The Novel Small Molecule 3hi2one-G4 Selectively Activates Homomeric GIRK4 Channels

Abstract

G protein-sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical therapeutic targets related to neuronal, cardiac and endocrine diseases. Although a number of GIRK channel modulators have been discovered in recent years, highly selective modulators are still lacking. GIRK channels function as either homomeric (i.e. GIRK2 and GIRK4) or heteromeric (e.g. GIRK1/2 and GIRK1/4) tetramers. Activators such as, ML297, ivermectin, and GAT1508 selectively activate GIRK1/2 over GIRK1/4 but not homomeric GIRK2 and GIRK4 channels. VU0529331 was discovered as the first homomeric GIRK channel activator, which shows weak selectivity for GIRK2 over GIRK4 homomeric channels. Here we report the first selective small molecule activator targeting GIRK4 homomeric channels that we refer to as 3hi2one-G4. 3hi2one-G4 selectively activates GIRK4 channels, but does not activate GIRK2, GIRK1/2 or GIRK1/4 channels. By using molecular modeling, mutagenesis, and electrophysiology, we report that the compound utilizes a binding site formed by the TM1, TM2 and slide helix regions of the channel, near the PIP2 site. We identify residue L77 in GIRK4, corresponding to residue I82 in GIRK2, as the selectivity determining residue. 3hi2one-G4 shares a similar binding site and activation mechanism to ivermectin, previously identified as a selective activator of GIRK1/2 heteromeric channels. 3hi2one-G4 could be a useful pharmaceutical probe molecule to study GIRK4 channel function. The newly identified binding site could be used in structure-based drug design studies to identify novel modulators of GIRK channels.