Gloeobacter Violaceus Ligand-gated Ion Channel Research Articles

A bupropion modulatory site in Gloeobacter violaceus ligand-gated ion channel

Bupropion is an atypical antidepressant and smoking cessation drug that causes adverse effects such as insomnia, irritability, and anxiety. Bupropion inhibits dopamine and norepinephrine reuptake transporters and eukaryotic cation-conducting pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine (nACh) and serotonin type 3A (5-HT3A) receptors, at clinically relevant concentrations. Here, we demonstrate that bupropion also inhibits a prokaryotic homolog of pLGICs, the Gloeobacter violaceus ligand-gated ion channel (GLIC). Using GLIC as a model, we used molecular docking to predict binding sites for bupropion. Several clusters of bupropion binding pose within the transmembrane domain were identified, with the predominant cluster being localized to the interface between transmembrane segments M1 and M3 of adjacent subunits. Residues W213, T214 and W217 in the first transmembrane segment, M1, and F267 and I271 in the third transmembrane segment, M3, most frequently reside within 4 Å distance from bupropion. We then used single amino acid substitutions at these positions and two-electrode voltage-clamp recordings to determine their impact on bupropion inhibitory effects. The substitution T214F alters bupropion potency by shifting the IC50 to a 13-fold higher value compared to wild-type GLIC. Residue T214 is found within a previously identified binding pocket for neurosteroids and lipids in GLIC. This intersubunit binding pocket is structurally conserved and almost identical to a binding pocket described for neurosteroids in GABAA receptors. Our data thus suggest that T214 that lines a previously identified lipophilic binding pocket in GLIC and GABAA receptors is also a modulatory site for bupropion interaction with GLIC.

Short-chain mono-carboxylates as negative modulators of allosteric transitions in Gloeobacter violaceus ligand-gated ion channel, and impact of a pre-β5 strand (Loop Ω) double mutation on crotonate, not butyrate effect.

Using the bacterial proton-activated pentameric receptor-channel Gloeobacter violaceus ligand-gated ion channel (GLIC): (1) We characterize saturated, mono-carboxylates as negative modulators of GLIC (as previously shown for crotonate; Alqazzaz etal., Biochemistry, 2016, 55, 5947). Butyrate and crotonate have indistinguishable properties regarding negative modulation of wt GLIC. (2) We identify a locus in the pre-β5 strand (Loop Ω) whose mutation inverses the effect of the mono-carboxylate crotonate from negative to positive modulation of the allosteric transitions, suggesting an involvement of the pre-β5 strand in coupling the extracellular orthotopic receptor to pore gating. (3) As an extension to the previously proposed "in series" mechanism, we suggest that a orthotopic/orthosteric site-vestibular site-Loop Ω-β5-β6 "sandwich"-Pro-Loop/Cys-Loop series may be an essential component of orthotopic/orthosteric compound-elicited gating control in this pentameric ligand-gated ion channel, on top of which compounds targeting the vestibular site may provide modulation.

Fumarate as positive modulator of allosteric transitions in the pentameric ligand-gated ion channel GLIC: requirement of an intact vestibular pocket.

Gloeobacter violaceus ligand-gated ion channel (GLIC) is a prokaryotic orthologue of brain pentameric neurotransmitter receptors. Using whole-cell patch-clamp electrophysiology in a host cell line, we show that short-chain dicarboxylate compounds are positive modulators of pHo5-evoked GLIC activity, with a rank order of action fumarate > succinate > malonate > glutarate. Potentiation by fumarate depends on intracellular pH, mainly as a result of a strong decrease of the pHo5-evoked current when intracellular pH decreases. The modulating effect of fumarate also depends on extracellular pH, as fumarate is a weak inhibitor at pHo6 and shows no agonist action at neutral pHo. A mutational analysis of residue dependency for succinate and fumarate effects, based on two carboxylate-binding pockets previously identified by crystallography (Fourati etal., 2020), shows that positive modulation involves both the inter-subunit pocket, homologous to the neurotransmitter-binding orthotopic site, and the intra-subunit (also called vestibular) pocket. An almost similar pattern of mutational impact is observed for the effect of caffeate, a known negative modulator. We propose, for both dicarboxylate compounds and caffeate, a model where the inter-subunit pocket is the actual binding site, and the region corresponding to the vestibular pocket is required either for inter-subunit binding itself, or for binding-to-gating coupling during the allosteric transitions involved in pore-gating modulation. KEY POINTS: Using a bacterial orthologue of brain pentameric neurotransmitter receptors, we show that the orthotopic/orthosteric agonist site and the adjacent vestibular region are functionally interdependent in mediating compound-elicited modulation. We propose that the two sites in the extracellular domain are involved 'in series', a mechanism which may have relevance for eukaryote receptors. We show that short-chain dicarboxylate compounds are positive modulators of the Gloeobacter violaceus ligand-gated ion channel (GLIC). The most potent compound identified is fumarate, known to occupy the orthotopic/orthosteric site in previously published crystal structures. We show that intracellular pH modulates GLIC allosteric transitions, as previously known for extracellular pH. We report a caesium to sodium permeability ratio (PCs /PNa ) of 0.54 for GLIC ion pore.

