Electrophysiological and computational analyses of anethole-induced inhibition of L-type calcium channels in rat tracheal smooth muscle.

Anethole, a naturally occurring phenylpropanoid, is a major constituent of several essential oils and exhibits a broad spectrum of pharmacological activities, including reduction of neuronal excitability, antispasmodic effects, and inhibition of Ca2+ influx. To elucidate the mechanism underlying calcium current inhibition, we investigated the effects of anethole on voltage-dependent calcium channels (VDCCs) in rat tracheal smooth muscle myocytes. Whole-cell patch-clamp recordings in dissociated tracheal myocytes were combined with molecular docking analyses. Ba2+ was used as the charge carrier because it generates larger and more stable inward currents through L-type VDCCs than Ca2+. At 0.67 mM (approximately the IC₅₀ for inhibition of tracheal contraction induced by 60 mM K+), anethole reduced the peak current density from -3.58 ± 0.39 to -1.53 ± 0.26 pA/pF (n = 18). In addition, anethole shifted the half-activation voltage (V1/2) from -6.46 to +1.41 mV and the half-inactivation voltage from -25.38 to -36.42 mV, indicating depolarizing and hyperpolarizing shifts in the steady-state activation and inactivation curves, respectively. Both effects reduced channel availability and favored inhibition of IBa. Computational analyses identified preferential interaction regions within the pore-forming and voltage-sensing domains of the channel, providing a plausible structural basis for both current inhibition and voltage-dependent modulation. Collectively, the electrophysiological and computational findings indicate that anethole inhibits VDCCs through complementary mechanisms involving pore interaction together with modulation of channel activation and inactivation. These effects occur at concentrations that also inhibit tracheal smooth muscle contraction, supporting the hypothesis that VDCC inhibition contributes to the antispasmodic action of anethole.