RESUMO
We present a nonlinear spectroelectrochemical technique to investigate photosynthetic protein complexes. The PEC2DES setup combines photoelectrochemical detection (PEC) that selectively probes the protein photogenerated charges output with two-dimensional electronic spectroscopy (2DES) excitation that spreads the nonlinear optical response of the system in an excitation-detection map. PEC allows us to distinguish the contribution of charge separation (CS) from other de-excitation pathways, whereas 2DES allows us to disentangle congested spectral bands and evaluate the exciton dynamics (decays and coherences) of the photosystem complex. We have developed in operando phase-modulated 2DES by measuring the photoelectrochemical reaction rate in a biohybrid electrode functionalized with a plant photosystem complex I-light harvesting complex I (PSI-LHCI) layer. Optimizing the photoelectrochemical current signal yields reliable linear spectra unequivocally associated with PSI-LHCI. The 2DES signal is validated by nonlinear features like the characteristic vibrational coherence at 750 cm-1. However, no energy transfer dynamics is observed within the 450 fs experimental window. These intriguing results are discussed in the context of incoherent mixing resulting in reduced nonlinear contrast for multichromophoric complexes, such as the 160 chlorophyll PSI. The presented PEC2DES method identifies generated charges unlike purely optical 2DES and opens the way to probe the CS channel in multichromophoric complexes.
RESUMO
The field of G protein-coupled receptor (GPCR) research has greatly benefited from the spatiotemporal resolution provided by light controllable, photoswitchable agents. Most of the developed tools have targeted the Rhodopsin-like family (Class A), the largest family of GPCRs. However, to date, all such Class A photoswitchable ligands were designed to act at the orthosteric binding site of these receptors. Herein, we report the development of the first photoswitchable allosteric modulators of Class A GPCRs, designed to target the M1 muscarinic acetylcholine receptor. The presented benzyl quinolone carboxylic acid (BQCA) derivatives, photo-BQCisA and photo-BQCtrAns, exhibit complementary photopharmacological behavior and allow reversible control over the receptor using light as an external stimulus. This makes them valuable tools to further investigate M1 receptor signaling and a proof of concept for photoswitchable allosteric modulators at Class A receptors.
RESUMO
Orexinergic neurons are critically involved in regulating arousal, wakefulness, and appetite. Their dysfunction has been associated with sleeping disorders, and non-peptide drugs are currently being developed to treat insomnia and narcolepsy. Yet, no light-regulated agents are available to reversibly control their activity. To meet this need, a photoswitchable peptide analogue of the endogenous neuroexcitatory peptide orexin-B was designed, synthesized, and tested in vitro and in vivo. This compound - photorexin - is the first photo-reversible ligand reported for orexin receptors. It allows dynamic control of activity in vitro (including almost the same efficacy as orexin-B, high nanomolar potency, and subtype selectivity to human OX2 receptors) and in vivo in zebrafish larvae by direct application in water. Photorexin induces dose- and light-dependent changes in locomotion and a reduction in the successive induction reflex that is associated with sleep behavior. Molecular dynamics calculations indicate that trans and cis photorexin adopt similar bent conformations and that the only discriminant between their structures and activities is the positioning of the N-terminus. This, in the case of the more active trans isomer, points towards the OX2 N-terminus and extra-cellular loop 2, a region of the receptor known to be involved in ligand binding and recognition consistent with a "message-address" system. Thus, our approach could be extended to several important families of endogenous peptides, such as endothelins, nociceptin, and dynorphins among others, that bind to their cognate receptors through a similar mechanism: a "message" domain involved in receptor activation and signal transduction, and an "address" sequence for receptor occupation and improved binding affinity.
