TMEM16A (tumor-amplified and overexpressed sequence 2) is an important component of CaCCs (Ca
2+ dependent Cl- channels). It is also known as Anoctamin-1 (ANO1) and is a protein that in humans is encoded by the ANO1 gene. Anoctamin-1 is thought to be responsible for a voltage-sensitive calcium-activated chloride current.
TMEM16A belongs to a family of ten mammalian paralogs (Tmem16 (a-h, j-k)) that are highly conserved membrane spanning proteins.
The gene is located on 11q13 region.
The primary structure of the TMEM16A protein has no similarity with other proteins having known function and, in particular, with other ion channels.
Examination of TMEM16A amino acid sequence with programs predicting structure and topology evidences at least eight putative transmembrane segments, with both NH2 and COOH termini protruding into the intracellular medium.
Because of the eight transmembrane segments and the anion selectivity, TMEM16A has been also named anoctamin-1 (ANO1).Based on mutagenesis experiments that result in altered ion selectivity, it has been proposed that the region between the fifth and the sixth transmembrane segment forms a reentrant loop that inserts into the plasma membrane and contributes to the formation of the channel pore.
Some tissues co-express multiple isoforms having variable levels of exons 6b or 15 skipping. Others show a preferential pattern of one isoform only. Interestingly, tissues appearing to preferentially skip exon 6b tend to include exon 15 and vice versa.
This coordinated pattern of splicing may suggest that segments b and d have mutually exclusive functional roles. In contrast, microexon 13 is always included, with a small degree of skipping in brain and skeletal muscle. The NH2 terminus of TMEM16A includes a region (segment a) that may be skipped when an alternative promoter is used. The resulting protein lacks the initial 116 amino acids. The transcript lacking segment a is also devoid of segments b, c, and d.
The corresponding isoform, called TMEM16A 0, is only 840 amino acids long compared with the longest one, TMEM16A (abcd), which has 1,008 amino acid residues.
Patch-clamp experiments have revealed that TMEM16A alternative splicing has a functional meaning. In particular, inclusion of segment b reduces the apparent affinity for Ca2+ of TMEM16A dependent channels. Accordingly, the Ca2+ sensitivity of isoforms TMEM16A (abc) and TMEM16 (ac) differ by nearly fourfold. On the other hand, the splicing of the four amino acids (Glu-Ala-Val-Lys) corresponding to segment c (exon13) alters the voltage dependence. p<>.
Recently two other important exon skipping events have been identified, exon 10 and exon 14, in mouse and human heart. Exon 10 encodes a hydrophobic region predicted to be the first transmembrane spanning domain of TMEM16A. Therefore, exon 10 exclusion would result in a change in the transmembrane topology. A PKC site is present in exon 14 at position S462 and one could speculate that TMEM16A transcripts lacking exon 14 may encode channels that differ in modulation by PKC.
Protein Aminoacids Percentage
By database analysis within the chromosome 11q13 region that is frequently amplified in various tumor types, Katoh and Katoh (FLJ10261 gene, located within the CCND1-EMS1 locus on human chromosome 11q13, encodes the eight-transmembrane protein homologous to C12orf3, C11orf25 and FLJ34272 gene products) identified a 5-prime truncated partial cDNA sequence derived from a previously uncharacterized cDNA clone, FLJ10261. Using in silico methods, they determined the complete coding sequence of the gene, TMEM16A, which encodes a deduced 986-amino acid protein. A shorter TMEM16A isoform results from alternative splicing. The human and mouse TMEM16A proteins share 89.9% sequence identity; both have 8 potential transmembrane regions with the N- and C-terminal tails facing the cytoplasm, 5 potential N-linked glycosylation sites, and a tyrosine phosphorylation site.
Subsequently Björn C. et al. (Expression cloning of TMEM16A as a calcium-activated chloride channel subunit, 2008) did an expression cloning of the Xenopus oocyte CaCC using oocytes from the physiologically polyspermic Axolotl (Ambystoma mexicanum) (Jego et al. Urodele egg jelly and fertilization, 1986) as expression system.
The xTMEM16A-induced current resembles the Xenopus oocyte CaCC in its anion selectivity, voltage dependence of calcium activation, and sensitivity to several chloride channel blockers. Moreover, mTMEM16A yielded CaCCs in the mammalian HEK293 cells and is broadly expressed in tissues known to contain native CaCCs.
