Phosphoinositides regulate ion channels

Abstract

Phosphoinositides serve as signature motifs for different cellular membranes and often are required for the function of membrane proteins. Here, we summarize clear evidence supporting the concept that many ion channels are regulated by membrane phosphoinositides. We describe tools used to test their dependence on phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, and consider mechanisms and biological meanings of phosphoinositide regulation of ion channels. This lipid regulation can underlie changes of channel activity and electrical excitability in response to receptors. Since different intracellular membranes have different lipid compositions, the activity of ion channels still in transit towards their final destination membrane may be suppressed until they reach an optimal lipid environment. This article is part of a Special Issue entitled Phosphoinositides.

Keywords: Calcium channel; G-protein coupled receptor (GPCR); Phosphatidylinositol 4,5-bisphosphate; Phospholipase C (PLC); Potassium channel; Transient receptor potential channel (TRP channel).

Fig. 1

Organelle phosphoinositide signature helps regulate ion channel function. Schematic representation of the predominant subcellular localization of phosphoinositide species in each organelle. PI(4,5)P₂ and PI(3,4,5)P₃ are concentrated at the plasma membrane (PM). PI(3,4)P₂ is found mostly in early endocytic pathways distal to the plasma membrane. PI(4)P is principally concentrated in the Golgi complex, but can also be found at the plasma membrane and secretory pathways (SGs). PI(3)P is located in early endosomes (EE) and PI(3,5)P₂ on late endosomal (LE), multi-vesicular body (MVB), and lysosomal compartments. PI is found in the endoplasmic reticulum (ER). Grey arrows represent the continuous flow of membrane between organelles. Note that heterotetrameric KCNQ2/3 ion channels are closed (no arrow) during trafficking and retrieval from the plasma membrane, but are open (with arrow) at the plasma membrane due to the presence of appropriate activating phosphoinositide (PI(4,5)P₂) and membrane voltage.

Fig. 2

Metabolic synthesis and cleavage of PI(4,5)P₂. A) Production of PI(4,5)P₂ from PI in two steps of phosphorylation. Lipid kinases transfer phosphates from ATP to the inositol 4- and 5-hydroxyls, and lipid phosphatases remove the phosphates in continuous steady-state cycles. B) G_q-couple receptors activate PLCβ, which cleaves PI(4,5)P₂ into soluble inositol 1,4,5-trisphosphate (IP₃) and membrane-bound diacylglycerol. IP₃ releases calcium from intracellular stores and diacylglycerol recruits and activates PKC.

Fig. 3

Structural basis of PI(4,5)P₂ activation of Kir2.2. A) X-Ray crystal structures of the Kir2.2 homotetrameric channel in the absence (left, blue α-carbon chain, Protein Data Base coordinates, PDB: 3JYC) and presence of PI(4,5)P₂ (right, gray, PDB: 3SPI) [12]. The Kir2.2 tetramer is viewed from the membrane side with the extracellular face up. The approximate position of the plasma membrane (PM) lipid bilayer is represented as grey bars. Purple spheres represent potassium ions on the axis of the pore. Each channel subunit is in complex with a single PI(4,5)P₂ molecule represented as spheres and colored according to atom type: carbon, grey; phosphorous, orange; and oxygen, red. Binding of PI(4,5)P₂ induces a translation of the cytoplasmic domains towards the transmembrane domains and overall 6 Å compression of the channel (compare red arrows) and an opening of the inner helix ‘activation’ gate. B) A single Kir2.2 subunit in complex with a single PI(4,5)P₂ molecule. The vertical line is the pore axis with four K⁺ ions visible. Inset: detailed view of the PI(4,5)P₂ binding site. Helices (shown as ribbon) are colored grey. All side chains are shown as sticks. Di-C₈-PI(4,5)P₂ is shown as sticks and colored according to atom type: carbon, grey; phosphorous, orange; oxygen, red; and nitrogen blue. N-O hydrogen bonds are dashed lines. Upon PI(4,5)P₂ binding, a flexible linker contracts to form a compact helical structure, the cytoplasmic domain translates and becomes tethered to the transmembrane domain, this causes the inner gate to open. Note the electrostatic map of the same subunit behind the Kir2.2 structure (blue is positive; red negative; white neutral). The PI(4,5)P₂ binding site comprises numerous basic residues (blue) that interact electrostatically with the negatively charged phosphates of PI(4,5)P₂.

Fig. 4

Depletion of plasma membrane PI(4,5)P₂ by different approaches closes KCNQ2/3 channels. A) Current traces of KCNQ2/3 channels activated by a depolarizing pulse from −60 mV to −20 mV in the whole-cell configuration before (black) and after (red) activation of muscarinic acetylcholine receptor type I (M₁R) by 10 μM oxotremorine methiodide (Oxo-M) in tsA-201 cells. Test pulse protocol is shown above the current traces. B) KCNQ2/3-mediated tail current amplitude against time in the same experiment as A. Orange bar indicates Oxo-M application. C-F) Same type of experiment as in A and B, but PI(4,5)P₂ was depleted either by a voltage-pulse to +100 mV to activate a voltage-sensitive lipid-5-phosphatase (VSP, panels C and D), or by recruitment of a lipid-5-phosphatase (pseudojanin) to an anchor protein at the plasma membrane (LDR) by addition of 5 μM rapamycin (panels E and F).

Fig. 5

Bidentate model of Ca_V2.2 channel regulation by PI(4,5)P₂ with different coexpressed Ca_V β subunits. A) Interactions between Ca_V2.2 and β2a subunit. The two palmitoyl fatty acyl chains of Ca_V β2a compete with the those of PI(4,5)P₂ at the channel, reducing the need for PI(4,5)P₂. See text. B). Interactions between Ca_V2.2, PI(4,5)P₂, and β3 subunit. Non lipidated Ca_V β3 subunits do not compete with PI(4,5)P₂ binding. The electrostatic interaction of PI(4,5)P₂ with the channel is disturbed after dephosphorylation by a lipid PI(4,5)P₂ 5-phosphatase. See text.