Crystal structures of LeuT, a bacterial homologue of mammalian neurotransmitter transporters, show a molecule of bound substrate that is essentially exposed to the extracellular space but occluded from the cytoplasm. Thus, there must exist an alternate conformation for LeuT in which the substrate is accessible to the cytoplasm and a corresponding mechanism that switches accessibility from one side of the membrane to the other. Here, we identify the cytoplasmic accessibility pathway of the alternate conformation in a mammalian serotonin transporter (SERT) (a member of the same transporter family as LeuT). We also propose a model for the cytoplasmic-facing state that exploits the internal pseudosymmetry observed in the crystal structure. LeuT contains two structurally similar repeats (TMs1-5 and TMs 6 -10) that are inverted with respect to the plane of the membrane. The conformational differences between them result in the formation of the extracellular pathway. Our model for the cytoplasm-facing state exchanges the conformations of the two repeats and thus exposes the substrate and ion-binding sites to the cytoplasm. The conformational change that connects the two states primarily involves the tilting of a 4-helix bundle composed of transmembrane helices 1, 2, 6, and 7. Switching the tilt angle of this bundle is essentially equivalent to switching the conformation of the two repeats. Extensive mutagenesis of SERT and accessibility measurements, using cysteine reagents, are accommodated by our model. These observations may be of relevance to other transporter families, many of which contain internal inverted repeats.alternating access mechanism ͉ neurotransmitter:sodium symporters ͉ serotonin ͉ structural modeling ͉ structural repeats

Ibogaine, a hallucinogenic alkaloid with purported anti-addiction properties, inhibited serotonin transporter (SERT) noncompetitively by decreasing V max with little change in the K m for serotonin (5-HT). Ibogaine also inhibited binding to SERT of the cocaine analog 2␤-2-carbomethoxy-3-(4-[ 125 I]iodophenyl)tropane. However, inhibition of binding was competitive, increasing the apparent K D without much change in B max . Ibogaine increased the reactivity of cysteine residues positioned in the proposed cytoplasmic permeation pathway of SERT but not at nearby positions out of that pathway. In contrast, cysteines placed at positions in the extracellular permeation pathway reacted at slower rates in the presence of ibogaine. These results are consistent with the proposal that ibogaine binds to and stabilizes the state of SERT from which 5-HT dissociates to the cytoplasm, in contrast with cocaine, which stabilizes the state that binds extracellular 5-HT.Ibogaine is a hallucinogenic alkaloid found in the roots of the West African shrub Tabernanthe iboga. In Europe and North America, ibogaine has been promoted as a treatment for addiction, although clinical evidence has been difficult to obtain, partly due to its listing as a Schedule I controlled substance (1, 2) and partly due to concerns about toxicity (3). Ibogaine has affinity for 2 receptors, N-methyl-D-aspartate receptors, -opioid receptors, and serotonin and dopamine transporters (4 -7). Ibogaine is demethylated to 12-hydroxyibogamine (noribogaine), which has been reported to persist in the blood for over 24 h and to have even higher affinity for 5-HT transporters than ibogaine (8).The 5-hydroxytryptamine (5-HT) 3 transporter (SERT) is responsible for reuptake of 5-HT released from serotonergic neurons (9). This protein is the target for antidepressant drugs, such as imipramine and fluoxetine, and psychostimulants, such as cocaine and 3,4-methylenedioxymethamphetamine. SERT couples the entry of 5-HT into the cell to the entry of Na ϩ and Cl Ϫ and the exit of K ϩ (10). The transport of 5-HT has been envisaged as a two-step process, where 5-HT, Na ϩ , and Cl Ϫ are transported in a single step and K ϩ is transported in a second step (11). These transport steps are proposed to interconvert two states of the transporter: an extracellular state that binds extracellular substrates and a cytoplasmic state that releases those substrates to the cytoplasm (9).SERT belongs to a large family of Na ϩ -dependent transporters in prokaryotes and animals designated the SLC6 or NSS family. The high resolution x-ray structure of the prokaryotic leucine transporter LeuT (12) appears to be a good model for other members of the family, and homology models for SERT and other neurotransmitter transporters have been generated using structure-based alignments (13). The utility of LeuT as a model for SERT has recently been validated by the identification of the Cl Ϫ binding site in SERT through analysis of the LeuT structure (14). This structure shows an aqueous pathway leading from the ...