Dynamic closed states of a ligand-gated ion channel captured by cryo-EM and simulations.

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high-quality structures in multiple, subtly distinct functional states. However, structural data in this family remain limited, particularly for resting and intermediate states on the activation pathway. Here, we report cryo-electron microscopy (cryo-EM) structures of the proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC) under three pH conditions. Decreased pH was associated with improved resolution and side chain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the β1-β2 and M2-M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryo-EM classes further indicated a low-pH population with an expanded pore. These results allow us to define distinct protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.

Heterogeneous Populations of Ligand-Gated Ion Channel, GLIC, Revealed by Cryo-Electron Microscopy and Simulations

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological development in this family relies on high-quality structural data in multiple, subtly distinct functional states; however, structural data remain limited, particularly for the native closed state. Here, we report cryo-electron microscopy structures of the proton-gated Gloeobacter violaceus ligand-gated ion channel (GLIC) under resting and activating conditions (pH 7, 5, and 3).

Solution Structure of the Gloeobacter Violaceus Ligand-Gated Ion Channel Probed by Small-Angle Neutron Scattering

Pentameric ligand-gated ion channels transform chemical signals into electrical ones, a process during which they undergo subtle conformational cycling resulting in rapid, reversible gating of an intrinsic transmembrane pore. The pH-gated bacterial channel GLIC has proved a valuable model system for this receptor family, in part due to its accessibility to X-ray crystallography - which has provided structures of closed, open, and intermediate conformations. However, such models raise critical questions as to whether the X-ray structures represent actual functional states, and if the cycle includes additional conformations such as highly contracted or expanded states. Small-angle neutron scattering (SANS) offers an approach to characterizing solution-phase structure, increasingly applicable to biomolecular problems on this scale. Here we characterized GLIC by SANS, in combination with inline size-exclusion chromatography (SEC), match-out deuterated detergents, and computational modeling of multiple putative structural models. SEC-SANS under resting conditions (pH 7) enabled the characterization of non-aggegated species which fit predictions from resting-state models, particularly following unrestrained molecular dynamics simulation of X-ray structures. Curves collected under activating conditions (pH 3) best fit a mixture of resting- and open-state models, whereas they diverged from expanded-pore models. in addition to supporting less-expanded models of the open state, this work demonstrates that SEC-SANS, in combination with molecular modeling and simulations, can distinguish solution-phase functional states of an ion channel. As neutron source brilliance increases and inline SEC-SANS set-ups become increasingly accessible, such methods will offer valuable tools for the elucidation of receptor conformational cycling and pharmaceutical development.

Modulation of the Gloeobacter violaceus Ion Channel by Fentanyl: A Molecular Dynamics Study.

Fentanyl is an opioid analgesic, which is routinely used in general surgery to suppress the sensation of pain and as the analgesic component in the induction and maintenance of anesthesia. Fentanyl is also used as the main component to induce anesthesia and as a potentiator to the general anesthetic propofol. The mechanism by which fentanyl induces its anesthetic action is still unclear, and we have therefore employed fully atomistic molecular dynamics simulations to probe this process by simulating the interactions of fentanyl with the Gloeobacter violaceus ligand-gated ion channel (GLIC). In this paper, we identify multiple extracellular fentanyl binding sites, which are different from the transmembrane general anesthetic binding sites observed for propofol and other general anesthetics. Our simulations identify a novel fentanyl binding site within the GLIC that results in conformational changes that inhibit conduction through the channel.

