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Sci Rep
2016 Dec 08;6:38788. doi: 10.1038/srep38788.
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Intermolecular Interactions in the TMEM16A Dimer Controlling Channel Activity.
Scudieri P
,
Musante I
,
Gianotti A
,
Moran O
,
Galietta LJ
.
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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.
Figure 2. Altered membrane currents in TMEM16A proteins with chimeric C-termini.(A) Representative membrane currents in HEK-293 cells expressing wild type TMEM16A or C-TERM/C-TERM4 mutants. Each panel reports superimposed currents elicited at different membrane voltages (−100 to +100 mV range) and at the indicated intracellular free Ca2+ concentrations. (B) Current-voltage relationships summarizing the results of experiments as those shown in (A). Each point is the mean ± SEM of 9–12 experiments.
Figure 4. Channel activity in double chimeric TMEM16A proteins.(A) Results from HS-YFP assay carried out on HEK-293 cells expressing the indicated constructs. Gray bars: anion transport in the absence of intracellular Ca2+ elevation. White bars: anion transport after stimulation with ionomycin (1 μM). #p < 0.05 vs. C-TERM1 (n = 6–9 experiments). (B) Representative traces from HS-YFP experiments. (C) Representative whole-cell membrane currents and current-voltage relationships from 6–9 experiments with the indicated TMEM16A constructs. Intracellular free Ca2+ concentration was 7 or 245 nM.
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