Transitioning from Faculty-Written Examinations to National Board of Medical Examiners Custom Examinations in Medical Education.

Purpose: A challenge for medical educators is choosing a method that best evaluates preclinical students' performance in preparation for Step 1. In previous years, block directors (BDs) of the 2nd year (MS2) neuroscience course at Texas Tech University Health Sciences Center School of Medicine issued faculty-written (FW) examinations during the course. In 2022, BDs replaced FW examinations with National Board of Medical Examiners (NBME) custom examinations. The rationale being that the customized NBME exams would better reflect the national neuroscience curriculum and enhance student preparedness for taking standardized exams. Methods: FW examinations (2021) were created by the faculty in the neuroscience course and reviewed by BDs. In contrast, questions that best aligned with the material covered for the 2022 course were selected by BDs using MyNBMESM Services Portal. The custom questions selected are assigned a "difficulty" score by NBME, generating a predicted national average score. At the end of the course, undergraduate medical students in the School of Medicine at Texas Tech University Health Sciences Center completed an online Qualtrics questionnaire to compare the transition of assessment type. Results: Participants reported greater satisfaction in their neuroscience education and block organization with NBME examinations. For example, there was a nearly twofold (1.83) increase in the number of students that strongly agreed with the statement "Overall, I am satisfied with the quality of my neuroscience education in this block." They were also less likely to report the workload as being "much too heavy." Overall, students expressed a preference for the customized NBME exams as opposed to faculty generated exams (88.1%). Conclusions: From the student perspective, building customized assessments through MyNBMESM Services Portal was found to be useful and preferable for evaluating student performance. From block directors' perspective, it is noted that time is saved assisting faculty in writing valid questions, time defending/justifying FW questions, and time expended generating exams. The only perceived negative regarding the NBME exams is the cost.

The Quality Improvement Review Board: An Innovative Approach to Oversight of Projects That Do Not Meet Criteria of Human Subject Research.

This article describes the development of an institutional quality improvement review board (QIRB) as an effective and efficient method for reviewing and overseeing institutional quality improvement (QI) initiatives. QI projects involve the systematic collection and analysis of data and the implementation of interventions designed to improve the quality of clinical care and/or educational programs for a distinct population in a specific setting. QI projects are fundamentally distinct from human subjects research (HuSR); however, the differences between them are subtle and highly nuanced. Determining whether a project meets the definition of QI or qualifies as HuSR, thus requiring institutional review board (IRB) review, can be confusing and frustrating. Nevertheless, this distinction is highly consequential due to the heavy regulatory requirements involved in HuSR and IRB oversight. Making the correct determination of a project's regulatory status is essential before the project begins. Project leaders may not realize that their work meets the definition of HuSR and, therefore, might conduct the project without appropriate IRB review. Therefore, best practices dictate that project leaders should not decide which type of institutional review is appropriate for their projects. In addition, when QI project teams attempt to disseminate the results of their work, documentation of formal review and approval is generally required by peer-reviewed journals and professional organizations. However, institutional review mechanisms are rarely available. Projects that do not meet the definition of HuSR fall outside the purview of IRBs and most institutions do not have an alternative review body. This creates frustration for both project leaders and IRB administrators. Apart from IRB review, a separate process for reviewing QI projects offers several benefits. These include (1) relieving the burden on busy IRB staff; (2) promoting scholarly activity; (3) protecting the institution, project leaders, and participants from HuSR conducted outside of appropriate IRB review; and (4) promoting rigorous QI methods.

Implantation Time Has No Effect on the Morphology and Extent of Previously Reported "Degradation" of Prolene Pelvic Mesh.