TMEM16A/CaCC: Role in Epithelia
One of the major sites for CaCC expression and function is represented by epithelial cells. CaCCs constitute a route for Cl- secretion across the apical membrane of epithelial cells of the airways, intestine, and exocrine glands. Elevation of intracellular free Ca2+ concentration, triggered by paracrine and autocrine mechanisms, leads to transient CaCC activation. In many epithelial cells, intracellular Cl- is accumulated by the coordinated activity of basolateral channels and transporters above the electrochemical equilibrium. Therefore, activation of CaCCs generates an efflux of Cl- in the apical membrane that is followed by Na+ through the paracellular pathway. The net secretion of NaCl drives transepithelial water transport. In the airways, local activation of CaCCs, through autocrine release of ATP and binding to purinergic receptors, may be a mechanism to increase water supply and hence mucociliary clearance. In agreement with the important role of CaCCs in epithelial cells, expression of TMEM16A protein or mRNA has been demonstrated in the airway surface epithelium and in the acinar cells of pancreas, salivary glands, and bronchial submucosal glands.
TMEM16A/CaCC: Role in Smooth Muscle Cells
CaCCs have been repeatedly identified by functional studies in smooth muscle cells (SMCs), particularly in blood vessels. Their role is considered essential in the mechanism of signal amplification leading to cell contraction. Indeed, cytosolic free Ca2+ elevation by paracrine mechanisms triggers CaCC activation. The resulting Cl- efflux causes membrane depolarization, opening of voltage-dependent Ca2+ channels, and hence further Ca2+ elevation. As in epithelial cells, the CaCC-dependent depolarization depends on a relatively high intracellular Cl- concentration.
In the gastrointestinal tract, TMEM16A is strongly expressed in the interstitial cells of Cajal (ICCs), which represent pacemaker cells controlling the contraction of the smooth muscle layers. The importance of TMEM16A in gastrointestinal motility is demonstrated by studies on knockout mice. These animals are devoid of slow waves, the rhythmic changes in membrane potential controlling contraction.
TMEM16A/CaCC: Role in Nervous System and Sensory Receptors
CaCCs also control the excitability of various types of neurons including olfactory sensory neurons, somatosensory neurons, photoreceptors, and spinal cord neurons. The opening of CaCCs generates membrane potential depolarization or hyperpolarization depending on whether the Cl- equilibrium potential is more positive or more negative than the resting potential, respectively. Somatosensory neurons, whose bodies constitute the root dorsal ganglia (DRG), transduce different stimuli such as skin temperature, pain, and touch. A subpopulation of DRG neurons show CaCC activity, thus indicating that these channels are involved in the transduction of specific sensory pathways. Gene silencing experiments demonstrated that CaCC activity in small DRG neurons depends on TMEM16A expression. It has been shown that CaCC currents in olfactory neurons are mediated by TMEM16B, a close homolog of TMEM16A.
TMEM16A Protein: A New Identity for Ca2
+ Dependent Cl-Channels
One of the intriguing characteristics of TMEM16A is its overexpression in some human cancers such as gastrointestinal stromal tumors GISTs and head and neck squamous cell carcinomas. Because of this relationship, TMEM16A protein is also known as DOG1 (discovered on gastrointestinal stromal tumor 1), TAOS2 (tumor amplified and overexpressed sequence 2), and ORAOV2 (oral cancer overexpressed 2). The overexpression of TMEM16A may imply that it is important for cancer development and metastasis. However, other hypotheses are also possible.
For example, TMEM16A upregulation may be a consequence of amplification of the genomic region (11q13) containing other genes with more relevance to cancer such as cyclin D1 and FADD. Alternatively, high TMEM16A expression may be a feature of the cells from which the tumor derived. For example, GISTs probably originate from or have a progenitor in common with ICCs.
TMEM16A and other members of the same family may represent novel drug targets for the treatment of various human diseases such as hypertension, gastrointestinal motility disorders, asthma, cancer and cystic fibrosis.
In fact the alternative splicing of TMEM16A is an important mechanism to regulate Ca2+ dependent Cl− channel properties. This mechanism has important physiological consequences in controlling Cl− transport in various organs. Such different characteristics may actually be caused by the expression of various TMEM16A isoforms. It may be speculated that changes in the status of a cell, such as those associated with proliferation, differentiation, hormonal stimulation, or pathological processes, may cause a shift in the splicing pattern of TMEM16A, thus modifying the properties of CaCCs.
The role of alternative splicing on channel characteristics also has important implications for TMEM16A as a possible pharmacological target. The presence of tissue-specific isoforms may allow the design of drugs with a specific action on a given physiological process thus reducing the possibility of side effects.
Regulation of TMEM16A Chloride Channel Properties by Alternative Splicing
Increased complexity of Tmem16a/Anoctamin 1 transcript alternative splicing