Effects of Cation Binding to the Intracellular Vestibule of TMEM16 Ion Transport Pathways

Abstract

TMEM16A and TMEM16F are prototype molecules for the two functional categories of TMEM16 family members, respectively, Ca2+-activated chloride channels and phospholipid scramblases. Upon binding of sub-µM to low µM intracellular Ca2+, TMEM16A conducts anionic currents while TMEM16F is permeable to both cations and anions. In this study, we found another divalent cation, Co2+, while unable to induce current in TMEM16A, inhibits the Ca2+-induced current via competing with Ca2+ for the high-affinity (sub-µM to low µM) channel activation sites. In addition, Co2+ also potentiates the TMEM16A current induced by Ca2+. All alkaline earth divalent cations, including Ca2+ itself, can generate this current potentiation with low affinities—at least hundreds of µM to mM concentrations are required for observing the effect. On the other hand, divalent cations at mM concentrations generate very little potentiation in TMEM16F, likely because the changes in local anion and cation concentrations caused by divalent cation bindings cancel out each other. Nonetheless, divalent cations change the cation/anion selectivity for the TMEM16F current conduction. Manipulating membrane phospholipids alters this low-affinity effect of divalent cations. Recent studies have shown that membrane phospholipids participate in various gating and permeation functions of TMEM16 molecules. Our results suggest that intracellular divalent cations bind to membrane phospholipids with a low affinity to alter the electrostatic potential near or in the ion-transport pathways of TMEM16 molecules. Monovalent cations and polyamine molecules also exert similar effects, although with different apparent affinities. The physiological significance of the TMEM16 current modulation by altering membrane surface potential will be discussed.