OBJECTIVES: Prolene polypropylene ("Prolene") meshes demonstrate no in vivo degradation, yet some claim degradation continues until no more Prolene polypropylene can be oxidized. We studied whether implantation time affects the morphology/extent of previously reported as cracking/degradation of completely cleaned Prolene explants. METHODS: Urogynecological explants (248 patients) were collected. After excluding non-Prolene/unknown meshes and those without known implantation times, completely cleaned explants (n = 205; 0.2-14.4 years implantation) were analyzed with light microscopy, scanning electron microscopy, and Fourier transform infrared spectroscopy. Based on implant times and storage (fixative or dry), representative specimens were randomly selected for comparison. Controls were unused ("exemplar") TVT specimens with and without intentional oxidation via ultraviolet light exposure. RESULTS: Prolene explants included 31 dry (18 TVT; 7 Prolift; 4 Gynemesh; 2 others) and 174 wet (87 TVT; 47 Prolift; 10 Gynemesh; 30 others) specimens. Specimens had similar morphologies before cleaning. Progressive cleaning removed tissue and cracked tissue-related material exposing smooth, unoxidized, and nondegraded fibers, with no visible gradient-type/ductile damage. Fourier transform infrared spectroscopy of the explants confirmed progressive loss of proteins. Cleaning intentionally oxidized exemplars did not remove oxidized carbonyl frequencies and showed deep cracks and gross fiber rupture/embrittlement, unlike the explants and nonoxidized exemplars. CONCLUSIONS: If in vivo Prolene degradation exists, there should be wide-ranging crack morphology and nonuniform crack penetration, as well as more cracking, degradation, and physical breakage for implants of longer implantation times, but this was not the case. There is no morphologic or spectral/chemical evidence of Prolene mesh degradation after up to 14.4 years in vivo.

Detection of in Situ Early Corrosion on Polymer-Coated Metal Substrates.

Protective coating systems (PCS) are a common and facile method to protect metal substrates from corrosion. The corrosion control performance of polymer-coated metal substrates is still predominantly evaluated by visual assessment. Unfortunately, for many decades, PCS material development and performance testing has basically been a complicated process of waiting to determine which coating, in relative terms, allows corrosion to occur first from an intentionally created breach through the coating. This type of testing provides only relative ratings between PCS performance. Electrochemical methods, such as electrochemical impedance spectroscopy, each have caveats and pitfalls for qualifying or quantifying polymer-coated metal substrates. When these data are studied carefully, these measurements result in many false positive and false negative results compared with real environmental testing and the paths to failure vary dramatically. The critical issue is that these methods do not result in a scientific basis for understanding either the pathway(s) or progressive milestones toward diminished PCS performance, failure, and the loss of substrate structural integrity for coated substrates. Data supports that ultimately all PCS fail to provide the necessary substrate protection. However, to make substantive gains, scientists and engineers require a rational basis to design, engineer, test, quantify, and/or estimate service life and remaining service life and repair future generations of PCS. Our research goal was to establish a quantifiable characterization protocol (CP) that directly detected, monitored, and ideally quantified the pathway(s) and important milestones of PCS corrosion spatially and temporally, with or without defects which related with testing and assessment variables regardless of environmental severity (real, laboratory, or accelerated). We report herein the CP and the results from an embedded pH-sensitive "turn-on" fluorescent probe blended with a simplified thermoplastic model PCS. The results support that the average-localized macroscopic pH is detected and tracked, and these "molecular titrations" result in values consistent with literature pH citations for premacroscopic corrosion processes, that is, before delamination and a detectable breach. The CP results are an improvement over visual corrosion detection and yet proportional to the steel substrate corrosion. The CP results deliver extreme early detection (within minutes), spatial and temporal tracking, and potentially quantifiable performance differences for the pathways and milestones toward failure of coated substrates with validated sensitivity to variables such as defect versus defect-free films, blending solvent type(s) influence, differences from varied degrees of annealing relative to Tg (thermoplastic films), substrate topography, and preparation differences. The CP utilizes small sample areas (25 mm spheres) and gathers data in a manner designed to improve statistical relevancy, provide results within short timeframes using real-time testing, diminish materials-testing timelines, and connect results with laboratory, accelerated, and real environmental severity differences. The results support that the CP directly measured the earliest possible in situ corrosion processes using defect and defect-free simplified model PCS.

Sequential and one-pot post-polymerization modification reactions of thiolactone-containing polymer brushes.

