We selected 4-PBA for these studies, because it is a clinically approved drug for treatment of a variety of human disorders originating or manifesting in the liver. These include urea cycle disorders, where 4-PBA is used short-term in dosages exceeding the concentration
used in this study. Importantly, this drug also ameliorates cell surface abundance of a number of misfolded and mislocalized membrane proteins with relevance to human liver disease. These include ABCB11 p.E297G and p.D482G, identified in PFIC2 patients, and CF transmembrane conductance regulator ΔF508, the most common mutation associated with CF.9, 22 Clinical trials demonstrated that 4-PBA treatment resulted
in increased chloride conductance in patients with CF.23 In our study, 4-PBA treatment stimulated the protein expression and/or cell surface abundance of ATP8B1 G308V, D454G, D554N, and I661T in vitro. Y-27632 cell line Amelioration of cell surface expression was most prominent for the latter mutant, with a dramatic increase in ATP8B1 I661T accessible for biotinylation. However, using multiple assays, we could not detect ATP8B1-mediated internalization of fluorescently Ibrutinib in vitro labeled phosphatidylserine upon coexpression of ATP8B1 WT and CDC50A (data not shown). This prohibited verification whether ATP8B1 I661T at the cell surface is functional. Furthermore, it is currently Glutamate dehydrogenase unclear to what level ATP8B1 cell surface abundance needs to be restored, and how much ATP8B1 catalytic activity is required, to relieve patients from an ATP8B1 deficiency phenotype. Given the episodic character of cholestatic attacks in BRIC1 patients, and the predominant absence of clinical symptoms in heterozygous carriers, we propose that partial restoration of cell surface expression by 4-PBA might already provide clinical improvement in BRIC1. A substantial proportion of BRIC1 patients carry at least one p.I661T allele, and many are homozygous for p.I661T. Whether the episodic
character of BRIC1 even precludes the need for long-term administration of 4-PBA in these patients, needs to be investigated in clinical trials once the in vivo efficacy has been ascertained. In conclusion, the results of this study investigating six missense and one nonsense mutation, indicate that future therapy aiming to restore ATP8B1 expression at the plasma membrane probably needs to be tailored to specific genetic defects, and emphasize the need for continued detailed and systematic analysis of ATP8B1 mutations that possibly result in folding defects. Treatment with pharmacological chaperones like 4-PBA, might present a clinically useful approach to increase the amount of ATP8B1 mutant protein at the cell surface, especially in patients with episodic presentation of ATP8B1 deficiency.