EFFECT OF CA2+ AND PH ON THE CATION CHANNEL ACTIVITY OF MUCOLIPIN-1 (TRP-ML1)

MONTALBETTI, N; TIMPANARO, G. A.; CANTERO, M. R.; CANTIELLO, H. F

Montevideo

Congreso; 6th Internacional Conference of Biological Physics, 5th Southern Cone Biophysical Society, 34th Annual Meeting of the Argentinean Biophysical Society; 2007

Mucolipidosis type IV (MLIV) is an autosomal recessive neurogenetic disorder characterized by developmental abnormalities of the brain and impaired neurological, ophthalmologic and gastric functions. MLIV is typified by accumulation of lipids and membranous materials in intracellular organelles, likely caused by altered membrane fusion in the late endosomal and lysosomal pathways of target cells. MLIV is caused by mutations in the gene, MCOLN1, encoding mucolipin-1 (ML1), a 65 kDa protein member of the TRP superfamily of cation channels (TRP-ML1). We recently established that ML1 is a Ca2+-permeable cation channel that is transiently modulated by changes in intracellular Ca2+. In this study, the role of pH on Ca2+ transport and regulation of ML1 was assessed by ion channel reconstitution. The data showed that ML1 is a multiple sub-conductance, non-selective cation channel. MLIV-causing mutations resulted in functional differences in the channel protein. In particular, the V446L and DeltaF408 mutations retained channel function but responded differently with regards to pH dependence and Ca2+ transport. While the wild-type protein was inhibited by Ca2+ transport, mutant ML1 was not. We conducted a kinetic analysis of spontaneous and pH-induced single channel current changes in ML1 and determined that the residence time of the sub-conductances states of the channel can be modified by lowering cytoplasmic pH, and changing voltage. These changes affected Ca2+ transport by ML1, providing further indication of regulation. We also used atomic force microscopy to image wild type and mutant ML1, and observed that changes in pH modify the aggregation and size of the ML1 channel complexes, which also has an impact on vesicular fusogenesis. Mutant-ML1 did not change in size on reduction of pH. This evidence provides further support for a novel role of ML1 cation channels in vesicular acidification and normal endosomal function, based on regulation of ML1 channel properties.