TMEM16A(a)/anoctamin-1 shares a homodimeric architecture with CLC chloride channels.
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.
Fallah
Ghada
G
Molecular Pharmacology, RWTH Aachen University, Aachen, Germany.
Römer
Thomas
T
Detro-Dassen
Silvia
S
Braam
Ursula
U
Markwardt
Fritz
F
Schmalzing
Günther
G
D000818:Animals; D000075369:Anoctamin-1; D018118:Chloride Channels / Q000737:chemistry*; D003432:Cross-Linking Reagents; D019281:Dimerization; D004594:Electrophysiology / Q000379:methods; D005091:Exons; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008957:Models, Genetic; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D020836:Protein Structure, Quaternary; D014982:Xenopus laevis
C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D018118:Chloride Channels; D003432:Cross-Linking Reagents