Thiolactone chemistry has garnered significant attention as a powerful post-polymerization modification (PPM) route to mutlifunctional polymeric materials. Here, we apply this versatile chemistry to the fabrication of ultrathin, multifunctional polymer surfaces via aminolysis and thiol-mediated double modifications of thiolactone-containing polymer brushes. Polymer brush surfaces were synthesized via microwave-assisted surface-initiated polymerization of DL-homocysteine thiolactone acrylamide. Aminolysis and thiol-Michael double modifications of the thiolactone-functional brush were explored using both sequential and one-pot reactions with bromobenzyl amine and 1H,1H-perfluoro-N-decyl acrylate. X-ray photoelectron spectroscopy and argon gas cluster ion sputter depth profiling enabled quantitative comparison of the sequential and one-pot PPM routes with regard to conversion and spatial distribution of functional groups immobilized throughout thickness of the brush. While one-pot conditions proved to be more effective in immobilizing the amine and acrylate within the brush, the sequenital reaction enabled the fabrication of multifunctional, micropattterned brush surfaces using reactive microcontact printing.

The antidepressant bupropion is a negative allosteric modulator of serotonin type 3A receptors.

The FDA-approved antidepressant and smoking cessation drug bupropion is known to inhibit dopamine and norepinephrine reuptake transporters, as well as nicotinic acetylcholine receptors (nAChRs) which are cation-conducting members of the Cys-loop superfamily of ion channels, and more broadly pentameric ligand-gated ion channels (pLGICs). In the present study, we examined the ability of bupropion and its primary metabolite hydroxybupropion to block the function of cation-selective serotonin type 3A receptors (5-HT3ARs), and further characterized bupropion's pharmacological effects at these receptors. Mouse 5-HT3ARs were heterologously expressed in HEK-293 cells or Xenopus laevis oocytes for equilibrium binding studies. In addition, the latter expression system was utilized for functional studies by employing two-electrode voltage-clamp recordings. Both bupropion and hydroxybupropion inhibited serotonin-gated currents from 5-HT3ARs reversibly and dose-dependently with inhibitory potencies of 87 µM and 112 µM, respectively. Notably, the measured IC50 value for hydroxybupropion is within its therapeutically-relevant concentrations. The blockade by bupropion was largely non-competitive and non-use-dependent. Unlike its modulation at cation-selective pLGICs, bupropion displayed no significant inhibition of the function of anion-selective pLGICs. In summary, our results demonstrate allosteric blockade by bupropion of the 5-HT3AR. Importantly, given the possibility that bupropion's major active metabolite may achieve clinically relevant concentrations in the brain, our novel findings delineate a not yet identified pharmacological principle underlying its antidepressant effect.

Spray-deposition and photopolymerization of organic-inorganic thiol-ene resins for fabrication of superamphiphobic surfaces.

Superamphiphobic surfaces, exhibiting high contact angles and low contact angle hysteresis to both water and low surface tension liquids, have attracted a great deal attention in recent years because of the potential of these materials in practical applications such as liquid-resistant textiles, self-cleaning surfaces, and antifouling/anticorrosion coatings. In this work, we present a simple strategy for fabricating of superamphiphobic coatings based on photopolymerization of hybrid thiol-ene resins. Spray-deposition and UV photopolymerization of thiol-ene resins containing hydrophobic silica nanoparticles and perfluorinated thiols provide a multiscale topography and low-energy surface that endows the surface with superamphiphobicity. The wettability and chemical composition of the surfaces were characterized by contact-angle goniometry and X-ray photoelectron spectroscopy, respectively. The hierarchical roughness features of the thiol-ene surfaces were investigated with field-emission scanning electron microscopy. Droplet impact and sandpaper abrasion tests indicate the coatings respectively possess a robust antiwetting behavior and good mechanical durability.

The monomeric state of the proton-coupled folate transporter represents the functional unit in the plasma membrane.

