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. 2010 Jun 2;588(Pt 17):3179–3185. doi: 10.1113/jphysiol.2010.192153

Phosphoinositides: lipid regulators of membrane proteins

Björn H Falkenburger 1, Jill B Jensen 1, Eamonn J Dickson 1, Byung-Chang Suh 1, Bertil Hille 1
PMCID: PMC2976013  PMID: 20519312

Abstract

Phosphoinositides are a family of minority acidic phospholipids in cell membranes. Their principal role is instructional: they interact with proteins. Each cellular membrane compartment uses a characteristic species of phosphoinositide. This signature phosphoinositide attracts a specific complement of functionally important, loosely attached peripheral proteins to that membrane. For example, the phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane attracts phospholipase C, protein kinase C, proteins involved in membrane budding and fusion, proteins regulating the actin cytoskeleton, and others. Phosphoinositides also regulate the activity level of the integral membrane proteins. Many ion channels of the plasma membrane need the plasma-membrane-specific PIP2 to function. Their activity decreases when the abundance of this lipid falls, as for example after activation of phospholipase C. This behaviour is illustrated by the suppression of KCNQ K+ channel current by activation of M1 muscarinic receptors; KCNQ channels require PIP2 for their activity. In summary, phosphoinositides contribute to the selection of peripheral proteins for each membrane and regulate the activity of the integral proteins.

Bertil Hille trained in Zoology and Biophysics (Yale, B.S.), and Life Sciences (Rockefeller University, Ph.D.). His postdoctoral work was with Alan L. Hodgkin and Richard D. Keynes in Cambridge. His major contributions have been in ion channels, first their existence, biophysical properties and pharmacology, and then their modulation by G-protein coupled receptors. His lab studies neurons, epithelial cells, endocrine cells, sperm, and expression systems using many electrical and optical methods associated with the patch clamp. He is the author of the book Ion Channels of Excitable Membranes. Byung-Chang Suh trained in neurophysiology (Pohang University, Ph.D.). His postdoctoral work was with K. T. Kim and Bertil Hille. He is now Research Assistant Professor. His interests have been in receptor-coupled signalling in cell biology and in modulation of K+ and Ca2+ channels by PIP2. He discovered PIP2 regulation of KCNQ channels.

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Phosphoinositide structure

Phosphoinositides are minority phospholipids of all eukaryotic cellular membranes. Like other phospholipids they have a glycerol backbone esterified to two fatty acid chains and a phosphate, and attached to a polar head group that extends into the cytoplasm (Fig. 1A). For phosphoinositides, the head group is the cyclic polyol myo-inositol, (CHOH)6. This inositol head group has free hydroxyl groups at positions D2 through D6, and those at positions D3, D4 and D5 are readily phosphorylated by cytoplasmic lipid kinases. This essay discusses the concept that the resulting seven combinatorially phosphorylated forms (Fig. 1B) of the inositol head group have informational content. Rather than playing a significant structural role in the lipid bilayer, polyphosphoinositides serve both as acidic address labels that identify different membranes and as instructions for certain proteins for how to behave at those membranes. Our essay focuses on ideas rather than on the large relevant literature, it gives only a few examples out of many, and it refers primarily to reviews. We emphasize here that many proteins bind lipids through lipid-binding domains.

Figure 1. Phosphoinositides: diversity, location and recognition.

Figure 1

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A, generic structure of phosphoinositides, showing three phosphorylatable positions, D3, D4, and D5, on the myo-inositol headgroup. B, the seven polyphosphoinositides and the parent phosphatidyl inositol, their dominant membrane location, and one of several protein domains that recognizes each one. ER: endoplasmic reticulum; MVB: multivesicular bodies.

The two classical plasma membrane phosphoinositides

The first phosphoinositide to receive a lot of attention in physiology was phosphatidylinositol 4,5-bisphosphate, often called PIP2 or more carefully, PI(4,5)P2. It is the principal substrate of receptor-stimulated phospholipases C (PLC). Over 50 hormone receptors that couple to the G protein Gq, as well as some receptor tyrosine kinases, stimulate PLCs; the PLC then cleaves PIP2 at the plasma membrane (Berridge & Irvine, 1984; Kirk et al. 1984; Smrcka et al. 1991). Figure 2A shows PLC being activated via M1 muscarinic receptors and Gq.

Figure 2. Phosphoinositide lipid regulation at the plasma membrane and the Golgi.