Assuntos
Luz , Receptores de Orexina , Orexinas , Peixe-Zebra , Receptores de Orexina/metabolismo , Receptores de Orexina/química , Animais , Orexinas/metabolismo , Humanos , Locomoção/efeitos dos fármacos , Simulação de Dinâmica Molecular , Larva/metabolismo , Larva/efeitos dos fármacos , Células HEK293 , LigantesRESUMO
A problem of systemic pharmacotherapy is off-target activity, which causes adverse effects. Outstanding examples include neuroinhibitory medications like antiseizure drugs, which are used against epilepsy and neuropathic pain but cause systemic side effects. There is a need of drugs that inhibit nerve signals locally and on-demand without affecting other regions of the body. Photopharmacology aims to address this problem with light-activated drugs and localized illumination in the target organ. Here, we have developed photoswitchable derivatives of the widely prescribed antiseizure drug carbamazepine. For that purpose, we expanded our method of ortho azologization of tricyclic drugs to meta/para and to N-bridged diazocine. Our results validate the concept of ortho cryptoazologs (uniquely exemplified by Carbazopine-1) and bring to light Carbadiazocine (8), which can be photoswitched between 400-590â nm light (using violet LEDs and halogen lamps) and shows good drug-likeness and predicted safety. Both compounds display photoswitchable activity in vitro and in translucent zebrafish larvae. Carbadiazocine (8) also offers in vivo analgesic efficacy (mechanical and thermal stimuli) in a rat model of neuropathic pain and a simple and compelling treatment demonstration with non-invasive illumination.
RESUMO
To interrogate neural circuits and crack their codes, in vivo brain activity imaging must be combined with spatiotemporally precise stimulation in three dimensions using genetic or pharmacological specificity. This challenge requires deep penetration and focusing as provided by infrared light and multiphoton excitation, and has promoted two-photon photopharmacology and optogenetics. However, three-photon brain stimulation in vivo remains to be demonstrated. We report the regulation of neuronal activity in zebrafish larvae by three-photon excitation of a photoswitchable muscarinic agonist at 50â pM, a billion-fold lower concentration than used for uncaging, and with mid-infrared light of 1560â nm, the longest reported photoswitch wavelength. Robust, physiologically relevant photoresponses allow modulating brain activity in wild-type animals with spatiotemporal and pharmacological precision. Computational calculations predict that azobenzene-based ligands have high three-photon absorption cross-section and can be used directly with pulsed infrared light. The expansion of three-photon pharmacology will deeply impact basic neurobiology and neuromodulation phototherapies.
Assuntos
Fótons , Peixe-Zebra , Animais , Raios Infravermelhos , LigantesRESUMO
Charge exchange is the fundamental process that sustains cellular respiration and photosynthesis by shuttling electrons in a cascade of electron transfer (ET) steps between redox cofactors. While intraprotein charge exchange is well characterized in protein complexes bearing multiple redox sites, interprotein processes are less understood due to the lack of suitable experimental approaches and the dynamic nature of the interactions. Proteins constrained between electrodes are known to support electron transport (ETp) through the protein matrix even without redox cofactors, as the charges housed by the redox sites in ET are furnished by the electrodes. However, it is unknown whether protein ETp mechanisms apply to the interprotein medium present under physiological conditions. We study interprotein charge exchange between plant photosystem I (PSI) and its soluble redox partner plastocyanin (Pc) and address the role of the Pc copper center. Using electrochemical scanning tunneling spectroscopy (ECSTS) current-distance and blinking measurements, we quantify the spatial span of charge exchange between individual Pc/PSI pairs and ETp through transient Pc/PSI complexes. Pc devoid of the redox center (Pcapo) can exchange charge with PSI at longer distances than with the copper ion (Pcholo). Conductance bursts associated with Pcapo/PSI complex formation are higher than in Pcholo/PSI. Thus, copper ions are not required for long-distance Pc/PSI ETp but regulate its spatial span and conductance. Our results suggest that the redox center that carries the charge in Pc is not necessary to exchange it in interprotein ET through the aqueous solution and question the canonical view of tight complex binding between redox protein partners.
Assuntos
Complexo de Proteína do Fotossistema I , Plastocianina , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Plastocianina/química , Plastocianina/metabolismo , Cobre , Transporte de Elétrons , Oxirredução , Plantas/metabolismoRESUMO
Clathrin-mediated endocytosis (CME) is an essential cellular process, conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of specific pharmacological agents to interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor targeting the AP2-ß-adaptin/ß-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP, demonstrating that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts. This is despite the ß-adaptin/ß-arrestin interaction not being conserved in yeast. Our data indicate that the AP2 α-adaptin is the functional target of activated TL2. We identified as interacting partners for the α-appendage, the Eps15 and epsin homologues Ede1 and Ent1. This demonstrates that endocytic cargo loading and sensing can be executed by conserved molecular interfaces, regardless of the proteins involved.
