Identification of two natural coumarin enantiomers for selective inhibition of heat-activated TRPV2 channels

Thermosensitive transient receptor potential vanil oid 2(thermo TRPV2) is a nonselective Ca2+-permeable cation channel broadly expressed, and is implicated in the pathology of diseases such as diabetes and pancreatitis.However, the physiological and pharmacological functions of TRPV2 channels have not been extensively investigated because of the absence of specific modulators and the channel with high-threshold of heat activation. In this study, we report a pair of natural coumarin derivative enantiomers(+)-murraxocin(B304-1) and(+)-murraxocin(B304-2) from the root of Murrayaexoyica for their selective inhibition of TRPV2 channels expressed in HEK293 cells and native TRPV2 currents in differentiated brown adipocytes. Both B304-1 and B304-2 were identified from screening of TRPV2-overexpressing HEK-293 cells in calcium imaging and fluorescence assays. Wholecell patch clamp recordings confirmed the enantiomers B304-1 and B304-2 could selectively inhibit the agonist 2-APB-mediated activation of TRPV2 current with IC_(50) values of(34.2±7.8) μmol·L~(-1) and(3.7±0.7) μmol·L~(-1), respectively.Molecular docking and site-directed mutagenesis revealed a key residue I600 of TRPV2 is critical for the binding of the enantiomers. Furthermore, B304-1 and B304-2 significantly reversed TRPV2 agonist-induced inhibition of mouse brown adipocyte differentiation. Taken together,our identification of two natural coumarin enantiomers provides valuable tools and chemical leads for further elucidation of TRPV2 channel function, and pharmacological modulation of thermo TRPV2 in brown adipocytes may represent a new therapeutic strategy for treatment of energy imbalance or metabolic disorders such as diabetes and obesity.