Figure 2

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Schematic diagram of some phosphoinositide-dependent processes discussed in the text. The cytoplasmic leaflet of the plasma membrane contains PIP2, and the cytoplasmic leaflet of the Golgi contains PI(4)P. A, open KCNQ channels require PIP2 and are transiently closed when M1 muscarinic receptors activate PLC to cleave and deplete PIP2. B, formation of clathrin-coated pits leading to endocytosis at the plasma membrane involves many PIP2-dependent proteins. C, formation of clathrin-coated pits and budding of vesicles from the Golgi involves a related set of proteins, but now they are PI(4)P-preferring.

The PLC pathway illustrates the theme of protein–lipid interaction. PLCβ is a soluble enzyme that is attracted to the membrane by a phosphoinositide-binding domain and by activated Gαq, Gαq.GTP. The resting PIP2 suffices to draw most of the PLCβ to the plasma membrane. The enzyme is co-activated by membrane Gαq.GTP and Ca2+. Both are required. The resulting cleavage products are two potent second messengers, the lipid diacylglycerol and soluble inositol 1,4,5-trisphosphate. Inositol trisphosphate releases Ca2+ from intracellular stores, creating a positive feedback loop to PLC. The newly formed, membrane-bound diacylglycerol lipid is recognized by the C1 domain of protein kinase C (PKC), as well as C1 domains of Munc13 and other proteins. For membrane-interacting proteins, the total interaction energy typically would be the sum of several terms, including, for PKC, at least the binding energy of the DAG-sensing C1 domain and that of the Ca2+- and phospholipid-binding C2 domain. The production of DAG tips the balance, and PKC translocates to the membrane and phosphorylates protein substrates at the plasma membrane on serine and threonine residues. For longer stimuli, PKC begins to phosphorylate proteins on other intracellular membranes (Gallegos et al. 2006).

PI(4,5)P2 is synthesized from phosphatidylinositol (PI) in two steps: a PI 4-kinase makes PI(4)P, followed by a PI(4)P 5-kinase at the plasma membrane. Corresponding to almost all lipid kinase reactions, there are lipid phosphatases that can remove the phosphate. Hence, the pools of phosphorylated phosphoinositides turn over continuously in a time frame of a few minutes. Although all phosphoinositides are minority lipids, the pool of unphosphorylated PI is by far the largest, whereas PI(4)P and PI(4,5)P2 each comprise only about 1% of the acidic phospholipids in the whole cell.

The second phosphoinositide to receive major attention in physiology was phosphatidylinositol 3,4,5-trisphosphate, often called PIP3 or more carefully PI(3,4,5)P3. PI(3,4,5)P3 is formed at the plasma membrane when growth factor receptors activate PI 3-kinase, which phosphorylates PI(4,5)P2 on the inositol D3 position (Auger et al. 1989). For example, PI(3,4,5)P3 is made in response to insulin, growth hormone, nerve growth factor, and epidermal growth factor. Comparable to diacylglycerol, the PI(3,4,5)P3 lipid and the related PI(3,4)P2 then recruit protein kinases to the cell membrane (in this case kinases like Akt/PKB, PDK1, Btk, Src and others), initiating signals of the growth-factor response pathway (Cantley, 2002). Only a small fraction of the plasma membrane PI(4,5)P2 is converted into PIP3, so the pool of PIP3 remains very small even during growth-factor stimulation.

Intracellular phosphoinositides

After recognition of the physiological importance of PI(4,5)P2 and PIP3 on the plasma membrane, it became apparent that the other polyphosphoinositides are components of specific intracellular membranes (Fig. 1B). Thus, PI(4)P is associated with the trans-Golgi and secretory vesicles, PI(3)P with late endosomes and multivesicular bodies, and so forth (Di Paolo & De Camilli, 2006; Simonsen et al. 2001). For each of these phosphoinositides there are specific lipid-binding domains on cytoplasmic proteins (Fig. 1B; Balla et al. 2009; Lemmon, 2008; Várnai & Balla, 2007). These ideas emerged and continue to be refined from at least three lines of investigation. (1) Identification of the different lipid kinase and phosphatase enzymes of PI metabolism showed discrete subcellular localizations (e.g. Doughman et al. 2003; Balla, 2007). (2) Analysis of the effects of mutation or knockout of lipid kinases and phosphatases on vesicular traffic and sorting showed defects at specific membrane trafficking steps (Schu et al. 1993; Simonsen et al. 2001). (3) Use of lipid-binding domains from proteins engineered as genetically expressible fluorescent labels inside living cells identified different lipid species in different organelles (Balla et al. 2009; Stauffer et al. 1998; Várnai & Balla, 1998, 2007).

