Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

Abstract

TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative porelining basic residues significantly altered the IC 50 of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore.calcium-activated channels | chloride channels | channel pharmacology | TMEM16A | ion channel biophysics

Atomistic insight into lipid translocation by a TMEM16 scramblase

“…Thus, we believe that oppositely charged residues at the extracellular ends of TM3 and TM6 are important for lipid interactions in nearly all family members, but it does not matter in which helix the basic and acidic residues reside. The arginine in TMEM16A (R511), which makes up one-half of the SE site, was previously shown to be critical for ion selectivity and pore blocker sensitivity (41). It was deduced that R511 forms part of the binding site for the pore blocker 1PBC (PubChem SID 49642647).…”

Characterization of the scrambling domain of the TMEM16 family

“…The region where the Se site is localized is well-conserved between a Cl − -channel TMEM16A and phospholipid-scramblase TMEM16F. The arginine residue in the third transmembrane segment of TMEM16A was shown to be critical for the Cl − -channel activity of mouse TMEM16A (27), indicating that the Cl − ion as well as PtdSer transports via the Se site. Thus, the ability of TMEM16 proteins to translocate phospholipids or Cl − seems to be determined by the Sc site or the SCRD.…”

Cryo-EM structures of the TMEM16A calcium-activated chloride channel

“…These observations are in agreement with mutagenesis studies implicating G640 as a flexible hinge that affects channel gating 13 . Additionally, the two structures differ in the orientation of TM3 and in the arrangement of the TM5-TM6 loop that is near the pore entrance and harbors K599 and R617 important for anion selectivity 25 , and the TM9-TM10 loop that harbors R784 important for anion selectivity 25 and packs against the TM5-TM6 loop (Extended Data Fig. 7g–l).…”

“…3a–c). Besides R511 on TM3 and K599 on the TM5-TM6 loop important for anion selectivity 25 , and K584 on TM5 that partially accounts for the selectivity for anions over cations 28 (Fig. 3b), we tested alanine substitutions of 24 other pore-lining residues including N542 and D550 on TM4, N587 and V595 on TM5, Q705 and F712 on TM7, and S635 on TM6 (Fig.…”

“…10). Unlike previous efforts searching among positively charged residues for those involved in anion permeation 12,25,28 , we tested residues facing water-filled potential pore structures to delineate the permeation pathway based on the involvement of pore-lining residues in anion selectivity. For ion permeation and channel gating, we classify pore-lining residues into two groups: one group is distributed along the entire pore and is important for anion selectivity, and the other group is clustered together and is important for gating.…”