Large Intracellular Domain-Dependent Effects of Positive Allosteric Modulators on Glycine Receptors.

Glycine receptors (GlyRs) are members of the pentameric ligand-gated ionic channel family (pLGICs) and mediate fast inhibitory neurotransmission in the brain stem and spinal cord. The function of GlyRs can be modulated by positive allosteric modulators (PAMs). So far, it is largely accepted that both the extracellular (ECD) and transmembrane (TMD) domains constitute the primary target for many of these PAMs. On the other hand, the contribution of the intracellular domain (ICD) to the PAM effects on GlyRs remains poorly understood. To gain insight about the role of the ICD in the pharmacology of GlyRs, we examined the contribution of each domain using a chimeric receptor. Two chimeras were generated, one consisting of the ECD of the prokaryotic homologue Gloeobacter violaceus ligand-gated ion channel (GLIC) fused to the TMD of the human α1GlyR lacking the ICD (Lily) and a second with the ICD (Lily-ICD). The sensitivity to PAMs of both chimeric receptors was studied using electrophysiological techniques. The Lily receptor showed a significant decrease in the sensitivity to four recognized PAMs. Remarkably, the incorporation of the ICD into the Lily background was sufficient to restore the wild-type α1GlyR sensitivity to these PAMs. Based on these data, we can suggest that the ICD is necessary to form a pLGIC having full sensitivity to positive allosteric modulators.

Probing the Binding Site(S) of Bupropion in Glic by Site-Directed Mutagenesis

Bupropion, an atypical antidepressant, has been shown to ameliorate the neurotransmission of both dopamine (DA) and norepinephrine (NE) by inhibiting their respective transporters in the brain. However, the clinical efficacy of bupropion is likely not due solely to its inhibition of DA and NE reuptake. The poor understanding of bupropion's mechanism of action (MOA) stems from the diversity of its molecular targets. Indeed, bupropion additionally inhibits key members of the Cys-loop superfamily, broadly known as pentameric ligand-gated ion channels (pLGIC). Allosteric inhibition by bupropion of some of the nicotinic acetylcholine receptors (nAChRs) has been implicated in its clinical efficacy as a smoking cessation aid. We recently reported for the first time that, in addition to bupropion's currently known actions, it is also an allosteric antagonist of the serotonin type 3A receptor (5-HT3AR). Our new finding adds another dimension to the complex conundrum of bupropion's MOA, and suggests an additional pharmacological principle underlying its efficacy as an antidepressant. In the continuum of exploring bupropion's actions on several members of pLGICs, we now are investigating bupropion's functional and binding interactions with the Gloeobacter violaceus ligand-gated ion channel (GLIC), a prokaryotic homologue of pentameric ligand-gated ion channels. To achieve the stated goals of our investigation, we have been utilizing an array of techniques including molecular docking, site-directed mutagenesis, cysteine modification, and two-electrode voltage clamp recording (TEVC). Further studies are in progress to identify specific interaction site(s) in GLIC, which can be conserved across the eukaryotic members of pLGICs.

Structural titration of receptor ion channel GLIC gating by HS-AFM

Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated, cation-selective channel, is a prokaryotic homolog of the pentameric Cys-loop receptor ligand-gated ion channel family. Despite large changes in ion conductance, small conformational changes were detected in X-ray structures of detergent-solubilized GLIC at pH 4 (active/desensitized state) and pH 7 (closed state). Here, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to perform structural titration experiments to visualize GLIC gating at the single-molecule level under native conditions. Reference-free 2D classification revealed channels in multiple conformational states during pH gating. We find changes of protein-protein interactions so far elusive and conformational dynamics much larger than previously assumed. Asymmetric pentamers populate early stages of activation, which provides evidence for an intermediate preactivated state.

