G protein–coupled receptors (GPCRs) play a fundamental role in the modulation of synaptic transmission. A pivotal example is provided by the metabotropic glutamate receptor type 4 (mGluR4), which inhibits glutamate release at presynaptic active zones (AZs). However, how GPCRs are organized within AZs to regulate neurotransmission remains largely unknown. Here, we applied two-color super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the nanoscale organization of mGluR4 at parallel fiber AZs in the mouse cerebellum. We find an inhomogeneous distribution, with multiple nanodomains inside AZs, each containing, on average, one to two mGluR4 subunits. Within these nanodomains, mGluR4s are often localized in close proximity to voltage-dependent CaV2.1 channels and Munc-18-1, which are both essential for neurotransmitter release. These findings provide previously unknown insights into the molecular organization of GPCRs at AZs, suggesting a likely implication of a close association between mGluR4 and the secretory machinery in modulating synaptic transmission.

γ-aminobutyric acid type A receptors (GABAARs) are the major mediators of synaptic inhibition in the brain. Aberrant GABAAR activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer’s and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review we introduce a new strategy to modulate GABAeric signaling: Targeting the intracellular protein interactors of GABAARs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABAAR subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABAAR-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABAAR numbers and clustering, modifying neuronal transmission. Interference with GABAAR trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABAAR to AP2 increase the surface concentration of GABAAR clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with nanometer resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABAAR accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABAAR modulators and following in-vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.

In fungi green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis, a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371) and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta, whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of White Collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids, especially by acetic acid and indole-3-acetic acid. In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis.