The prion protein family member Shadoo induces spontaneous ionic currents in cultured cells

Abstract

Some mutant forms of the cellular prion protein (PrPC) carrying artificial deletions or point mutations associated with familial human prion diseases are capable of inducing spontaneous ionic currents across the cell membrane, conferring hypersensitivity to certain antibiotics to a wide range of cultured cells and primary cerebellar granular neurons (CGNs). These effects are abrogated when the wild type (WT) form is co-expressed, suggesting that they might be related to a physiological activity of PrPC. Interestingly, the prion protein family member Shadoo (Sho) makes cells hypersensitive to the same antibiotics as mutant PrP-s, an effect that is diminished by the co-expression of WT-PrP. Here, we report that Sho engages in another mutant PrP-like activity: it spontaneously induces large ionic currents in cultured SH-SY5Y cells, as detected by whole-cell patch clamping. These currents are also decreased by the co-expression of WT-PrP. Furthermore, deletion of the N-terminal (RXXX)8 motif of Sho, mutation of the eight arginine residues of this motif to glutamines, or replacement of the hydrophobic domain by that of PrP, also diminish Sho-induced ionic currents. Our results suggest that the channel activity that is also characteristic to some pathogenic PrP mutants may be linked to a physiological function of Sho.

Membrane Domain Localization and Interaction of the Prion-Family Proteins, Prion and Shadoo with Calnexin

“…In addition, studies by our group demonstrated that, contrary to wild type PrP C , Sho expression sensitizes human SH-SY5Y and HEK293 cells to certain drugs, similar to the toxic, central region (CR)-deletion mutant, ∆ , PrP∆CR-a phenotype that could be rescued by the wild type PrP C [63]. Additionally, we showed that the expression of Sho produces spontaneous large inward currents in cell cultures, a similar effect that is exerted by the HD-deletion mutant PrP∆HD [64], which could also be rescued by PrP C in both cases. In animal models, Sho and PrP are observed to be involved in embryonic development; however, a cross regulation between the levels of the two proteins is not apparent in the adult state [47].…”

“…In the case of Sho, two bands appear at around the expected weight of Sho-EYFP (~45-49 kDa), recognized by both α-Sho and α-GFP antibodies in Sho-EYFP cells, which are absent in parental N2a cell samples (Figure 2B, lanes 1-2 and 4-5). It should be noted that there are no unequivocally good anti-Shadoo antibodies available and that cross-reactive bands are frequently apparent on blots of various cells by anti-Sho antibodies [47,60,63,64,82]. However, since α-GFP shows similar bands, these might be two forms of Sho.…”

Regulation of sub-compartmental targeting and folding properties of the Prion-like protein Shadoo

“…The recently proposed channel activity of Sho 41 , which is also characteristic to some pathogenic PrP mutants, may be linked to a physiological function of Sho.…”

The study of the membrane microdomain localization of the prion-family proteins, prion and Shadoo and their interaction with calnexin

“…These cells are well characterized and broadly used as a cellular model system in the study of physiology of cellular and the pathological prion protein 90,156 . N2a cells possess detectable amounts of endogenous PrP but not Sho; at least the endogenous Shadoo expression cannot be detected in these cells with the currently available shadoo antibodies 70,86,96,97,157 . To compare Sho and PrP, we generated N2a cells stably expressing either Sho or PrP in fusion with a fluorescent protein (EYFP for Sho and EGFP for PrP) tag by using the plasmid constructs described in the Materials and methods section for PrP (for the plasmid Map see Appendix-I, Figure A1) and earlier by our group for Sho 69 .…”

“…On the other hand, opposing roles for Sho and wild type PrP had also been reported: expression of Sho caused drug hypersensitivity in certain type of cells, while expression of wild-type PrP rescued cells against the effect of the same drugs 96 . Also, Sho induces large spontaneous ionic currents similarly to the toxic PrP mutant associated with familial human prion diseases, whereas expression of wild type PrP rescues the same cells against these effects 97 . Altogether, these demonstrate that while Sho and PrP may act similarly then may undertake opposing roles as well depending on the experimental setting and the process studied, and more data are required to understand their exact relation and interplay.…”