Manganese is a trace element that is an essential co-factor in many enzymes critical to diverse biological pathways. However, excess Mn2+ leads to neurotoxicity, with psychiatric and motor dysfunction resembling parkinsonism. The liver is the main organ for Mn2+ detoxification by excretion into bile. Although many pathways of cellular Mn2+ uptake have been established, efflux mechanisms remain essentially undefined. In this study, we evaluated a potential role in Mn2+ detoxification by the Secretory Pathway Ca2+, Mn2+-ATPase in rat liver and a liver-derived cell model WIF-B that polarizes to distinct bile canalicular and sinusoidal domains in culture. Of two known isoforms, only secretory pathway Ca2+-ATPase isoform 1 (SPCA1) was expressed in liver and WIF-B cells. As previously observed in non-polarized cells, SPCA1 showed overlapping distribution with TGN38, consistent with Golgi/TGN localization. However, a prominent novel localization of SPCA1 to an endosomal population close to, but not on the basolateral membrane was also observed. This was confirmed by fractionation of rat liver homogenates which revealed dual distribution of SPCA1 to the Golgi/TGN and a fraction that included the early endosomal marker, EEA1. We suggest that this novel pool of endosomes may serve to sequester Mn2+ as it enters from the sinusoidal/basolateral domains. Isoform-specific partial knockdown of SPCA1 delayed cell growth and formation of canalicular domain by about 30% and diminished viability upon exposure to Mn2+. Conversely, overexpression of SPCA1 in HEK 293T cells conferred tolerance to Mn2+ toxicity. Taken together, our findings suggest a role for SPCA1 in Mn2+ detoxification in liver.
Fig. S1Selective role of SPCA1 in Mn2+ detoxification. (A) RT-PCR (left and middle panel) and Western blot (right panel) showing redundant expression and specific knockdown of SPCA1 and SPCA2 in HEK 293T cells. SPCA2 expression was evaluated by Western blot analysis of microsomes. (B) Cell viability, evaluated by MTT assay, of WIF-B cells after exposure to CuCl2. Note the lack of significant effect of SPCA1 knockdown. Data was normalized to growth in the absence of added Cu2+. (C) Viability of HEK 293T cells following exposure to Mn2+ or Cu2+. Note the lack of effect of SPCA1 knockdown. Data are representative of two independent experiments with similar results. (JPEG 177 kb)
Twenty-five 0.32 mm optical sections of WIF-B cells co-stained for SPCA1 (green) and HA321 (red) were obtained by confocal microscopy, as described under Experimental Procedures, and combined to generate a three-dimensional representation. Although labeling of SPCA1 appears close to the basolateral membrane, no significant co-localization with HA321 is observed (MOV 535 kb)