Na+ and Ca2+ Channels in the Lysosome: Opening the Gate to the Cell's Recycling Center.

Abstract

Lysosomes primarily serve as the cell’s “garbage disposal and recycling center”, and are recently found to be involved in many important cellular functions. Lysosomes are also ion stores enriched with H+, Ca2+, and Na¬+. While it’s well known that the lysosomal ionic homeostasis is essential for its proper functions, the properties of ion transporters and channels residing on lysosomal membranes are barely understood, largely due to the lack of a reliable functional assay. Recently our lab has established a unique lysosomal patch-clamp method to directly record from native lysosomal membrane. Taking advantage of the technique, I discovered two novel lysosomal Na+-selective channels (Two-Pore-Channels TPC1 and TPC2), which are previously thought to be Ca2+ release channels, triggered by the second messenger NAADP. Using an integrative approach, I further demonstrated that TPCs are not activated by NAADP, but instead by PI(3,5)P2, a lysosome-specific phosphoinositide that regulates lysosomal ion homeostasis and membrane trafficking. TPCs represent the first intracellular Na+-selective channels, although their functions are not characterized. In addition, my colleagues and I found that PI(3,5)P2 also activates TRPML1, a principle lysosomal Ca2+ channel. Loss-of-function mutations in human TRPML1 cause type IV Mucolipidosis (ML4), a childhood neurodegenerative disease. My results showed that increasing TRPML1’s activity alleviated lysosomal trafficking defects in PI(3,5)P2-deficient cells, suggesting that PI(3,5)P2 controls Ca2+-dependent membrane trafficking by regulating TRPML1. To study the role of TRPML1 in membrane trafficking, I focused on the involvement of TRPML1 in Ca2+-dependent lysosomal exocytosis, a universal process important for many cellular functions, including cellular clearance, plasma membrane repair and phagocytosis. I found that gain-of-function mutations of TRPML1 caused a dramatic increase in lysosomal exocytosis. During particle uptake in macrophages, lysosomal exocytosis is required to provide membrane supplies to facilitate phagosome formation. By whole-cell recordings and newly developed whole-phagosome recordings, I found that upon particle binding, TRPML1-associated lysosomes are delivered to the newly-formed phagosomes via lysosomal exocytosis in a Ca2+-dependent manner. Overall, my thesis work has characterized two types of important channels (TPCs and TRPMLs) in the lysosome, identified their first endogenous activator PI(3,5)P2, and explored their functions in lysosomal biology.