Molecular and Functional Characterization of Mucolipin-1

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The primary goal of this Mentored Career Development Award is to prepare the applicant for a career as an independent investigator in neurodegenerative and Ca(2+) signaling research. The applicant has shown a firm commitment to a biomedical research career and has acquired extensive prior training and expertise in cell and molecular biology, biochemistry, and gene targeting of lysosomal storage diseases, however, she believes that a period of additional training in the outlined imaging and electrophysiological techniques in the context of this proposed study will dramatically improve her competitiveness as an independent scientist. The applicant's environment is ideally suited to support her continued career development, with access to state-of-the-artcore facilities and on-site scientific experts. Her mentor, Dr. Muallem, is an expert in Ca(2+) signaling and in the regulation of ion channel functions and has had extensive experience in mentoring young investigators. The candidate's immediate goal is to elucidate the cellular functions of TRP-ML1 (ML1), and to determine how defects in this lysosomal membrane ion channel lead to mucolipidosis type IV (MLIV), a lipid storage neurodegenerative disorder. MLIV is defined at the cellular level by a lysosomal accumulation of lipids and by a block in the formation of mature lysosomes from late endosomes/hybrid organelles. The exact function of ML1 is unknown. In preliminary studies, we have shown that ML1 is a H+ selective cation channel that is specifically cleaved in the lysosomes. The cleaved form is predominantly seen in native cells and tissues and may function to regulate lipase activity. We now hypothesize that ML1 is a dual function protein that regulates lysosomal lipid metabolism by mediating alterations in lysosomal pH. In the absence of a functional ML1, lipid metabolism is aberrant. The following specific aims will test this hypothesis: 1) Characterize the molecular determinants of lysosomal targeting and proteolytic cleavage, 2) Identify which lipases are activated by ML1, 3) Characterize the interactions of ML1 with MLS and with other proteins that may function in a complex. Results from these studies will provide the cellular and therapeutic basis of this disease.