RESUMO
Ratiometric fluorescent nanothermometers with near-infrared emission play an important role in in vivo sensing since they can be used as intracellular thermal sensing probes with high spatial resolution and high sensitivity, to investigate cellular functions of interest in diagnosis and therapy, where current approaches are not effective. Herein, the temperature-dependent fluorescence of organic nanoparticles is designed, synthesized, and studied based on the dual emission, generated by monomer and excimer species, of the tris(2,4,6-trichlorophenyl)methyl radical (TTM) doping organic nanoparticles (TTMd-ONPs), made of optically neutral tris(2,4,6-trichlorophenyl)methane (TTM-αH), acting as a matrix. The excimer emission intensity of TTMd-ONPs decreases with increasing temperatures whereas the monomer emission is almost independent and can be used as an internal reference. TTMd-ONPs show a great temperature sensitivity (3.4% K-1 at 328 K) and a wide temperature response at ambient conditions with excellent reversibility and high colloidal stability. In addition, TTMd-ONPs are not cytotoxic and their ratiometric outputs are unaffected by changes in the environment. Individual TTMd-ONPs are able to sense temperature changes at the nano-microscale. In vivo thermometry experiments in Caenorhabditis elegans (C. elegans) worms show that TTMd-ONPs can locally monitor internal body temperature changes with spatio-temporal resolution and high sensitivity, offering multiple applications in the biological nanothermometry field.
Assuntos
Nanopartículas , Termometria , Animais , Caenorhabditis elegans , TemperaturaRESUMO
It has been recently shown that electron transfer between mitochondrial cytochrome c and the cytochrome c1 subunit of the cytochrome bc1 can proceed at long-distance through the aqueous solution. Cytochrome c is thought to adjust its activity by changing the affinity for its partners via Tyr48 phosphorylation, but it is unknown how it impacts the nanoscopic environment, interaction forces, and long-range electron transfer. Here, we constrain the orientation and separation between cytochrome c1 and cytochrome c or the phosphomimetic Y48pCMF cytochrome c, and deploy an array of single-molecule, bulk, and computational methods to investigate the molecular mechanism of electron transfer regulation by cytochrome c phosphorylation. We demonstrate that phosphorylation impairs long-range electron transfer, shortens the long-distance charge conduit between the partners, strengthens their interaction, and departs it from equilibrium. These results unveil a nanoscopic view of the interaction between redox protein partners in electron transport chains and its mechanisms of regulation.
Assuntos
Respiração Celular , Citocromos c , Transporte de Elétrons , Fosforilação , OxirreduçãoRESUMO
The transduction of odorant binding into cellular signaling by olfactory receptors (ORs) is not understood and knowing its mechanism would enable developing new pharmacology and biohybrid electronic detectors of volatile organic compounds bearing high sensitivity and selectivity. The electrical characterization of ORs in bulk experiments is subject to microscopic models and assumptions. We have directly determined the nanoscale electrical properties of ORs immobilized in a fixed orientation, and their change upon odorant binding, using electrochemical scanning tunneling microscopy (EC-STM) in near-physiological conditions. Recordings of current versus time, distance, and electrochemical potential allows determining the OR impedance parameters and their dependence with odorant binding. Our results allow validating OR structural-electrostatic models and their functional activation processes, and anticipating a novel macroscopic biosensor based on ORs.
