Ion permeation in TMEM16A and TMEM16F
TMEM16A and TMEM16F belong to the transmembrane protein 16 family. TMEM16A functions as a Ca2+-activated Cl− channel, whereas TMEM16F functions as a phospholipid scramblase that also conducts ionic current. The anion-selective TMEM16A has a linear current-voltage relationship, whereas the non-selective TMEM16F has an outwardly rectified current-voltage relationship. I used electrophysiological and biomolecular techniques to study the molecular basis of ion permeation in TMEM16A and TMEM16F. Manipulating the charge of the pore-lining K584 in TMEM16A alters the channel’s current-voltage relationship, demonstrating an electrostatic effect. The current-voltage relationship of TMEM16A becomes more outwardly rectified as the charge of this residue becomes more negatively charged. Mutating the analogous residue in TMEM16F, Q599, also alters the current-voltage relationship of TMEM16F. However, there is no correlation between the charge of this residue and the current-voltage relationship of TMEM16F. Interestingly, aromatic mutants of Q559 exhibit a more linear current-voltage relationship and is more resistant to current rundown than the wild-type protein. Since the current rundown in TMEM16A and TMEM16F has been attributed to the depletion of phosphatidylinositol 4,5-bisphosphate from the cell membrane, and the ion permeation (or the phospholipids transport) pathway of TMEM16 proteins is partially exposed to membrane phospholipids, I speculated that mutating Q559 in TMEM16F influences ion permeation by altering the conformation of phospholipids nearby. Therefore, there is a possibility that divalent cations binding to phospholipids near the intracellular vestibule of TMEM16A and TMEM16F may affect ion permeation in these proteins. Indeed, mM concentration of divalent cations potentiate TMEM16A current and reduce TMEM16F selectivity for Na+ over Cl−. Tens of mM concentrations of monovalent cations also affect the interaction between phospholipids and divalent cations. Lowering the concentration of NaCl enhances the potentiation of TMEM16A by divalent cations and enhances the ability of divalent cations to alter the ion selectivity of TMEM16F. The sensitivity of ion selectivity to ionic conditions explains the controversial relative ion permeability of TMEM16F reported in the literature. Also, sequestering negatively charged phospholipids with poly-L-lysine alters the interaction between divalent cations and these two proteins, consistent with the idea that divalent cations can interact with membrane phospholipids.