PROJECT SUMMARY Lysosomes are small, membrane-bound organelles that participate in numerous critical processes including macromolecular degradation, secretion, membrane repair, signaling, nutrient sensing and cellular metabolism. Central to lysosomal function is its acidic lumen, which can reach pH values as low as 4.5. The low pH activates degradative enzymes that break down proteins, damaged organelles and other macromolecules into building blocks that can be recycled for cellular use. In neurons, defects in lysosomal function can lead to accumulation of potentially cytotoxic macromolecules such as ab, a-synuclein, tau and others. Consequently, lysosomal dysregulation is associated with numerous human diseases, including many neurodegenerative diseases. In this application, we propose experiments that will elucidate the molecular mechanisms of two lysosomal ion transport proteins, TMEM175 and CLC-7, whose mutation can lead to defects in lysosomal homeostasis and are associated with disease in humans and mice. TMEM175 is a lysosomal K+ channel that was identified in as a highly potent risk-factor for the development of Parkinson’s Disease. In cells, TMEM175 establishes a membrane potential between the lysosomal lumen and the cytosol and is critical for lysosomal and cellular homeostasis. Loss of TMEM175 leads to dysregulation of lysosomal pH, deficiencies in autophagy and mitophagy and an increased susceptibility to cytotoxic stress. Despite its importance in Parkinson’s Disease and cellular homeostasis, both the molecular details of TMEM175 function and its physiological roles are only starting to be understood. We recently determined cryo-EM structures of TMEM175 in open and closed states that demonstrated that TMEM175 is structurally unrelated to other K+ channels. Moreover, the structure confirmed that its gating, permeation and selectivity mechanisms are distinct from those characterized for other K+ channels. Through a combination of biophysical, biochemical and structural analyses, we will determine the permeation, gating and selectivity mechanisms underlying TMEM175 function and gain insights into how its mutation can lead to lysosome dysfunction and disease. CLC-7 is a member of the CLC family of Cl- channels and Cl-/H+ exchangers that requires a b-subunit, OSTM1, to function in lysosomes and the ruffled border of osteoclasts. CLC-7 is a lysosomal Cl-/H+ exchanger whose mutation can lead osteopetrosis, lysosomal storage disease and developmental delay. Notably, several of the mutations associated with associated with disease in humans result in changes in gating. While studies of prokaryotic and eukaryotic CLC proteins have established a framework for the transport cycles, the gating of CLCs is poorly understood at the molecular level. Using structural and electrophysiological approaches, we will elucidate mechanisms by which pH and ligands regulate the gating of CLC-7. These studies will serve as a foundation for better understandin...