BPS2026 – Arachidonic acid inhibition of TMEM16A channels

Transmembrane protein 16A (TMEM16A) is a calcium-activated chloride channel that regulates processes, such as vascular tone. Although its physiological importance is established, the mechanisms that regulate TMEM16A in vivo remain poorly understood. We have found that arachidonic acid (AA), a polyunsaturated fatty acid (PUFA), inhibits TMEM16A, but the mechanism of inhibition remains unclear. To investigate this, we applied fatty acids with different structural features to inside-out patches from Xenopus laevis oocytes. Application of 10 μM AA inhibited ∼50% of TMEM16A currents within 44 seconds ( N = 8), whereas the saturated fatty acid stearic acid (SA) had no effect ( N = 7). Across the fatty acids tested, at least one unsaturated bond was required for inhibition, with AA producing the fastest effect. Molecular dynamics simulations revealed AA and SA enrichment around TMEM16A compared to bulk, but a 10-fold increase in AA lipid density over SA at the permeation pore. Supporting our finding that AA and SA may compete for similar sites, co-application of the lipids significantly slowed AA-mediated inhibition of TMEM16A currents. This also supports our experimental findings that unsaturated bonds are crucial for channel inhibition. To assess physiological consequences, we tested whether AA altered two TMEM16A-dependent processes: arterial constriction and fertilization-induced depolarization in X. laevis eggs. Wire myography of mouse mesenteric arteries showed that AA inhibited α1-adrenergic-induced constriction in a dose-dependent manner, reducing contraction by 80% at 100 nM and completely abolishing it at higher concentrations. Whole-cell recordings of X. laevis eggs during fertilization showed that 1 μM AA prevented the TMEM16A-dependent depolarization that mediates the fast polyspermy block. Importantly, embryos still developed, indicating that AA blocked TMEM16A activation without preventing fertilization. Together, these findings establish AA as a direct inhibitor of TMEM16A, with broad physiological consequences.