Electrical synapse structure requires distinct isoforms of a postsynaptic scaffold.

Electrical synapses are neuronal gap junction (GJ) channels associated with a macromolecular complex called the electrical synapse density (ESD), which regulates development and dynamically modifies electrical transmission. However, the proteomic makeup and molecular mechanisms utilized by the ESD that direct electrical synapse formation are not well understood. Using the Mauthner cell of zebrafish as a model, we previously found that the intracellular scaffolding protein ZO1b is a member of the ESD, localizing postsynaptically, where it is required for GJ channel localization, electrical communication, neural network function, and behavior. Here, we show that the complexity of the ESD is further diversified by the genomic structure of the ZO1b gene locus. The ZO1b gene is alternatively initiated at three transcriptional start sites resulting in isoforms with unique N-termini that we call ZO1b-Alpha, -Beta, and -Gamma. We demonstrate that ZO1b-Beta and ZO1b-Gamma are broadly expressed throughout the nervous system and localize to electrical synapses. By contrast, ZO1b-Alpha is expressed mainly non-neuronally and is not found at synapses. We generate mutants in all individual isoforms, as well as double mutant combinations in cis on individual chromosomes, and find that ZO1b-Beta is necessary and sufficient for robust GJ channel localization. ZO1b-Gamma, despite its localization to the synapse, plays an auxiliary role in channel localization. This study expands the notion of molecular complexity at the ESD, revealing that an individual genomic locus can contribute distinct isoforms to the macromolecular complex at electrical synapses. Further, independent scaffold isoforms have differential contributions to developmental assembly of the interneuronal GJ channels. We propose that ESD molecular complexity arises both from the diversity of unique genes and from distinct isoforms encoded by single genes. Overall, ESD proteomic diversity is expected to have critical impacts on the development, structure, function, and plasticity of electrical transmission.

Neurobeachin controls the asymmetric subcellular distribution of electrical synapse proteins.

The subcellular positioning of synapses and their specialized molecular compositions form the fundamental basis of neural circuits. Like chemical synapses, electrical synapses are constructed from an assortment of adhesion, scaffolding, and regulatory molecules, yet little is known about how these molecules localize to specific neuronal compartments. Here, we investigate the relationship between the autism- and epilepsy-associated gene Neurobeachin, the neuronal gap junction channel-forming Connexins, and the electrical synapse scaffold ZO1. Using the zebrafish Mauthner circuit, we find Neurobeachin localizes to the electrical synapse independently of ZO1 and Connexins. By contrast, we show Neurobeachin is required postsynaptically for the robust localization of ZO1 and Connexins. We demonstrate that Neurobeachin binds ZO1 but not Connexins. Finally, we find Neurobeachin is required to restrict electrical postsynaptic proteins to dendrites, but not electrical presynaptic proteins to axons. Together, the results reveal an expanded understanding of electrical synapse molecular complexity and the hierarchical interactions required to build neuronal gap junctions. Further, these findings provide novel insight into the mechanisms by which neurons compartmentalize the localization of electrical synapse proteins and provide a cell biological mechanism for the subcellular specificity of electrical synapse formation and function.

Isolation of Immunocomplexes from Zebrafish Brain.

Zebrafish is an excellent model to study vertebrate neurobiology, but its synaptic components that mediate and regulate fast electrical synaptic transmission are largely unidentified. Here, we describe methods to solubilize and immunoprecipitate adult zebrafish brain homogenate under conditions to preserve electrical synapse protein complexes. The methods presented are well-suited to probe electrical synapse immunocomplexes, and potentially other brain-derived immunocomplexes, for candidate interactors from zebrafish brain.

The disconnect2 mutation disrupts the tjp1b gene that is required for electrical synapse formation.

To investigate electrical synapse formation in vivo we used forward genetics to disrupt genes affecting Mauthner cell electrical synapses in larval zebrafish. We identify the disconnect2 ( dis2 ) mutation for its failure to localize neural gap junction channels at electrical synapses. We mapped this mutation to chromosome 25 and identified a splice-altering mutation in the tjp1b gene. We demonstrated that the dis2 mutation disrupts tjp1b function using complementation analysis with CRISPR generated mutants. We conclude that the dis2 mutation disrupts the tjp1b gene that is required for electrical synapse formation.

Transformation of an early-established motor circuit during maturation in zebrafish.

Locomotion is mediated by spinal circuits that generate movements with a precise coordination and vigor. The assembly of these circuits is defined early during development; however, whether their organization and function remain invariant throughout development is unclear. Here, we show that the first established fast circuit between two dorsally located V2a interneuron types and the four primary motoneurons undergoes major transformation in adult zebrafish compared with what was reported in larvae. There is a loss of existing connections and establishment of new connections combined with alterations in the mode, plasticity, and strength of synaptic transmission. In addition, we show that this circuit no longer serves as a swim rhythm generator, but instead its components become embedded within the spinal escape circuit and control propulsion following the initial escape turn. Our results thus reveal significant changes in the organization and function of a motor circuit as animals develop toward adulthood.

