David D. Mowrey

Asymmetric Ligand Binding Facilitates Conformational Transitions in Pentameric Ligand-Gated Ion Channels

The anesthetic propofol inhibits the currents of the homopentameric ligand-gated ion channel GLIC, yet the crystal structure of GLIC with five propofol molecules bound symmetrically shows an open-channel conformation. To address this dilemma and determine if the symmetry of propofol binding sites affects the channel conformational transition, we performed a total of 1.5 μs of molecular dynamics simulations for different GLIC systems with propofol occupancies of 0, 1, 2, 3, and 5. GLIC without propofol binding or with five propofol molecules bound symmetrically, showed similar channel conformation and hydration status over multiple replicates of 100-ns simulations. In contrast, asymmetric binding to one, two or three equivalent sites in different subunits accelerated the channel dehydration, increased the conformational heterogeneity of the pore-lining TM2 helices, and shifted the lateral and radial tilting angles of TM2 toward a closed-channel conformation. The results differentiate two groups of systems based on the propofol binding symmetry. The difference between symmetric and asymmetric groups is correlated with the variance in the propofol-binding cavity adjacent to the hydrophobic gate and the force imposed by the bound propofol. Asymmetrically bound propofol produced greater variance in the cavity size that could further elevate the conformation heterogeneity. The force trajectory generated by propofol in each subunit over the course of a simulation exhibits an ellipsoidal shape, which has the larger component tangential to the pore. Asymmetric propofol binding creates an unbalanced force that expedites the channel conformation transitions. The findings from this study not only suggest that asymmetric binding underlies the propofol functional inhibition of GLIC, but also advocate for the role of symmetry breaking in facilitating channel conformational transitions.

Isoflurane Alters the Structure and Dynamics of GLIC

Pentameric ligand-gated ion channels are targets of general anesthetics. Although the search for discrete anesthetic binding sites has achieved some degree of success, little is known regarding how anesthetics work after the events of binding. Using the crystal structures of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), which is sensitive to a variety of general anesthetics, we performed multiple molecular dynamics simulations in the presence and absence of the general anesthetic isoflurane. Isoflurane bound to several locations within GLIC, including the transmembrane pocket identified crystallographically, the extracellular (EC) domain, and the interface of the EC and transmembrane domains. Isoflurane also entered the channel after the pore was dehydrated in one of the simulations. Isoflurane disrupted the quaternary structure of GLIC, as evidenced in a striking association between the binding and breakage of intersubunit salt bridges in the EC domain. The pore-lining helix experienced lateral and inward radial tilting motion that contributed to the channel closure. Isoflurane binding introduced strong anticorrelated motions between different subunits of GLIC. The demonstrated structural and dynamical modulations by isoflurane aid in the understanding of the underlying mechanism of anesthetic inhibition of GLIC and possibly other homologous pentameric ligand-gated ion channels.

NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the β2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., [32]). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions.

NMR structures of the transmembrane domains of the α4β2 nAChR.

The α4β2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4β2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and β2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and β2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na(+) flux assay revealed that α4β2 formed Na(+) permeable channels in lipid vesicles. Efflux of Na(+) through the α4β2 channels reduced intra-vesicle Sodium Green™ fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4β2. The study provides structural insight into the TM domains of the α4β2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4β2 nAChR and for designing new therapeutic modulators.

Insights into Distinct Modulation of α7 and α7β2 Nicotinic Acetylcholine Receptors by the Volatile Anesthetic Isoflurane

Background: What determines hypersensitivity or insensitivity of β2-containing or α7 nAChRs to volatile anesthetics remains unclear. Results: Isoflurane binds to the EC end of the TM domain, modulates channel dynamics, and inhibits channel current in β2 not α7. Conclusion: The dynamic and structural differences between β2 and α7 affect isoflurane binding and inhibition. Significance: Both structure and dynamics are critical for drug action. Nicotinic acetylcholine receptors (nAChRs) are targets of general anesthetics, but functional sensitivity to anesthetic inhibition varies dramatically among different subtypes of nAChRs. Potential causes underlying different functional responses to anesthetics remain elusive. Here we show that in contrast to the α7 nAChR, the α7β2 nAChR is highly susceptible to inhibition by the volatile anesthetic isoflurane in electrophysiology measurements. Isoflurane-binding sites in β2 and α7 were found at the extracellular and intracellular end of their respective transmembrane domains using NMR. Functional relevance of the identified β2 site was validated via point mutations and subsequent functional measurements. Consistent with their functional responses to isoflurane, β2 but not α7 showed pronounced dynamics changes, particularly for the channel gate residue Leu-249(9′). These results suggest that anesthetic binding alone is not sufficient to generate functional impact; only those sites that can modulate channel dynamics upon anesthetic binding will produce functional effects.

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.

NMR structure and dynamics of a designed water-soluble transmembrane domain of nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828-1833, 2004; Ma et al., PNAS, 105: 16537-42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ~83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T(2) dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.

