SUMMARY The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium-and chloride-dependent recycling of serotonin into presynaptic neurons1–3. Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors (SSRIs), small molecules that competitively block substrate binding, prolonging neurotransmitter action2,4. The dopamine and norepinephrine transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. Cocaine and amphetamines inhibit or modulate the transport activities of NSSs2,3 and genetic variants are associated with multiple neuropsychiatric disorders including attention deficit hyperactivity disorder, autism, and bipolar disorder2,5. Studies of bacterial NSS homologs, including LeuT, have shown how transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane1,6–12. However, the conformational changes associated with transport in eukaryotic NSSs remain obscure. To elucidate structure-based mechanisms for transport in SERT, we turned to complexes with ibogaine, a centuries old hallucinogenic natural product with psychoactive and anti-addictive properties13,14 (Fig. 1a). Interestingly, ibogaine displays non-competitive inhibition of transport, yet it exhibits competitive binding toward SSRIs15,16. Here we report cryo-EM structures of SERT-ibogaine complexes captured in outward-open, occluded, and inward-open conformations. Ibogaine binds to the central binding site and closure of the extracellular gate largely involves movements of TMs 1b and 6a. Opening of the intracellular gate involves a hinge-like movement of TM1a and partial unwinding of TM5, which together create a permeation pathway enabling substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from outward-open to the inward-open conformations, providing insight into the mechanism of neurotransmitter transport and ibogaine inhibition.

No abstract

Enalapril was associated with increased odds of developing anemia at one year. Those with periods of time with incident anemia had the poorest survival, followed by those with prevalent anemia, then those without anemia. Enalapril was protective of overall mortality after adjusting for incident anemia and in those with prevalent anemia.

BackgroundNext Generation Sequencing (NGS) technology generates tens of millions of short reads for each DNA/RNA sample. A key step in NGS data analysis is the short read alignment of the generated sequences to a reference genome. Although storing alignment information in the Sequence Alignment/Map (SAM) or Binary SAM (BAM) format is now standard, biomedical researchers still have difficulty accessing this information.ResultsWe have developed a Graphical User Interface (GUI) software tool named SAMMate. SAMMate allows biomedical researchers to quickly process SAM/BAM files and is compatible with both single-end and paired-end sequencing technologies. SAMMate also automates some standard procedures in DNA-seq and RNA-seq data analysis. Using either standard or customized annotation files, SAMMate allows users to accurately calculate the short read coverage of genomic intervals. In particular, for RNA-seq data SAMMate can accurately calculate the gene expression abundance scores for customized genomic intervals using short reads originating from both exons and exon-exon junctions. Furthermore, SAMMate can quickly calculate a whole-genome signal map at base-wise resolution allowing researchers to solve an array of bioinformatics problems. Finally, SAMMate can export both a wiggle file for alignment visualization in the UCSC genome browser and an alignment statistics report. The biological impact of these features is demonstrated via several case studies that predict miRNA targets using short read alignment information files.ConclusionsWith just a few mouse clicks, SAMMate will provide biomedical researchers easy access to important alignment information stored in SAM/BAM files. Our software is constantly updated and will greatly facilitate the downstream analysis of NGS data. Both the source code and the GUI executable are freely available under the GNU General Public License at http://sammate.sourceforge.net.

Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric N-ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes. We now report that PA also induces conformational changes in Sec18 protomers and that hexameric Sec18 cannot bind PA membranes. Molecular dynamics (MD) analyses revealed that the D1 and D2 domains of Sec18 contain PA-binding sites and that the residues needed for PA binding are masked in hexameric Sec18. Importantly, these simulations also disclosed that a major conformational change occurs in the linker region between the D1 and D2 domains, which is distinct from the conformational changes that occur in hexameric Sec18 during SNARE priming. Together, these findings indicate that PA regulates Sec18 function by altering its architecture and stabilizing membrane-bound Sec18 protomers.Membrane fusion is necessary for all eukaryotes to effectively transport cellular components between organelles. The trafficking and fusion of vesicles is carried out through a series of events that are highly conserved across eukarya (1). Although many proteins that drive the process may differ between eukaryotic species, they all perform similar roles allowing compartment contact, bilayer fusion, and luminal content mixing (2). The final stage of membrane fusion, and luminal content mixing, is catalyzed by SNARE 3 proteins. Each participating membrane contributes either an R-SNARE or three Q-SNARE coils that wrap around each other to form a parallel four-helical trans-SNARE complex that brings membranes into close apposition. The formation of such complexes releases free energy that is transmitted to the membranes to trigger fusion. Once fusion occurs and membranes are merged, the four-helical SNARE bundle, now a cis-SNARE complex, is inactive and requires disassembly to undergo a new round of fusion.The disassembly of cis-SNAREs, also known as Priming, is carried out by the AAA ϩ protein Sec18/NSF and its adaptor protein Sec17/␣-SNAP (3) (Fig. 1A). Current models suggest that NSF primes cis-SNAREs through a "loaded spring" mechanism triggered by cis-SNARE recognition and ATP hydrolysis (4). NSF binds to cis-SNAREs with the help of ␣-SNAP to form what is known as the 20S complex (5-8). Although NSF was originally isolated as a trimer or tetramer, it can only prime SNAREs as a homohexamer that surrounds the cis-SNAREs and ␣-SNAP proteins to form the 20S particle (9 -11). Association with cis-SNARE-␣-SNAP complexes triggers ATP hydrolysis, which leads to a large conformational change in the protein, with the major change occurring at the N terminus where it folds back over the D1-D2 rings (8). This generates eno...