Probing the Transmembrane Topology of TMEM16A(A)/Anoctamin-1 by Cysteine Scanning Mutagenesis

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TMEM16A(a)/Anoctamin-1 (Ano1) is a calcium-activated chloride channel. Using blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking, we have demonstrated that the recombinantly expressed mouse Ano1 (mAno1) assembles as an obligate homodimer in both Xenopus laevis oocytes and HEK293 cells (Fallah et al Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.004697, 1-10, 2011). Here we replaced individual residues by cysteines in the four putative mAno1 ectodomains, whose boundaries were based on the predictions of the MEMSAT3 topology program (Jones, Bioinformatics 23, 538-544. 2007). As a quantifiable readout we examined the accessibility of the introduced cysteine residue at the extracellular membrane surface of intact X. laevis oocytes for a thiol-reactive fluorescent maleimide dye. The mAno1 mutants were purified by Ni-NTA affinity chromatography, resolved by BN-PAGE and SDS-PAGE, and visualized by scanning of the metabolically incorporated [35S]methionine and the thiol-bound dye fluorescence. All the mutants examined appeared in the oocyte plasma membrane in the homodimeric state. Based on a total of 42 mAno1 cysteine mutants analyzed so far, the experimentally determined boundaries of the four mAno1 ectodomains (EC1-EC4) in terms of the residues positions are: 358-406 (EC1; predicted 359-402), 511-531 (EC2, predicted 513-527), 598-630 (EC3, predicted 598-707) and 791-848 (EC4, predicted 790-748). The major deviation pertains to EC3, which according to cysteine accessibility is much shorter, comprising residues 598-629 instead of residues 598-707 predicted by the MEMSAT3 program. Our data suggest that TM6 is represented by residues 630-650 (and not residues 708-732 predicted by MEMSAT3). In contrast, the predicted position of TM7 (residues 765-789) is in line with our experimental data. Our data confirm and extent earlier findings of Yu, Duran, Qu, Cui and Hartzell (Circ. Res. 110: 990-999, 2012).