Stimulation of G protein–coupled receptors (GPCRs) that couple to G i/o leads to the activation of G protein–activated inward rectifier K + (GIRK) channels and membrane hyperpolarization in excitable cells. GIRK channel availability can determine the success of optogenetic silencing strategies based on G i/o -coupled GPCRs. To selectively enhance G i/o -mediated GIRK currents, we created expression cassettes that coupled the activity of a GIRK subunit to the light-activated G i/o -coupled GPCRs Opn4L (an opsin also known as melanopsin), longwave-sensitive opsin Opn1, parapinopsin, or Opn7b. We used the GIRK1 F137S mutant because of its fast activation, generation of large currents, and formation of homomeric channels, which reduced the size of the expression cassette. We demonstrated that light-mediated activation of GIRK1 F137S through the opsins induced robust GIRK currents in HEK293 cells, cardiomyocytes, and cerebellar Purkinje neurons. Moreover, coupling GIRK1 F137S activation to the promiscuous Opn4L changed the net effect of G protein signaling from G q/11 -mediated excitation to G i/o -mediated inhibition in cerebellar Purkinje neurons and cardiomyocytes. Thus, our tools optimize the use of GIRK activity in the optogenetic control of the G i/o pathway in excitable cells.
Many signals contribute to the selective assembly of neural circuits alongside the developing brain vasculature. In this issue of Science Signaling , Lu et al. identify endothelial PRMT5 as an epigenetic brake that restrains Angptl4 expression to limit inhibitory synapse formation in the cerebellum, linking angiocrine signaling to synaptic specificity and circuit function.
Brain vasculature is essential for central nervous system function. Here, we found a cerebrovascular signaling axis in which the arginine methyltransferase PRMT5 in endothelial cells promoted proper inhibitory synaptogenesis and motor coordination in male mice. Cerebrovascular endothelial cell–specific deletion of Prmt5 in mice led to excessive inhibitory synaptic input onto Purkinje cells and progressive motor deficits. Mechanistically, Prmt5 deficiency epigenetically increased the expression of the gene encoding the secreted angiocrine ANGPTL4 through reduced symmetric dimethylation of Arg 8 in histone H3 and Arg 3 in histone H4, which coincided with increased acetylation of Lys 9 in histone H3, at the Angptl4 promoter. Angptl4 deletion in brain endothelium normalized inhibitory synaptic input onto Purkinje cells and partially restored motor coordination in mice with a conditional Prmt5 deficiency. Together, these findings define the endothelial PRMT5-ANGPTL4 axis as a key regulator of cerebellar inhibitory circuitry and motor function, highlighting cerebrovascular mechanisms as potential therapeutic targets for motor coordination disorders.
Chemokine receptors are G protein–coupled receptors (GPCRs) that drive cell migration through interactions with chemokines. Even in the absence of ligand, chemokine receptors spend a portion of their time in active conformational states, rendering them constitutively active. Here, we identified and characterized two mutations in the third intracellular loop of atypical chemokine receptor 3 (ACKR3) that inhibited or promoted its constitutive activity as assessed by ligand-independent β-arrestin recruitment and receptor internalization. Single-molecule fluorescence resonance energy transfer experiments showed that these effects on activity corresponded to changes in the conformational equilibrium of the receptor, which also affected the kinetics of chemokine binding. The potencies of chemokines and small-molecule agonists for β-arrestin recruitment were differentially affected by the mutations, showing that the effects of an altered receptor conformational equilibrium on receptor activation were ligand dependent. Together, our findings reveal an inside-out allosteric mechanism for ACKR3 in which intracellular, conformation-altering mutants affect the ability of extracellular ligands to bind to and activate the receptor.
Bifunctional antibodies linking a GPCR with the transferrin receptor lead to GPCR down-regulation.
