SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Specificity of PIP2 signalling
  4. PIP2 affinity of membrane proteins
  5. Technical considerations
  6. Perspectives
  7. References
  8. Appendix

Phosphatidylinositol 4,5-bisphosphate (PIP2)-mediated signalling is a new and rapidly developing area in the field of cellular signal transduction. With the extensive and growing list of PIP2-sensitive membrane proteins (many of which are ion channels and transporters) and multiple signals affecting plasma membrane PIP2 levels, the question arises as to the cellular mechanisms that confer specificity to PIP2-mediated signalling. In this review we critically consider two major hypotheses for such possible mechanisms: (i) clustering of PIP2 in membrane microdomains with restricted lateral diffusion, a hypothesis providing a mechanism for spatial segregation of PIP2 signals and (ii) receptor-specific buffering of the global plasma membrane PIP2 pool via Ca2+-mediated stimulation of PIP2 synthesis or release, a concept allowing for receptor-specific signalling with free lateral diffusion of PIP2. We also discuss several other technical and conceptual intricacies of PIP2-mediated signalling.

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 or, in this review, PIP2) is a major phosphoinositide of the plasma membrane that comprises about 1% of plasma membrane phospholipids. Calculated as a concentration in cells (as if all the PIP2 were dissolved in the cytosol), [PIP2] is ∼10 μm, and as an abundance, there are ∼3–5000 molecules of PIP2 per μm2 of plasma membrane (McLaughlin et al. 2002; Xu et al. 2003). The known functions of PIP2 in mammalian cells are steadily increasing and include the following: (1) PIP2 is the precursor of the second messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) (Berridge, 1984); (2) it serves as a ‘lipid anchor’ that attaches the cytoskeleton to the plasma membrane and shapes processes that require membrane reorganization (e.g. endo- and exocytosis) (Itoh et al. 2001), and (3) it stabilizes or activates many membrane proteins, such as ion channels and transporters (Suh & Hille, 2005). In this last role, PIP2 is widely seen as an intracellular signalling molecule itself. Thus, membrane PIP2 abundance is recognized as a dynamic entity, with its depletion by phospholipase C (PLC) activity induced by stimulation of Gq/11-coupled or growth-factor receptors causing modulation of a wide spectrum of ion channels (Suh & Hille, 2005; Rohacs, 2007). In addition to lipid signals that involve altered PIP2 binding due to its depletion are allosteric interactions in which the affinity of the channels for phosphoinositides are dynamically regulated by other signalling molecules, the prototypic example being Kir3 channels (Xie et al. 2006). By now, the physiological role of PIP2 as a major intracellular messenger in the G protein-mediated modulation of M-type K+, N- and P/Q-type Ca2+, and Kir3 K+ channels is well established and have been extensively discussed (Delmas & Brown, 2005; Delmas et al. 2005; Suh & Hille, 2005; reports herein). In this paper, we focus on some inherent difficulties of the concept of PIP2 signalling that have yet to be overcome.

Specificity of PIP2 signalling

  1. Top of page
  2. Abstract
  3. Specificity of PIP2 signalling
  4. PIP2 affinity of membrane proteins
  5. Technical considerations
  6. Perspectives
  7. References
  8. Appendix

Given the countless physiological roles of PIP2 signals that are being established, a conundrum arises as to the mechanism (or mechanisms) of spatio-temporal segregation of these roles that confer specificity within the plasma membrane. In other words, there must be ways of preventing the uniform triggering of all PIP2-dependent processes in the cell by fluctuations of global PIP2 levels. For this review, we will only consider putative mechanisms of such segregation of signalling towards PIP2-sensitive transmembrane proteins such as ion channels, but our reasoning should be applicable to the spatio-temporal isolation of any PIP2-dependent process.

Accumulating data on PIP2-sensitive ion channels and transporters soon led to the intriguing observation that they do not respond to PIP2 hydrolysis from stimulation of all Gq/11-coupled receptors in native cells. Thus, N-type Ca2+ channels (Gamper et al. 2004; cf. Lechner et al. 2005) and Kir3 K+ channels (Winks et al. 2005) in rat superior cervical ganglion (SCG) sympathetic neurons are inhibited by stimulation of Gq/11-coupled muscarinic M1, but not bradykinin B2, receptors, even though robust PIP2 hydrolysis is induced by stimulation of both receptors. Similar divergent sensitivity of Kir3 channels to different PLC-coupled receptors has been revealed in atrial myocytes, in which bradykinin was found to be almost ineffective in inducing channel inhibition. However, adrenergic agonists and angiotensin II (acting via α1 and AT1 receptors, respectively) produced a moderate effect, and endothelin-1 and prostaglandin F2α induced strong inhibition of the channels (Cho et al. 2005b). All of these experiments show that not all receptors that induce PIP2 hydrolysis can reach all PIP2-sensitive targets within the same cell type.

