Anoctamin/TMEM16 family members are Ca2+-activated Cl- channels

Abstract

Ca(2+)-activated Cl- channels (CaCCs) perform many important functions in cell physiology including secretion of fluids from acinar cells of secretory glands, amplification of olfactory transduction, regulation of cardiac and neuronal excitability, mediation of the fast block to polyspermy in amphibian oocytes, and regulation of vascular tone. Although a number of proteins have been proposed to be responsible for CaCC currents, the anoctamin family (ANO, also known as TMEM16) exhibits characteristics most similar to those expected for the classical CaCC. Interestingly, this family of proteins has previously attracted the interest of both developmental and cancer biologists. Some members of this family are up-regulated in a number of tumours and functional deficiency in others is linked to developmental defects.

Figure 1. Ca²⁺-activated Cl⁻ currents

Comparison of Ca²⁺-activated Cl⁻ currents induced by ANO1 transfected in HEK293 cells and in native IMCD cells. Recordings were performed as previously described (Qu et al. 2003a). The voltage clamp protocol is shown above A. A and B, currents from HEK-cells transfected with ANO1 cDNA (provided by Uhtaek Oh, Seoul National University) (A) and native IMCD cells (B). C and D, I–V curves of currents measured at the end of the test pulse in A and B, respectively.

Figure 2. Phylogeny of the ANO family

Human ANO1 was subjected at NCBI to position-specific iterated BLAST (2.2.18+) (http://www.ncbi.nlm.nih.gov/blast) (Altschul et al. 1990) with a threshold of 0.005 against all mammalian sequences in the reference proteins database (ref_seq_protein) through 6 iterations to find all of the mammalian members of the family. The tree was constructed using the fast minimal evolution method (Desper & Gascuel, 2004) using distances computed according to Grishin (1995).

Figure 3. Topology of ANO1.The human ANO1 protein containing all of its potential alternatively spliced exons (the ‘abcd’ form, 1008 amino acids, Caputo et al. 2008)

The ANO1 protein sequence was aligned with the ANO7 sequence and then ANO1 was mapped onto the topology of the ANO7 sequence determined by Das et al. (2008). Alternatively spliced segments a, b, c and d are shown with a chartreuse background. Single amino acid codes are in circles coloured according to the physical properties of the amino acid (green, hydrophilic; pale blue, hydrophobic; red, acidic; magenta, basic; cyan, other ionizable (tyrosine and histidine); yellow, proline; gold, glycine; pink, cysteine). Amino acids shown as filled circles have been mutated (Caputo et al. 2008; Yang et al. 2008): filled red amino acids have altered ionic permeability or gating, grey are wild-type, pink have altered sensitivity to MTSET. Glyco is a conserved N-linked glycosylation site. Purple: C404 in this sequence corresponds to C356 in ANO5 that is mutated to cause GDD. The green box shows the region including the re-entrant loop that is not conserved in ANO8 and ANO10.