Stellera chamaejasme L. (Thymelaeaceae) is a toxic perennial herb and widespread in Mongolia and the northern parts of China. Previous studies have revealed that Neochamaejasmin A (NCA), one of the main active ingredients in the plant roots, has many bioactivities such as inhibiting the P-gp-mediated efflux. But whether NCA affects ion channels is unknown. Here the whole cell patch clamp technique was used to investigate whether NCA affects ion channels, especially how it inhibits KV1.4. Mutagenesis and structure-based molecular simulation were used for analysis of inhibition mechanism and identification of binding site. Among all the channels assayed, KV1.4 stood out as the one on which NCA showed strongest inhibition activity with IC50 of 7.55 µM. Compared with NCA's isomerides, neochamaejasmin B (NCB) and chamaechromone (CMC), NCA also exhibited superior inhibition ability on KV1.4. Three mutations, V549A, A553V and V560A, occurred inside the pore, were found to significantly alleviate the NCA blocking effects, suggesting that they are the important binding sites of NCA. Structure-based modelling showed that the phenolic hydroxyl group of NCA can form hydrogen bonds with main chains of Val549 and Ala553 in IS6 and IVS6 segment respectively, which support our in vitro results. In conclusion, data suggest that NCA might inhibit KV1.4 channels via direct binding to the pore domain.
Keywords: Inhibition; Ion channel; K(V)1.4; Neochamaejasmin A; Thymelaeaceae; Whole cell patch clamp.
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