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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.
Highlights
We performed a side-by-side comparison of the electrophysiological properties of the TMEM16F wt and Q559K mutant using both whole-cell and inside-out configurations with the same ionic solutions. We found that both the time dependence of Ca2+ activation and ion selectivity depend on the recording configuration
Ca2+ concentrations varying from 3.8 to 500 μM showed that the time necessary to activate the TMEM16F wt current significantly increased with lower intracellular Ca2+, ranging from about 2.5 min at 100 and 500 μM Ca2+ to about 20 min at 3.8 μM Ca2+ (Figure 1C)
Since it has been recently reported that PNa /PCl of TMEM16F wt in inside-out patches may depend on Ca2+ concentration [36], we investigated this possibility by reducing Ca2+
Summary
The TMEM16 ( named anoctamin or ANO) family is a heterogeneous functional group of ten transmembrane proteins [1,2,3,4]. TMEM16B (ANO2), encode for Ca2+ -activated Clchannels [5,6,7,8,9] and are involved in several relevant physiological functions, such as control of fluid secretion, modulation of smooth muscle cell excitability, regulation of neuronal signalling in various brain regions and in olfactory and vomeronasal sensory neurons [1,10,11,12,13,14]. TMEM16F (ANO6) and most of the other members of the TMEM16 family, with the exception of TMEM16H (ANO8), function as both Ca2+ -activated lipid scramblases and ion channels [15,16]
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