RIM and RIM-binding protein localize synaptic CaV2 channels to differentially regulate transmission in neuronal circuits.

At chemical synapses, voltage-gated Ca2+-channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca2+ microdomains they elicit must be located precisely to primed SVs, to evoke rapid transmitter release. Localization is mediated by Rab3 interacting molecule (RIM) and RIM-binding proteins (RIM-BPs), which interact and bind to the C-terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction (NMJ) of Caenorhabditis elegans hermaphrodites. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Even though postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABAA or GABAB receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus RIMB-1 function differentially affects excitation/inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission in mixed circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 ß-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerges from VGCC regulation.Significance statement The machinery for chemical synaptic transmission is organized in a precise spatial arrangement in order to enable efficient and temporally accurate coupling of action potentials with the rise of the Ca2+ concentration through CaV2 P/Q-type voltage gated Ca2+ channels. This triggers the fusion of synaptic vesicles with the plasma membrane and the release of transmitters. Here, we analyzed the molecular and functional interplay of proteins of the active zone scaffold, RIM and RIM-binding protein (RIMB-1), with the CaV2 channel in the C. elegans neuromuscular junction, a tripartite synapse with cholinergic and GABAergic neuronal input. Our work shows a differential requirement of RIMB-1 in cholinergic vs. GABAergic neurons, that affects the regulation of excitation-inhibition balance at circuit, cellular and ultrastructural levels.

Cryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment.

Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H2R) in an active conformation with bound histamine and in complex with Gs heterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion of the receptor into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. Structural comparison with the inactive conformation of H2R and the inactive and Gq-coupled active state of H1R together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized H2R, its agonist-dependent internalization and its interaction with endogenously synthesized H1R and H2R in HEK293 cells by applying a recently developed nanotransfer technique.

Cell-Free Expression of GPCRs into Nanomembranes for Functional and Structural Studies.

Despite their importance in many essential physiological processes of living cells, G protein-coupled receptors (GPCRs) are often difficult to express and purify in sufficient quality and quantity. We demonstrate cell-free protein synthesis as an interesting alternative to classical cell-based expression systems. We focus on a recently developed detergent-free expression mode by co-translational integration of nascent GPCRs into provided nanodisc membranes of defined composition. The protocol is in particular suitable for detergent sensitive targets and allows the synthesis of full-length as well as modified GPCRs. As a basic blueprint for the cell-free synthesis of GPCRs and potentially other membrane proteins as well, we describe the production of the human endothelin-B receptor. Subsequent purification strategies are streamlined by implementing complementary affinity chromatography steps. We further show the evaluation and optimization of the final GPCR samples for homogeneity and activity through a radioligand binding assay.

Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells.

Nanodiscs are emerging to serve as transfer vectors for the insertion of recombinant membrane proteins into membranes of living cells. In combination with cell-free expression technologies, this novel process opens new perspectives to analyze the effects of even problematic targets such as toxic, hard-to-express, or artificially modified membrane proteins in complex cellular environments of different cell lines. Furthermore, transferred cells must not be genetically engineered and primary cell lines or cancer cells could be implemented as well. We have systematically analyzed the basic parameters of the nanotransfer approach and compared the transfer efficiencies from nanodiscs with that from Salipro particles. The transfer of five membrane proteins was analyzed: the prokaryotic proton pump proteorhodopsin, the human class A family G-protein coupled receptors for endothelin type B, prostacyclin, free fatty acids type 2, and the orphan GPRC5B receptor as a class C family member. The membrane proteins were cell-free synthesized with a detergent-free strategy by their cotranslational insertion into preformed nanoparticles containing defined lipid environments. The purified membrane protein/nanoparticles were then incubated with mammalian cells. We demonstrate that nanodiscs disassemble and only lipids and membrane proteins, not the scaffold protein, are transferred into cell membranes. The process is detectable within minutes, independent of the nanoparticle lipid composition, and the transfer efficiency directly correlates with the membrane protein concentration in the transfer mixture and with the incubation time. Transferred membrane proteins insert in both orientations, N-terminus in and N-terminus out, in the cell membrane, and the ratio can be modulated by engineering. The viability of cells is not notably affected by the transfer procedure, and transferred membrane proteins stay detectable in the cell membrane for up to 3 days. Transferred G-protein coupled receptors retained their functionality in the cell environment as shown by ligand binding, induction of internalization, and specific protein interactions. In comparison to transfection, the cellular membrane protein concentration is better controllable and more uniformly distributed within the analyzed cell population. A further notable difference to transfection is the accumulation of transferred membrane proteins in clusters, presumably determined by microdomain structures in the cell membranes.

Ingestion and Toxicity of Polystyrene Microplastics in Freshwater Bivalves.

The ubiquity of microplastics in aquatic ecosystems has raised concerns over their interaction with biota. However, microplastics research on freshwater species, especially mollusks, is still scarce. We, therefore, investigated the factors affecting microplastics ingestion in the freshwater mussel Dreissena polymorpha. Using polystyrene spheres (5, 10, 45, 90 µm), we determined the body burden of microplastics in the mussels in relation to 1) exposure and depuration time, 2) body size, 3) food abundance, and 4) microplastic concentrations. D. polymorpha rapidly ingested microplastics and excreted most particles within 12 h. A few microplastics were retained for up to 1 wk. Smaller individuals had a higher relative body burden of microplastics than larger individuals. The uptake of microplastics was concentration-dependent, whereas an additional food supply (algae) reduced it. We also compared the ingestion of microplastics by D. polymorpha with 2 other freshwater species (Anodonta anatina, Sinanodonta woodiana), highlighting that absolute and relative uptake depends on the species and the size of the mussels. In addition, we determined toxicity of polystyrene fragments (≤63 µm, 6.4-100 000 p mL-1 ) and diatomite (natural particle, 100 000 p mL-1 ) in D. polymorpha after 1, 3, 7, and 42 d of exposure, investigating clearance rate, energy reserves, and oxidative stress. Despite ingesting large quantities, exposure to polystyrene fragments only affected the clearance rate of D. polymorpha. Further, results of the microplastic and diatomite exposure did not differ significantly. Therefore, D. polymorpha is unaffected by or can compensate for polystyrene fragment toxicity even at concentrations above current environmental levels. Environ Toxicol Chem 2021;40:2247-2260. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.