The paucity of specific GIRK channel modifiers motivated a de novo search for new GIRK modulators. In 2013, ML297 was identified in a high-throughput screening (HTS) assay, which was based on a thallium-flux measurement in HEK293 cells expressing GIRK1/GIRK2 channels [24]. Kaufmann and colleagues performed chemical optimization of an initial promising compound identified in the HTS assay (VU0032230), which led to the discovery of ML297 (Figure 2A, Table 1) [25]. ML297 was the first potent activator of GIRK1/GIRK2, with an EC50 of 160 nM. ML297 exhibited modest selectivity towards GIRK1/GIRK2 subunits, relative to GIRK1/GIRK3 and GIRK1/GIRK4 (around 6-fold range), and an absence of activity on GIRK1-lacking channels. Two amino acids in mouse GIRK1 (mGIRK1), F137 in the pore helix and D173 in the second membrane-spanning domain (Figure 1B), were subsequently identified as required for ML297 activity [26]. ML297 activation of GIRK1/GIRK2 required the presence of the membrane phospholipid, Phosphatidylinositol 4,5-bisphosphate (PIP2), but not G protein Gβγ subunits, therefore exhibiting a GPCR-independent mechanism of channel activation [25]. ML297 activated native GIRK channels expressed in hippocampal mouse neurons in acutely prepared brain slices (most likely GIRK1/GIRK2 channels) (Table 1) [26, 27].