Are there local PIP2 microdomains within the plasma membrane? Probably the simplest way to account for divergent PIP2 signalling within a restricted area of plasma membrane would be to allow for the existence of autonomous membrane microdomains in which the PIP2 abundance can change independently, so that activation of a given PLC-coupled receptor affects only those targets that are partitioned in the same microdomain (pool, raft, etc. Fig. 1). Such independent pools of PIP2, which would govern distinct functions and probably even utilize separate PIP2 turnover machinery, were indeed suggested early in the development of the concept of phosphoinositide signalling (Hinchliffe et al. 1998; Simonsen et al. 2001; reviewed in Janmey & Lindberg, 2004), but the exact mechanisms of formation and maintenance of such pools, indeed even their very existence, remain controversial (e.g. van Rheenen et al. 2005). Among the mechanisms of enrichment of membrane domains with PIP2 have been suggested the following: (1) spontaneous aggregation of PIP2 molecules due to formation of hydrogen bonds between PI(4,5)P2 head groups (Redfern & Gericke, 2005); (2) partitioning of PIP2 molecules into cholesterol-rich membrane rafts (Pike & Casey, 1996; Pike & Miller, 1998); (3) localized production of phosphoinositides (Janmey & Lindberg, 2004); (4) spatial sequestration of PIP2 by local membrane curvature (Janmey & Lindberg, 2004); (5) electrostatic sequestration of PIP2 by basic/aromatic regions of natively unfolded proteins such as myristoylated alanine-rich C-kinase substrate (MARCKS, Gambhir et al. 2004; McLaughlin & Murray, 2005). All of these mechanisms are predicated on a low lateral mobility of PIP2 in the membrane, as recently suggested (Cho et al. 2005a, 2006). However, hitherto no consensus on a prevailing principle of local PIP2 enrichment has been achieved. Thus, the idea of spontaneous PIP2 clustering via hydrogen bond formation has been challenged both theoretically and experimentally (Fernandes et al. 2006). Partitioning of PIP2 into lipid rafts has been criticized on the basis of energetic considerations: PIP2 has a polyunsaturated acyl side chain (arachidonic acid) that would not spontaneously partition into cholesterol-rich rafts (McLaughlin et al. 2002). There is still a possibility that PIP2 is enriched in rafts by binding to raft-localized proteins, but it is then unclear how this bound PIP2 can contribute to signal transduction, which requires fast lateral diffusion (see below). Moreover, using a combination of patch-clamp recording and imaging, van Rheenen et al. (2005) demonstrated homogeneous distribution of PIP2 within the membrane of HEK293 cells and found that two distinct PLC-coupled receptors, one of which localized to membrane rafts (neurokinin A receptors) and the other not (endothelin receptors), share the same global pool of PIP2 in those cells. Might the principles of PIP2 segregation be cell-type dependent? Indeed, low lateral mobility of PIP2 has been demonstrated in atrial myocytes (Cho et al. 2005a) but not for HEK293 cells (van Rheenen et al. 2005; Cho et al. 2006) or fibroblasts (Haugh et al. 2000). This is also very unlikely to be the case for sympathetic neurons (Delmas et al. 2005; see below). The idea of localized PIP2 production is supported by the concentration of enzymes involved in PIP2 synthesis at the sites of actin polymerization (Rozelle et al. 2000; Coppolino et al. 2002; Ling et al. 2002) but again, it requires some mechanism of PIP2 retention to achieve its local enrichment, otherwise, as noted by McLaughlin et al.‘PIP2 will diffuse away faster than it can be produced’ (McLaughlin et al. 2002).

image

Figure 1. Schematic representation of two opposing views on the effect of PLC-coupled receptor stimulation on global PIP2 abundance, as discussed in the text PIP2 molecules within the bilayer are shown as red ovals. On the left is depicted the scenario of restricted PIP2 diffusion, resulting in local PIP2 microdomains. A hypothetical microdomain is shaded grey. Stimulation of the PLC-coupled receptor within the microdomain causes a drop of local [PIP2] without appreciably affecting global membrane PIP2 abundance. In this case, triggering of the receptor only affects spatially co-localized channels. Depicted on the right is the opposing scenario in which free lateral diffusion of PIP2 is permitted within the entire membrane area. Strong stimulation of PLC-coupled receptors in this case depletes [PIP2] globally, affecting all PIP2-sensitive membrane targets. L, ligand of the Gq/11-coupled receptor; PLC, phospholipase C.

