An outer-pore gate modulates the pharmacology of the TMEM16A channel

TMEM16A Ca²⁺-activated chloride channels are involved in multiple cellular functions and are proposed targets for diseases such as hypertension, stroke, and cystic fibrosis. This therapeutic endeavor, however, suffers from paucity of selective and potent modulators. Here, exploiting a synthetic small molecule with a biphasic effect on the TMEM16A channel, anthracene-9-carboxylic acid (A9C), we shed light on sites of the channel amenable for pharmacological intervention. Mutant channels with the intracellular gate constitutively open were generated. These channels were entirely insensitive to extracellular A9C when intracellular Ca²⁺ was omitted. However, when physiological Ca²⁺ levels were reestablished, the mutants regained sensitivity to A9C. Thus, intracellular Ca²⁺ is mandatory for the channel response to an extracellular modulator. The underlying mechanism is a conformational change in the outer pore that enables A9C to enter the pore to reach its binding site. The explanation of this structural rearrangement highlights a critical site for pharmacological intervention and reveals an aspect of Ca²⁺ gating in the TMEM16A channel.