Cellular Zinc Levels Are Modulated by TRPML1–TMEM163 Interaction

Abstract

Mucolipidosis type IV (MLIV) is caused by loss of function mutations in the TRPML1 ion channel. We previously reported that tissue zinc levels in MLIV were abnormally elevated; however, the mechanism behind this pathologic accumulation remains unknown. Here, we identify transmembrane (TMEM)-163 protein, a putative zinc transporter, as a novel interacting partner for TRPML1. Evidence from yeast two-hybrid, tissue expression pattern, co-immunoprecipitation, mass spectrometry, and confocal microscopy studies confirmed the physical association of TMEM163 with TRPML1. This interaction is disrupted when a part of TMEM163's N-terminus was deleted. Further studies to define the relevance of their interaction revealed that the plasma membrane (PM) levels of TMEM163 significantly decrease when TRPML1 is co-expressed in HEK-293 cells, while it mostly localizes within the PM when co-expressed with a mutant TRPML1 that distributes mostly in the PM. Meanwhile, co-expression of TMEM163 does not alter TRPML1 channel activity, but its expression levels in MLIV patient fibroblasts are reduced, which correlate with marked accumulation of zinc in lysosomes when these cells are acutely exposed to exogenous zinc (100 μM). When TMEM163 is knocked down or when TMEM163 and TRPML1 are co-knocked down in HEK-293 cells treated overnight with 100 nM zinc, the cells have significantly higher intracellular zinc levels than untreated control. Overall, these findings suggest that TMEM163 and TRPML1 proteins play a critical role in cellular zinc homeostasis, and thus possibly explain a novel mechanism for the pathological overload of zinc in MLIV disease.

PAX5 is the transcriptional activator of mucolipin-2 (MCOLN2) gene

“…Despite recent advances in the field, the cellular function of the TRPML proteins remains obscure. Nevertheless, there are indications that these proteins are involved in endosomal-lysosomal biogenesis, vesicle fusion, membrane trafficking, organelle acidification, autophagy, exocytosis, and metal homeostasis (LaPlante et al, 2006; Song et al, 2006; Karacsonyi et al, 2007; Dong et al, 2008; Miedel et al, 2008; Vergarajauregui et al, 2008; Martina et al, 2009; Eichelsdoerfer et al, 2010; Samie et al, 2013; Cuajungco et al, 2014). …”

ROS and intracellular ion channels

“…It is possible that this is an additional mechanism through which cells increase their transition metal clearance capacity in response to transition metal exposure and oxidative stress. Finally, it was proposed recently that TRPML1 may determine the correct localization of transition metal transporters and in the absence of which transition metals may accumulate to cause ROS build up [125]. …”

The mucolipin-1 TRPML1 ion channel transmembrane-163 TMEM163 protein and lysosomal zinc handling

“…As commonly known, such organelles include components of the Golgi network, mitochondria, and the “secretory vesicles.” The observations in neuronal cells, in breast tissue, and in certain model system points out that lysosomes are also involved in the Zn 2+ handling. Such evidence is based on Zn 2+ accumulation in the lysosomes of cells exposed to Zn 2+ , or in cells undergoing processes associated with large Zn 2+ transitions, such as cell death at the onset of mammary gland involution and pathological events (Eichelsdoerfer et al, 2010; Kelleher et al, 2011; Seo et al, 2011; McCormick and Kelleher, 2012; Kukic et al, 2013; Cuajungco et al, 2014; Kukic et al, 2014). The lysosomal involvement in Zn 2+ handling is interesting from several perspectives.…”

“…A buildup of Zn 2+ in the lysosomes of cells exposed to oxidative stress or high Zn 2+ levels to induce Zn 2+ uptake across the plasma membrane has been shown in various cell culture models using the high affinity dye, Fluozin-3 (Hwang et al, 2008; Chung et al, 2009; Lee et al, 2009; Eichelsdoerfer et al, 2010; Kukic et al, 2013; Cuajungco et al, 2014). Such buildup has led to lysosomal permeabilization followed by the release of the lysosomal digestive enzymes and cell death by the autophagic scenario.…”

“…Such a Zn 2+ overload then triggers cell death (Cuajungco and Lees, 1997; Choi and Koh, 1998; Frederickson et al, 2004). Interestingly, a significant increase in cerebral Zn 2+ levels of Mucolipin-1 knockout ( Mcoln1 −/− ) mice, a model for Mucolipidosis type IV (MLIV), has been reported (Eichelsdoerfer et al, 2010; Kukic et al, 2013; Cuajungco et al, 2014). It was surmised that lysosomal Zn 2+ overload could potentially contribute to MLIV pathology, as well as cause progressive neuronal and retinal cell degeneration (Eichelsdoerfer et al, 2010).…”