Lysosomal stress impairs calcium signaling by TRPML in RPE cells

Abstract

Purpose: 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: 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: 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.

Conclusions: 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 />