| Transient Receptor Potential (TRP) genes of the TRPML subfamily (TRPML1-3, MCOLN1-3) are presumed to encode ion channel proteins of intracellular endosomes and lysosomes. Mutations in human TRPML1 (mucolipin1/MCOLN1) result in Mucolipidosis Type IV, a severe inherited neurodegenerative disease associated with defective lysosomal biogenesis and trafficking. A mutation in mouse TRPML3 (A419P; TRPML3Va) results in the Varitint-waddler (Va) phenotype. Va mice are deaf, exhibit circling behavior due to vestibular defects, and have variegated/dilute coat color as a result of pigmentation defects. The Va mutation produces a gain-of-function that allows TRPML3, as well as TRPML1 and TRPML2 to be measured and identified as inwardly rectifying, proton-impermeant, Ca2+-permeant cation channels. Despite the progress of understanding the pathological mechanism underlying MLIV and the biological functions of TRPML1, the physiological roles of TRPML3 remain unknown. In this dissertation, I first present data showing that TRPML3 is highly expressed in normal melanocytes. Melanocyte markers are lost in the Va mouse, suggesting that their variegated and hypopigmented fur is caused by severe alteration of melanocyte function or cell death. TRPML3 Va expression in melanocyte cell lines results in rounded, poorly adherent cells. These results suggest a general role of TRPML channels in lysosomal Ca2+ signaling and that the Va phenotype is caused by a mutation-induced TRPML3 gain-of-function, resulting in cell death. Second, 1 show that wild-type TRPML3 is also highly expressed in epithelial cells in the kidney and in the intestine. The loss of TRPML3 protein or its channel activity in a polarized epithelial cell line causes the missorting of polarized membrane proteins, such as transferrin receptors. In zebrafish, the depletion of TRPML3 leads to a polycystic kidney and an abnormal intestinal tissue architecture. I hypothesize that these phenotypes in TRPML3-depleted zebrafish are caused by the dysregulation of membrane polarity in epithelial cells.;My dissertation shows that an endosomal TRP ion channel, TRPML3, regulates polarized membrane trafficking in epithelial cells, presumably by regulating Ca2+ release from endosomes to the cytoplasm during vesicle fusion/fission events. |
