Abstract
Intracellular divalent cations control the molecular function of transmembrane protein 16 (TMEM16) family members. Both anion channels (such as TMEM16A) and phospholipid scramblases (such as TMEM16F) in this family are activated by intracellular Ca2+ in the low µM range. In addition, intracellular Ca2+ or Co2+ at mM concentrations have been shown to further potentiate the saturated Ca2+-activated current of TMEM16A. In this study, we found that all alkaline earth divalent cations in mM concentrations can generate similar potentiation effects in TMEM16A when applied intracellularly, and that manipulations thought to deplete membrane phospholipids weaken the effect. In comparison, mM concentrations of divalent cations minimally potentiate the current of TMEM16F but significantly change its cation/anion selectivity. We suggest that divalent cations may increase local concentrations of permeant ions via a change in pore electrostatic potential, possibly acting through phospholipid head groups in or near the pore. Monovalent cations appear to exert a similar effect, although with a much lower affinity. Our findings resolve controversies regarding the ion selectivity of TMEM16 proteins. The physiological role of this mechanism, however, remains elusive because of the nearly constant high cation concentrations in cytosols.
Highlights
The transmembrane protein 16 (TMEM16) family encompasses transmembrane proteins functioning as Ca2+ sensitive Cl- channels and phospholipid scramblases [1,2]
Recent structural-functional studies of TMEM16 molecules have revealed that the homodimeric architecture is conserved between anion channels and phospholipid scramblases, and helices 3–8 appear to form a conduit thought to be the pathway for ion and/or phospholipid transport [12,13,14,15,16,17]
These results demonstrate that the current of TMEM16A and the PNa /PCl ratio in TMEM16F are both modulated by membrane phospholipid manipulation
Summary
The TMEM16 family encompasses transmembrane proteins functioning as Ca2+ sensitive Cl- channels and phospholipid scramblases [1,2]. The same high concentrations of divalent cations had a minimal effect in potentiating the current of TMEM16F, but changed its PNa /PCl ratio, as reported by Ye et al [35]. We suggest that divalent and monovalent cations congregate around phospholipid head groups near or within the ion-transport pathway of TMEM16 proteins with low affinities, changing the electrostatic potential near the pore region. These observations are significant because they explain the controversy in the literature regarding the cation versus anion selectivity of TMEM16 molecules. Co2+ and cations, whereas Ipeak and ICo represent the peak current after the application of Co2+ and the quasi-steady-state current at the end of the Co2+ application, respectively
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