G protein–gated inwardly rectifying potassium (GIRK) channels control neuronal excitability in the brain and are implicated in several different neurological diseases. The anionic phospholipid phosphatidylinositol 4,5 bisphosphate (PIP 2 ) is an essential cofactor for GIRK channel gating, but the precise mechanism by which PIP 2 opens GIRK channels remains poorly understood. Previous structural studies have revealed several highly conserved, positively charged residues in the “tether helix” (C-linker) that interact with the negatively charged PIP 2 . However, these crystal structures of neuronal GIRK channels in complex with PIP 2 provide only snapshots of PIP 2 ’s interaction with the channel and thus lack details about the gating transitions triggered by PIP 2 binding. Here, our functional studies reveal that one of these conserved basic residues in GIRK2, Lys200 (6′K), supports a complex and dynamic interaction with PIP 2 . When Lys200 is mutated to an uncharged amino acid, it activates the channel by enhancing the interaction with PIP 2 . Atomistic molecular dynamic simulations of neuronal GIRK2 with the same 6′ substitution reveal an open GIRK2 channel with PIP 2 molecules adopting novel positions. This dynamic interaction with PIP 2 may explain the intrinsic low open probability of GIRK channels and the mechanism underlying activation by G protein Gβγ subunits and ethanol.

Steeply voltage-dependent block by intracellular polyamines underlies the strong inward rectification properties of Kir2.1 and other Kir channels. Mutagenesis studies have identified several negatively charged pore-lining residues (D172, E224, and E299, in Kir2.1) in the inner cavity and cytoplasmic domain as determinants of the properties of spermine block. Recent crystallographic determination of the structure of the cytoplasmic domains of Kir2.1 identified additional negatively charged residues (D255 and D259) that influence inward rectification. In this study, we have characterized the kinetic and steady-state properties of spermine block in WT Kir2.1 and in mutations of the D255 residue (D255E, A, K, R). Despite minimal effects on steady-state blockade by spermine, D255 mutations have profound effects on the blocking kinetics, with D255A marginally, and D255R dramatically, slowing the rate of block. In addition, these mutations result in the appearance of a sustained current (in the presence of spermine) at depolarized voltages. These features are reproduced with a kinetic model consisting of a single open state, two sequentially linked blocked states, and a slow spermine permeation step, with residue D255 influencing the spermine affinity and rate of entry into the shallow blocked state. The data highlight a “long-pore” effect in Kir channels, and emphasize the importance of considering blocker permeation when assessing the effects of mutations on apparent blocker affinity.