CRYO-EM Structures of the GIRK2 Channel Reveal Mechanisms for Lipid Modulation

Abstract

G protein-gated inwardly rectifying potassium (GIRK) channels regulate cellular excitability in the heart and brain. GIRK channels have emerged as a candidate drug target for modulating activity in the brain and treating diseases such as epilepsy or alcoholism. However, structural determinants of gating regulation of GIRK channels by various ligands are not fully understood. Two regions have been identified as important molecular determinants of PIP2 and Gβγ gating: the inner helix bundle crossing (HBC) formed by the M2 TM domain, and the G loop in the cytoplasmic terminal domain (CTD). Our previous work indicated that cholesterol acts as a positive allosteric modulator (PAM) for PIP2. X-ray crystal structures of GIRK channels have revealed several distinct conformations with modulators such as PIP2 and Gβγ. Here, we present the first cryo-EM structures of GIRK2 in the absence and presence of the lipid modulator PIP2 and the cholesterol analog cholesteryl hemisuccinate (CHS). In the absence of PIP2 the apo GIRK2 structure has a cytoplasmic terminal domain (CTD) that is disengaged from the transmembrane domain (TM), in contrast to the apo GIRK2 crystal structure where the CTD is engaged with the TM domain. In the presence of PIP2, we resolve a structural binding domain for CHS, located on the opposite side of the PIP2-channel interface. The diameter of the HBC in the GIRK2/PIP2/CHS structure is larger than in the crystal structure with PIP2 alone. These new structural models suggest a gating mechanism where CHS enhances the PIP2 affinity for the channel, increasing the diameter of the HBC and lowering the threshold for channel activation. These data strongly suggest that CHS acts as a PAM of the channel agonist, PIP2. Funding by NIAAA-AA018734