Folic acid is an essential vitamin required for de novo biosynthesis of nucleotides and amino acids. The proton-coupled folate transporter (PCFT; SLC46A1) has been identified as the major contributor for intestinal folate uptake. It is also involved in folate transport across the blood-brain barrier and into solid tumors. PCFT belongs to the major facilitator superfamily. Major facilitator superfamily members can exist in either monomeric or homo-oligomeric form. Here, we utilized blue native polyacrylamide gel electrophoresis (BN/PAGE) and crosslinking with bi-functional chemicals to investigate the quaternary structure of human PCFT after heterologous expression in Xenopus laevis oocytes and CHO cells. PCFT was expressed in the plasma membrane in both expression systems. The functionality of the utilized PCFT construct was confirmed in oocytes by folic acid induced currents at acidic pH. For both the oocyte and CHO expression system [(3)H]folic acid uptake studies indicated that PCFT was functional. To analyze the oligomeric state of PCFT in the plasma membrane, plasma membranes were isolated by polymerization with colloidal silica and polyacrylic acid and subsequent centrifugation. The digitonin-solubilized non-denatured PCFT migrated during BN/PAGE as a monomer, as judged by comparison with a membrane protein (5-HT(3A) receptor) of known pentameric assembly that was used to create a molecular sizing ladder. The chemical crosslinkers glutaraldehyde and dimethyl adipimidate were not able to covalently link potential higher order PCFT structures to form oligomers that were stable following SDS treatment. Together, our results demonstrate that plasma-membrane PCFT functions as a monomeric protein.

Structural sensitivity of a prokaryotic pentameric ligand-gated ion channel to its membrane environment.

Although the activity of the nicotinic acetylcholine receptor (nAChR) is exquisitely sensitive to its membrane environment, the underlying mechanisms remain poorly defined. The homologous prokaryotic pentameric ligand-gated ion channel, Gloebacter ligand-gated ion channel (GLIC), represents an excellent model for probing the molecular basis of nAChR sensitivity because of its high structural homology, relative ease of expression, and amenability to crystallographic analysis. We show here that membrane-reconstituted GLIC exhibits structural and biophysical properties similar to those of the membrane-reconstituted nAChR, although GLIC is substantially more thermally stable. GLIC, however, does not possess the same exquisite lipid sensitivity. In particular, GLIC does not exhibit the same propensity to adopt an uncoupled conformation where agonist binding is uncoupled from channel gating. Structural comparisons provide insight into the chemical features that may predispose the nAChR to the formation of an uncoupled state.

Bupropion binds to two sites in the Torpedo nicotinic acetylcholine receptor transmembrane domain: a photoaffinity labeling study with the bupropion analogue [(125)I]-SADU-3-72.

Bupropion, a clinically used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analogue, (±)-2-(N-tert-butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone (SADU-3-72). Based on inhibition of [(125)I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC(50) = 0.8 µM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with 3-fold higher affinity in the desensitized (IC(50) = 1.2 µM) than in the resting state. Photolabeling of Torpedo nAChRs with [(125)I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [(125)I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, ßV8-22/23K, and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within ßV8-22/23K, γV8-24K, and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu(265), ßLeu(257)) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr(213) in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket.

(±)-2-(N-tert-Butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone: a dopamine transporter and nicotinic acetylcholine receptor photoaffinity ligand based on bupropion (Wellbutrin, Zyban).

Towards addressing the knowledge gap of how bupropion interacts with the dopamine transporter (DAT) and nicotinic acetylcholine receptors (nAChRs), a ligand was synthesized in which the chlorine of bupropion was isosterically replaced with an iodine and a photoreactive azide was added to the 4'-position of the aromatic ring. Analog (±)-3 (SADU-3-72) demonstrated modest DAT and α4ß2 nAChR affinity. A radioiodinated version was shown to bind covalently to hDAT expressed in cultured cells and affinity-purified, lipid-reincorporated human α4ß2 neuronal nAChRs. Co-incubation of (±)-[(125)I]-3 with non-radioactive (±)-bupropion or (-)-cocaine blocked labeling of these proteins. Compound (±)-[(125)I]-3 represents the first successful example of a DAT and nAChR photoaffinity ligand based on the bupropion scaffold. Such ligands are expected to assist in mapping bupropion-binding pockets within plasma membrane monoamine transporters and ligand-gated nAChR ion channels.

Structural characterization and agonist binding to human α4ß2 nicotinic receptors.

The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4ß2 neuronal nicotinic acetylcholine receptor. We show that human α4ß2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and ß-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition - particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4ß2 and Torpedo receptors, we begin to delineate their structural similarities and differences.