If all cellular membranes derive from and traffic to other membranes, how do organelles maintain distinct dominant phosphoinositide compositions? One mechanism is the subcellular distribution of lipid phosphatases and kinases. For example the plasma membrane has a high PIP 5-kinase activity that maintains PI(4,5)P2 in the face of ongoing PIP2 5-phosphatase activity (Mao & Yin, 2007), and similarly the Golgi has PI 4-kinase (Wang et al. 2003). Further, the concept has arisen that at the moment of budding or transition to a new compartment, cytoplasmic enzymes are recruited that act as gatekeepers to convert the phosphoinositides in the membrane to the new appropriate molecular species.

For example, consider the clathrin-mediated endocytosis of fast recycling synaptic vesicles at nerve terminals (Fig. 2B). In very simplified form: a cytoplasmic clathrin-adapter molecule AP-2 is recruited to the plasma membrane; sometimes cytoplasmic epsin sorts cargo proteins into the complex; cytoplasmic clathrin binds to the AP-2 adapter and to epsin, shaping the invaginating pit; the cytoplasmic GTPase dynamin is drawn in to pinch off the neck of the bud; and a cytoplasmic lipid phosphatase synaptojanin is brought into the complex to modify the phosphoinositide (Cremona & De Camilli, 2001; Wenk & De Camilli, 2004). Four of these proteins, AP-2, epsin1–3, dynamin I, and synaptojanin, use PIP2-binding motifs to aid in their association. Synaptojanin is the gatekeeper enzyme that dephosphorylates the PIP2 of the bud on the D5 position and possibly also on the D4 position. Once the PIP2 is gone, clathrin and all the PIP2-requiring members of the complex drop off again. In knockout mice lacking synaptojanin, clathrin-coated pits accumulate in synaptic terminals (Cremona & De Camilli, 2001). Since synaptic vesicles normally can be recycled in seconds (Dittman & Ryan, 2009), all of these events and the subsequent refilling with neurotransmitter and docking are quick. In the end, the nascent synaptic vesicle has PI(4)P as its dominant phosphoinositide, but since synaptojanin has both 5-phosphatase and 4-phosphatase structural domains, it is still not sure whether the PIP2 of the original pit membrane is stably converted directly to the final PI(4)P or is processed in a more circuitous route (Wenk & De Camilli, 2004).

Other membranes use clathrin for budding, including the trans-Golgi, which buds off secretory granules (Fig. 2C). Here the precursor and the product membrane have an identical dominant phosphoinositide, PI(4)P, the adaptor proteins are AP-1 and epsin4, and the pinching protein is dynamin II (Hirst et al. 2003; Wang et al. 2003). As expected, AP-1 and epsin4 recognize PI(4)P rather than PIP2.

Cytoplasmic proteins bind phosphoinositides

We have mentioned numerous cytoplasmic proteins that use lipid recognition as one of several combinatorial signals that direct them to specific membranes (Fig. 1B). To get an idea of the ubiquity of this mechanism, consider a proteomic study (Catimel et al. 2008). Liposomes and beads that displayed either of two structurally similar lipids, PI(3,5)P2 or PI(4,5)P2, were used as bait to pull proteins out of an extract of cytoplasmic proteins. The bound proteins were identified by mass spectrometry. The result was that 96 cytoplasmic proteins recognized both bisphosphoinositide isomers, 105 recognized only PI(3,5)P2, and 187 recognized only PI(4,5)P2. Among these 388 proteins there were many with known phosphoinositide-binding domains, including PH (pleckstrin homology), PX (phagocyte oxidase homology), ENTH (epsin N-terminal homology), C2, and other domains. Proteins captured this way included 50 GTPases and GTPase regulators, 67 proteins involved in cargo transport and membrane trafficking, 37 kinases and phosphatases, and 49 proteins associated with regulation of the actin cytoskeleton. Similarly, from a genomic analysis (Lemmon, 2008), many human proteins contain sequences for potential lipid-binding domains: 258 with PH domains, 125 with C2 domains, 35 with PX domains, etc. For both of these reports, further functional evaluation would be needed to decide how many of the in vitro and in silico predictions correspond to events in living cells. For example, although there are many PH domains with unique lipid specificity (Fig. 1B), there also are PH domains that do not bind lipids at all (Lemmon, 2008).

