Member Of TMEM16 Family Research Articles

TMEM16E regulates endothelial cell procoagulant activity and thrombosis.

Endothelial cells (ECs) normally form an anticoagulant surface under physiological conditions, but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid "scramblases," such as TMEM16F. TMEM16F-dependent PS externalization is well characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified 2 TMEM16 family members, TMEM16F and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall-dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.

Structural and functional investigations of CLCA proteins as potentiators of TMEM16 family members

The calcium-activated chloride channel regulator (CLCA) family of proteins have been suggested as drug targets for a number of diseases including asthma, COPD, cystic fibrosis, Crohn’s disease, infectious diarrhea, and some cancers. However, a detailed understanding of their biological functions has lagged far behind their association with diseases. CLCA proteins were originally presumed to be chloride channels but work by ourselves and others have demonstrated them to be secreted regulators that act on endogenous channels.

Effects of Cation Binding to the Intracellular Vestibule of TMEM16 Ion Transport Pathways

TMEM16A and TMEM16F are prototype molecules for the two functional categories of TMEM16 family members, respectively, Ca2+-activated chloride channels and phospholipid scramblases. Upon binding of sub-µM to low µM intracellular Ca2+, TMEM16A conducts anionic currents while TMEM16F is permeable to both cations and anions. In this study, we found another divalent cation, Co2+, while unable to induce current in TMEM16A, inhibits the Ca2+-induced current via competing with Ca2+ for the high-affinity (sub-µM to low µM) channel activation sites. In addition, Co2+ also potentiates the TMEM16A current induced by Ca2+. All alkaline earth divalent cations, including Ca2+ itself, can generate this current potentiation with low affinities—at least hundreds of µM to mM concentrations are required for observing the effect. On the other hand, divalent cations at mM concentrations generate very little potentiation in TMEM16F, likely because the changes in local anion and cation concentrations caused by divalent cation bindings cancel out each other. Nonetheless, divalent cations change the cation/anion selectivity for the TMEM16F current conduction. Manipulating membrane phospholipids alters this low-affinity effect of divalent cations. Recent studies have shown that membrane phospholipids participate in various gating and permeation functions of TMEM16 molecules. Our results suggest that intracellular divalent cations bind to membrane phospholipids with a low affinity to alter the electrostatic potential near or in the ion-transport pathways of TMEM16 molecules. Monovalent cations and polyamine molecules also exert similar effects, although with different apparent affinities. The physiological significance of the TMEM16 current modulation by altering membrane surface potential will be discussed.

Comparison of ion transport determinants between a TMEM16 chloride channel and phospholipid scramblase.

Two TMEM16 family members, TMEM16A and TMEM16F, have different ion transport properties. Upon activation by intracellular Ca2+, TMEM16A-a Ca2+-activated Cl- channel-is more selective for anions than cations, whereas TMEM16F-a phospholipid scramblase-appears to transport both cations and anions. Under saturating Ca2+ conditions, the current-voltage (I-V) relationships of these two proteins also differ; the I-V curve of TMEM16A is linear, while that of TMEM16F is outwardly rectifying. We previously found that mutating a positively charged lysine residue (K584) in the ion transport pathway to glutamine converted the linear I-V curve of TMEM16A to an outwardly rectifying curve. Interestingly, the corresponding residue in the outwardly rectifying TMEM16F is also a glutamine (Q559). Here, we examine the ion transport functions of TMEM16 molecules and compare the roles of K584 of TMEM16A and Q559 of TMEM16F in controlling the rectification of their respective I-V curves. We find that rectification of TMEM16A is regulated electrostatically by the side-chain charge on the residue at position 584, whereas the charge on residue 559 in TMEM16F has little effect. Unexpectedly, mutation of Q559 to aromatic amino acid residues significantly alters outward rectification in TMEM16F. These same mutants show reduced Ca2+-induced current rundown (or desensitization) compared with wild-type TMEM16F. A mutant that removes the rundown of TMEM16F could facilitate the study of ion transport mechanisms in this phospholipid scramblase in the same way that a CLC-0 mutant in which inactivation (or closure of the slow gate) is suppressed was used in our previous studies.

Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase.

The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several TMEM16 family members have been identified as Ca2+-activated scramblases, but the mechanisms underlying their Ca2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here, we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs. These structures reveal that Ca2+-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. These structures, together with functional experiments, suggest that activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement.

Preparation of Proteoliposomes with Purified TMEM16 Protein for Accurate Measures of Lipid Scramblase Activity.

The distribution of different lipid species between the two leaflets is tightly regulated and underlies the concerted action of distinct catalytic entities. While flippases and floppases establish membrane asymmetry, scramblases randomize the lipid distribution and play pivotal roles during blood clotting, apoptosis, and in processes such as N-linked glycosylation of proteins. The recent discovery of TMEM16 family members acting as scramblases has led to an increasing demand for developing protocols tailored for TMEM16 proteins to enable functional investigations of their scrambling activity. Here we describe a protocol for the expression, purification, and functional reconstitution of TMEM16 proteins into preformed liposomes and measurement of their scrambling activity using fluorescence-labeled lipid derivatives. The reconstitution involves extrusion of liposomes through a membrane, destabilization of liposomes using Triton X-100, and stepwise detergent removal by adsorption on styryl-beads. The scrambling assay is based on the selective bleaching of nitrobenzoxadiazol fluorescent lipids on the outer leaflet of liposomes by the membrane-impermeant reducing agent sodium dithionite. The assay allows conclusions on the substrate specificity and on the kinetics of the transported lipids as shown with the example of a Ca2+-activated TMEM16 scramblase from the fungus Nectria haematococca (nhTMEM16).

An ancestral TMEM16 homolog from Dictyostelium discoideum forms a scramblase.

TMEM16 proteins are a recently identified protein family comprising Ca2+-activated Cl- channels that generate outwardly rectifying ionic currents in response to intracellular Ca2+ elevations. Some TMEM16 family members, such as TMEM16F/ANO6 are also essential for Ca2+-dependent phospholipid scrambling. TMEM16-like genes are present in the genomes of most eukaryotic species, the function(s) of TMEM16 family members from evolutionary ancient eukaryotes is not completely clear. Here, we provide insight into the evolution of these TMEM16 proteins by similarity searches for ancestral sequences. All eukaryotic genomes contain TMEM16 homologs, but only vertebrates have the full repertoire of ten distinct subtypes. TMEM16 homologs studied so far belong to the opisthokont branch of the phylogenetic tree, which includes the animal and fungal kingdoms. An organism outside this group is Dictyostelium discoideum, a representative of the amoebozoa group that diverged from the metazoa before fungi. We here functionally investigated the TMEM16 family member from Dictyostelium discoideum. When recombinantly expressed in HEK293 cells, DdTMEM16 induces phospholipid scrambling. However, in several electrophysiological experiments we did not find evidence for a Ca2+-activated Cl- channel function of DdTMEM16.

Mathematical Models of Protein Induced Membrane Deformation

I will describe recent advances we have made in using fast, continuum elasticity theory to describe membrane deformations around proteins. I will show that our calculations match the deformations predicted from all atom molecular dynamics simulations for two proteins: gramicidin (a small antibiotic ion channel) and nhTMEM16 (a member of the calcium activated chloride channel family). Our calculations reveal that nhTMEM16 produces large distortions in the membrane potentially related to its ability to scramble lipids from one leaflet to the other. This hypothesis is supported by atomistic simulations in which we observe lipids flipping from one leaflet to the other. Experiments to test the lipid flipping mechanism in a mammalian TMEM16 family member will also be discussed.

