Abstract
TMEM16A and TMEM16B are plasma membrane proteins with Ca2+-dependent Cl− channel function. By replacing the carboxy-terminus of TMEM16A with the equivalent region of TMEM16B, we obtained channels with potentiation of channel activity. Progressive shortening of the chimeric region restricted the “activating domain” to a short sequence close to the last transmembrane domain and led to TMEM16A channels with high activity at very low intracellular Ca2+ concentrations. To elucidate the molecular mechanism underlying this effect, we carried out experiments based on double chimeras, Forster resonance energy transfer, and intermolecular cross-linking. We also modeled TMEM16A structure using the Nectria haematococca TMEM16 protein as template. Our results indicate that the enhanced activity in chimeric channels is due to altered interaction between the carboxy-terminus and the first intracellular loop in the TMEM16A homo-dimer. Mimicking this perturbation with a small molecule could be the basis for a pharmacological stimulation of TMEM16A-dependent Cl− transport.
Highlights
TMEM16A and TMEM16B, known as ANO1 and ANO2, are plasma membrane proteins with Cl− channel function[1,2,3]
The C-terminus of TMEM16A was totally or in part replaced by equivalent regions of TMEM16B and the activity was determined with the halide-sensitive yellow fluorescent protein (HS-YFP) assay[1,11]
Besides replacing segments of TMEM16A with those of TMEM16B, we truncated the C-terminus at different levels
Summary
TMEM16A and TMEM16B, known as ANO1 and ANO2, are plasma membrane proteins with Cl− channel function[1,2,3]. With more divergent sequence, could be responsible for the different biophysical properties To identify such regions, we previously generated chimerae in which domains of TMEM16A were replaced with equivalent portions of TMEM16B11. The constitutive activity is not an acquisition of a TMEM16B feature since this channel is even less Ca2+-sensitive than TMEM16A. Such findings suggest that the chimeric C-terminus generates a perturbation of TMEM16A structure that leads to pore opening. We restricted the critical region to a segment of 14 amino acids and found experimental and theoretical evidence that the C-terminus interacts with the first cytosolic loop of TMEM16A protein
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