Introduction:
Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid (PUFA), exerts beneficial cardiovascular effects, including vasodilation and blood pressure reduction, although the underlying mechanisms remain incompletely understood. In vascular smooth muscle, TMEM16A chloride channels contribute to membrane depolarisation and contraction in response to vasoconstrictor stimuli. DHA has previously been reported to inhibit cloned TMEM16A channels REF[PT2] .
Aim:
To determine whether inhibition of TMEM16A contributes to the vasorelaxant actions of DHA and to investigate the underlying mechanisms.
Methods:
Whole-cell patch-clamp electrophysiology was used to assess the effects of DHA on TMEM16A currents in HEK293T cells expressing TMEM16A alone or together with α1-adrenoceptors or Danio rerio voltage-sensitive phosphatase (DrVSP). Vascular effects were examined by wire myography in mouse aortic and carotid artery rings. Intracellular Ca²⁺ responses were measured using GCaMP6-based calcium imaging.
Results:
DHA inhibited heterologously expressed TMEM16A currents with an IC₅₀ of 15.4 ± 1.5 µM and a Hill coefficient of 1.1 ± 0.1 (n = 10). In cells co-expressing TMEM16A and α1-adrenoceptors, DHA (70 µM) markedly suppressed phenylephrine-induced current activation, reducing current amplitude at +95 mV from 371.3 ± 66.9 (n = 7) to 37.3 ± 14.0 (n = 10; P < 0.005).
DHA inhibited TMEM16A with similar potency in cells expressing either inactive DrVSP(C302S) (IC₅₀ = 15.9 ± 3.9 µM, n = 7) or wild-type DrVSP (IC₅₀ = 16.7 ± 1.0 µM, n = 6), indicating that inhibition is independent of membrane PIP₂ depletion. Calcium imaging demonstrated that DHA did not alter phenylephrine-induced intracellular Ca²⁺ release, with peak fluorescence increases of approximately 2.5-fold (ΔF/F₀) observed under both control and DHA-treated conditions.
In carotid artery rings, DHA (70 µM) reduced U46619-induced contraction by ~40% (n = 10), whereas the selective TMEM16A inhibitor Ani9 (2 µM) reduced contraction by ~20% (n = 12). Combined treatment produced ~70% inhibition (n = 9; P < 0.005). In aortic rings, DHA reduced contraction by ~90% (n = 12; P < 0.005), whereas Ani9 alone produced only ~10% inhibition (n = 12). The combination of DHA and Ani9 was not more effective than DHA alone.
DHA also modulated additional ion channels involved in vascular excitability. At 70 µM, DHA inhibited CaV1.2 and Kv2.1 currents while enhancing KATP channel activity, effects expected to favour vasorelaxation.
Conclusions:
TMEM16A inhibition contributes significantly to the vasorelaxant actions of DHA but does not fully account for them. DHA acts through coordinated modulation of multiple ion channels, including TMEM16A, CaV1.2, Kv2.1 and KATP channels, with the relative contribution of each pathway varying between vascular beds. These findings identify TMEM16A as a novel vasorelaxant target and provide new mechanistic insight into the cardiovascular benefits of omega-3 PUFAs.