Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2

Abstract

The regulation of ion channel activity by specific lipid molecules is widely recognized as an integral component of electrical signalling in cells. In particular, phosphatidylinositol 4,5-bisphosphate (PIP(2)), a minor yet dynamic phospholipid component of cell membranes, is known to regulate many different ion channels. PIP(2) is the primary agonist for classical inward rectifier (Kir2) channels, through which this lipid can regulate a cell's resting membrane potential. However, the molecular mechanism by which PIP(2) exerts its action is unknown. Here we present the X-ray crystal structure of a Kir2.2 channel in complex with a short-chain (dioctanoyl) derivative of PIP(2). We found that PIP(2) binds at an interface between the transmembrane domain (TMD) and the cytoplasmic domain (CTD). The PIP(2)-binding site consists of a conserved non-specific phospholipid-binding region in the TMD and a specific phosphatidylinositol-binding region in the CTD. On PIP(2) binding, a flexible expansion linker contracts to a compact helical structure, the CTD translates 6 Å and becomes tethered to the TMD and the inner helix gate begins to open. In contrast, the small anionic lipid dioctanoyl glycerol pyrophosphatidic acid (PPA) also binds to the non-specific TMD region, but not to the specific phosphatidylinositol region, and thus fails to engage the CTD or open the channel. Our results show how PIP(2) can control the resting membrane potential through a specific ion-channel-receptor-ligand interaction that brings about a large conformational change, analogous to neurotransmitter activation of ion channels at synapses.

Figure 1. Effect of a short chain PIP₂ on Kir2.2

a, Endogenous PIP₂ depletion causes “run down” of Kir2.2 channels in an excised inside out patch from Xenopus oocytes as shown by the three macroscopic current traces recorded with a voltage ramp from−80 to +80 mV immediately (green), 30 min (blue), and 50 min (black) after patch excision. b, The short chain PIP₂ added to the bath solution (solid line with concentration indicated below) 30 min after patch excision partially rescued Kir2.2 channel activity. The bath was then perfused (dashed line) at time 40 min with ~1 ml/min bath solution for 3 min. c, X-ray crystal structures of apo (left, pdb 3JYC) and PIP₂ bound (right, pdb 3SPI) Kir 2.2 tetramer (grey α-carbon traces) viewed from the side with the extracellular solution above. The lipid bilayer boundaries are shown as grey bars. Four PIP₂ molecules are shown as sticks and colored according to atom type: carbon, yellow; phosphorous, orange; and oxygen, red. One PIP₂ molecule in the same orientation as in figure 2a is outlined by a black box. Upon PIP₂ binding the flexible linker between CTD and TMD consisting of two strands (highlighted green for one subunit, dotted line indicating disordered region in the crystal structure) form helical structures, and the CTD translates towards the TMD by 6 Å. A set of reference atoms (Asp72 and Lys220 α-carbons) are highlighted as blue spheres in each structure.

Figure 2. PIP₂ binding site

a, A detailed view of the PIP₂ binding site is shown in the same orientation as outlined in figure 1c. Helices (shown as ribbon) from different subunits are colored orange and cyan interior. Residues hydrogen bonded (dashed lines) to PIP₂ are colored green, and residues stabilizing the PIP₂ binding site in the CTD while lacking direct contact are colored blue. All side chains are shown as sticks. PIP₂ is shown as sticks and colored according to atom type: carbon, yellow; phosphorous, orange; and oxygen, red. b, An amino acid sequence alignment of selected eukaryotic Kirs showing residues predicted from literature (blue outline) and not predicted (purple outline) to interact with PIP₂9,28. Residues with direct bonding interactions to PIP₂ and with a structural role are highlighted with green and blue shade, respectively. The two residues serving as the inner helix gate are also highlighted with grey shade.

Figure 3. Conserved non-specific lipid binding site in Kirs

a, A grey α-carbon representation of Kir2.2 tetramer in complex with PPA, a small anionic lipid lacking an inositol ring. PPA bound Kir2.2 assumes a closed conformation similar to apo (pdb 3JYC) with the flexible linker elongated and the CTD unengaged. The four PPA molecules are shown as sticks and colored according to atom type: carbon, yellow; phosphorous, orange; and oxygen, red. b, A close-up view of the PPA binding site. PPA contacts Kir at the cytoplasmic end of the outer helix making strong interactions with the guanidiniums of R78 and R80 and the backbone amide nitrogens of the helix turn; similar to the interactions of the 1′ phosphate of PIP₂. However, residues (blue sticks) for interacting with the PIP₂ inositol 4′,5′ phosphate remain distant to the lipid binding site; R186 orients with its side chain pointing towards the ion conduction pathway. c, Superposition of PPA (colored the same as in panel a) and PIP₂ (grey). The position of the 1′ phosphate in PIP₂ is between the pyrophosphate of PPA.

Figure 4. A proposed mechanism of Kir2.2 activation by PIP₂

a, Superposition of the TMD inner helices of the PIP₂ bound (blue ribbon) and apo (red ribbon ) Kir2.2 structures. PIP₂ binding results in a splaying of the helices near the helix bundle activation gate. b and c, comparison of the inner helix bundle gate in PPA bound Kir2.2 (b) and PIP₂ bound Kir2.2 (c) viewed from the extracellular side. Side chains of the residues in the bundle crossing are represented as either grey sticks or space filling CPK models (carbon, yellow; and sulfur, green). d, A proposed mechanism for Kir2.2 activation by PIP₂. PIP₂(purple sphere) binds at an interface between the TMD (grey cylinder) and the CTD (grey rectangle) and induces a large conformational change: a flexible linker (green line) contracts to a compact helical structure (green cylinder), the CTD translates towards and becomes tethered to the TMD, the G-loop (cyan wedge) inserts into the TMD, and the inner helix activation gate opens.