Characterization of the scrambling domain of the TMEM16 family

The TMEM16 protein family has 10 members, each of which carries 10 transmembrane segments. TMEM16A and 16B are Ca2+-activated Cl- channels. Several other members, including TMEM16F, promote phospholipid scrambling between the inner and outer leaflets of a cell membrane in response to intracellular Ca2+ However, the mechanism by which TMEM16 proteins translocate phospholipids in plasma membranes remains elusive. Here we show that Ca2+-activated, TMEM16F-supported phospholipid scrambling proceeds at 4 °C. Similar to TMEM16F and 16E, seven TMEM16 family members were found to carry a domain (SCRD; scrambling domain) spanning the fourth and fifth transmembrane segments that conferred scrambling ability to TMEM16A. By introducing point mutations into TMEM16F, we found that a lysine in the fourth transmembrane segment of the SCRD as well as an arginine in the third and a glutamic acid in the sixth transmembrane segment were important for exposing phosphatidylserine from the inner to the outer leaflet. However, their role in internalizing phospholipids was limited. Our results suggest that TMEM16 provides a cleft containing hydrophilic "stepping stones" for the outward translocation of phospholipids.

New Insights on the Regulation of Ca2+‐Activated Chloride Channel TMEM16A

TMEM16A, also known as anoctamin 1, is a recently identified Ca2+ -activated chloride channel and the first member of a 10-member TMEM16 family. TMEM16A dysfunction is implicated in many diseases such as cancer, hypertension, and cystic fibrosis. TMEM16A channels are well known to be dually regulated by voltage and Ca2+ . In addition, recent studies have revealed that TMEM16A channels are regulated by many molecules such as calmodulin, protons, cholesterol, and phosphoinositides, and a diverse range of stimuli such as thermal and mechanical stimuli. A better understanding of the regulatory mechanisms of TMEM16A is important to understand its physiological and pathological role. Recently, the crystal structure of a TMEM16 family member from the fungus Nectria haematococcaten (nhTMEM16) is discovered, and provides valuable information for studying the structure and function of TMEM16A. In this review, we discuss the structure and function of TMEM16A channels based on the crystal structure of nhTMEM16A and focus on the regulatory mechanisms of TMEM16A channels. J. Cell. Physiol. 232: 707-716, 2017. © 2016 Wiley Periodicals, Inc.

Functional Characterization of Calcium-activated Phospholipid Scramblase Activity of nhTMEM16

Asymmetric lipid distribution between the two leaflets of bilayer is a physiologically important feature of biological membranes. Dissipation of lipid asymmetry by lipid scrambling is a ubiquitous cellular mechanism essential for membrane biogenesis, cell signaling, and bacterial cell wall assembly. The recently crystallized structure of a TMEM16 family member nhTMEM16 provided the first structural insight into the mechanism of lipid scramblases, which facilitate the passive bidirectional transport of diverse lipids between the two leaflets of the membrane. It identified a conserved Ca2+-binding site harbored in a hydrophilic crevice predicted to face the hydrophobic core of the membrane and involve in lipid scrambling, although no lipids were resolved in the structure. In order to investigate the Ca2+-mediated scrambling mechanism, identify the lipid transport pathway, and characterize the role of Ca2+, we have performed extended equilibrium molecular dynamics (MD) simulations with nhTMEM16 embedded in various lipid bilayers (POPC, POPE, POPS, and mixtures thereof) in the presence or absence of Ca2+. Our simulations reveal a previously unidentified spiral membrane-traversing hydrophilic track around the protein surface, connecting the inner and outer leaflets. Especially in the Ca2+-bound systems, lipids from the inner and outer leaflets spontaneously bind to the hydrophilic track with their headgroups and propagate toward the center of the membrane. In the Ca2+-free systems, on the other hand, the hydrophilic track is observed to substantially narrow allowing only smaller species such as water and ions to bind. Furthermore, umbrella sampling (US) simulations are used to characterize the complete lipid transport pathway and to identify key residues and energetics of the lipid translocation. Our study provides crucial atomic details of the scrambling pathway for the first time, and uncovers the nature of Ca2+-dependence by revealing the structural rearrangements of the hydrophilic track for lipid translocation.