Expansion of a Transmembrane Cavity Facilitates Anesthetic Potentiation of a Pentameric Ligand Gated Ion Channel

Ligand-gated ion channels are important molecular targets of general anesthetics, some of which have been in use since the mid 19th century. However, the molecular mechanism of these drug-receptor interactions is poorly understood, creating barriers to the development of new anesthetics. Biochemical and electrophysiological studies have shown that general anesthetics bind to small pockets in Cys-loop receptors, also known as pentameric ligand-gated ion channels. In addition, recent X-ray structures of the Gloeobacter violaceus ligand-gated ion channel show differential binding of the common anesthetic propofol in the presence of sensitizing mutations. In this work, we used molecular dynamics simulations to investigate how anesthetic binding relates to receptor structure. We found a correlation between anesthetic occupancy and binding site expansion. This correlation was removed by a mutation that increased functional sensitivity to anesthetic potentiation. Contraction of the apo binding site from the expanded conformation was associated with a pinching motion of two transmembrane helices, this motion being less pronounced in the mutant than in the wild-type channel. The sensitizing mutation also influenced flexibility of the mutated amino acid side chain and the bound anesthetic. These novel dynamic observations enabled us to refine an allosteric mechanism for bimodal anesthetic modulation in the Cys-loop receptor family, and provided testable hypotheses for further structure-function experiments.

Full mutational mapping of titratable residues helps to identify proton-sensors involved in the control of channel gating in the Gloeobacter violaceus pentameric ligand-gated ion channel.

The Gloeobacter violaceus ligand-gated ion channel (GLIC) has been extensively studied by X-ray crystallography and other biophysical techniques. This provided key insights into the general gating mechanism of pentameric ligand-gated ion channel (pLGIC) signal transduction. However, the GLIC is activated by lowering the pH and the location of its putative proton activation site(s) still remain(s) unknown. To this end, every Asp, Glu, and His residue was mutated individually or in combination and investigated by electrophysiology. In addition to the mutational analysis, key mutations were structurally resolved to address whether particular residues contribute to proton sensing, or alternatively to GLIC-gating, independently of the side chain protonation. The data show that multiple residues located below the orthosteric site, notably E26, D32, E35, and D122 in the lower part of the extracellular domain (ECD), along with E222, H235, E243, and H277 in the transmembrane domain (TMD), alter GLIC activation. D122 and H235 were found to also alter GLIC expression. E35 is identified as a key proton-sensing residue, whereby neutralization of its side chain carboxylate stabilizes the active state. Thus, proton activation occurs allosterically to the orthosteric site, at the level of multiple loci with a key contribution of the coupling interface between the ECD and TMD.

A membrane-embedded pathway delivers general anesthetics to two interacting binding sites in the Gloeobacter violaceus ion channel

General anesthetics exert their effects on the central nervous system by acting on ion channels, most notably pentameric ligand-gated ion channels. Although numerous studies have focused on pentameric ligand-gated ion channels, the details of anesthetic binding and channel modulation are still debated. A better understanding of the anesthetic mechanism of action is necessary for the development of safer and more efficacious drugs. Herein, we present a computational study identifying two anesthetic binding sites in the transmembrane domain of the Gloeobacter violaceus ligand-gated ion channel (GLIC) channel, characterize the putative binding pathway, and observe structural changes associated with channel function. Molecular simulations of desflurane reveal a binding pathway to GLIC via a membrane-embedded tunnel using an intrasubunit protein lumen as the conduit, an observation that explains the Meyer-Overton hypothesis, or why the lipophilicity of an anesthetic and its potency are generally proportional. Moreover, employing high concentrations of ligand led to the identification of a second transmembrane site (TM2) that inhibits dissociation of anesthetic from the TM1 site and is consistent with the high concentrations of anesthetics required to achieve clinical effects. Finally, asymmetric binding patterns of anesthetic to the channel were found to promote an iris-like conformational change that constricts and dehydrates the ion pore, creating a 13.5 kcal/mol barrier to ion translocation. Together with previous studies, the simulations presented herein demonstrate a novel anesthetic binding site in GLIC that is accessed through a membrane-embedded tunnel and interacts with a previously known site, resulting in conformational changes that produce a non-conductive state of the channel.

Structure and Pharmacologic Modulation of Inhibitory Glycine Receptors.

