6A and Supporting Information). Substitutions p.L127P and p.G1040R are predicted to result in structural changes within the transmembrane domains of ATP8B1 (Fig. 6B,F). The p.G308V mutation causes a destabilizing rearrangement in the ATP8B1 Actuator domain (Fig. 6C), which likely influences the association between the Actuator selleck chemicals llc and Phosphorylation domains (indicated by A and P in Fig. 6A), two ATP8B1 structural domains that are highly conserved in all P-type ATPases. The residues D454 and D554 are close together in the cytosolic core of the protein, and are critical for the catalytic cycle of P-type ATPases (Fig. 6D). I661

is a fully exposed residue, located in the Nucleotide-binding domain (N-domain) (Fig. 6E). The I661T mutation does not seem to result in major structural changes within ATP8B1, CH5424802 in line with the relatively mild clinical consequences of this mutation.11 ATP8B1 R1164X lacks three helical turns

of the last transmembrane helix (shown green in Fig. 6A) and 80 C-terminal residues, whose structure could not be reliably predicted. Together, these modeling data support the hypothesis that most of the studied mutations result in significant structural alterations. We investigated whether treatment with the pharmacological chaperone 4-PBA ameliorated the low expression of ATP8B1 mutants. ATP8B1 G308V protein expression was significantly increased by 4-PBA treatment in a dose-dependent manner (Fig. 7A). Total cellular expression of ATP8B1 G308V, D454G, D554N, and R1164X was induced two-fold to five-fold by 4-PBA treatment (Fig. 7B). Interestingly, protein expression of ATP8B1 I661T and G1040R showing

only mildly reduced expression levels in control conditions, also poorly responded to 4-PBA treatment. ATP8B1 WT expression was not stimulated by 4-PBA, suggesting specific up-regulation selleck inhibitor of otherwise misfolded proteins. Subsequently, cell surface biotinylation was performed to determine whether 4-PBA stimulated the trafficking of ATP8B1 mutants to the cell surface. Neither ATP8B1 nor the transferrin receptor (used as a loading control) was detected when biotin was omitted, indicating the specificity of the signal for cell surface resident proteins. ATP8B1 G308V, D454G, and D554N showed a 1.5-fold to 2-fold increase in plasma membrane expression upon 4-PBA treatment (Fig. 8B). Despite increased protein expression upon 4-PBA treatment, no ATP8B1 R1164X signal was detectable at the cell surface in either condition. Interestingly, ATP8B1 I661T abundance in the biotinylated fraction was strongly enhanced (5-fold to 10-fold) upon 4-PBA treatment, suggesting markedly improved trafficking to the plasma membrane (Fig. 8A,B). The reverse occurred when cells were cultured at 40°C. This temperature increase resulted in a significant decrease in the amount of ATP8B1 I661T, but not WT protein at the cell surface (Fig. 8C).