Battle of the sections: Student outcomes and course feedback support combined prosection and dissection laboratory formats to maximize student success.

Gross anatomy laboratories frequently utilize dissection or prosection formats within medical curricula. Practical examination scores are consistent across the formats, yet these examinations assessed larger anatomical structures. In contrast, a single report noted improved scores when prosection was used in the hand and foot regions, areas that are more difficult to dissect. The incorporation of prosected donors within "Head and Neck" laboratories provided an opportunity to further characterize the impact of prosection in a structurally complex area. Retrospective analysis of 21 Head and Neck practical examination questions was completed to compare scores among cohorts that utilized dissection exclusively or incorporated prosection. Mean scores of practical examination questions were significantly higher in the prosection cohort (84.27% ± 12.69) as compared with the dissection cohort (75.59% ± 12.27) (p < 0.001). Of the 12 questions that performed better in the prosection cohort (88.42% ± 8.21), 10 items mapped to deeper anatomical regions. By comparison, eight of nine questions in the dissection cohort outperformed (88.44% ± 3.34) the prosection cohort (71.74% ± 18.11), and mapped to anatomically superficial regions. Despite the mean score increase with positional location of the questions, this effect was not statically significant across cohorts (p = 1.000), suggesting that structure accessibility in anatomically complex regions impacts performance. Student feedback cited structure preservation (71.5%) and time savings (55.8%) as advantages to prosection; however, dissection was the perceived superior and preferred laboratory format (88.6%). These data support combined prosection and dissection formats for improving student recognition of deeply positioned structures and maximizing student success.

Analysis of Nitroxide-Based Distance Measurements in Cell Extracts and in Cells by Pulsed ESR Spectroscopy.

Measurements of distances in cells by pulsed ESR spectroscopy afford tremendous opportunities to study proteins in native environments that are irreproducible in vitro. However, the in-cell environment is harsh towards the typical nitroxide radicals used in double electron-electron resonance (DEER) experiments. A systematic examination is performed on the loss of the DEER signal, including contributions from nitroxide decay and nitroxide side-chain cleavage. In addition, the possibility of extending the lifetime of the nitroxide radical by use of an oxidizing agent is investigated. Using this oxidizing agent, DEER distance measurements are performed on doubly nitroxide-labeled GB1, the immunoglobulin-binding domain of protein G, at varying incubation times in the cellular environment. It is found that, by comparison of the loss of DEER signal to the loss of the CW spectrum, cleavage of the nitroxide side chain contributes to the loss of DEER signal, which is significantly greater in cells than in cell extracts. Finally, local spin concentrations are monitored at varying incubation times to show the time required for molecular diffusion of a small globular protein within the cellular milieu.

Structural Basis of Alcohol Inhibition of the Pentameric Ligand-Gated Ion Channel ELIC.

The structural basis for alcohol modulation of neuronal pentameric ligand-gated ion channels (pLGICs) remains elusive. We determined an inhibitory mechanism of alcohol on the pLGIC Erwinia chrysanthemi (ELIC) through direct binding to the pore. X-ray structures of ELIC co-crystallized with 2-bromoethanol, in both the absence and presence of agonist, reveal 2-bromoethanol binding in the pore near T237(6') and the extracellular domain (ECD) of each subunit at three different locations. Binding to the ECD does not appear to contribute to the inhibitory action of 2-bromoethanol and ethanol as indicated by the same functional responses of wild-type ELIC and mutants. In contrast, the ELIC-α1ß3GABAAR chimera, replacing the ELIC transmembrane domain (TMD) with the TMD of α1ß3GABAAR, is potentiated by 2-bromoethanol and ethanol. The results suggest a dominant role of the TMD in modulating alcohol effects. The X-ray structures and functional measurements support a pore-blocking mechanism for inhibitory action of short-chain alcohols.

Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac.

Voltage-gated sodium channels (NaV) play an important role in general anesthesia. Electrophysiology measurements suggest that volatile anesthetics such as isoflurane inhibit NaV by stabilizing the inactivated state or altering the inactivation kinetics. Recent computational studies suggested the existence of multiple isoflurane binding sites in NaV, but experimental binding data are lacking. Here we use site-directed placement of 19F probes in NMR experiments to quantify isoflurane binding to the bacterial voltage-gated sodium channel NaChBac. 19F probes were introduced individually to S129 and L150 near the S4-S5 linker, L179 and S208 at the extracellular surface, T189 in the ion selectivity filter, and all phenylalanine residues. Quantitative analyses of 19F NMR saturation transfer difference (STD) spectroscopy showed a strong interaction of isoflurane with S129, T189, and S208; relatively weakly with L150; and almost undetectable with L179 and phenylalanine residues. An orientation preference was observed for isoflurane bound to T189 and S208, but not to S129 and L150. We conclude that isoflurane inhibits NaChBac by two distinct mechanisms: (i) as a channel blocker at the base of the selectivity filter, and (ii) as a modulator to restrict the pivot motion at the S4-S5 linker and at a critical hinge that controls the gating and inactivation motion of S6.