Assuntos
Técnicas Biossensoriais , Receptores Odorantes , Compostos Orgânicos Voláteis , Ligantes , Odorantes , Receptores Odorantes/química , Compostos Orgânicos Voláteis/químicaRESUMO
Understanding the dopaminergic system is a priority in neurobiology and neuropharmacology. Dopamine receptors are involved in the modulation of fundamental physiological functions, and dysregulation of dopaminergic transmission is associated with major neurological disorders. However, the available tools to dissect the endogenous dopaminergic circuits have limited specificity, reversibility, resolution, or require genetic manipulation. Here, we introduce azodopa, a novel photoswitchable ligand that enables reversible spatiotemporal control of dopaminergic transmission. We demonstrate that azodopa activates D1-like receptors in vitro in a light-dependent manner. Moreover, it enables reversibly photocontrolling zebrafish motility on a timescale of seconds and allows separating the retinal component of dopaminergic neurotransmission. Azodopa increases the overall neural activity in the cortex of anesthetized mice and displays illumination-dependent activity in individual cells. Azodopa is the first photoswitchable dopamine agonist with demonstrated efficacy in wild-type animals and opens the way to remotely controlling dopaminergic neurotransmission for fundamental and therapeutic purposes.
Assuntos
Animais Selvagens , Peixe-Zebra , Animais , Dopamina , Ligantes , Camundongos , Transmissão SinápticaRESUMO
Photosynthesis is a fundamental process that converts photons into chemical energy, driven by large protein complexes at the thylakoid membranes of plants, cyanobacteria, and algae. In plants, water-soluble plastocyanin (Pc) is responsible for shuttling electrons between cytochrome b6f complex and the photosystem I (PSI) complex in the photosynthetic electron transport chain (PETC). For an efficient turnover, a transient complex must form between PSI and Pc in the PETC, which implies a balance between specificity and binding strength. Here, we studied the binding frequency and the unbinding force between suitably oriented plant PSI and Pc under redox control using single molecule force spectroscopy (SMFS). The binding frequency (observation of binding-unbinding events) between PSI and Pc depends on their respective redox states. The interaction between PSI and Pc is independent of the redox state of PSI when Pc is reduced, and it is disfavored in the dark (reduced P700) when Pc is oxidized. The frequency of interaction between PSI and Pc is higher when at least one of the partners is in a redox state ready for electron transfer (ET), and the post-ET situation (PSIRed-PcOx) leads to lower binding. In addition, we show that the binding of ET-ready PcRed to PSI can be regulated externally by Mg2+ ions in solution.
Assuntos
Complexo de Proteína do Fotossistema I , Plastocianina , Complexo Citocromos b6f/química , Complexo Citocromos b6f/metabolismo , Transporte de Elétrons , Elétrons , Luz , Oxirredução , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Plastocianina/química , Plastocianina/metabolismo , Análise Espectral , Água/metabolismoRESUMO
The interest in the photochromism and functional applications of donor-acceptor Stenhouse adducts (DASAs) soared in recent years owing to their outstanding advantages and flexible design. However, their low solubility and irreversible conversion in aqueous solutions hampered exploring DASAs for biology and medicine. It is notably unknown whether the barbiturate electron acceptor group retains the pharmacological activity of drugs such as phenobarbital, which targets γ-aminobutyric acid (GABA)-type A receptors (GABAARs) in the brain. Here, we have developed the model compound DASA-barbital based on a scaffold of red-switching second-generation DASAs, and we demonstrate that it is active in GABAARs and alters the neuronal firing rate in a physiological medium at neutral pH. DASA-barbital can also be reversibly photoswitched in acidic aqueous solutions using cyclodextrin, an approved ingredient of drug formulations. These findings clarify the path toward the biological applications of DASAs and to exploit the versatility displayed in polymers and materials science.
Assuntos
Barbital , Água , Barbital/farmacologia , Neurônios , Polímeros , Ácido gama-AminobutíricoRESUMO
Artificial control of neuronal activity enables the study of neural circuits and restoration of neural functions. Direct, rapid, and sustained photocontrol of intact neurons could overcome the limitations of established electrical stimulation such as poor selectivity. We have developed fast photoswitchable ligands of glutamate receptors (GluRs) to enable neuronal control in the auditory system. The new photoswitchable ligands induced photocurrents in untransfected neurons upon covalently tethering to endogenous GluRs and activating them reversibly with visible light pulses of a few milliseconds. As a proof of concept of these molecular prostheses, we applied them to the ultrafast synapses of auditory neurons of the cochlea that encode sound and provide auditory input to the brain. This drug-based method afforded the optical stimulation of auditory neurons of adult gerbils at hundreds of hertz without genetic manipulation that would be required for their optogenetic control. This indicates that the new photoswitchable ligands are also applicable to the spatiotemporal control of fast spiking interneurons in the brain.
