Transmembrane protein 16A (TMEM16A) is a Ca2+-regulated Cl- secretory channel in mouse airways

Abstract

For almost two decades, it has been postulated that calcium-activated Cl(-) channels (CaCCs) play a role in airway epithelial Cl(-) secretion, but until recently, the molecular identity of the airway CaCC(s) was unknown. Recent studies have unequivocally identified TMEM16A as a glandular epithelial CaCC. We have studied the airway bioelectrics of neonatal mice homozygous for a null allele of Tmem16a (Tmem16a(-/-)) to investigate the role of this channel in Cl(-) secretion in airway surface epithelium. When compared with wild-type tracheas, the Tmem16a(-/-) tracheas exhibited a >60% reduction in purinoceptor (UTP)-regulated CaCC activity. Other members of the Tmem16 gene family, including Tmem16f and Tmem16k, were also detected by reverse transcription-PCR in neonatal tracheal epithelium, suggesting that other family members could be considered as contributing to the small residual UTP response. TMEM16A, however, appeared to contribute little to unstimulated Cl(-) secretion, whereas studies with cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice and wild-type littermates revealed that unstimulated Cl(-) secretion reflected approximately 50% CFTR activity and approximately 50% non-Tmem16a activity. Interestingly, the tracheas of both the Tmem16a(-/-) and the CFTR(-/-) mice exhibited similar congenital cartilaginous defects that may reflect a common Cl(-) secretory defect mediated by the molecularly distinct Cl(-) channels. Importantly, the residual CaCC activity in Tmem16a(-/-) mice appeared inadequate for normal airway hydration because Tmem16a(-/-) tracheas exhibited significant, neonatal, lumenal mucus accumulation. Our data suggest that TMEM16A CaCC-mediated Cl(-) secretion appears to be necessary for normal airway surface liquid homeostasis.

FIGURE 1.

Bioelectric and expession patterns of TMEMs and CFTR neonatal tracheas. A, tracheal bioelectrics of WT (solid bars, n = 7), Tmem16a^+/– (hatched bars, n = 28), and Tmem16a^–/– pups (open bars, n = 7). Unstim. indicates the unstimulated I_sc before drug application. Then, amiloride (Amil) 10^–4 m was added apically, and the magnitude of the resulting change is shown. The residual I_sc is the I_sc remaining after amiloride application. Then, UTP (UTP 10^–4) was added apically followed by apical forskolin (Forsk)10^–5 m, and then bumetanide 10^–4 (Bumet) was added basolaterally. The UTP response differed (**, p ≤ 0.01) from the other two groups, *, p ≤ 0.05 versus ^+/+ and ^+/–. Data are means ± S.E. (error bars). B, expression of mRNA from TMEM isoforms in epithelium and mesenchyme isolated from trachea at postnatal day 1. Tracheae were separated into sections, RNA was isolated, and RT-PCR was performed as described under “Experimental Procedures.” Whole adult trachea and lung were used as positive controls for Tmem16 a, Tmem16f, Tmem16j, and Tmem16k. For Tmem16b, mouse eye and brain were used because Tmem16b was not detected in adult trachea or lung (not shown). Blank = water control; MW = molecular weight marker. Approximate sizes of products are given (in bp). C, tracheal bioelectrics of WT (solid bars, n = 29) and CF (open bars, n = 31) pups. Drug additions and symbols are the same as in panel A, with the exception that forskolin was added before UTP. **, p ≤ 0.01 from WT. Data are means ± S.E (error bars).

FIGURE 2.

Histology of WT and mutant tracheas (A, B, E, and F are stained with hematoxylin and eosin, and C and D are stained with AB-PAS). A, WT (littermate to B). In the normal trachea, the cartilage is continuous except along the posterior membrane (at the top of the figure), giving the normal trachea a rounded appearance (see also C and E). Due to the plane of section, the cartilage can appear discontinuous in the WT tissue (as in C). B, Tmem16a^–/– trachea exhibiting cartilaginous defects on the ventral (bottom of figure) side. Evaginations of the epithelial layer are evident, and the normal rounded appearance of the lumen is lost. C, WT trachea stained with AB-PAS. D, Tmem16a^–/– trachea stained with AB-PAS exhibiting significant lumenal mucus accumulation. E, trachea of WT (littermate to F). F, CF trachea exhibits a cartilaginous defect on the ventral side that results in discontinuous cartilaginous rings. Epithelial evaginations are also evident. All were photographed at same magnification; see scale in F.