Abstract
TMEM16F is activated by elevated intracellular Ca2+, and functions as a small-conductance ion channel and as a phospholipid scramblase. In contrast to its paralogs, the TMEM16A/B calcium-activated chloride channels, mouse TMEM16F has been reported as a cation-, anion-, or non-selective ion channel, without a definite conclusion. Starting with the Q559K mutant that shows no current rundown and less outward rectification in excised patch, we found that the channel shifted its ion selectivity in response to the change of intracellular Ca2+ concentration, with an increased permeability ratio of Cl- to Na+ (PCl-/PNa+) at a higher Ca2+ level. The gradual shift of relative ion permeability did not correlate with the channel activation state. Instead, it was indicative of an alteration of electrostatic field in the permeation pathway. The dynamic change of ion selectivity suggests a charge-screening mechanism for TMEM16F ion conduction, and it provides hints to further studies of TMEM16F physiological functions.
Highlights
Mammalian TMEM16F (Anoctamin-6, ANO6) is a membrane protein with dual functions of phospholipid scrambling and ion conduction, both activated by elevation of intracellular Ca2+ (Whitlock and Hartzell, 2017; Falzone et al, 2018; Bevers and Williamson, 2016; Pedemonte and Galietta, 2014)
Recording from inside-out membrane patch held at +80 mV revealed that wild-type mouse TMEM16F current was activated by intracellular Ca2+ in a dose-dependent manner
The change of the permeability ratio PNa+/PCl- with rising Ca2+ in this double mutant was similar to that of Q559K TMEM16F (Figure 4E,F). This double mutant revealed that in the presence of Q559K mutation, TMEM16F channel in 15 mM Ca2+ was modestly more permeable to Na+, but it became more permeable to Cl- in 1 mM Ca2+ regardless of TM6 stabilization. These results suggest that TMEM16F ion selectivity more likely depends on intracellular Ca2+ concentration directly rather than its open state(s), it remains possible that TMEM16F might undergo miniscule conformational changes at different Ca2+ concentrations that contribute to the transition of relative permeability
Summary
Mammalian TMEM16F (Anoctamin-6, ANO6) is a membrane protein with dual functions of phospholipid scrambling and ion conduction, both activated by elevation of intracellular Ca2+ (Whitlock and Hartzell, 2017; Falzone et al, 2018; Bevers and Williamson, 2016; Pedemonte and Galietta, 2014). Many groups have reported that the TMEM16F whole-cell current is activated several minutes after cytoplasmic Ca2+ elevation, and it displays less cation-selectivity (Yu et al, 2015) or even higher permeability to Cl- than to Na+ (Grubb et al, 2013; Scudieri et al, 2015; Shimizu et al, 2013; Tian et al, 2012). This disagreement hampers our further understanding of the functions of this membrane protein
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