Download figure to PowerPoint

A very attractive idea is spatial sequestration of PIP2 by electrostatic interactions with natively unfolded proteins such as MARCKS, growth-associated protein 43 (GAP43) or cytoskeleton-associated protein (CAP23) which can not only bind PIP2, but possibly even function as ‘PIPmodulins’ (Laux et al. 2000) and release PIP2 locally in response to certain stimuli such as PKC phosphorylation or Ca2+–calmodulin (CaM) action (Arbuzova et al. 1998). MARCKS is a ubiquitous protein expressed in the majority of cells at micromolar concentrations (Aderem, 1992), whereas GAP43 (neuromodulin) and CAP23 have mostly neuronal distribution. Intracellularly, MARCKS localizes to the inner leaflet of the plasma membrane, presumably forming ‘clusters’, and sequesters PIP2 by electrostatic interactions via its basic effector domain (McLaughlin et al. 2002; McLaughlin & Murray, 2005). Importantly, MARCKS translocates to the cytosol upon PKC phosphorylation or binding to Ca2+–CaM, presumably freeing sequestered PIP2 (Arbuzova et al. 1998; see McLaughlin et al. 2002; McLaughlin & Murray, 2005 for reviews). MARCKS also binds to the actin cytoskeleton (Aderem, 1992). Developing the analogy with Ca2+ signalling further, one can envision the existence of ‘PIP2somes’– membrane-anchored clusters of MARCKS (or related proteins) that release bound PIP2 in response to phosphorylation or [Ca2+]i rises. However, amongst the complications of this hypothesis is the fact that MARCKS-bound PIP2 is apparently inaccessible to PLC (Wang et al. 2002; Gambhir et al. 2004), so then if we wish to apply this concept to modulation of PIP2-sensitive ion channels by PIP2 depletion, we would have to consider that our channels and PLC-coupled receptors are localized outside of such PIP2somes, returning us back to the beginning of our discussion. In addition, the suggested release of PIP2 in response to [Ca2+]i transients (due to binding of Ca2+–CaM to MARCKS) would suggest up-regulation of PIP2-sensitive channels by stimulation of PLC-coupled receptors, whereas we usually observe the opposite effect (however, see below). To summarize the above, one of the major complications with PIP2-enriched microdomains is that PIP2 is thought to usually have fast lateral diffusion within the bilayer; to restrict such lateral mobility, we need to invoke the binding of PIP2 to some lipid-sequestering proteins or structural molecules, but then we will need to explain how such bound PIP2 could dynamically participate in signalling.

To further illustrate this difficulty we return back to the modulation of N-type Ca2+ and M-type K+ channels in sympathetic ganglia neurons. As was discovered some 15 years ago, the inhibition by muscarinic agonists of both types of channels isolated in the tips of cell-attached pipettes can be induced by external application of receptor agonists (Bernheim et al. 1991; Selyanko et al. 1992). Such inhibition is relatively fast (some 100 s), reversible and was interpreted at the time as being mediated by a ‘diffusible cytosolic second messenger’, because of the logical requirement for a mediator capable of migrating between the receptor situated outside of the patch and the channel inside the patch. After much investigation, there is wide agreement that the Gq/11-coupled muscarinic modulation of both N-type Ca2+ and M-type K+ channels is mediated by receptor-induced depletion of PIP2 (Suh & Hille, 2002; Ford et al. 2003; Zhang et al. 2003; reviewed in Delmas & Brown, 2005; Suh & Hille, 2005 and reports herein). Thus, the messenger loses its attribute of being ‘cytosolic’, but the requirement for being diffusible nevertheless remains. Indeed, if a given channel inside the membrane patch is inhibited by PIP2 hydrolysis outside of it, free lateral diffusion of PIP2 away from the patch is required to cause the inhibition (Fig. 2). Therefore, at least in the case of sympathetic neurons, we cannot rely on PIP2-rich microdomains and different partitioning of receptors and channels into such microdomains to explain receptor-specific inhibition of Ca2+ channels, M channels and Kir3 channels. Again, one must consider the possibility that the lateral mobility of PIP2 depends on the cell type, since external application of the Gq/11-coupled receptor agonist, endothelin-1, does not inhibit Kir3 channels isolated in cell-attached patches excised from atrial myocytes (Cho et al. 2005a).

image

Figure 2. Depiction of how channels isolated in the patch of membrane in the cell-attached pipette can be inhibited within the context of PIP2 as a diffusible messenger PIP2 molecules within the bilayer are shown as red ovals. We assume that neither ligand-bound receptors nor activated PLC can diffuse through the membrane into the pipette tip, an assumption supported by previous work (Soejima & Noma, 1984). Stimulation of Gq/11-coupled receptors in the membrane outside of the patch results in global lowering of [PIP2], a gradient of [PIP2] between the membrane patch and the membrane outside of the patch, diffusion of PIP2 from the patch to the rest of the cell membrane, lowered [PIP2] in the membrane patch, and unbinding of PIP2 from the channel, resulting in its inhibition. Note that such a result is predicated on free diffusion of PIP2 within the lipid bilayer. L, ligand of the Gq/11-coupled receptor; PLC, phospholipase C.

