| We examined the gating mechanism of G protein-activated K+ channels (GIRK) by using a combination of genetic selection and mutagenesis. GIRK2 (Kir3.2) is a major contributor to G protein-activated inward-rectifier potassium channels in the mammalian brain. GIRK channels are mostly closed in the basal state, but activate upon binding the Gbetagamma subunits of G proteins. This implies that, at the molecular level, GIRK channels undergo a conformational change between a closed, or non-conducting, state and an open state-a process referred to as gating. A central goal of ion channel research is to understand how ion channels gate in response to different stimuli.; Saccharomyces cerevisiae that lack the high affinity K-uptake transporters, Trk1 and Trk2, require the expression of a highly active inward rectifier potassium channel to grow on low potassium medium. The inward rectifier, GIRK2, does not complement the potassium-uptake deficient phenotype, in part, because it has an open probability (Po) <1% making it possible to select for mutations that increase the probability of opening by expressing GIRK2 mutants in yeast. We screened a large library of randomly mutated GIRK2 for gating mutations. Five mutations at four positions increased the open probability of GIRK2 while producing dramatic effects on the single channel kinetics. GIRK2, in the absence of stimulation, showed short, flickery openings. The mutant channels, however, displayed bursts of openings similar those observed when GIRK2 is stimulated by Gbetagamma. Interestingly, all of these mutations are located within the core transmembrane domain of GIRK2: N94H in M1; E152D in the P loop; S177T and V188 to A and G in M2.; By substituting all twenty amino acids into each of the positions identified, we were able to deduce features of the open and closed state of GIRK2. In agreement with the KcsA structure where the equivalent residue is buried in the M2-M2 interface; GIRK2 V188 is likely buried in protein in the closed state. In the open state, however, our analysis suggested that V188 resides in a pore-lining position consistent with the open state model of IRK1. We, therefore, propose that the gating of GIRK2 can be modeled as a conformational change from a closed state similar to KcsA to an open state resembling IRK1. |