Hydrophobic photolabeling studies identify the lipid-protein interface of the 5-HT3A receptor.

A HEK-293 cell line that stably expresses mouse 5-HT(3A)Rs containing a C-terminal extension that confers high-affinity binding of alpha-bungarotoxin (alphaBgTx) was established (alphaBgTx-5-HT(3A)Rs) and used to purify alphaBgTx-5-HT(3A)Rs in a lipid environment for use in structural studies using photoaffinity labeling. alphaBgTx-5-HT(3A)Rs were expressed robustly (60 pmol of [(3)H]BRL-43694 binding sites (approximately 3 microg of receptor) per milligram of protein) and displayed the same functional properties as wild-type receptors (serotonin EC(50) = 5.3 +/- 0.04 microM). While [(125)I]alphaBgTx bound to the alphaBgTx-5-HT(3A)Rs with high affinity (K(d) = 11 nM), application of nonradioactive alphaBgTx (up to 300 microM) had no effect on serotonin-induced current responses. alphaBgTx-5-HT(3A)Rs were purified on an alphaBgTx-derivatized affinity column from detergent extracts in milligram quantities and at approximately 25% purity. The hydrophobic photolabel 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) was used to identify the amino acids at the lipid-protein interface of purified and lipid-reconstituted alphaBgTx-5-HT(3A)Rs. [(125)I]TID photoincorporation into the alphaBgTx-5-HT(3A)R subunit was initially mapped to subunit proteolytic fragments of 8 kDa, containing the M4 transmembrane segment and approximately 60% of incorporated (125)I, and 17 kDa, containing the M1-M3 transmembrane segments. Within the M4 segment, [(125)I]TID labeled Ser(451), equivalent to the [(125)I]TID-labeled residue Thr(422) at the lipid-exposed face of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha1M4 alpha-helix. These results provide a first definition of the surface of the 5-HT(3A)R M4 helix that is exposed to lipid and establish that this surface is equivalent to the surface exposed to lipid in the Torpedo nAChR.

[(3)H]Epibatidine photolabels non-equivalent amino acids in the agonist binding site of Torpedo and alpha4beta2 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo alpha(2)betagammadelta and expressed human alpha4beta2 nAChRs with [(3)H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of alphaTyr(198) in agonist binding site Segment C of the principal (+) face in both alpha subunits and of gammaLeu(109) and gammaTyr(117) in Segment E of the complementary (-) face, with no labeling detected in the delta subunit. For affinity-purified alpha4beta2 nAChRs, [(3)H]epibatidine photolabeled alpha4Tyr(195) (equivalent to Torpedo alphaTyr(190)) in Segment C as well as beta2Val(111) and beta2Ser(113) in Segment E (equivalent to Torpedo gammaLeu(109) and gammaTyr(111), respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the alpha-gamma transmitter binding site, whereas it binds in two distinct orientations in the alpha4beta2 nAChR.

Photoaffinity labeling the agonist binding domain of alpha4beta4 and alpha4beta2 neuronal nicotinic acetylcholine receptors with [(125)I]epibatidine and 5[(125)I]A-85380.

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human alpha4beta2 and rat alpha4beta4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [(125)I]epibatidine and 5[(125)I]A-85380. Our results show that both agonists photoincorporated into the beta4 subunit with little or no labeling of the beta2 and alpha4 subunits respectively. [(125)I]epibatidine labeling in the beta4 subunit was mapped to two overlapping proteolytic fragments that begin at beta4V102 and contain Loop E (beta4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that beta4Q117 in Loop E is the principal site of [(125)I]epibatidine labeling. This was accomplished by substituting residues in the beta2 subunit with the beta4 homologs and finding [(125)I]epibatidine labeling in beta4 and beta2F119Q subunits with little, if any, labeling in alpha4, beta2, or beta2S113R subunits. Finally, functional studies established that the beta2F119/beta4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.

Probing the structure of the affinity-purified and lipid-reconstituted torpedo nicotinic acetylcholine receptor.

