed antibodies specific for mouse TMEM16A, as evidenced by the absence of immunoreactivity in TMEM16A knockout mice. Here, we sho ...[more]

PMID: 19965375

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Project description:The transmembrane 16 (TMEM16) family contains 10 subtypes, and the function of each protein is different. TMEM16A is a calcium-activated chloride channel involved in physiological and pathological situations. Liquiritigenin is an aglycone derived from Glycyrrhiza glabra, and it is generated via the metabolism of enterobacterial flora. It has been known that liquiritigenin reduces pain sensation involving TMEM16A activation in primary sensory neurons. In addition, other pharmacological effects of liquiritigenin in physiological functions involving TMEM16A have been reported. However, the relationship between TMEM16A and liquiritigenin is still unknown. Therefore, we hypothesized that TMEM16A is inhibited by liquiritigenin. To confirm this hypothesis, we investigated the effect of liquiritigenin on TMEM16A currents evoked by intracellular free calcium in HEK293T cells transfected with TMEM16A. In this study, we found that liquiritigenin inhibited the mouse and human TMEM16A currents. To further confirm its selectivity, we also investigated its pharmacological effects on other ion channels, including transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), which are non-selective cation channels involved in pain sensation. However, liquiritigenin did not inhibit the currents of TRPV1 and TRPA1 induced by capsaicin and allyl isothiocyanate, respectively. Therefore, our findings indicate that selective TMEM16A inhibition could be one molecular mechanism that explains liquiritigenin-induced pain reduction. Additionally, we also investigated the inhibitory effects of estrogens on TMEM16A because liquiritigenin reportedly binds to the estrogen receptor. In this study, a pregnancy-dependent estrogen, estriol, significantly inhibited TMEM16A. However, the efficacy was weak. Although there is a possibility that TMEM16A activity could be suppressed during pregnancy, the physiological significance seems to be small. Thus, the inhibitory effect of estrogen might not be significant under physiological conditions. Furthermore, we investigated the effect of dihydrodaidzein, which is an analog of liquiritigenin that has a hydroxyphenyl at different carbon atom of pyranose. Dihydrodaidzein also inhibited mouse and human TMEM16A. However, the inhibitory effects were weaker than those of liquiritigenin. This suggests that the efficacy of TMEM16A antagonists depends on the hydroxyl group positions. Our finding of liquiritigenin-dependent TMEM16A inhibition could connect the current fragmented knowledge of the physiological and pathological mechanisms involving TMEM16A and liquiritigenin.

Project description:Niclosamide and benzbromarone have been described as inhibitors of the calcium activated chloride channel, TMEM16A, and on this basis have been considered and tested as clinical candidates for the treatment of airway diseases. However, both compounds have previously demonstrated activity on a range of additional biological targets and it is unclear from the literature to what extent any activity on TMEM16A may contribute to efficacy in these models of airway disease. The aim of the present study was therefore to examine the pharmacology and selectivity of these clinical candidates together with a structurally unrelated TMEM16A blocker, Ani9, in a range of functional assays to better appreciate the putative role of TMEM16A in the regulation of both epithelial ion transport and the development of an airway epithelial mucus secretory phenoptype. Benzbromarone and Ani9 both attenuated recombinant TMEM16A activity in patch clamp studies, whereas in contrast, niclosamide induced a paradoxical potentiation of the TMEM16A-mediated current. Niclosamide and benzbromarone were also demonstrated to attenuate receptor-dependent increases in intracellular Ca2+ levels ([Ca2+]i) which likely contributed to their concomitant attenuation of the Ca2+-stimulated short-circuit current responses of FRT-TMEM16A and primary human bronchial epithelial (HBE) cells. In contrast, Ani9 attenuated the Ca2+-stimulated short-circuit current responses of both cell systems without influencing [Ca2+]i which supports a true channel blocking mechanism for this compound. Additional studies using HBE cells revealed effects of both niclosamide and benzbromarone on global ion transport processes (absorptive and secretory) as well as signs of toxicity (elevated LDH levels, loss of transepithelial resistance) that were not shared by Ani9. Ani9 also failed to influence the IL-13 induced differentiation of HBE towards a goblet cell rich, mucus hypersecreting epithelium, whereas niclosamide and benzbromarone attenuated numbers of both goblet and multiciliated cells, that would be consistent with cellular toxicity. Together these data challenge the description of niclosamide as a TMEM16A blocker and illustrate a range of off-target effects of both niclosamide and benzbromarone which may contribute to the reported activity in models of airway function.

Dataset Information

Studies on expression and function of the TMEM16A calcium-activated chloride channel.

Publications

Studies on expression and function of the TMEM16A calcium-activated chloride channel.

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