Although ML297 also activates GIRK1-containing channels [25], there are several important differences to consider. In addition to differences in the method of discovery (Figure 2), GiGA1 showed different kinetics of modulation. While ML297 has a slow off-rate (t1/2 ≈ 20 s), GiGA1 exhibits fast (alcohol-like) activation and deactivation rates (2–3 s). This difference might be attributable to different binding mechanisms and affinity. It has been previously demonstrated that two amino acids in the membrane-spanning region of GIRK1 are critical for ML297 action on GIRK1/GIRK2 channels [26] (Figure 1B). Mutation of two amino acids in GIRK2 to the equivalent amino acid in GIRK1 enabled activation by ML297, though only in the context of co-expression with wild-type GIRK2 [26]. However, neither of these positions appeared to be important for GiGA1 activation. Since GiGA1 identification was based on the GIRK2 alcohol pocket, mutagenesis studies and related molecular dynamics simulations revealed critical residues in the alcohol pocket of GIRK1/GIRK2 channel for GiGA1 action (Figure 1B). In particular, a pair of amino acids (R43 on mGIRK1 and D346 on mGIRK2) located in the subunit interface region seems to contribute to the subunit specificity of GiGA1 [46].