The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical and biophysical techniques, detergent solubilization, purification, and lipid reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) to compare the state-dependent photolabeling of the Torpedo nAChR before and after purification and reincorporation into lipid. For the purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9, and M2-13, the agonist-enhanced labeling of deltaThr274 (deltaM2-18) within the delta subunit helix bundle, and the labeling at the lipid-protein interface (alphaMu4) were the same as for the nAChR in native membranes. However, addition of agonist did not enhance [(125)I]TID photolabeling of deltaIle288 within the deltaM2-M3 loop. These results indicate that after purification and reconstitution of the Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the deltaM2-M3 loop. To further characterize the pharmacology of [(125)I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [(125)I]TID photolabeling. PCP inhibited [(125)I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6) while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the delta subunit helix bundle.

Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-azidoimipramine photolabeling and molecular dynamics studies.

Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [3H]imipramine binding to resting (closed) and desensitized nAChRs. [3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with approximately 25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the alpha subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a approximately 20 kDa Staphylococcus aureus V8 protease fragment (alphaV8-20; Ser173-Glu338). To further map the labeling site, the alphaV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (alphaMet243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel.

Identifying the lipid-protein interface of the alpha4beta2 neuronal nicotinic acetylcholine receptor: hydrophobic photolabeling studies with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine.

Using an acetylcholine-derivatized affinity column, we have purified human alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) from a stably transfected HEK-293 cell line. Both the quantity and the quality of the purified receptor are suitable for applying biochemical methods to directly study the structure of the alpha4beta2 nAChR. In this first study, the lipid-protein interface of purified and lipid-reconstituted alpha4beta2 nAChRs was directly examined using photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID photoincorporated into both alpha4 and beta2 subunits, and for each subunit the labeling was initially mapped to fragments containing the M4 and M1-M3 transmembrane segments. For both the alpha4 and beta2 subunits, approximately 60% of the total labeling was localized within fragments that contain the M4 segment, which suggests that the M4 segment has the greatest exposure to lipid. Within M4 segments, [125I]TID labeled homologous amino acids alpha4-Cys582/beta2-Cys445, which are also homologous to the [125I]TID-labeled residues alpha1-Cys418 and beta1-Cys447 in the lipid-exposed face of Torpedo nAChR alpha1M4 and beta1M4, respectively. Within the alpha4M1 segment, [125I]TID labeled residues Cys226 and Cys231, which correspond to the [125I]TID-labeled residues Cys222 and Phe227 at the lipid-exposed face of the Torpedo alpha1M1 segment. In beta2M1, [125I]TID labeled beta2-Cys220, which is homologous to alpha4-Cys226. We conclude from these studies that the alpha4beta2 nAChR can be purified from stably transfected HEK-293 cells in sufficient quantity and purity for structural studies and that the lipid-protein interfaces of the neuronal alpha4beta2 nAChR and the Torpedo nAChR display a high degree of structural homology.

The hypothalamic-pituitary-thyroid (HPT) axis in fish and its role in fish development and reproduction.

Bony fishes represent the largest vertebrate class and are a very diverse animal group. This chapter provides a thorough review of the available scientific literature on the thyroid system in these important vertebrate animals. The molecular components of the hypothalamic-pituitary-thyroid (HPT) axis in this group correspond closely to those of mammals. The thyroid tissue in the fishes is organized as diffuse follicles, with a few exceptions, rather than as an encapsulated gland as is found in most other vertebrate species. The features of this diffuse tissue in fishes are reviewed with an emphasis on feedback relationships within the HPT axis, the molecular biology of the thyroid system in fishes, and comparisons versus the thyroid systems of other vertebrate taxa. A review of the role of thyroid hormone in fish development and reproduction is included. Available information about the HPT axis in fishes is quite detailed for some species and rather limited or absent in others. This review focuses on species that have been intensively studied for their value as laboratory models in assays to investigate disruption in normal function of the thyroid system. In addition, in vitro and in vivo assay methods for screening chemicals for their potential to interfere with the thyroid system are reviewed. It is concluded that there are currently no in vitro or in vivo assays in fish species that are sufficiently developed to warrant recommendation for use to efficiently screen chemicals for thyroid disruption. Methods are available that can be used to measure thyroid hormones, although our ability to interpret the causes and implications of potential alterations in T4 or T3 levels in fishes is nonetheless limited without further research.