The TMEM16 protein family: a new class of chloride channels?

Abstract

Cl(-) channels play important roles in many physiological processes, including transepithelial ion absorption and secretion, smooth and skeletal muscle contraction, neuronal excitability, sensory perception, and cell volume regulation. The molecular identity of many types of Cl(-) channels is still unknown. Recently, three research groups have arrived independently at the identification of TMEM16A (also known as anoctamin-1) as a membrane protein strongly related to the activity of Ca(2+)-activated Cl(-) channels (CaCCs). Site-specific mutagenesis of TMEM16A alters the properties of the channels, thus suggesting that TMEM16A forms, at least in part, the CaCC. TMEM16A is a member of a family that includes nine other membrane proteins. All TMEM16 proteins have a similar structure, with eight putative transmembrane domains and cytosolic amino- and carboxy-termini. TMEM16B expression also evokes the appearance of CaCCs, but with biophysical characteristics (voltage dependence, unitary conductance) different from those associated to TMEM16A. The roles of the other TMEM16 proteins are still unknown. The study of TMEM16 proteins may lead to identification of novel molecular mechanisms underlying ion transport and channel gating by voltage and Ca(2+).

Figure 1

Physiological roles of CaCCs. In epithelial cells, activation of CaCCs by intracellular Ca²⁺ elevation leads to Cl⁻ secretion followed by transepithelial transport of Na⁺ and water. In smooth muscle cells, activation of CaCCs is part of an amplification mechanism. Intracellular Ca²⁺ increase by extracellular stimuli activates CaCCs and Cl⁻ efflux. The resulting membrane depolarization opens voltage-dependent Ca²⁺ channels that cause a further intracellular Ca²⁺ increase, thus potentiating contraction. Another amplification mechanism occurs in olfactory receptors, where the initial Ca²⁺ increase is triggered by cAMP-gated channels. CaCC is also involved in phototransduction and regulation of neuronal excitability.

Figure 2

Identification of TMEM16A by functional genomics. Differentiated human bronchial epithelial cells respond to a 24-h IL-4 treatment with an upregulation of Ca²⁺-depedent Cl⁻ secretion, presumably by hyperexpression of the gene coding for CaCC. Global gene expression analysis was carried out by microarrays to identify CaCC candidates, i.e., genes upregulated by IL-4 and coding for putative membrane proteins with unknown function. Candidate genes were silenced with siRNA in epithelial cells with endogenous CaCC activity. Functional analysis by short-circuit current (Isc) recording across whole epithelia or by the whole-cell patch-clamp technique showed that silencing of TMEM16A caused reduction of CaCC activity.

Figure 3

Structure of TMEM16A protein. The protein has eight putative transmembrane domains and intracellular N- and C-termini. Various isoforms are generated by inclusion or skipping of four alternative segments labeled a (116 residues), b (22 residues), c (4 residues), and d (26 residues). The full protein, TMEM16A(abcd), containing all four segments, is 1008 amino acids long. The sequence of segments b, c, and d is shown. Colored circles show amino acids whose mutagenesis leads to alteration of ion channel properties (voltage dependence and/or ion selectivity). Blue circles, R563 and Q757 in the third and sixth transmembrane domains (17); red circles, R669, K693, and K716 (R621, K645, and K668 in Yang et al. (15)). According to results obtained for these three amino acids (15), the region between the fifth and sixth transmembrane domains may not be entirely extracellular, as shown in the figure, but may form a reentrant loop that constitutes part of the channel pore.