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.