Assuntos
Cóclea , Optogenética , Cóclea/fisiologia , Ligantes , Neurônios , Optogenética/métodos , Próteses e ImplantesRESUMO
Charge separation and transport through the reaction center of photosystem I (PSI) is an essential part of the photosynthetic electron transport chain. A strategy is developed to immobilize and orient PSI complexes on gold electrodes allowing to probe the complex's electron acceptor side, the chlorophyll special pair P700. Electrochemical scanning tunneling microscopy (ECSTM) imaging and current-distance spectroscopy of single protein complex shows lateral size in agreement with its known dimensions, and a PSI apparent height that depends on the probe potential revealing a gating effect in protein conductance. In current-distance spectroscopy, it is observed that the distance-decay constant of the current between PSI and the ECSTM probe depends on the sample and probe electrode potentials. The longest charge exchange distance (lowest distance-decay constant ß) is observed at sample potential 0 mV/SSC (SSC: reference electrode silver/silver chloride) and probe potential 400 mV/SSC. These potentials correspond to hole injection into an electronic state that is available in the absence of illumination. It is proposed that a pair of tryptophan residues located at the interface between P700 and the solution and known to support the hydrophobic recognition of the PSI redox partner plastocyanin, may have an additional role as hole exchange mediator in charge transport through PSI.
Assuntos
Clorofila , Complexo de Proteína do Fotossistema I , Clorofila/química , Clorofila/metabolismo , Transporte de Elétrons , Cinética , Oxirredução , Fotossíntese , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismoRESUMO
Tricyclic chemical structures are the core of many important drugs targeting all neurotransmitter pathways. These medicines enable effective therapies to treat from peptic ulcer disease to psychiatric disorders. However, when administered systemically, they cause serious adverse effects that limit their use. To obtain localized and on-demand pharmacological action using light, we have designed photoisomerizable ligands based on azobenzene that mimic the tricyclic chemical structure and display reversibly controlled activity. Pseudo-analogues of the tricyclic antagonist pirenzepine demonstrate that this is an effective strategy in muscarinic acetylcholine receptors, showing stronger inhibition upon illumination both in vitro and in cardiac atria ex vivo. Despite the applied chemical modifications to make pirenzepine derivatives sensitive to light stimuli, the most potent candidate of the set, cryptozepine-2, maintained a moderate but promising M1 vs M2 subtype selectivity. These photoswitchable "crypto-azologs" of tricyclic drugs might open a general way to spatiotemporally target their therapeutic action while reducing their systemic toxicity and adverse effects.
Assuntos
Desenho de Fármacos , Antagonistas Muscarínicos/farmacologia , Pirenzepina/farmacologia , Receptores Muscarínicos/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Antagonistas Muscarínicos/síntese química , Antagonistas Muscarínicos/química , Pirenzepina/síntese química , Pirenzepina/química , Relação Estrutura-AtividadeRESUMO
The ability to control neural activity is essential for research not only in basic neuroscience, as spatiotemporal control of activity is a fundamental experimental tool, but also in clinical neurology for therapeutic brain interventions. Transcranial-magnetic, ultrasound, and alternating/direct current (AC/DC) stimulation are some available means of spatiotemporal controlled neuromodulation. There is also light-mediated control, such as optogenetics, which has revolutionized neuroscience research, yet its clinical translation is hampered by the need for gene manipulation. As a drug-based light-mediated control, the effect of a photoswitchable muscarinic agonist (Phthalimide-Azo-Iper (PAI)) on a brain network is evaluated in this study. First, the conditions to manipulate M2 muscarinic receptors with light in the experimental setup are determined. Next, physiological synchronous emergent cortical activity consisting of slow oscillations-as in slow wave sleep-is transformed into a higher frequency pattern in the cerebral cortex, both in vitro and in vivo, as a consequence of PAI activation with light. These results open the way to study cholinergic neuromodulation and to control spatiotemporal patterns of activity in different brain states, their transitions, and their links to cognition and behavior. The approach can be applied to different organisms and does not require genetic manipulation, which would make it translational to humans.