Activation of the Ca2+-Activated Chloride Channel TMEM16A

TMEM16A is a Ca2+-activated chloride channel that is involved in various physiological processes. The functional behavior of the murine ion channel mTMEM16A has been characterized by electrophysiology. mTMEM16A forms anion-selective channels that are activated by Ca2+-binding from the intracellular side. Channel activation by Ca2+ was shown to be voltage-dependent with an increase of the EC50 at negative potentials. Although studies have identified residues contributing to Ca2+ binding, a mechanistic understanding of channel activation and the relationship to the voltage-dependence remained unknown.To address this question, we have determined the structure of nhTMEM16, a fungal member of TMEM16 family, which functions as lipid scramblase. Due to the close relationships within the family, however, this protein also provides a valuable framework to investigate structure-function relationships in mTMEM16A. The structure of nhTMEM16 reveals a highly conserved Ca2+-binding site within each subunit of the dimeric protein. In this site, up to two Ca2+ ions are coordinated by six residues, five of which carry a negative charge. The location of the site within the transmembrane region provides an explanation for the voltage-dependence of Ca2+ activation observed in TMEM16A. To probe the importance of the corresponding residues, single mutants of TMEM16A were expressed in HEK293T cells and studied by excised patch electrophysiology. The Ca2+ dose-response relationships of all mutants show a shift to higher Ca2+ concentrations, indicating that the interaction of these residues with Ca2+ is key to channel activation. The structure also hints at a region in the protein, termed the ‘subunit cavity’, as a potential site of ion permeation. The proximity of the ‘cavity’ to the Ca2+-binding site and the presence of residues previously shown to affect ion selectivity of the channel, suggests this region as the likely candidate for the ion conduction path in TMEM16 channels.

X-ray structure of a calcium-activated TMEM16 lipid scramblase.

The TMEM16 family of proteins, also known as anoctamins, features a remarkable functional diversity. This family contains the long sought-after Ca(2+)-activated chloride channels as well as lipid scramblases and cation channels. Here we present the crystal structure of a TMEM16 family member from the fungus Nectria haematococca that operates as a Ca(2+)-activated lipid scramblase. Each subunit of the homodimeric protein contains ten transmembrane helices and a hydrophilic membrane-traversing cavity that is exposed to the lipid bilayer as a potential site of catalysis. This cavity harbours a conserved Ca(2+)-binding site located within the hydrophobic core of the membrane. Mutations of residues involved in Ca(2+) coordination affect both lipid scrambling in N. haematococca TMEM16 and ion conduction in the Cl(-) channel TMEM16A. The structure reveals the general architecture of the family and its mode of Ca(2+) activation. It also provides insight into potential scrambling mechanisms and serves as a framework to unravel the conduction of ions in certain TMEM16 proteins.

Functional Swapping between Transmembrane Proteins TMEM16A and TMEM16F

The transmembrane proteins TMEM16A and -16F each carry eight transmembrane regions with cytoplasmic N and C termini. TMEM16A carries out Ca(2+)-dependent Cl(-) ion transport, and TMEM16F is responsible for Ca(2+)-dependent phospholipid scrambling. Here we established assay systems for the Ca(2+)-dependent Cl(-) channel activity using 293T cells and for the phospholipid scramblase activity using TMEM16F(-/-) immortalized fetal thymocytes. Chemical cross-linking analysis showed that TMEM16A and -16F form homodimers in both 293T cells and immortalized fetal thymocytes. Successive deletion from the N or C terminus of both proteins and the swapping of regions between TMEM16A and -16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F) and C-terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and -16F mutants with point mutations in the pore region (located between the fifth and sixth transmembrane regions) indicated that the pore region is essential for both the Cl(-) channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited the Cl(-) channel activity of TMEM16A and the scramblase activity of TMEM16F with an opposite preference. These results indicate that TMEM16A and -16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains significantly differ.