ELIC-α7 Nicotinic Acetylcholine Receptor (α7nAChR) Chimeras Reveal a Prominent Role of the Extracellular-Transmembrane Domain Interface in Allosteric Modulation

Background: Allosteric modulators bound to the transmembrane domain (TMD) of the α7 nicotinic acetylcholine receptor (α7nAChR) can potentiate channel function. Results: ELIC-α7nAChR showed potentiation only when the extracellular-transmembrane domain (ECD-TMD) interface matched that of α7nAChR. Conclusion: PAM modulation through the TMD requires a more specific ECD-TMD interface than agonist activation. Significance: The study provides insight into the basis for positive allosteric modulation of α7nAChR. The native α7 nicotinic acetylcholine receptor (α7nAChR) is a homopentameric ligand-gated ion channel mediating fast synaptic transmission and is of pharmaceutical interest for treatment of numerous disorders. The transmembrane domain (TMD) of α7nAChR has been identified as a target for positive allosteric modulators (PAMs), but it is unclear whether modulation occurs through changes entirely within the TMD or changes involving both the TMD and the extracellular domain (ECD)-TMD interface. In this study, we constructed multiple chimeras using the TMD of human α7nAChR and the ECD of a prokaryotic homolog, ELIC, which is not sensitive to these modulators, and for which a high resolution structure has been solved. Functional ELIC-α7nAChR (EA) chimeras were obtained when their ECD-TMD interfaces were modified to resemble either the ELIC interface (EAELIC) or α7nAChR interface (EAα7). Both EAα7 and EAELIC show similar activation response and desensitization characteristics, but only EAα7 retained the unique pharmacology of α7nAChR evoked by PAMs, including potentiation by ivermectin, PNU-120596, and TQS, as well as activation by 4BP-TQS. This study suggests that PAM modulation through the TMD has a more stringent requirement at the ECD-TMD interface than agonist activation.

Unresponsive Correlated Motion in α7 nAChR to Halothane Binding Explains Its Functional Insensitivity to Volatile Anesthetics

Neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated as targets for general anesthetics, but the functional responses to anesthetic modulation vary considerably among different subtypes of nAChRs. Inhaled general anesthetics, such as halothane, could effectively inhibit the channel activity of the alpha4beta2 nAChR but not the homologous alpha7 nAChR. To understand why alpha7 is insensitive to inhaled general anesthetics, we performed multiple sets of 20 ns molecular dynamics (MD) simulations on the closed- and open-channel alpha7 in the absence and presence of halothane and critically compared the results with those from our studies on the alpha4beta2 nAChR (Liu et al. J. Phys. Chem. B 2009, 113, 12581 and Liu et al. J. Phys. Chem. B 2010, 114, 626). Several halothane binding sites with fairly high binding affinities were identified in both closed- and open-channel alpha7, suggesting that a lack of sensitive functional responses of the alpha7 nAChR to halothane in the previous experiments was unlikely due to a lack of halothane interaction with alpha7. The binding affinities of halothane in alpha7 seemed to be protein conformation-dependent. Overall, halothane affinity was higher in the closed-channel alpha7. Halothane binding to alpha7 did not induce profound changes in alpha7 structure and dynamics that could be related to the channel function. In contrast, correlated motion of the open-channel alpha4beta2 was reduced substantially in the presence of halothane, primarily due to the more susceptible nature of beta2 to anesthetic modulation. The amphiphilic extracellular and transmembrane domain interface of the beta2 subunit is attractive to halothane and is susceptible to halothane perturbation, which may be why alpha4beta2 is functionally more sensitive to halothane than alpha7.

Signal Transduction Pathways in the Pentameric Ligand- Gated Ion Channels

The mechanisms of allosteric action within pentameric ligand-gated ion channels (pLGICs) remain to be determined. Using crystallography, site-directed mutagenesis, and two-electrode voltage clamp measurements, we identified two functionally relevant sites in the extracellular (EC) domain of the bacterial pLGIC from Gloeobacter violaceus (GLIC). One site is at the C-loop region, where the NQN mutation (D91N, E177Q, and D178N) eliminated inter-subunit salt bridges in the open-channel GLIC structure and thereby shifted the channel activation to a higher agonist concentration. The other site is below the C-loop, where binding of the anesthetic ketamine inhibited GLIC currents in a concentration dependent manner. To understand how a perturbation signal in the EC domain, either resulting from the NQN mutation or ketamine binding, is transduced to the channel gate, we have used the Perturbation-based Markovian Transmission (PMT) model to determine dynamic responses of the GLIC channel and signaling pathways upon initial perturbations in the EC domain of GLIC. Despite the existence of many possible routes for the initial perturbation signal to reach the channel gate, the PMT model in combination with Yens algorithm revealed that perturbation signals with the highest probability flow travel either via the β1–β2 loop or through pre-TM1. The β1–β2 loop occurs in either intra- or inter-subunit pathways, while pre-TM1 occurs exclusively in inter-subunit pathways. Residues involved in both types of pathways are well supported by previous experimental data on nAChR. The direct coupling between pre-TM1 and TM2 of the adjacent subunit adds new insight into the allosteric signaling mechanism in pLGICs.