Electrical synaptic transmission requires a postsynaptic scaffolding protein.

Electrical synaptic transmission relies on neuronal gap junctions containing channels constructed by Connexins. While at chemical synapses neurotransmitter-gated ion channels are critically supported by scaffolding proteins, it is unknown if channels at electrical synapses require similar scaffold support. Here, we investigated the functional relationship between neuronal Connexins and Zonula Occludens 1 (ZO1), an intracellular scaffolding protein localized to electrical synapses. Using model electrical synapses in zebrafish Mauthner cells, we demonstrated that ZO1 is required for robust synaptic Connexin localization, but Connexins are dispensable for ZO1 localization. Disrupting this hierarchical ZO1/Connexin relationship abolishes electrical transmission and disrupts Mauthner cell-initiated escape responses. We found that ZO1 is asymmetrically localized exclusively postsynaptically at neuronal contacts where it functions to assemble intercellular channels. Thus, forming functional neuronal gap junctions requires a postsynaptic scaffolding protein. The critical function of a scaffolding molecule reveals an unanticipated complexity of molecular and functional organization at electrical synapses.

Neurons 'talk' with each another at junctions called synapses, which can either be chemical or electrical. Communication across a chemical synapse involves a 'sending' neuron releasing chemicals that diffuse between the cells and subsequently bind to specialized receptors on the receiving neuron. These complex junctions involve a large number of well-studied molecular actors. Electrical synapses, on the other hand, are believed to be simpler. There, neurons are physically connected via channels formed of 'connexin' proteins, which allow electrically charged ions to flow between the cells. However, it is likely that other proteins help to create these structures. In particular, recent evidence shows that without a structurally supporting 'scaffolding' protein called ZO1, electrical synapses cannot form in the brain of a tiny freshwater fish known as zebrafish. As their name implies, scaffolding proteins help cells organize their internal structure, for example by anchoring other molecules to the cell membrane. By studying electrical synapses in zebrafish, Lasseigne, Echeverry, Ijaz, Michel et al. now show that these structures are more complex than previously assumed. In particular, the experiments reveal that ZO1 proteins are only present on one side of electrical synapses; despite their deceptively symmetrical anatomical organization, these junctions can be asymmetric, like their chemical cousins. The results also show that ZO1 must be present for connexins to gather at electrical synapses, whereas the converse is not true. This suggests that when a new electrical synapse forms, ZO1 moves into position first: it then recruits or stabilizes connexins to form the channels connecting the two cells. In many animals with a spine, electrical synapses account for about 20% of all neural junctions. Understanding how these structures form and work could help to find new treatments for disorders linked to impaired electrical synapses, such as epilepsy.

Asymmetry of an Intracellular Scaffold at Vertebrate Electrical Synapses.

Neuronal synaptic connections are either chemical or electrical, and these two types of synapses work together to dynamically define neural circuit function [1]. Although we know a great deal about the molecules that support chemical synapse formation and function, we know little about the macromolecular complexes that regulate electrical synapses. Electrical synapses are created by gap junction (GJ) channels that provide direct ionic communication between neurons [2]. Although they are often molecularly and functionally symmetric, recent work has found that pre- and postsynaptic neurons can contribute different GJ-forming proteins, creating molecularly asymmetric channels that are correlated with functional asymmetry at the synapse [3, 4]. Associated with the GJs are structures observed by electron microscopy termed the electrical synapse density (ESD) [5]. The ESD has been suggested to be critical for the formation and function of the electrical synapse, yet the biochemical makeup of these structures is poorly understood. Here we find that electrical synapse formation in vivo requires an intracellular scaffold called Tight Junction Protein 1b (Tjp1b). Tjp1b is localized to the electrical synapse, where it is required for the stabilization of the GJs and for electrical synapse function. Strikingly, we find that Tjp1b protein localizes and functions asymmetrically, exclusively on the postsynaptic side of the synapse. Our findings support a novel model of electrical synapse molecular asymmetry at the level of an intracellular scaffold that is required for building the electrical synapse. We propose that such ESD asymmetries could be used by all nervous systems to support molecular and functional asymmetries at electrical synapses.

Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.

Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.

NMDA receptor structures reveal subunit arrangement and pore architecture.

N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present X-ray crystal structures of the Xenopus laevis GluN1-GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

Crystal structure of the ATP-gated P2X(4) ion channel in the closed state.

P2X receptors are cation-selective ion channels gated by extracellular ATP, and are implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Because P2X receptors are not related to other ion channel proteins of known structure, there is at present no molecular foundation for mechanisms of ligand-gating, allosteric modulation and ion permeation. Here we present crystal structures of the zebrafish P2X(4) receptor in its closed, resting state. The chalice-shaped, trimeric receptor is knit together by subunit-subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in beta-strands, have large acidic patches that may attract cations, through fenestrations, to vestibules near the ion channel. In the transmembrane pore, the 'gate' is defined by an approximately 8 A slab of protein. We define the location of three non-canonical, intersubunit ATP-binding sites, and suggest that ATP binding promotes subunit rearrangement and ion channel opening.