Guiding G protein signaling by target enhancement of GPCRs

Activation of G protein coupled receptors coupling to the G_i/opathway leads to the activation of G protein-activated inward rectifier potassium channels (GIRK) in a fast membrane-delimited manner in excitable cells. Activation of GIRK causes the hyperpolarization of the cell membrane, where hyperpolarization is dependent on te availability of G_i/ocoupled GPCRs and GIRK. In particular, in optogenetic and chemogenetic experiments neuronal silencing depends on downstream targets of G_i/o-coupled GPCRs. To selectively enhance G_i/omediated GIRK currents, we created expression cassettes consisting of a homomer forming GIRK subunit and various light-activated G_i/o-coupled GPCRs (Melanopsin, Long-wave-sensitive opsin 1, Parapinopsin or Opsin 7b). We demonstrate that light-activation of the GIRK/GPCR constructs induces robust GIRK currents in human embryonic kidney 293 cells, cardiomyocytes and cerebellar Purkinje cells and changes the net effect of G protein signaling of the promiscuous Opn4L from a G_q/11mediated excitation towards an G_i/omediated inhibition. Thus, our tools enhance target selectivity and improve optogenetic control of the G_i/opathway by light in excitable cells.