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Pharmacogenomic testing is viewed as an integral part of precision medicine. To achieve this, we originated The 1200 Patients Project which offers broad, preemptive pharmacogenomic testing to patients at our institution. We analyzed enrollment, genotype, and encounter-level data from the first year of implementation to assess utility of providing pharmacogenomic results. Results were delivered via a genomic prescribing system (GPS) in the form of traffic lights: green (favorable), yellow (caution), and red (high risk). Additional supporting information was provided as a virtual pharmacogenomic consult, including citation to relevant publications. Currently, 812 patients have participated, representing 90% of those approached; 608 have been successfully genotyped across a custom array. A total of 268 clinic encounters have occurred at which results were accessible via the GPS. At 86% of visits, physicians accessed the GPS, receiving 367 result signals for medications patients were taking: 57% green lights, 41% yellow lights, and 1.4% red lights. Physician click frequencies to obtain clinical details about alerts varied according to color severity (100% of red were clicked, 72% yellow, 20% green). For 85% of visits, clinical pharmacogenomic information was available for at least one drug the patient was taking, suggesting relevance of the delivered information. We successfully implemented an individualized health care model of preemptive pharmacogenomic testing, delivering results along with pharmacogenomic decision support. Patient interest was robust, physician adoption of information was high, and results were routinely utilized. Ongoing examination of a larger number of clinic encounters and inclusion of more physicians and patients is warranted.

Purpose Appendiceal neoplasms are heterogeneous and are often treated with chemotherapy similarly to colorectal cancer (CRC). Genomic profiling was performed on 703 appendiceal cancer specimens to compare the mutation profiles of appendiceal subtypes to CRC and other cancers, with the ultimate aim to identify potential biomarkers and novel therapeutic targets. Methods Tumor specimens were submitted to a Clinical Laboratory Improvement Amendments–certified laboratory (Foundation Medicine, Cambridge, MA) for hybrid-capture–based sequencing of 3,769 exons from 315 cancer-related genes and 47 introns of 28 genes commonly rearranged in cancer. Interactions between genotype, histologic subtype, treatment, and overall survival (OS) were analyzed in a clinically annotated subset of 76 cases. Results There were five major histopathologic subtypes: mucinous adenocarcinomas (46%), adenocarcinomas (30%), goblet cell carcinoids (12%), pseudomyxoma peritonei (7.7%), and signet ring cell carcinomas (5.2%). KRAS (35% to 81%) and GNAS (8% to 72%) were the most frequent alterations in epithelial cancers; APC and TP53 mutations were significantly less frequent in appendiceal cancers relative to CRC. Low-grade and high-grade tumors were enriched for GNAS and TP53 mutations, respectively (both χ2 P < .001). GNAS and TP53 were mutually exclusive (Bonferroni corrected P < .001). Tumor grade and TP53 mutation status independently predicted OS. The mutation status of GNAS and TP53 strongly predicted OS (median, 37.1 months for TP53 mutant v 75.8 GNAS- TP53 wild type v 115.5 GNAS mutant; log-rank P = .0031) and performed as well as grade in risk stratifying patients. Conclusion Epithelial appendiceal cancers and goblet cell carcinoids show differences in KRAS and GNAS mutation frequencies and have mutation profiles distinct from CRC. This study highlights the benefit of performing molecular profiling on rare tumors to identify prognostic and predictive biomarkers and new therapeutic targets.