Natural and synthetic flavonoid modulation of TRPC5 channels

Abstract

Background and PurposeThe TRPC5 proteins assemble to create calcium‐permeable, non‐selective, cationic channels. We sought novel modulators of these channels through studies of natural products.Experimental ApproachIntracellular calcium measurements and patch clamp recordings were made from cell lines. Compounds were generated by synthetic chemistry.Key ResultsThrough a screen of natural products used in traditional Chinese medicines, the flavonol galangin was identified as an inhibitor of lanthanide‐evoked calcium entry in TRPC5 overexpressing HEK 293 cells (IC50 0.45 μM). Galangin also inhibited lanthanide‐evoked TRPC5‐mediated current in whole‐cell and outside‐out patch recordings. In differentiated 3T3‐L1 cells, it inhibited constitutive and lanthanide‐evoked calcium entry through endogenous TRPC5‐containing channels. The related natural flavonols, kaempferol and quercetin were less potent inhibitors of TRPC5. Myricetin and luteolin lacked effect, and apigenin was a stimulator. Based on structure–activity relationship studies with natural and synthetic flavonols, we designed 3,5,7‐trihydroxy‐2‐(2‐bromophenyl)‐4H‐chromen‐4‐one (AM12), which inhibited lanthanide‐evoked TRPC5 activity with an IC50 of 0.28 μM. AM12 also inhibited TRPC5 activity evoked by the agonist (−)‐Englerin A and was effective in excised outside‐out membrane patches, suggesting a relatively direct effect. It inhibited TRPC4 channels similarly, but its inhibitory effect on TRPC1–TRPC5 heteromeric channels was weaker.Conclusions and ImplicationsThe data suggest that galangin (a natural product from the ginger family) is a TRPC5 inhibitor and that other natural and synthetic flavonoids contain antagonist or agonist capabilities at TRPC5 and closely related channels depending on the substitution patterns of both the chromone core and the phenyl ring.

Potent, selective, and subunit‐dependent activation of TRPC5 channels by a xanthine derivative

“…CHO cells stably expressing http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=510 were maintained in Ham's Nutrient Mixture F12 (ThermoFisher Scientific, Loughborough, UK) supplemented with 10% FBS, penicillin–streptomycin, and 1 mg·ml −1 G418 (InvivoGen, San Diego, California, USA). For experiments using hTRPC3 and hTRPC6, WT HEK 293 cells were transfected with hTRPC3 (Naylor et al, ) or hTRPC6 (cloned into pcDNA3) using jetPRIME® transfection reagent (VWR, Lutterworth, UK). Cells were assayed 48 hr after transfection.…”

“…The TRPC proteins are widely expressed in excitable and non-excitable cells, and TRPC channels are modulated by many physiological mechanisms and chemical and physical factors, including G-protein signalling, receptor tyrosine kinase signalling, temperature, heavy metal ions, lipids, and dietary compounds. This has led to the idea that the channels are complex integrators of multiple physiological and environmental stimuli (Bon & Beech, 2013;Clapham, 2003;Mederos y Schnitzler, Gudermann, & Storch, 2018;Naylor et al, 2016;Storch et al, 2017).…”

(−)-Englerin A-evoked Cytotoxicity Is Mediated by Na+ Influx and Counteracted by Na+/K+-ATPase

“…HEK T-REx TM -293 (HEK T-REx, Invitrogen) cell lines stably expressing human TRPC4β or human TRPC3 under a tetracycline-inducible promoter have been described previously (7, 22). An HEK T-REx cell line stably expressing a human TRPC4β-TRPC1 chimera was generated similarly and validated by Western blotting and biophysical analysis.…”

“…Human TRPC4 and TRPC1 were cloned upstream and downstream, respectively, of a four-amino acid linker (ASAS) flanked by AgeI and SacII restriction endonuclease sites that had previously been introduced into pcDNA TM 4/TO (22). TRPC4β, including an N-terminal Kozak sequence, was inserted between BamHI and AgeI restriction sites using hTRPC4β/pcDNA4/TO (7) as a PCR template (forward primer, 5′-AGTCGGATCCGCCACCATGGCTCAGTTCTATTACAAAAG-3′; reverse primer, 5′-AGTTACCGGTCAATCTTGTGGTCACGTAATCTTC-3′).…”

A Short Guide to Electrophysiology and Ion Channels

“…This mode is desirable when studying small channel populations or single channels and to manipulate the cell to various bathing solutions for rapid perfusion. In addition, this mode enables the study of extracellular ligandgated ion channels (13). The main disadvantage is that an outside-out configuration is more difficult to achieve as several steps are involved in the patching process.…”