Glycine receptors (GlyR) are inhibitory Cys-loop ion channels that contribute to the control of excitability along the central nervous system (CNS). GlyR are found in the spinal cord and brain stem, and more recently they were reported in higher regions of the CNS such as the hippocampus and nucleus accumbens. GlyR are involved in motor coordination, respiratory rhythms, pain transmission, and sensory processing, and they are targets for relevant physiologic and pharmacologic modulators. Several studies with protein crystallography and cryoelectron microscopy have shed light on the residues and mechanisms associated with the activation, blockade, and regulation of pentameric Cys-loop ion channels at the atomic level. Initial studies conducted on the extracellular domain of acetylcholine receptors, ion channels from prokaryote homologs-Erwinia chrysanthemi ligand-gated ion channel (ELIC), Gloeobacter violaceus ligand-gated ion channel (GLIC)-and crystallized eukaryotic receptors made it possible to define the overall structure and topology of the Cys-loop receptors. For example, the determination of pentameric GlyR structures bound to glycine and strychnine have contributed to visualizing the structural changes implicated in the transition between the open and closed states of the Cys-loop receptors. In this review, we summarize how the new information obtained in functional, mutagenesis, and structural studies have contributed to a better understanding of the function and regulation of GlyR.

Direct interaction of the resistance to inhibitors of cholinesterase type 3 protein with the serotonin receptor type 3A intracellular domain.

Pentameric ligand-gated ion channels (pLGIC) are expressed in both excitable and non-excitable cells that are targeted by numerous clinically used drugs. Assembly from five identical or homologous subunits yields homo- or heteromeric pentamers, respectively. The protein known as Resistance to Inhibitors of Cholinesterase (RIC-3) was identified to interfere with assembly and functional maturation of pLGICs. We have shown previously for serotonin type 3A homopentamers (5-HT3A ) that the interaction with RIC-3 requires the intracellular domain (ICD) of this pLGIC. After expression in Xenopus laevis oocytes RIC-3 attenuated serotonin-induced currents in 5-HT3A wild-type channels, but not in functional 5-HT3A glvM3M4 channels that have the 115-amino acid ICD replaced by a heptapeptide. In complementary experiments we have shown that engineering the Gloeobacter violaceus ligand-gated ion channel (GLIC) to contain the 5-HT3A -ICD confers sensitivity to RIC-3 in oocytes to otherwise insensitive GLIC. In this study, we identify endogenous RIC-3 protein expression in X.laevis oocytes. We purified RIC-3 to homogeneity after expression in Echericia coli. By using heterologously over-expressed and purified RIC-3 and the chimera consisting of the 5-HT3A -ICD and the extracellular and transmembrane domains of GLIC in pull-down experiments, we demonstrate a direct and specific interaction between the two proteins. This result further underlines that the domain within 5-HT3 A R that mediates the interaction with RIC-3 is the ICD. Importantly, this is the first experimental evidence that the interaction between 5-HT3 A R-ICD and RIC-3 does not require other proteins. In addition, we demonstrate that the pentameric assembly of the GLIC-5-HT3A -ICD chimera interacts with RIC-3. We hypothesized that pentameric ligand-gated ion channels (pLGICs) associate directly with the chaperone protein RIC-3 (resistance to inhibitors of cholinesterase type 3), and that the interaction does not require other protein factors. We found that the two proteins indeed interact directly, that the pLGIC intracellular domain is required for the effect, and that pLGICs in their pentameric form associate with RIC-3. These results provide the basis for future studies aimed at investigating which motifs provide the interaction surfaces, and at delineating the mechanism(s) of RIC-3 modulation of functional pLGIC surface expression.

Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors

Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC.

Elucidating Proton Sensitivity and Activation in the Gloeobacter Violaceus Ligand-Gated Ion Channel