Subdued, a TMEM16 family Ca2+-activated Cl− channel in Drosophila melanogaster with an unexpected role in host defense

TMEM16A and TMEM16B are calcium-activated chloride channels (CaCCs) with important functions in mammalian physiology. Whether distant relatives of the vertebrate TMEM16 families also form CaCCs is an intriguing open question. Here we report that a TMEM16 family member from Drosophila melanogaster, Subdued (CG16718), is a CaCC. Amino acid substitutions of Subdued alter the ion selectivity and kinetic properties of the CaCC channels heterologously expressed in HEK 293T cells. This Drosophila channel displays characteristics of classic CaCCs, thereby providing evidence for evolutionarily conserved biophysical properties in the TMEM16 family. Additionally, we show that knockout flies lacking subdued gene activity more readily succumb to death caused by ingesting the pathogenic bacteria Serratia marcescens, suggesting that subdued has novel functions in Drosophila host defense. DOI: http://dx.doi.org/10.7554/eLife.00862.001.

Calcium-dependent Phospholipid Scramblase Activity of TMEM16 Protein Family Members

TMEM16A and 16B work as Cl(-) channel, whereas 16F works as phospholipid scramblase. The function of other TMEM16 members is unknown. Using TMEM16F(-/-) cells, TMEM16C, 16D, 16F, 16G, and 16J were shown to be lipid scramblases. Some TMEM16 members are divided into two Cl(-) channels and five lipid scramblases. Learning the biochemical function ofTMEM16family members is essential to understand their physiological role. Asymmetrical distribution of phospholipids between the inner and outer plasma membrane leaflets is disrupted in various biological processes. We recently identified TMEM16F, an eight-transmembrane protein, as a Ca(2+)-dependent phospholipid scramblase that exposes phosphatidylserine (PS) to the cell surface. In this study, we established a mouse lymphocyte cell line with a floxed allele in the TMEM16F gene. When TMEM16F was deleted, these cells failed to expose PS in response to Ca(2+) ionophore, but PS exposure was elicited by Fas ligand treatment. We expressed other TMEM16 proteins in the TMEM16F(-/-) cells and found that not only TMEM16F, but also 16C, 16D, 16G, and 16J work as lipid scramblases with different preference to lipid substrates. On the other hand, a patch clamp analysis in 293T cells indicated that TMEM16A and 16B, but not other family members, acted as Ca(2+)-dependent Cl(-) channels. These results indicated that among 10 TMEM16 family members, 7 members could be divided into two subfamilies, Ca(2+)-dependent Cl(-) channels (16A and 16B) and Ca(2+)-dependent lipid scramblases (16C, 16D, 16F, 16G, and 16J).

Ca2+-Dependent Ion and Lipid Transport Mediated by a Fungal TMEM16 Homologue

Ca2+-activated Cl- channels (CaCCs) play crucial roles in human physiology, from epithelial secretion to nociception and sensory transduction. Recent work showed that two members of the TMEM16 family, TMEM16A and B, encode for CaCCs. The TMEM16 family is comprised of 10 human homologues whose malfunction has been implicated in several human diseases. Despite their physiological relevance the function of the other TMEM16s is unclear and controversial. For example, TMEM16F has been reported to be a Ca2+-dependent cation channel, three different Cl- channels and to be involved in lipid scrambling (lipid transport between membrane leaflets). These data raise the possibility that not all TMEM16 proteins are CaCCs and that some might be scramblases or regulators of scrambling activity.To differentiate between these hypotheses we expressed, purified and reconstituted several TMEM16 family members and discovered that a fungal homologue simultaneously mediates ion movement and lipid scrambling. Reconstitution in planar lipid bilayers shows a non-selective ion channel of high conductance, ∼300 pS. Both transport functions are tightly regulated by Ca2+: in its absence ion channel activity is abolished and lipid scrambling is severely diminished. The apparent Km of Ca2+ for transport is ∼400 nM, a value comparable to that of other TMEM16s. We mutated a highly conserved di-acidic motif previously shown to be important for Ca2+-regulation in TMEM16A and F. Simultaneous charge-neutralization of these residues eliminates Ca2+ dependent activation of ion and lipid transport, suggesting that a single Ca2+ site regulates both.Our results demonstrate for the first time that a member of the TMEM16 family is simultaneously a Ca2+ dependent ion channel and a Ca2+ dependent lipid scramblase. This suggests that other family members, such as TMEM16F, might also be dual function proteins thus resolving the confusion regarding their function.