Common Anesthetic-binding Site for Inhibition of Pentameric Ligand-gated Ion Channels.

BACKGROUND: Identifying functionally relevant anesthetic-binding sites in pentameric ligand-gated ion channels (pLGICs) is an important step toward understanding the molecular mechanisms underlying anesthetic action. The anesthetic propofol is known to inhibit cation-conducting pLGICs, including a prokaryotic pLGIC from Erwinia chrysanthemi (ELIC), but the sites responsible for functional inhibition remain undetermined. METHODS: We photolabeled ELIC with a light-activated derivative of propofol (AziPm) and performed fluorine-19 nuclear magnetic resonance experiments to support propofol binding to a transmembrane domain (TMD) intrasubunit pocket. To differentiate sites responsible for propofol inhibition from those that are functionally irrelevant, we made an ELIC-γ-aminobutyric acid receptor (GABAAR) chimera that replaced the ELIC-TMD with the α1ß3GABAAR-TMD and compared functional responses of ELIC-GABAAR and ELIC with propofol modulations. RESULTS: Photolabeling showed multiple AziPm-binding sites in the extracellular domain (ECD) but only one site in the TMD with labeled residues M265 and F308 in the resting state of ELIC. Notably, this TMD site is an intrasubunit pocket that overlaps with binding sites for anesthetics, including propofol, found previously in other pLGICs. Fluorine-19 nuclear magnetic resonance experiments supported propofol binding to this TMD intrasubunit pocket only in the absence of agonist. Functional measurements of ELIC-GABAAR showed propofol potentiation of the agonist-elicited current instead of inhibition observed on ELIC. CONCLUSIONS: The distinctly different responses of ELIC and ELIC-GABAAR to propofol support the functional relevance of propofol binding to the TMD. Combining the newly identified TMD intrasubunit pocket in ELIC with equivalent TMD anesthetic sites found previously in other cationic pLGICs, we propose this TMD pocket as a common site for anesthetic inhibition of pLGICs.

Direct Pore Binding as a Mechanism for Isoflurane Inhibition of the Pentameric Ligand-gated Ion Channel ELIC.

Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but molecular mechanisms underlying anesthetic action remain debatable. We found that ELIC, a pLGIC from Erwinia chrysanthemi, can be functionally inhibited by isoflurane and other anesthetics. Structures of ELIC co-crystallized with isoflurane in the absence or presence of an agonist revealed double isoflurane occupancies inside the pore near T237(6') and A244(13'). A pore-radius contraction near the extracellular entrance was observed upon isoflurane binding. Electrophysiology measurements with a single-point mutation at position 6' or 13' support the notion that binding at these sites renders isoflurane inhibition. Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation. This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC.

Structural rearrangements underlying functional transitions of pentameric ligand-gated ion channels (pLGICs) are not fully understood. Using (19)F nuclear magnetic resonance and electron spin resonance spectroscopy, we found that ELIC, a pLGIC from Erwinia chrysanthemi, expanded the extracellular end and contracted the intracellular end of its pore during transition from the resting to an apparent desensitized state. Importantly, the contraction at the intracellular end of the pore likely forms a gate to restrict ion transport in the desensitized state. This gate differs from the hydrophobic gate present in the resting state. Conformational changes of the TM2-TM3 loop were limited to the N-terminal end. The TM4 helices and the TM3-TM4 loop appeared relatively insensitive to agonist-mediated structural rearrangement. These results indicate that conformational changes accompanying functional transitions are not uniform among different ELIC regions. This work also revealed the co-existence of multiple conformations for a given state and suggested asymmetric conformational arrangements in a homomeric pLGIC.

Atomistic insights into human Cys-loop receptors by solution NMR.

Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) mediating fast neurotransmission in the central and peripheral nervous systems. They are important targets for many currently used clinical drugs, such as general anesthetics, and for allosteric modulators with potential therapeutic applications. Here, we provide an overview of advances in the use of solution NMR in structural and dynamic characterization of ion channels, particularly human Cys-loop receptors. We present challenges to overcome and realistic solutions for achieving high-resolution structural information for this family of receptors. We discuss how subtle structural differences among homologous channels define unique channel pharmacological properties and advocate the necessity to determine high-resolution structures for individual receptor subtypes. Finally, we describe drug binding to the TMDs of Cys-loop receptors identified by solution NMR and the associated dynamics changes relevant to channel functions.