Assuntos
Encéfalo/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Animais , Furões , Camundongos , Camundongos Endogâmicos C57BL , Modelos AnimaisRESUMO
Photosynthetic reactions in plants, algae, and cyanobacteria are driven by photosystem I and photosystem II complexes, which specifically reduce or oxidize partner redox biomolecules. Photosynthetic complexes can also bind synthetic organic molecules, which inhibit their photoactivity and can be used both to study the electron transport chain and as herbicides and algicides. Thus, their development, characterization, and sensing bears fundamental and applied interest. Substantial efforts have been devoted to developing photosensors based on photosystem II to detect compounds that bind to the plastoquinone sites of this complex. In comparison, photosystem I based sensors have received less attention and could be used to identify novel substances displaying phytotoxic effects, including those obtained from natural product extracts. We have developed a robust procedure to functionalize gold electrodes with photo- and redox-active photosystem I complexes based on transparent gold and a thiolate self-assembled monolayer, and we have obtained reproducible electrochemical photoresponses. Chronoamperometric recordings have allowed us to measure photocurrents in the presence of the viologen derivative paraquat at concentrations below 100 nM under lock-in operation and a sensor dynamic range spanning six orders of magnitude up to 100 mM. We have modeled their time course to identify the main electrochemical processes and limiting steps in the electron transport chain. Our results allow us to isolate the contributions from photosystem I and the redox mediator, and evaluate photocurrent features (spectral and power dependence, fast transient kinetics) that could be used as a sensing signal to detect other inhibitors and modulators of photosystem I activity.
Assuntos
Técnicas Biossensoriais , Complexo de Proteína do Fotossistema I , Transporte de Elétrons , Paraquat , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismoRESUMO
Adrenoceptors are ubiquitous and mediate important autonomic functions as well as modulating arousal, cognition, and pain on a central level. Understanding these physiological processes and their underlying neural circuits requires manipulating adrenergic neurotransmission with high spatio-temporal precision. Here we present a first generation of photochromic ligands (adrenoswitches) obtained via azologization of a class of cyclic amidines related to the known ligand clonidine. Their pharmacology, photochromism, bioavailability, and lack of toxicity allow for broad biological applications, as demonstrated by controlling locomotion in zebrafish and pupillary responses in mice.
Assuntos
Adrenérgicos/farmacologia , Compostos Cromogênicos/farmacologia , Receptores Adrenérgicos/metabolismo , Adrenérgicos/síntese química , Adrenérgicos/química , Animais , Compostos Cromogênicos/síntese química , Compostos Cromogênicos/química , Ligantes , Camundongos , Camundongos Nus , Estrutura Molecular , Peixe-ZebraRESUMO
Photopharmacology is a unique approach that through a combination of photochemistry methods and advanced life science techniques allows the study and control of specific biological processes, ranging from intracellular pathways to brain circuits. Recently, a first photochromic channel blocker of anion-selective GABAA receptors, the azobenzene-nitrazepam-based photochromic compound (Azo-NZ1), has been described. In the present study, using patch-clamp technique in heterologous system and in mice brain slices, site-directed mutagenesis and molecular modeling we provide evidence of the interaction of Azo-NZ1 with glycine receptors (GlyRs) and determine the molecular basis of this interaction. Glycinergic synaptic neurotransmission determines an important inhibitory drive in the vertebrate nervous system and plays a crucial role in the control of neuronal circuits in the spinal cord and brain stem. GlyRs are involved in locomotion, pain sensation, breathing, and auditory function, as well as in the development of such disorders as hyperekplexia, epilepsy, and autism. Here, we demonstrate that Azo-NZ1 blocks in a UV-dependent manner the activity of α2 GlyRs (GlyR2), while being barely active on α1 GlyRs (GlyR1). The site of Azo-NZ1 action is in the chloride-selective pore of GlyR at the 2' position of transmembrane helix 2 and amino acids forming this site determine the difference in Azo-NZ1 blocking activity between GlyR2 and GlyR1. This subunit-specific modulation is also shown on motoneurons of brainstem slices from neonatal mice that switch during development from expressing "fetal" GlyR2 to "adult" GlyR1 receptors.