The bile salt export pump (BSEP/ABCB11) is responsible for the transport of bile salts from hepatocytes into bile canaliculi. Malfunction of this transporter results in progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2) and intrahepatic cholestasis of pregnancy (ICP). Over the past few years, several small molecular weight compounds have been identified, which hold the potential to treat these genetic diseases (chaperones and potentiators). As the treatment response is mutation-specific, genetic analysis of the patients and their families is required. Furthermore, some of the mutations are refractory to therapy, with the only remaining treatment option being liver transplantation. In this review, we will focus on the molecular structure of ABCB11, reported mutations involved in cholestasis and current treatment options for inherited BSEP deficiencies.

The multispecific efflux transporter, P-glycoprotein, plays an important role in drug disposition. Substrate translocation occurs along the interface of its transmembrane domains. The rotational C2 symmetry of ATP-binding cassette transporters implies the existence of two symmetry-related sets of substrate-interacting amino acids. These sets are identical in homodimeric transporters, and remain evolutionary related in full transporters, such as P-glycoprotein, in which substrates bind preferentially, but nonexclusively, to one of two binding sites. We explored the role of pore-exposed tyrosines for hydrogen-bonding interactions with propafenone type ligands in their preferred binding site 2. Tyrosine 953 is shown to form hydrogen bonds not only with propafenone analogs, but also with the preferred site 1 substrate rhodamine123. Furthermore, an accessory role of tyrosine 950 for binding of selected propafenone analogs is demonstrated. The present study demonstrates the importance of domain interface tyrosine residues for interaction of small molecules with P-glycoprotein.

Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site. ABC transporters are one of the largest membrane protein superfamilies, present in all organisms from archaea to humans. They transport a wide range of molecules including amino acids, sugars, vitamins, nucleotides, peptides, lipids, metabolites, antibiotics, and xenobiotics. ABC transporters energize substrate transport by hydrolyzing ATP in two symmetrically arranged nucleotide binding sites (NBSs). The human multidrug resistance transporter ABCB1 has two active NBSs, and it is generally believed that integrity and cooperation of both sites are needed for transport. Several human ABC transporters, such as the bile salt transporter ABCB11, have one degenerate NBS, which has significantly reduced ATPase activity. Interestingly, unilateral mutations affecting one of the two NBSs completely abolish the function of symmetrical ABC transporters. Here we engineered an ABCB1 variant with a degenerate, ABCB11-like NBS1, which can nevertheless transport substrates. Our results indicate that ABCB1 can mediate active transport with a single active site, questioning the validity of models assuming strictly alternating catalysis.

In recent years, microfluidic chip-based sperm sorting has emerged as an alternative tool to centrifugation-based conventional techniques for in vitro fertilization. This prospective study aims to compare the effects of density gradient centrifugation and microfluidic chip sperm preparation methods on embryo development in patient populations with astheno-teratozoospermia. In the study, the semen samples of the patients were divided into two groups for preparation with either the microfluidic or density gradient methods. Selected spermatozoa were then used to fertilize mature sibling oocytes and the semen parameters and embryo development on days 3 and 5 were assessed. While the density gradient group was associated with a higher sperm concentration, motility (progressive and total) was significantly higher in the microfluidic chip group. No significant differences were observed in the fertilization rates or grade 1 (G1) and grade 2 (G2) proportions of the third-day embryos. Furthermore, while the proportions of the poor, fair and good blastocysts on day 5 did not differ significantly, excellent blastocysts (indicating high-quality embryos) were observed in a significantly higher proportion of the microfluidic chip group. When compared to the classical density gradient method, the microfluidic chip sperm preparation yielded sperm with higher motility and higher quality blastocysts at day 5; in patients with astheno-teratozoospermia.