An optogenetic method for the controlled release of single molecules.

We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for a light dose-dependent delivery of functional proteins to the cytosol and plasma membrane in amounts compatible with single-molecule imaging, greatly simplifying access to single-molecule microscopy of any protein in live cells. We were able to reconstitute ion conductance by delivering BK and LRRC8/volume-regulated anion channels to the plasma membrane. Finally we were able to induce NF-kB signaling in T lymphoblasts stimulated by interleukin-1 by controlled release of a signaling protein that had been knocked out. We observed light-induced formation of functional inflammatory signaling complexes that triggered phosphorylation of the inhibitor of nuclear factor kappa-B kinase only in activated cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single-molecule level.

Controlling the interaction between CaMKII and Calmodulin with a photocrosslinking unnatural amino acid.

Using unnatural amino acid mutagenesis, we made a mutant of CaMKII that forms a covalent linkage to Calmodulin upon illumination by UV light. Like wild-type CaMKII, the L308BzF mutant stoichiometrically binds to Calmodulin, in a calcium-dependent manner. Using this construct, we demonstrate that Calmodulin binding to CaMKII, even under these stochiometric conditions, does not perturb the CaMKII oligomeric state. Furthermore, we were able to achieve activation of CaMKII L308BzF by UV-induced binding of Calmodulin, which, once established, is further insensitive to calcium depletion. In addition to the canonical auto-inhibitory role of the regulatory segment, inter-subunit crosslinking in the absence of CaM indicates that kinase domains and regulatory segments are substantially mobile in basal conditions. Characterization of CaMKIIL308BzF in vitro, and its expression in mammalian cells, suggests it could be a promising candidate for control of CaMKII activity in mammalian cells with light.

CaMKII autophosphorylation can occur between holoenzymes without subunit exchange.

The dodecameric protein kinase CaMKII is expressed throughout the body. The alpha isoform is responsible for synaptic plasticity and participates in memory through its phosphorylation of synaptic proteins. Its elaborate subunit organization and propensity for autophosphorylation allow it to preserve neuronal plasticity across space and time. The prevailing hypothesis for the spread of CaMKII activity, involving shuffling of subunits between activated and naive holoenzymes, is broadly termed subunit exchange. In contrast to the expectations of previous work, we found little evidence for subunit exchange upon activation, and no effect of restraining subunits to their parent holoenzymes. Rather, mass photometry, crosslinking mass spectrometry, single molecule TIRF microscopy and biochemical assays identify inter-holoenzyme phosphorylation (IHP) as the mechanism for spreading phosphorylation. The transient, activity-dependent formation of groups of holoenzymes is well suited to the speed of neuronal activity. Our results place fundamental limits on the activation mechanism of this kinase.

Mechanism of Calcium Permeation in a Glutamate Receptor Ion Channel.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are neurotransmitter-activated cation channels ubiquitously expressed in vertebrate brains. The regulation of calcium flux through the channel pore by RNA-editing is linked to synaptic plasticity while excessive calcium influx poses a risk for neurodegeneration. Unfortunately, the molecular mechanisms underlying this key process are mostly unknown. Here, we investigated calcium conduction in calcium-permeable AMPAR using Molecular Dynamics (MD) simulations with recently introduced multisite force-field parameters for Ca2+. Our calculations are consistent with experiment and explain the distinct calcium permeability in different RNA-edited forms of GluA2. For one of the identified metal binding sites, multiscale Quantum Mechanics/Molecular Mechanics (QM/MM) simulations further validated the results from MD and revealed small but reproducible charge transfer between the metal ion and its first solvation shell. In addition, the ion occupancy derived from MD simulations independently reproduced the Ca2+ binding profile in an X-ray structure of an NaK channel mimicking the AMPAR selectivity filter. This integrated study comprising X-ray crystallography, multisite MD, and multiscale QM/MM simulations provides unprecedented insights into Ca2+ permeation mechanisms in AMPARs, and paves the way for studying other biological processes in which Ca2+ plays a pivotal role.