The lipid-recognizing domains approach the bilayer from the cytoplasm and frequently see only the negatively charged polar head group of the lipid. For several proteins, the crystal structure with the appropriate inositol phosphate (i.e. the head group of the target phosphoinositide) shows a characteristic fold for each domain type. The fold brings basic residues, widely separated in the sequence, together to form a structured, superficial binding pocket for the acidic lipid. In other proteins, including the small GTPases, a cytoplasmic polybasic region rather than a recognizable known domain fold interacts with the phosphoinositides (Heo et al. 2006).

In summary, the cytoplasm contains hundreds of proteins that can be drawn to membranes as loosely bound peripheral proteins when offered the appropriate combination of lipid and other signals. To each membrane they bring enzymatic and signalling functions appropriate for that compartment (Di Paolo & De Camilli, 2006). These proteins are responsive to the remodelling of lipids that occurs during membrane traffic. We mentioned, for example, the recruitment and loss of cytoplasmic proteins during clathrin-mediated endocytosis to make a synaptic vesicle, but in a larger sense, every transition from one dominant phosphoinositide to another will cause the membrane to relinquish one set of proteins and recruit another. In this way, each compartment will have a unique set of peripheral proteins associated with its cytoplasmic face, organized in part by its membrane phosphoinositides.

The distribution of lipid-binding proteins is also responsive to receptor-induced changes in lipid composition. We have mentioned the depletion of PIP2 and the synthesis of DAG and PIP3 at the plasma membrane (Fig. 1B). Other examples are the stimulation of PIP 5-kinase by the Rho family of small G-proteins (Doughman et al. 2003), and the calcium-induced stimulation of PIP 4-kinase (D’Angelo et al. 2008). The latter enzyme is localized primarily in the Golgi, suggesting that receptor-induced changes in phosphoinositide composition are not limited to the plasma membrane.

Some integral membrane proteins need phosphoinositides

Phosphoinositides also play a strong instructive role for integral membrane proteins such as ion channels and ion transporters. What is known is that phosphoinositides regulate protein function. In most cases they are cofactors needed or at least helpful for full function. The first suggestion came from the finding that KATP channels and the Na+/Ca2+ exchanger run down in giant excised patches from heart, and the run-down could be reversed or accentuated by manipulations that would change the PI(4,5)P2 on the cytoplasmic face of the membrane patch (Hilgemann & Ball, 1996). Adding ATP, which allows PIP2 synthesis, was favourable for the channels, and adding polycations or PIP2 antibodies to chelate PIP2 was unfavourable. Similar results were found for other channels and transporters, and up to now more than 40 PIP2-regulated integral proteins have been identified (Hilgemann et al. 2001; Suh & Hille, 2005). Among others, the list includes many TRP and Kir channels and a few voltage-gated K+ and Ca2+ channels, such as KCNQ, L-type Cav1.3, and N-type Cav2.2.

We have shown that regulation by receptors of some ion channels in neurons could be explained this way. The M-current, now called KCNQ after its subunit genes, is a K+ current in sympathetic ganglion cells that can be turned off by muscarinic agonists (Fig. 3A and B; Brown & Adams, 1980). The signal for suppression of the current requires the M1 muscarinic receptor, Gq, and PLC (Delmas & Brown, 2005). Thus, PIP2 hydrolysis begins, myriad potent second messengers are made, and the KCNQ channel turns off. What is the signal? We and others found that KCNQ channels require PIP2 to be functional (Suh & Hille, 2002; Zhang et al. 2003; Suh et al. 2006; Falkenburger et al. 2010b). They are inactive in its absence. The principal muscarinic signal is therefore the depletion of plasma membrane PIP2 by PLC rather than the formation of inhibitory messages (Fig. 2A; Delmas & Brown, 2005). Actually, the many activated second messenger signals also act to decrease the current, but the requirement for PIP2 is absolute and overriding. The time course of inhibition depends on the receptor-activated rate of PIP2 depletion and the time course of recovery depends on the slower rate of enzymatic PIP2 regeneration from the pool of PI. In an expression system, we showed that PIP2 is depleted by M1 receptor activation (Horowitz et al. 2005), and current is suppressed (Fig. 3A; Suh & Hille, 2002); the subsequent recovery of current is blocked by omitting intracellular ATP (Fig. 3A) or by inhibiting PI 4-kinase; the recovery of current is accelerated by increasing PI 4-kinase or PIP 5-kinase activity; and depletion of PIP2 and suppression of current can be initiated by suddenly activating specially introduced PIP2 5-phosphatases, including a voltage-sensitive 5-phosphatase (Fig. 3B; Suh et al. 2006; Falkenburger et al, 2010b). We have measured and developed quantitative kinetic descriptions that include the known biochemical steps of G protein signalling and phosphoinositide metabolism (Suh et al. 2004; Jensen et al. 2009; Falkenburger et al. 2010a,b;). These models of biochemical transformations also capture the temporal features of muscarinic inhibition seen in Fig. 2A.

