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Mol Cell Proteomics
2011 Feb 01;102:M110.004697. doi: 10.1074/mcp.M110.004697.
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TMEM16A(a)/anoctamin-1 shares a homodimeric architecture with CLC chloride channels.
Fallah G
,
Römer T
,
Detro-Dassen S
,
Braam U
,
Markwardt F
,
Schmalzing G
.
???displayArticle.abstract??? TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca(2+)-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca(2+)-induced measured macroscopic Cl(-) currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl(-) channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca(2+)-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.
Fig. 1. Functional characterization of mTMEM16A(a) in X. laevis oocytes. A, Topological model of mTMEM16A, as drawn with TeXtopo (44). The indicated location of the transmembrane segments (TMI-TMVIII) was calculated with the topology prediction program MEMSAT3.0 (56). The C-terminal His tag (green) and the positions of the five luminally located N-glycosylation sequons (NXS/T) are also indicated. Human TMEM16A(a) isoforms are generated by the inclusion or omission of three alternative segments, b-d (14), of which the sequence of segment c (EAVK) was identified in the mouse genome following a BLAST search of DNA databases. B, Current responses were elicited in uninjected control oocytes and mTMEM16A(a)-expressing oocytes by the addition of 1 μm A23187 for the time indicated by the horizontal bar. The time-dependent outward conductances are the mean ± S.E. of recordings from five to six oocytes per data point. C, The bars represent the mean ± S.E. (5 to 17 oocytes per bar) of the maximum outward conductance from the A23187-elicited current response in oocytes expressing the indicated mTMEM16A(a) constructs, recorded as in B.
Fig. 2. Total and cell-surface expression of the Clâ-conducting channels mTMEM16A(a), hCLC-1, and hGlyR α1. A, B, The indicated [35S]-methionine- and Cy5-labeled proteins were purified by Ni-NTA chromatography and resolved with both reducing and nonreducing SDS-urea-PAGE. The incorporated [35S]-methionine (A) and Cy5 fluorescence (B) was visualized with phosphorimaging and Typhoon fluorescence scanning to display the total and cell-surface pools of the proteins, respectively. N and C in the figure legend indicate whether the His tag was N- or C-terminally positioned, respectively. C, D, Aliquots of the same protein samples used in A and B were deglycosylated with Endo H and PNGase F, as indicated, and were found to exist exclusively in the complex-glycosylated form in the plasma membrane (D). In contrast, the [35S]-methionine-labeled protein was partially Endo H-sensitive (C, red arrowheads).
Fig. 3. BN-PAGE analysis of the oligomeric structure of mTMEM16A(a). The indicated channel proteins were purified by nondenaturing Ni-NTA chromatography from digitonin extracts of X. laevis oocytes, resolved by BN-PAGE, and visualized in the [35S]-methionine-labeled total form or the Cy5-labeled plasma membrane-bound form with phosphorimaging (upper panels) or Typhoon fluorescence scanning (lower panels), respectively. Protein migration is shown both under native conditions and after partial denaturation following a 1-h incubation with SDS and/or DTT at 37 °C, as indicated. A, B, The yellow and blue ovals schematically illustrate the oligomeric states (protomers to pentamers) of the partially denatured hGlyR α1 and m5HT3R, respectively. C, D, The orange ovals schematically illustrate the native homodimeric and the denatured protomeric states of the hCLC-1 channel; the homodimeric structure of prokaryotic CLCs was verified by x-ray crystallography (54). The number in the right margin indicates the mass of the partially disassembled hGlyR α1, which resolved in the BN-PAGE gel as a ladder of proteins consisting of one to five 52-kDa protomers (cf. Figs. 3A and 3B, lane 1).
Fig. 4. Analysis of the oligomeric structure of mTMEM16A(a) following chemical cross-linking. mTMEM16A(a)-expressing X. laevis oocytes were metabolically labeled with [35S]-methionine overnight. After an additional 24-h chase, the outer oocyte surface was labeled with Cy5-NHS-ester. Immediately thereafter, the oocytes were homogenized in the presence of the indicated concentration of dimethyl adipimidate (DMA) or glutaraldehyde (GA). The proteins were purified by nondenaturing Ni-NTA chromatography and resolved in the fully SDS-denatured state with reducing SDS-urea-PAGE (A, B) and in the nondenatured and partially SDS-denatured state with BN-PAGE (C, D). The upper and lower panels present the [35S]-methionine-labeled total form and the Cy5-labeled plasma membrane-bound form of the polypeptides. Note that no protein could be isolated following treatment with 100 mm glutaraldehyde. The concatenated mTMEM16A(a) dimer was also analyzed. The orange and green double ovals indicate the position(s) of the concatenated mTMEM16A(a) homodimer following no treatment or treatment with 10 mm glutaraldehyde, respectively. Lanes 12â14 were taken at threefold higher image gain settings than the other lanes.
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