The human multispecific drug efflux transporter P-glycoprotein (P-gp) causes drug resistance and modulates the pharmacological profile of systemically administered medicines. It has arisen from a homodimeric ancestor by gene duplication. Crystal structures of mouse MDR1A indicate that P-gp shares the overall architecture with two homodimeric bacterial exporters, Sav1866 and MsbA, which have complete rotational symmetry. For ATP-binding cassette transporters, nucleotide binding occurs in two symmetric positions in the motor domains. Based on the homology with entirely symmetric half-transporters, the present study addressed the key question: can biochemical evidence for the existence of dual drug translocation pathways in the transmembrane domains of P-gp be found? P-gp was photolabeled with propafenone analogs, purified, and digested proteolytically, and peptide fragments were identified by highresolution mass spectrometry. Labeling was assigned to two regions in the protein by projecting data into homology models. Subsequently, symmetric residue pairs in the putative translocation pathways were identified and replaced by site-directed mutagenesis. Transport assays corroborated the existence of two pseudosymmetric translocation pathways. Although rhodamine123 has a preference to take one path, verapamil, propafenones, and vinblastine preferentially use the other. Two major findings ensued from this study: the existence of two solute translocation pathways in P-gp as a reflection of evolutionary origin from a homodimeric ancestor and selective but not exclusive use of one of these pathways by different P-gp solutes. The pseudosymmetric behavior reconciles earlier kinetic and thermodynamic data, suggesting an alternative concept of drug transport by P-gp that will aid in understanding the off-target quantitative structure activity relationships of P-gp interacting drugs.

Background: Intron gains reportedly are very rare during evolution of vertebrates, and the mechanisms underlying their creation are largely unknown. Previous investigations have shown that, during metazoan radiation, the exon-intron patterns of serpin superfamily genes were subject to massive changes, in contrast to many other genes.

By alternative use of four RSL (reactive site loop) coding exon cassettes, the serpin (serine protease inhibitor) gene Spn4 from Drosophila melanogaster was proposed to enable the synthesis of multiple protease inhibitor isoforms, one of which has been shown to be a potent inhibitor of human furin. Here, we have investigated the inhibitory spectrum of all Spn4 RSL variants. The analyses indicate that the Spn4 gene encodes inhibitors that may inhibit serine proteases of the subtilase family (S8), the chymotrypsin family (S1), and the papain-like cysteine protease family (C1), most of them at high rates. Thus a cohort of different protease inhibitors is generated simply by grafting enzyme-adapted RSL sequences on to a single serpin scaffold, even though the target proteases contain different types and/or a varying order of catalytic residues and are descendents of different phylogenetic lineages. Since all of the Spn4 RSL isoforms are produced as intracellular residents and additionally as variants destined for export or associated with the secretory pathway, the Spn4 gene represents a versatile defence tool kit that may provide multiple antiproteolytic functions.

Lancelets are considered to take a key position in the evolution of lineages leading to vertebrates. Herein, a serpin from the lancelet Branchiostoma lanceolatum, Bl-Spn1, was identified that inhibits the PCs (proprotein convertases) PC1/3 and furin. The inhibitor forms SDS-stable complexes with either of its targets. Analysis of the inhibitor/furin reaction products by mass spectroscopy assigns the enzyme's cleavage position C-terminally to Met-Met-Lys-Arg downward arrow in the reactive site loop of Spn1, in concordance with the classical recognition/cleavage site of the principal vertebrate PCs. The inhibitor is equipped with a canonical ER (endoplasmic reticulum) retrieval signal, Lys-Asp-Glu-Leu (KDEL), marking the inhibitor as a guardian of the cellular secretory routes. Deletion of the ER retrieval signal results in the export of the inhibitor into the medium of transfected COS-7 cells, consistent with the assigned intracellular location. These results identify Bl-Spn1 as the first serpin that may inhibit PC1/3-like subtilases at their natural sites of action. Phylogenetic comparisons support a concept implying a general role for ER-residing serpins in the surveillance of subtilase-like enzymes along the constitutive and regulated secretory pathways of metazoans including a role in the defence of intruders that turn PCs to their propagation.

An alternatively spliced form of human sulfonylurea receptor (SUR) 1 mRNA lacking exon 2 (SUR1Δ2) has been identified. The omission of exon 2 caused a frame shift and an immediate stop codon in exon 3 leading to translation of a 5.6-kDa peptide that comprises the N-terminal extracellular domain and the first transmembrane helix of SUR1. Based on a weak first splice acceptor site in the human SUR1 gene (ABCC8), RT-PCR revealed a concurrent expression of SUR1Δ2 and SUR1. The SUR1Δ2/(SUR1 + SUR1Δ2) mRNA ratio differed between tissues, and was lowest in pancreas (46%), highest in heart (88%) and negatively correlated with alternative splice factor/splicing factor 2 (ASF/SF2) expression. In COS-7 cells triple transfected with SUR1Δ2/SUR1/Kir6.2, the SUR1Δ2 peptide co-immunoprecipitated with Kir6.2, thereby displacing two of four SUR1 subunits on the cell surface. The ATP sensitivity of these hybrid ATP-sensitive potassium channels (K(ATP)) channels was reduced by about sixfold, as shown with single-channel recordings. RINm5f rat insulinoma cells, which genuinely express SUR1 but not SUR1Δ2, exhibited a strongly increased K(ATP) channel current upon transfection with SUR1Δ2. This led to inhibition of glucose-induced depolarization, calcium flux, insulin release and glibenclamide action. A non-mutagenic SNP on nucleotide position 333 (Pro69Pro) added another exonic splicing enhancer sequence detected by ASF/SF2, reduced relative abundance of SUR1Δ2 and slightly protected from non-insulin dependent diabetes in homozygotic individuals. Thus, SUR1Δ2 represents an endogenous K(ATP)-channel modulator with prodiabetic properties in islet cells. Its predominance in heart may explain why high-affinity sulfonylurea receptors are not found in human cardiac tissue.

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