The pentameric ligand-gated ion channel (pLGIC) family plays an important role in the chemical-to-electrical transduction of neuronal signaling. Although human pLGICs are endogenously activated by acetylcholine, serotonin, zinc, glycine, or gamma-aminobutyric acid, some of them notably glycine and gamma-aminobutyric acid receptors are modulated by protons. The Gloeobacter violaceus ligand-gated ion channel (GLIC) is a prokaryotic proton-gated ion channel of the family and was the first pLGIC to be structurally resolved in both an open and closed conformation offering a structural perspective to the mechanism of activation for the pLGIC family. Determining the proton modulatory site(s) of receptors, including acid-sensing and ATP-sensitive K+ channels, may be a complicated endeavor. Although residues have been identified in these receptors as key proton-sensitive sites the underlying molecular mechanism of modulation is not always clear. GLIC has been shown to be a robust model of the pLGIC family, and understanding how a single or several proton(s) can stabilize the open conformation would shed light on the intricate mechanism involved in channel opening of human pLGICs. This comprehension might also pave the way to reveal proton modulatory mechanisms of other receptors. Here we represent a systematic functional evaluation of titratable residues in GLIC to pinpoint the key proton sensitive sites that play a role in the extracellular initiation of the activation mechanism. We have found two key inter-subunit interacting regions, distinct from the ligand-binding pocket, which play key roles in the opening of the channel.

Insights into Gating Motions of GLIC via Perturbation of Critical Prolines with Non-Canonical Amino Acid Probes

Pentameric ligand-gated ion channels (pLGICs) are membrane proteins involved in fast synaptic transmission, and have been implicated in a number of neurodegenerative and psychological diseases in humans. These proteins have been the focus of extensive biochemical investigation, but high-resolution structures of mammalian receptors have only recently become available. The bacterial pLGIC Gloeobacter violaceus ligand-gated ion channel (GLIC), whose structure was first published in 2008, has served as a valuable model. There are now over 40 published structures obtained under various conditions, and these suggest specific conformational transitions that may occur during channel activation. If these gating motions are conserved throughout the pLGIC family, a detailed understanding of the process holds promise for the development of strategies to tune receptor activity. Here we discuss recent work in which we explore the role of prolines in the gating transitions of GLIC. Receptors were expressed in Xenopus oocytes, and examined using whole-cell electrophysiology. We first probed the importance of each proline residue in the receptor with an alanine scan. Pro119 in the Cys-loop, Pro198 and Pro203 in the M1 helix, and Pro299 in the M4 helix were sensitive to substitution. At these positions, we then used in vivo non-canonical amino acid mutagenesis with a range of proline analogs to determine specific structural requirements. The data revealed unique requirements at each position, and show that Pro119, Pro203 and Pro299 obey previously observed phenotypes for cis-preferring, bulge-inducing, and kink-inducing prolines respectively. However, an unusual phenotype was displayed by Pro198, a non-conserved site, and possible interpretations will be discussed.

A chimeric prokaryotic pentameric ligand-gated channel reveals distinct pathways of activation.

Recent high resolution structures of several pentameric ligand-gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron-electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand-gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand-gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators.

Ethanol Modulation is Quantitatively Determined by the Transmembrane Domain of Human α1 Glycine Receptors.

Mutagenesis and labeling studies have identified amino acids from the human α1 glycine receptor (GlyR) extracellular, transmembrane (TM), and intracellular domains in mediating ethanol (EtOH) potentiation. However, limited high-resolution structural data for physiologically relevant receptors in this Cys-loop receptor superfamily have made pinpointing the critical amino acids difficult. Homologous ion channels from lower organisms provide conserved models for structural and functional properties of Cys-loop receptors. We previously demonstrated that a single amino acid variant of the Gloeobacter violaceus ligand-gated ion channel (GLIC) produced EtOH and anesthetic sensitivity similar to that of GlyRs and provided crystallographic evidence for EtOH binding to GLIC. We directly compared EtOH modulation of the α1 GlyR and GLIC to a chimera containing the TM domain from human α1 GlyRs and the ligand-binding domain of GLIC using 2-electrode voltage-clamp electrophysiology of receptors expressed in Xenopus laevis oocytes. EtOH potentiated α1 GlyRs in a concentration-dependent manner in the presence of zinc-chelating agents, but did not potentiate GLIC at pharmacologically relevant concentrations. The GLIC/GlyR chimera recapitulated the EtOH potentiation of GlyRs, without apparent sensitivity to zinc chelation. For chimera expression in oocytes, it was essential to suppress leakage current by adding 50μM picrotoxin to the media, a technique that may have applications in expression of other ion channels. Our results are consistent with a TM mechanism of EtOH modulation in Cys-loop receptors. This work highlights the relevance of bacterial homologs as valuable model systems for studying ion channel function of human receptors and demonstrates the modularity of these channels across species.