Expression patterns of the Tmem16 gene family during cephalic development in the mouse

Tmem16a, Tmem16c, Tmem16f, Tmem16h and Tmem16k belong to the newly identified Tmem16 gene family encoding eight-pass transmembrane proteins. We have analyzed the expression patterns of these genes during mouse cephalic development. In the central nervous system, Tmem16a transcripts were abundant in the ventricular neuroepithelium, whereas the other Tmem16 family members were readily detectable in the subventricular zone and differentiating fields. In the rostral spinal cord, Tmem16f expression was highest in the motor neuron area. In the developing eye, the highest amounts of Tmem16a transcripts were detected in the lens epithelium, hyaloid plexus and outer layer of the retina, while the other family members were abundant in the retinal ganglionic cell layer. Interestingly, throughout development, Tmem16a expression in the inner ear was robust and restricted to a subset of cells within the epithelium, which at later stages formed the organ of Corti. The stria vascularis was particularly rich in Tmem16a and Tmem16f mRNA. Other sites of Tmem16 expression included cranial nerve and dorsal root ganglia, meningeal precursors and the pituitary. Tmem16c and Tmem16f transcripts were also patent in the submandibular autonomic ganglia. A conspicuous feature of Tmem16a was its expression along the walls of blood vessels as well as in cells surrounding the trigeminal and olfactory nerve axons. In organs developing through epithelial–mesenchymal interactions, such as the palate, tooth and tongue, the above five Tmem16 family members showed interesting dynamic expression patterns as development proceeded. Finally and remarkably, osteoblasts and chondrocytes were particularly loaded with Tmem16a, Tmem16c and Tmem16f transcripts.

Expression Cloning of TMEM16A as a Calcium-Activated Chloride Channel Subunit

Calcium-activated chloride channels (CaCCs) are major regulators of sensory transduction, epithelial secretion, and smooth muscle contraction. Other crucial roles of CaCCs include action potential generation in Characean algae and prevention of polyspermia in frog egg membrane. None of the known molecular candidates share properties characteristic of most CaCCs in native cells. Using Axolotl oocytes as an expression system, we have identified TMEM16A as the Xenopus oocyte CaCC. The TMEM16 family of "transmembrane proteins with unknown function" is conserved among eukaryotes, with family members linked to tracheomalacia (mouse TMEM16A), gnathodiaphyseal dysplasia (human TMEM16E), aberrant X segregation (a Drosophila TMEM16 family member), and increased sodium tolerance (yeast TMEM16). Moreover, mouse TMEM16A and TMEM16B yield CaCCs in Axolotl oocytes and mammalian HEK293 cells and recapitulate the broad CaCC expression. The identification of this new family of ion channels may help the development of CaCC modulators for treating diseases including hypertension and cystic fibrosis.

The transmembrane protein TMEM16A is required for normal development of the murine trachea

Pathological collapsibility of the upper airways, caused by many different genetic and environmental insults, is known as tracheomalacia in humans. We determined that Tmem16a, a member of an evolutionarily conserved family of predicted transmembrane proteins, is expressed in the developing trachea. We report that all mice homozygous for a null allele of Tmem16a died within one month of birth and exhibited severe tracheomalacia with gaps in the tracheal cartilage rings along the entire length of the trachea. In addition, the development of the trachealis muscle that spans the dorsal aspect of the trachea was abnormal in Tmem16a mutants. Since the chondrogenic mesenchyme does not express Tmem16a at any time, we propose that the cartilage ring defect observed in Tmem16a mutants is secondary to an expansion of the embryonic trachea that might result from improper stratification of the embryonic tracheal epithelium or the abnormal trachealis muscle. Our data identify Tmem16a as a novel regulator of epithelial and smooth muscle cell organization in murine development. This mutant, the first knockout of a vertebrate TMEM16 family member, provides a mouse model of tracheomalacia.