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Nestor Mas Gomez, Wennan Lu, Jason C Lim, Alan M Laties, Kirill Kiselyov, Claire H Mitchell; Lysosomal stress impairs calcium signaling by TRPML in RPE cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5437.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Calcium signaling controls many responses in RPE cells. Lysosomes have recently been recognized as a main source of intracellular Ca2+, with Ca2+ released into the cytoplasm through TRPML channels. Here we examine the rise of cytoplasmic Ca2+ in response to TRPML activation, and ask whether lysosomal stress affects this Ca2+ signal.<br />
Methods: Lysosomal Ca2+ channel TRPML was activated by the agonist MLSA1 in mouse RPE cells and cultured human ARPE-19 cells. Ca2+ levels were determined with the dye fura-2 from individual cells using a microscope based system and from cells in 96-well plates using a fluorometer.
Results: The TRPML agonist MLSA1 raised cytoplasmic Ca2+ in mouse RPE cells and ARPE-19 cells. The rise in calcium was gradual and continuous with MLSA1 exposure up to 10 min, and slowly reversed upon removal of the drug. The rise in cytoplasmic Ca2+ in response to MLSA1 was unchanged by the removal of extracellular calcium or by the depletion of calcium in the endoplasmic reticulum by thapsigargin. Similar results were found in mouse RPE cells. Treatment of cells with U18666A for 24 hrs or chloroquine for 7 days reduced the response to MLSA1; when U1866A and chloroquine we given together, the rise in Ca2+ was largely prevented.
Conclusion: Activation of TRPML channels with MLSA1 elevated cytoplasmic Ca2+ in RPE cells, consistent with release from lysosomal stores. This response is reduced upon elevation of lysosome PH with chloroquine as lysosomal cholesterol with U18666A. Together these results suggest that lysosomal stress can interfere with Ca2+ signaling through TRPML channels in RPE cells.<br />
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