Phosphoinositide control of membrane protein function: a frontier led by studies on ion channels

Abstract

Anionic phospholipids are critical constituents of the inner leaflet of the plasma membrane, ensuring appropriate membrane topology of transmembrane proteins. Additionally, in eukaryotes, the negatively charged phosphoinositides serve as key signals not only through their hydrolysis products but also through direct control of transmembrane protein function. Direct phosphoinositide control of the activity of ion channels and transporters has been the most convincing case of the critical importance of phospholipid-protein interactions in the functional control of membrane proteins. Furthermore, second messengers, such as [Ca(2+)]i, or posttranslational modifications, such as phosphorylation, can directly or allosterically fine-tune phospholipid-protein interactions and modulate activity. Recent advances in structure determination of membrane proteins have allowed investigators to obtain complexes of ion channels with phosphoinositides and to use computational and experimental approaches to probe the dynamic mechanisms by which lipid-protein interactions control active and inactive protein states.

Keywords: P2; P2-induced gating; PI(4,5); ion channels; modulation; phosphoinositides; phosphorylation.

Figure 1

PIPs, their metabolism, and protein domains that bind them. PI is phosphorylated by specific kinases at the 3′, 4′, or 5′ positions to yield monophosphorylated PIPs. PIP kinases recognize the monophosphorylated species and phosphorylate them to yield the diphosphorylated PIPs. PIP₃ can be formed by phosphorylation of PI(4,5)P₂ by PI3K. The reverse reactions are catalyzed by phosphatases acting at the 3′, 4′, or 5′ positions (5′ phosphatase, e.g., SHIPs; 3′ phosphatase, e.g., PTEN). Phospholipid-binding domains that recognize specific PIPs are also shown. Modified from Reference . Abbreviations: ENTH domain, epsin N-terminal homology domain; FYVE domain, domain common to the Fab1, YOTB, Vac1, and EEA1 proteins; PH domain, pleckstrin homology domain; PHD fingers, plant homeodomain fingers; PI, phosphatidylinositol; PI(3,4)P₂, phosphatidylinositol 3,4-bisphosphate; PI(3,4)P₂5K, phosphatidylinositol 3,4-bisphosphate 5-kinase; PI(3,4,5)P₃, phosphatidylinositol 3,4, 5-trisphosphate; PI(3,5)P₂, phosphatidylinositol 3,5-bisphosphate; PI(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; PI3K, phosphatidylinositol 3-kinase; PI3P, phosphatidylinositol 3-phosphate phosphatase; PI3P4K, phosphatidylinositol 3-phosphate 4-kinase; PI3P5K, phosphatidylinositol 3-phosphate 5-kinase; PI4K, phosphatidylinositol 4-kinase; PI4P, phosphatidylinositol 4-phosphate phosphatase; PI4P5K, phosphatidylinositol 4-phosphate 5-kinase; PI5K, phosphatidylinositol 5-kinase; PI5P, phosphatidylinositol 5-phosphate phosphatase; PI5P4K, phosphatidylinositol 5-phosphate 4-kinase; PIPs, phosphoinositides; PROPPINS, β-propellers that bind polyphosphoinositides; PTEN, phosphatase and tensin homolog; PTB domain, phosphotyrosine-binding domain; SHIPs, SH2-containing inositol 5-phosphatase.

Figure 2

Putative interaction site of the Kir3.1 chimera with PI(4,5)P₂. (a) PI(4,5)P₂ molecules are colored yellow, orange, and red (denoting carbon, phosphorus, and oxygen atoms, respectively). PI(4,5)P₂ is shown in the context of the whole GIRK2 channel (gray). The thick black lines indicate the approximate boundaries of the plasma membrane, and the black box highlights the region of the close-up view in panel b. (b) Close-up view of PI(4,5)P₂ interactions. The main coordinating residues are shown as sticks. Residues K90 and R92 were modeled as alanines due to a lack of electron density but probably contribute to the positive electrostatics of the binding site. The important gating residue of the inner helix (or helix bundle crossing), F192, is also shown for reference. (c) The juxtamembrane Kir3.1 chimera region, where a nonylglucoside (red) with its contoured omit map (cyan mesh) is cocrystallized with the channel. Channel subunits and selected side chains near the detergent molecule are colored green and yellow, respectively. Residues 303–308 in the G-loop are colored cyan. Side chains colored white alter activation by PI(4,5)P₂ when mutated. Among them, side chains of K188, K189, E192, and R219 were disordered and poorly defined in the electron-density map, and residues 67 and 68 on the N-terminal side of the interfacial helix (yellow) were omitted for clarity. Abbreviations: CTD, C-terminal domain; PI(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; TM, transmembrane. Panels a and b adapted from Reference 51 with permission from Elsevier. Panel c adapted from Reference 53 with permission from the European Molecular Biology Organization.

Figure 3

Structure of the Kir3.1 chimera and its activation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. (a) Superposition of two crystallized conformations of the chimeric crystal. For clarity, only two opposing subunits of the homotetramer are shown. The cytoplasmic pore in one channel (blue) is dilated, whereas the other (red) is constricted. Residues 302–309 in the G-loop are highlighted. Panel a adapted from Nishida et al. (53). (b) Surface representations of the subunits shown in panel a, showing the movement of the cytosolic domains as the G-loop gate transitions from a constricted (closed, orange) to a dilated (open, blue) conformation. (c) Similar snapshot as in panel a, with all three gates labeled (in red). The blue dots represent the position of residues that, when mutated, alter the channel’s PI(4,5)P₂ sensitivity (see text). Abbreviations: HBC, helix bundle crossing; SF, selectivity filter.(d) Summary of the major results of Nishida et al. (53) and Meng et al. (61). Transitioning from closed to open, the secondary structure elements switch their close interactions from adjacent elements on one side to the elements on the other side. PI(4,5)P₂ stabilizes the conformation by direct interactions with the CD-loop and N terminus. Ethanol and Gβγ act by lodging themselves in the DE-LM cleft to stabilize the LM-loop interactions with the N terminus. Panel d adapted from Mahajan et al. (84).

Figure 4

Regulation of PI(4,5)P₂ interactions with the SK2/CaM complex by phosphorylation. (a) The putative PI(4,5)P₂-binding site includes the CaM linker and the SK2 channel fragment before the CaM-binding domain. (b) Molecular dynamics simulations showing the interactions of the PI(4,5)P₂ phosphates with the positively charged K77 (CaM) and K402 and K405 (SK2). (c) Phosphomimetic T79D mutation of CaM decreases the interaction between the PI(4,5)P₂-binding site and the PI(4,5)P₂ head group (atomic distances between interacting residues are shown in angstroms). Abbreviation: CaM, calmodulin; PI(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; SK2, small-conductance K⁺ channel subtype 2.