Department of Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Department of Biological Science, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
FASEB J. 2018 Feb;32(2):782-794. doi: 10.1096/fj.201700220RR. Epub 2018 Jan 4.
The transient receptor potential cation channel mucolipin 1 (TRPML1) channel is a conduit for lysosomal calcium efflux, and channel activity may be affected by lysosomal contents. The lysosomes of retinal pigmented epithelial (RPE) cells are particularly susceptible to build-up of lysosomal waste products because they must degrade the outer segments phagocytosed daily from adjacent photoreceptors; incomplete degradation leads to accumulation of lipid waste in lysosomes. This study asks whether stimulation of TRPML1 can release lysosomal calcium in RPE cells and whether such release is affected by lysosomal accumulations. The TRPML agonist ML-SA1 raised cytoplasmic calcium levels in mouse RPE cells, hesRPE cells, and ARPE-19 cells; this increase was rapid, robust, reversible, and reproducible. The increase was not altered by extracellular calcium removal or by thapsigargin but was eliminated by lysosomal rupture with glycyl-l-phenylalanine-β-naphthylamide. Treatment with desipramine to inhibit acid sphingomyelinase or YM201636 to inhibit PIKfyve also reduced the cytoplasmic calcium increase triggered by ML-SA1, whereas RPE cells from TRPML1 mice showed no response to ML-SA1. Cotreatment with chloroquine and U18666A induced formation of neutral, autofluorescent lipid in RPE lysosomes and decreased lysosomal Ca release. Lysosomal Ca release was also impaired in RPE cells from the ATP-binding cassette, subfamily A, member 4 mouse model of Stargardt's retinal dystrophy. Neither TRPML1 mRNA nor total lysosomal calcium levels were altered in these models, suggesting a more direct effect on the channel. In summary, stimulation of TRPML1 elevates cytoplasmic calcium levels in RPE cells, but this response is reduced by lysosomal accumulation.-Gómez, N. M., Lu, W. Lim, J. C., Kiselyov, K., Campagno, K. E., Grishchuk, Y., Slaugenhaupt, S. A., Pfeffer, B., Fliesler, S. J., Mitchell, C. H. Robust lysosomal calcium signaling through channel TRPML1 is impaired by lysosomal lipid accumulation.
瞬时受体电位阳离子通道黏蛋白 1 (TRPML1) 通道是溶酶体钙外排的通道，通道活性可能受溶酶体内容物的影响。视网膜色素上皮 (RPE) 细胞的溶酶体特别容易积聚溶酶体废物，因为它们必须降解每天从相邻光感受器吞噬的外节；不完全降解会导致溶酶体中脂质废物的积累。本研究探讨了 TRPML1 的刺激是否可以释放 RPE 细胞中的溶酶体钙，以及这种释放是否受溶酶体积累的影响。TRPML 激动剂 ML-SA1 增加了小鼠 RPE 细胞、hesRPE 细胞和 ARPE-19 细胞的细胞质钙水平；这种增加是快速、强大、可逆和可重复的。细胞外钙去除或 thapsigargin 对其没有影响，但用甘氨酰-L-苯丙氨酸-β-萘基酰胺破坏溶酶体后则消除了增加。用去甲丙咪嗪抑制酸性鞘磷脂酶或 YM201636 抑制 PIKfyve 处理也减少了 ML-SA1 触发的细胞质钙增加，而 TRPML1 小鼠的 RPE 细胞对 ML-SA1 没有反应。氯喹和 U18666A 的共同处理诱导 RPE 溶酶体中中性、自发荧光脂质的形成，并减少溶酶体钙释放。Stargardt 视网膜营养不良的 ABCA4 小鼠模型的 RPE 细胞中，溶酶体钙释放也受损。这些模型中 TRPML1 mRNA 或总溶酶体钙水平均未改变，表明对通道有更直接的影响。总之，TRPML1 的刺激可提高 RPE 细胞的细胞质钙水平，但溶酶体积累会降低这种反应。