Figure 3. Modulation of KCNQ current by M1 receptor activation and by direct dephosphorylation of PIP2.

Figure 3

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A, metabolic steps that alter PIP2 levels. B, modulation by activation of PLC. Open circles, suppression and recovery of the K+ current when the muscarinic agonist oxotremorine M (Oxo-M, 10 μm) is applied and removed. The whole-cell pipette contains 3 mm ATP to allow PIP2 resynthesis. Filled circles, muscarinic suppression but no recovery when the pipette contains 4 mm of a non-hydrolysable ATP analogue (trinitrophenyl-ATP, TNP-ATP). Without ATP, PIP2 cannot be resynthesized. These experiments use tsA-201 cells as an expression system studied in whole-cell recording. The cells are transfected with the M1 receptor and two channel subunits, KCNQ2 and KCNQ3. (Unpublished data. Methods as in Suh & Hille, 2002.) C, decrease of PIP2 and suppression of current by brief activation of a voltage-sensitive PIP2 5-phosphatase, VSP. The phosphatase is expressed by transfection and activated by a depolarizing voltage pulse at time zero. Filled circles, loss and recovery of PIP2 measured with fluorescence resonance energy transfer (FRET) between CFP and YFP labelled PH domains. Open circles, parallel suppression and recovery of the K+ current. (Unpublished data. Methods as in Falkenburger et al. 2010b.)

The KCNQ channels (Delmas & Brown, 2005; Hernandez et al. 2008), Kir channels (Logothetis et al. 2007), and TRP channels (Rohacs, 2009) are now the best characterized with respect to PIP2 effects. For some of the channels, current suppression is not complete when PIP2 is strongly depleted, or it may be possible to substitute PIP2 with other acidic phospholipids. Nevertheless, in general, they function best in the presence of PIP2.

Significance of phosphoinositide requirements

What is the physiological significance of a PIP2 requirement for plasma membrane proteins? Is this a physiological mechanism for concerted shutting down of many kinds of channels and transporters when hormones activate PLC? It seems unlikely. Hilgemann proposed a more interesting and wide-reaching alternative hypothesis (Hilgemann et al. 2001). Noting that plasma membrane proteins are synthesized in the ER, traffic through the Golgi, and traffic to the plasma membrane, he suggested that phosphoinositide recognition keeps membrane proteins from being active until they arrive at the cell surface. This would explain why these channels and transporters do not dump Ca2+ from the ER or establish a membrane potential where there should be none. It would also help to explain why in almost all cases PIP2 has evolved as a positive cofactor rather than as an inhibitor of plasma membrane channels. If PIP2 were used as an inhibitor, the absence of PIP2 in other membranes would permit activation of this ‘plasma membrane’ protein in the ‘wrong’ place.

Extending the phosphoinositide hypothesis, we suggest that the phosphoinositides of organelle membranes might play a similar permissive role for the integral proteins that function on their membranes. Several tests of that hypothesis would require new tools, including direct patch clamping of organelles. However, perhaps the plasma membrane contains silenced integral proteins that normally function on other membranes whose function might be restored in a plasma-membrane excised patch by adding some other phosphoinositide.

We anticipate that phosphoinositide recognition may extend to integral proteins other than ion channels and transporters. If one could design online assays for plasma membrane receptors and enzymes in situ, it would be straightforward to test the effects of rapid PIP2 depletion on them with the voltage-sensitive PIP2 phosphatase or dimerizable PIP2 phosphatase tools (Suh et al. 2006; Falkenburger et al. 2010a,b;).

Finally there is a possibility that the local phosphoinositide instructs some membrane proteins to stop trafficking or makes them more stable. While bound to the lipid, the protein may stop displaying protein targeting, transport, or turnover signals. Thus, the lipid might assist in physical localization as well as functional localization.

Acknowledgments

We thank Lea M. Miller for technical help and the NIH for decades of support from grant NS08174 and associated ARRA funds.

Glossary

Abbreviations

PI

phosphatidylinositol

PIP2 and PI(4,5)P2

phosphatidylinositol 4,5-bisphosphate

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