Asymmetry and Ion Selectivity Properties of Bacterial Channel NaK Mutants Derived from Ionotropic Glutamate Receptors.

Ionotropic glutamate receptors are ligand-gated cation channels that play essential roles in the excitatory synaptic transmission throughout the central nervous system. A number of open-pore structures of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid (AMPA)-type glutamate receptors became available recently by cryo-electron microscopy (cryo-EM). These structures provide valuable insights into the conformation of the selectivity filter (SF), the part of the ion channel that determines the ion selectivity. Nonetheless, due to the moderate resolution of the cryo-EM structures, detailed information such as ion occupancy of monovalent and divalent cations as well as positioning of the side-chains in the SF is still missing. Here, in an attempt to obtain high-resolution information about glutamate receptor SFs, we incorporated partial SF sequences of the AMPA and kainate receptors into the bacterial tetrameric cation channel NaK, which served as a structural scaffold. We determined a series of X-ray structures of NaK-CDI, NaK-SDI and NaK-SELM mutants at 1.42-2.10 Å resolution, showing distinct ion occupation of different monovalent cations. Molecular dynamics (MD) simulations of NaK-CDI indicated the channel to be conductive to monovalent cations, which agrees well with our electrophysiology recordings. Moreover, previously unobserved structural asymmetry of the SF was revealed by the X-ray structures and MD simulations, implying its importance in ion non-selectivity of tetrameric cation channels.

Targeted sensors for glutamatergic neurotransmission.

Optical report of neurotransmitter release allows visualisation of excitatory synaptic transmission. Sensitive genetically-encoded fluorescent glutamate reporters operating with a range of affinities and emission wavelengths are available. However, without targeting to synapses, the specificity of the fluorescent signal is uncertain, compared to sensors directed at vesicles or other synaptic markers. We fused the state-of-the-art reporter iGluSnFR to glutamate receptor auxiliary proteins in order to target it to postsynaptic sites. Chimeras of Stargazin and gamma-8 that we named SnFR-γ2 and SnFR-γ8, were enriched at synapses, retained function and reported spontaneous glutamate release in rat hippocampal cells, with apparently diffraction-limited spatial precision. In autaptic mouse neurons cultured on astrocytic microislands, evoked neurotransmitter release could be quantitatively detected at tens of synapses in a field of view whilst evoked currents were recorded simultaneously. These experiments revealed a specific postsynaptic deficit from Stargazin overexpression, resulting in synapses with normal neurotransmitter release but without postsynaptic responses. This defect was reverted by delaying overexpression. By working at different calcium concentrations, we determined that SnFR-γ2 is a linear reporter of the global quantal parameters and short-term synaptic plasticity, whereas iGluSnFR is not. On average, half of iGluSnFR regions of interest (ROIs) showing evoked fluorescence changes had intense rundown, whereas less than 5% of SnFR-γ2 ROIs did. We provide an open-source analysis suite for extracting quantal parameters including release probability from fluorescence time series of individual and grouped synaptic responses. Taken together, postsynaptic targeting improves several properties of iGluSnFR and further demonstrates the importance of subcellular targeting for optogenetic actuators and reporters.

Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling.

Channelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H+, Na+, K+ and anions have been discovered or engineered, Ca2+-selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca2+ permeability and improve their Ca2+ affinity by targeted mutagenesis at the central selectivity filter. The engineered channels, termed CapChR1 and CapChR2 for calcium-permeable channelrhodopsins, exhibit reduced sodium and proton conductance in connection with strongly improved Ca2+ permeation at negative voltage and low extracellular Ca2+ concentrations. In cultured cells and neurons, CapChR2 reliably increases intracellular Ca2+ concentrations. Moreover, CapChR2 can robustly trigger Ca2+ signalling in hippocampal neurons. When expressed together with genetically encoded Ca2+ indicators in Drosophila melanogaster mushroom body output neurons, CapChRs mediate light-evoked Ca2+ entry in brain explants.

