Distinct modulation of calcium-activated chloride channel TMEM16A by drug-binding sites

TMEM16A is a calcium-activated chloride channel with significant role in epithelial fluid secretion, sensory transduction, and smooth muscle contraction. Several TMEM16A inhibitors have been identified; however, their binding sites and inhibitory mechanisms remain unclear. Using magnolol and honokiol, the two regioisomeric inhibitors, as chemical probes, we have identified a drug-binding site distinct from the pore region, in TMEM16A, which is described here. With electrophysiology, unbiased molecular docking and clustering, molecular dynamics simulations, and experimental validation with mutant cycle analysis, we show that magnolol and honokiol utilize different drug-binding sites, pore and nonpore pockets. The pore blocker utilizes amino acids crucial for chloride passage, whereas the nonpore blocker allosterically modulates the pore residues to hinder ion permeation. Among 17 inhibitors tested, 11 were pore blockers and 6 were nonpore blockers, indicating the importance of this nonpore pocket. Our study provides insights into drug-binding mechanism in TMEM16A together with a rationale for future drug development.