3.2 Lysine normalizes PQ-induced STIM1-TRPC1 association and intracellular calcium burden
The above observations suggested that a precise reduction of PQ-induced intracellular calcium burden but not calcium influx depletion might be beneficial for treating PQ-raised pulmonary fibrosis. Our previous work revealed that PQ targets STIM1, which facilitates the exposure of poly-K region in STIM1 and ultimately leads to an increase of intracellular calcium levels. Specifically, the STIM1-TRPC1 axis mediates PQ toxicity, from which the poly-K region in STIM1 and the D639&D640 residues in TRPC1 are mainly required for STIM1-TRPC1 interaction in response to PQ stimulation(Yang et al., 2022). We therefore hypothesized that addition of exogenous lysine would compete with the poly-K region for binding to D639&D640 residues and results in a blockage of PQ-raised STIM1-TRPC1 association. We then monitored whether lysine would affect the STIM1-TRPC1 association enhanced by PQ stimulation. As shown in Fig. 2A, PQ-increased STIM1-TRPC1 association was suppressed by lysine co-treatment in a time-dependent manner. Secondly, we found that PQ-induced NFAT luciferase production, a well-established downstream of calcium signaling, was also largely reduced by administration of lysine in a dose dependent manner, with a normalization to basal level when cells treated with high amount of lysine (Fig. 2B). Furthermore, we examined the effect of lysine on PQ-induced extracellular calcium entry and found that PQ-raised extracellular calcium influx was dramatically declined with lysine treatment in a dose dependent manner (Fig. 2C). As a result, PQ-induced intracellular calcium burden was significantly reduced with lysine treatment both in A549 cells and MLE-12 cells, two type of AT II cells (Fig. 2D&2E). Taken together, these results together suggest that lysine largely suppresses PQ-raised STIM1-TRPC1 association and thus intracellular calcium burden.