Selective endocytosis of Ca2+-permeable AMPARs by the Alzheimer's disease risk factor CALM bidirectionally controls synaptic plasticity.

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission, and the plastic modulation of their surface levels determines synaptic strength. AMPARs of different subunit compositions fulfill distinct roles in synaptic long-term potentiation (LTP) and depression (LTD) to enable learning. Largely unknown endocytic mechanisms mediate the subunit-selective regulation of the surface levels of GluA1-homomeric Ca2+-permeable (CP) versus heteromeric Ca2+-impermeable (CI) AMPARs. Here, we report that the Alzheimer's disease risk factor CALM controls the surface levels of CP-AMPARs and thereby reciprocally regulates LTP and LTD in vivo to modulate learning. We show that CALM selectively facilitates the endocytosis of ubiquitinated CP-AMPARs via a mechanism that depends on ubiquitin recognition by its ANTH domain but is independent of clathrin. Our data identify CALM and related ANTH domain-containing proteins as the core endocytic machinery that determines the surface levels of CP-AMPARs to bidirectionally control synaptic plasticity and modulate learning in the mammalian brain.

The action of Con-ikot-ikot toxin on single AMPA-type glutamate receptors.

Conotoxins are a large group of naturally occurring toxic peptides produced by the predatory sea snails of the genus Conus. Many of these toxins target ion channels, often with high specificity and affinity. As such, they have proven to be invaluable for basic research, as well as acting as leads for therapeutic strategies. Con-ikot-ikot is the only conotoxin so far identified that targets AMPA-type glutamate receptors, the main mediators of excitatory neurotransmission in the vertebrate brain. Here, we describe how the toxin modifies the activity of AMPA receptors at the single-channel level. The toxin binds to the AMPA receptor with EC50 of 5 nM, and once bound takes minutes to wash out. As shown previously, it effectively blocks desensitization of AMPA receptors; however, compared to other desensitization blockers, it is a poor stabilizer of the open channel because toxin-bound AMPA receptors undergo frequent brief closures. We propose that this is a direct consequence of the toxin's unique binding mode to the ligand-binding domains (LBDs). Unlike other blockers of desensitization, which stabilize individual dimers within an AMPA receptor tetramer, the toxin immobilizes all four LBDs of the tetramer. This result further emphasizes that quaternary reorganization of independent LBD dimers is essential for the full activity of AMPA receptors.

Seeing glutamate at central synapses.

Glutamate is the major fast excitatory neurotransmitter in the nervous system, and researchers have for decades pursued tools for monitoring glutamate release in real time. Optical reporters, primarily genetically-encoded glutamate sensors, have developed into the predominant method, particularly following the advent of the intensity-based glutamate-sensing fluorescent reporter (iGluSnFR). Several variants have been developed to improve the performance of iGluSnFR in brightness, kinetics and spectral range. Following the targeting principle of other sensors for neurotransmission, iGluSnFR mutants tethered to pre- or postsynaptic targeting proteins should show enhanced properties for detecting glutamate release at central synapses.

Slow excitatory synaptic currents generated by AMPA receptors.

