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Anion and Cation Permeability of the Mouse TMEM16F Calcium-Activated Channel
Stefano Stabilini1, Anna Menini1, Simone Pifferi1,2
1Neurobiology Group, SISSA, International School for Advanced Studies, 34136 Trieste, Italy.
Abstract:
TMEM16F is involved in several physiological processes, such as blood coagulation, bone development and virus infections. This protein acts both as a Ca2+-dependent phospholipid scramblase and a Ca2+-activated ion channel but several studies have reported conflicting results about the ion selectivity of the TMEM16F-mediated current. Here, we have performed a detailed side-by-side comparison of the ion selectivity of TMEM16F using the whole-cell and inside-out excised patch configurations to directly compare the results. In inside-out configuration, Ca2+-dependent activation was fast and the TMEM16F-mediated current was activated in a few milliseconds, while in whole-cell recordings full activation required several minutes. We determined the relative permeability between Na+ and Cl¯ (PNa/PCl) using the dilution method in both configurations. The TMEM16F-mediated current was highly nonselective, but there were differences depending on the configuration of the recordings. In whole-cell recordings, PNa/PCl was approximately 0.5, indicating a slight preference for Cl¯ permeation. In contrast, in inside-out experiments the TMEM16F channel showed a higher permeability for Na+ with PNa/PCl reaching 3.7. Our results demonstrate that the time dependence of Ca2+ activation and the ion selectivity of TMEM16F depend on the recording configuration.
Insights
The transmembrane protein 16 family member F (TMEM16F) ion channel
Area of Science:
- Membrane biophysics
- Ion channel physiology
- Molecular biology
Background:
- Transmembrane protein 16 family member F (TMEM16F) is implicated in critical physiological functions, including blood coagulation, bone development, and viral infections.
- TMEM16F exhibits dual functionality as a Ca2+-dependent phospholipid scramblase and a Ca2+-activated ion channel.
- Previous research has yielded conflicting data regarding the ion selectivity of the TMEM16F-mediated current.
Purpose of the Study:
- To conduct a direct, side-by-side comparison of TMEM16F ion selectivity using whole-cell and inside-out excised patch configurations.
- To investigate the influence of recording configuration on TMEM16F activation kinetics and ion permeation properties.
Main Methods:
- Utilized whole-cell and inside-out patch-clamp electrophysiology to record TMEM16F-mediated currents.
- Employed the dilution method to determine the relative ion permeability (PNa/PCl) in both recording configurations.
- Analyzed Ca2+-dependent activation kinetics in different experimental setups.
Main Results:
- Observed significantly different Ca2+ activation kinetics between configurations: rapid (milliseconds) in inside-out versus slow (minutes) in whole-cell.
- TMEM16F exhibited high non-selectivity for ions, but with configuration-dependent differences in relative permeability (PNa/PCl).
- Whole-cell recordings showed PNa/PCl ≈ 0.5 (slight Cl− preference), while inside-out recordings revealed PNa/PCl ≈ 3.7 (significant Na+ preference).
Conclusions:
- The Ca2+ activation time course of TMEM16F is highly dependent on the electrophysiological recording configuration.
- TMEM16F ion selectivity is also influenced by the recording configuration, challenging previous assumptions.
- These findings highlight the importance of experimental conditions in characterizing TMEM16F channel properties and suggest potential mechanisms for its diverse physiological roles.
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