Abstract
Kir2.1 channels are uniquely activated by phosphoinositide 4,5-bisphosphate (PI(4,5)P2) and can be inhibited by other phosphoinositides (PIPs). Using biochemical and computational approaches, we assess PIP-channel interactions and distinguish residues that are energetically critical for binding from those that alter PIP sensitivity by shifting the open-closed equilibrium. Intriguingly, binding of each PIP is disrupted by a different subset of mutations. In silico ligand docking indicates that PIPs bind to two sites. The second minor site may correspond to the secondary anionic phospholipid site required for channel activation. However, 96-99% of PIP binding localizes to the first cluster, which corresponds to the general PI(4,5)P2 binding location in recent Kir crystal structures. PIPs can encompass multiple orientations; each di- and triphosphorylated species binds with comparable energies and is favored over monophosphorylated PIPs. The data suggest that selective activation by PI(4,5)P2 involves orientational specificity and that other PIPs inhibit this activation through direct competition.
Highlights
Kir2.1 channels are uniquely activated by PI[4,5]P2 and can be inhibited by other PIPs
We speculate that cluster 2 may reflect the secondary anionic phospholipid site that we described previously [5]
Residues Arg189, Arg218, and Lys219, which are located some distance from the crystal structure binding site and from the identified sites in the docking analysis may be influencing binding indirectly, potentially by disruption of the extensive hydrogen bonding network that links the cytoplasmic domain of one subunit to the slide helix of the neighboring subunit [19, 41]
Summary
Kir2.1 channels are uniquely activated by PI[4,5]P2 and can be inhibited by other PIPs. The atomic structures of Kir2.2 [19] and Kir3.2 bound to PI[4,5]P2 [41] have been solved These reveal one specific site, formed at the interface of N- and C-terminal domains, just beyond the transmembrane segments and clearly involving some of the key residues previously identified as controlling PI[4,5]P2 sensitivity. These structures provide no insight into the energetic contributions of the various residues to ligand binding nor explain how multiple residues outside the binding pocket may affect activation.
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