Decades of literature indicate that the AMPA-type glutamate receptor is among the fastest acting of all neurotransmitter receptors. These receptors are located at excitatory synapses, and conventional wisdom says that they activate in hundreds of microseconds, deactivate in milliseconds due to their low affinity for glutamate and also desensitize profoundly. These properties circumscribe AMPA receptor activation in both space and time. However, accumulating evidence shows that AMPA receptors can also activate with slow, indefatigable responses. They do so through interactions with auxiliary subunits that are able promote a switch to a high open probability, high-conductance 'superactive' mode. In this review, we show that any assumption that this phenomenon is limited to heterologous expression is false and rather that slow AMPA currents have been widely and repeatedly observed throughout the nervous system. Hallmarks of the superactive mode are a lack of desensitization, resistance to competitive antagonists and a current decay that outlives free glutamate by hundreds of milliseconds. Because the switch to the superactive mode is triggered by activation, AMPA receptors can generate accumulating 'pedestal' currents in response to repetitive stimulation, constituting a postsynaptic mechanism for short-term potentiation in the range 5-100 Hz. Further, slow AMPA currents span 'cognitive' time intervals in the 100 ms range (theta rhythms), of particular interest for hippocampal function, where slow AMPA currents are widely expressed in a synapse-specific manner. Here, we outline the implications that slow AMPA receptors have for excitatory synaptic transmission and computation in the nervous system.

Slow AMPA receptors in hippocampal principal cells.

Glutamate receptor ion channels, including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, mediate fast excitatory neurotransmission in the CNS. Previous work suggested that AMPA receptors produce a synaptic current with a millisecond duration. However, we find that about two-thirds of principal cells in the hippocampal CA1 region also express AMPA receptors with reduced desensitization that can stay active for half a second after repetitive stimuli. These slow AMPA receptors are expressed at about half of the synapses, with a flat spatial distribution. The increased charge transfer from slow AMPA receptors allows short-term potentiation from a postsynaptic locus and reliable triggering of action potentials. Biophysical and pharmacological observations imply slow AMPA receptors incorporate auxiliary proteins, and their activation lengthens miniature synaptic currents. These data indicate that AMPA receptors are a major source of synaptic diversity. Synapses harboring slow AMPA receptors could have unique roles in hippocampal function.

Crosslinking glutamate receptor ion channels.

Combining crosslinking strategies with electrophysiology, biochemistry, and structural in silico analysis is a powerful tool to study transient movements of ion channels during gating. This chapter describes crosslinking in living cells using cysteine and photoactive unnatural amino acids (UAAs) that we have used on glutamate receptor ion channels. Here, we share the protocol for building a perfusion tool to enable rapid chemical modification of glutamate-gated AMPA receptors, optimized for their fast activation. This system can be used to perform state-dependent crosslinking in receptors modified by cysteines or UAA incorporation on the millisecond timescale. Introducing UAAs results in receptors with lower expression levels relative to the introduction of cysteine residues. Reduced expression is principally a challenge for biochemical studies, and we share here our approach to capture the light driven oligomerization of AMPA receptors containing UAA crosslinkers. Finally, we describe strategies for computational analysis to make sense of the crosslinking results in terms of structure and function.

NMDA Receptors Singled out for Delayed Activation.

In this issue of Neuron, Amin et al. (2021) recorded single NMDA receptors in synaptic-like conditions to reveal how unreliable coupling between agonist binding and channel opening depends on structured linkers. After neurotransmitter binding, a complicated molecular "discussion" ensues, dividing fast synaptic events from delayed openings and failures.

Nonselective cation permeation in an AMPA-type glutamate receptor.

Fast excitatory synaptic transmission in the central nervous system relies on the AMPA-type glutamate receptor (AMPAR). This receptor incorporates a nonselective cation channel, which is opened by the binding of glutamate. Although the open pore structure has recently became available from cryo-electron microscopy (Cryo-EM), the molecular mechanisms governing cation permeability in AMPA receptors are not understood. Here, we combined microsecond molecular dynamic (MD) simulations on a putative open-state structure of GluA2 with electrophysiology on cloned channels to elucidate ion permeation mechanisms. Na+, K+, and Cs+ permeated at physiological rates, consistent with a structure that represents a true open state. A single major ion binding site for Na+ and K+ in the pore represents the simplest selectivity filter (SF) structure for any tetrameric cation channel of known structure. The minimal SF comprised only Q586 and Q587, and other residues on the cytoplasmic side formed a water-filled cavity with a cone shape that lacked major interactions with ions. We observed that Cl- readily enters the upper pore, explaining anion permeation in the RNA-edited (Q586R) form of GluA2. A permissive architecture of the SF accommodated different alkali metals in distinct solvation states to allow rapid, nonselective cation permeation and copermeation by water. Simulations suggested Cs+ uses two equally populated ion binding sites in the filter, and we confirmed with electrophysiology of GluA2 that Cs+ is slightly more permeant than Na+, consistent with serial binding sites preferentially driving selectivity.

