Abstract
The TRPC5 proteins assemble to create calcium-permeable, non-selective, cationic channels. We sought novel modulators of these channels through studies of natural products. Intracellular calcium measurements and patch clamp recordings were made from cell lines. Compounds were generated by synthetic chemistry. Through 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 0.45μM). 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 0.28μM. 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. The 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.
Highlights
In mammals, twenty-eight genes encode transient receptor potential (TRP) proteins (Damann et al, 2008)
The transient receptor potential canonical 5 (TRPC5) current–voltage relationship (I-V) characteristically showed inward rectification at negative voltages and outward rectification at positive voltages with a plateau between 0 and +40 mV, which gave an approximate inverted S-shape and seat-like effect at positive voltages (Figure 1D). This signature I-V was suppressed by galangin, consistent with it acting as a TRPC5 channel inhibitor (Figure 1D)
Galangin was effective against TRPC5 current stimulated by S1P, suggesting that its effect was not restricted to inhibition of the Gd3+ effect (Figure 1e)
Summary
Twenty-eight genes encode transient receptor potential (TRP) proteins (Damann et al, 2008). There is a view that TRPC5 forms a receptor-activated channel and that this is its physiological purpose, but there is the view that it is activated by stress factors without the need for receptor activation (Birnbaumer, 2009; Jiang et al, 2011) Both may be true and, TRPC5 channels, like several other TRP channels, show what is variously described as versatility, promiscuity or multiplicity of activation (Birnbaumer, 2009; Jiang et al, 2011; Vriens et al, 2008; Zeng et al, 2004).
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