Improving optogenetic control over G_i/o-coupled receptor signaling by optimized GIRK channel expression

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.