Abstract
Zinc is a vital trace element crucial for the proper function of some 3,000 cellular proteins. Specifically, zinc is essential for key physiological processes including nucleic acid metabolism, regulation of gene expression, signal transduction, cell division, immune- and nervous system functions, wound healing, and apoptosis. Consequently, impairment of zinc homeostasis disrupts key cellular functions resulting in various human pathologies. Mammalian zinc transport proceeds via two transporter families ZnT and ZIP. However, the detailed mechanism of action of ZnT2, which is responsible for vesicular zinc accumulation and zinc secretion into breast milk during lactation, is currently unknown. Moreover, although the putative coupling of zinc transport to the proton gradient in acidic vesicles has been suggested, it has not been conclusively established. Herein we modeled the mechanism of action of ZnT2 and demonstrated both computationally and experimentally, using functional zinc transport assays, that ZnT2 is indeed a proton-coupled zinc antiporter. Bafilomycin A1, a specific inhibitor of vacuolar-type proton ATPase (V-ATPase) which alkalizes acidic vesicles, abolished ZnT2-dependent zinc transport into intracellular vesicles. Moreover, using LysoTracker Red and Lyso-pHluorin, we further showed that upon transient ZnT2 overexpression in intracellular vesicles and addition of exogenous zinc, the vesicular pH underwent alkalization, presumably due to a proton-zinc antiport; this phenomenon was reversed in the presence of TPEN, a specific zinc chelator. Finally, based on computational energy calculations, we propose that ZnT2 functions as an antiporter with a stoichiometry of 2H+/Zn2+ ion. Hence, ZnT2 is a proton motive force-driven, electroneutral vesicular zinc exchanger, concentrating zinc in acidic vesicles on the expense of proton extrusion to the cytoplasm.
Highlights
Divalent zinc ions are key and integral components of a multitude of proteins involved in a plethora of essential physiological processes including metabolism of nucleic acids, regulation of gene expression, signal transduction, cell division, immune- and nervous-system functions, wound healing, as well as apoptosis [1,2,3,4]
ZnT2 functions as a proton-dependent antiporter exhibiting a 2:1 proton:zinc stoichiometry electrostatic and pKa calculations as well as zinc binding free-energy curves
Upon integration of our calculation results, we conclude that ZnT2 functions as an antiporter with a 2H+/Zn2+ stoichiometry, construct a Monte Carlo model to test this mode of ZnT2 transport activity, and validate our computational results experimentally using live human breast epithelial cells
Summary
Divalent zinc ions are key and integral components of a multitude of proteins involved in a plethora of essential physiological processes including metabolism of nucleic acids, regulation of gene expression, signal transduction, cell division, immune- and nervous-system functions, wound healing, as well as apoptosis [1,2,3,4]. Mammalian zinc transporters belong to two families, ZnT and ZIP, canonically exporting and importing zinc, respectively. ZnT2 plays a crucial role in concentrating zinc within secretory vesicles which were suggested to release zinc into breast milk during lactation [5]. Exclusively breastfed infants nursed by mothers harboring loss-of-function mutations in ZnT2 suffer from transient neonatal zinc deficiency (TNZD), which leads to severe zinc deficiency in these infants ZnT2 was shown to have a critical role in the development and normal function of the mouse mammary gland [13], as well as in involution [14,15]. Taken together, expanding our mechanistic understanding of ZnT2 function, will provide valuable information with important physiological and possible therapeutic implications
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