Electrical synapse molecular diversity revealed by proximity-based proteomic discovery.

Neuronal circuits are composed of synapses that are either chemical, where signals are transmitted via neurotransmitter release and reception, or electrical, where signals pass directly through interneuronal gap junction channels. While the molecular complexity that controls chemical synapse structure and function is well appreciated, the proteins of electrical synapses beyond the gap-junction-forming Connexins are not well defined. Yet, electrical synapses are expected to be molecularly complex beyond the gap junctions.

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

To investigate electrical synapse formation we used forward genetics to disrupt genes affecting Mauthner cell electrical synapses in larval zebrafish. We identify the ( ) 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 gene.

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.

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.

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].

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.

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.

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 protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways.

Cyclic adenosine 3', 5'-monophosphate (cAMP) is a ubiquitous mediator of intracellular signalling events. It acts principally through stimulation of cAMP-dependent protein kinases (PKAs) but also activates certain ion channels and guanine nucleotide exchange factors (Epacs). Metabolism of cAMP is catalysed by phosphodiesterases (PDEs).

PKA-phosphorylation of PDE4D3 facilitates recruitment of the mAKAP signalling complex.

mAKAP (muscle-selective A-kinase-anchoring protein) co-ordinates a cAMP-sensitive negative-feedback loop comprising PKA (cAMP-dependent protein kinase) and the cAMP-selective PDE4D3 (phosphodiesterase 4D3). In vitro and cellular experiments demonstrate that PKA-phosphorylation of PDE4D3 on Ser-13 increases the affinity of PDE4D3 for mAKAP. Our data suggest that activation of mAKAP-anchored PKA enhances the recruitment of PDE4D3, allowing for quicker signal termination.

Intracellular targeting of protein kinases and phosphatases.

Compartmentalization of kinases and phosphatases is a key determinant in the specificity of second messenger-mediated signaling events. Localization of the cAMP-dependent protein kinase (PKA) and other signaling enzymes is mediated by interaction with A-kinase anchoring proteins (AKAPs). This study focused on recent advances that further our understanding of AKAPs, with particular emphasis on the bidirectional regulation of signaling events by AKAP signaling complexes and their contribution to the control of actin reorganization events.