Measurements of the Timescale and Conformational Space of AMPA Receptor Desensitization.

Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the central nervous system. Desensitization of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype after glutamate binding appears critical for brain function and involves rearrangement of the ligand binding domains (LBDs). Recently, several full-length structures of ionotropic glutamate receptors in putative desensitized states were published. These structures indicate movements of the LBDs that might be trapped by cysteine cross-links and metal bridges. We found that cysteine mutants at the interface between subunits A and C and lateral zinc bridges (between subunits C and D or A and B) can trap freely desensitizing receptors in a spectrum of states with different stabilities. Consistent with a close approach of subunits during desensitization processes, the introduction of bulky amino acids at the A-C interface produced a receptor with slow recovery from desensitization. Further, in wild-type GluA2 receptors, we detected the population of a stable desensitized state with a lifetime around 1 s. Using mutations that progressively stabilize deep desensitized states (E713T and Y768R), we were able to selectively protect receptors from cross-links at both the diagonal and lateral interfaces. Ultrafast perfusion enabled us to perform chemical modification in less than 10 ms, reporting movements associated to desensitization on this timescale within LBD dimers in resting receptors. These observations suggest that small disruptions of quaternary structure are sufficient for fast desensitization and that substantial rearrangements likely correspond to stable desensitized states that are adopted relatively slowly on a timescale much longer than physiological receptor activation.

Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis.

Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission throughout the vertebrate nervous system. Conformational changes of the transmembrane domain (TMD) underlying ion channel activation and desensitization remain poorly understood. Here, we explored the dynamics of the TMD of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type iGluRs using genetically encoded unnatural amino acid (UAA) photocross-linkers, p-benzoyl-l-phenylalanine (BzF) and p-azido-l-phenylalanine (AzF). We introduced these UAAs at sites throughout the TMD of the GluA2 receptor and characterized the mutants in patch-clamp recordings, exposing them to glutamate and ultraviolet (UV) light. This approach revealed a range of optical effects on the activity of mutant receptors. We found evidence for an interaction between the Pre-M1 and the M4 TMD helix during desensitization. Photoactivation at F579AzF, a residue behind the selectivity filter in the M2 segment, had extraordinarily broad effects on gating and desensitization. This observation suggests coupling to other parts of the receptor and like in other tetrameric ion channels, selectivity filter gating.

Neurotransmitter Funneling Optimizes Glutamate Receptor Kinetics.

Ionotropic glutamate receptors (iGluRs) mediate neurotransmission at the majority of excitatory synapses in the brain. Little is known, however, about how glutamate reaches the recessed binding pocket in iGluR ligand-binding domains (LBDs). Here we report the process of glutamate binding to a prototypical iGluR, GluA2, in atomistic detail using unbiased molecular simulations. Charged residues on the LBD surface form pathways that facilitate glutamate binding by effectively reducing a three-dimensional diffusion process to a spatially constrained, two-dimensional one. Free energy calculations identify residues that metastably bind glutamate and help guide it into the binding pocket. These simulations also reveal that glutamate can bind in an inverted conformation and also reorient while in its pocket. Electrophysiological recordings demonstrate that eliminating these transient binding sites slows activation and deactivation, consistent with slower glutamate binding and unbinding. These results suggest that binding pathways have evolved to optimize rapid responses of AMPA-type iGluRs at synapses.