Download figure to PowerPoint

Can we reconcile receptor-specific PIP2 signalling with free lateral diffusion of PIP2? We recently suggested an alternative mechanism of target-specific PIP2 signalling that merges the ideas of membrane microdomains with free lateral diffusion of PIP2 (Gamper et al. 2004; Delmas et al. 2005). This mechanism is based on the observation in SCG neurons that bradykinin B2 and purinergic P2Y, but not muscarinic M1 nor angiotensin AT1, receptors induce significant rises in [Ca2+]i via IP3-mediated release, a selectivity hypothesized to arise from the co-localization of the former, but not the latter, receptors with IP3 receptors (Shapiro et al. 1994; Cruzblanca et al. 1998; Delmas & Brown, 2002; Delmas et al. 2002; Gamper & Shapiro, 2003; Zaika et al. 2006). Such Ca2+ rises can stimulate PIP2 synthesis, concurrently with its hydrolysis, via Ca2+-sensitive neuronal calcium sensor-1 (NCS-1) stimulation of PI4-kinase (Koizumi et al. 2002; Winks et al. 2005); therefore, bradykinin and purinergic agonists not only induce PIP2 hydrolysis, but also PIP2 synthesis, preventing a significant drop in PIP2 abundance (Fig. 3). In this scheme, B2 and P2Y receptor triggering inhibits M-type K+ channels by Ca2+–CaM action, rather then by PIP2 depletion (Cruzblanca et al. 1998; Bofill-Cardona et al. 2000; Gamper & Shapiro, 2003; Gamper et al. 2005; Zaika et al. 2007) whereas N- and P/Q-type Ca2+ or Kir3 channels are not affected (Gamper et al. 2004; Delmas et al. 2005). An alternative mechanism could be receptor-specific release of PIP2 from MARCKS-bound pools (McLaughlin & Murray, 2005). In this scenario, Ca2+ rises induced by Gq/11-coupled receptors co-localized with IP3 receptors release PIP2 from such pools and thus compensate for PIP2 depletion by PLC, preventing N-type Ca2+ or Kir3 channel inhibition, whereas M channels are inhibited by Ca2+–CaM action. Although both these ideas provide a satisfactory explanation for receptor-specific PIP2 signalling in SCG neurons, it is, however, not clear how we can integrate earlier data indicating that prevention of IP3-mediated Ca2+ signals reduces inhibition of M channels by bradykinin or purinergic agonists (Cruzblanca et al. 1998; Bofill-Cardona et al. 2000). Indeed, in the absence of such Ca2+ signals, not only Ca2+–CaM action, but also stimulation of PIP2 synthesis should be prevented, allowing depletion of PIP2 and M-current suppression, as by muscarinic stimulation. Perhaps differential affinities of released Ca2+ for its Ca2+-binding protein partners can provide an explanation. Clearly, further work is needed to test these ideas.

image

Figure 3. ‘Buffering’ of global plasma membrane PIP2 pool Hypothesis of ‘buffering’ of global plasma membrane PIP2 pool by concurrent stimulation of PIP2 hydrolysis and PIP2 synthesis (or release from locally sequestered PIP2 domains, such as proposed MARCKS–PIP2 clusters) by those PLC-coupled receptors that induce Ca2+ transients (such as bradykinin B2 and purinergic P2Y receptors in SCG neurons). See text for details. L, ligand of the Gq/11-coupled receptor; PLC, phospholipase C; ER, endoplasmatic reticulum; IP3, inositol 1,4,5-trisphosphate.

Download figure to PowerPoint

PIP2 affinity of membrane proteins

  1. Top of page
  2. Abstract
  3. Specificity of PIP2 signalling
  4. PIP2 affinity of membrane proteins
  5. Technical considerations
  6. Perspectives
  7. References
  8. Appendix

Another perspective on the same issue of specificity in PIP2-mediated signalling is the question of PIP2 affinity of its target proteins. The (already) remarkable list of PIP2-sensitive ion channels and transporters (see Suh & Hille, 2005) continues to grow, thus creating the impression that the majority of them are, at least to some extent, sensitive to PIP2. This creates an additional difficulty for the concept of target-specific signalling. It is, however, clear that different membrane proteins bind PIP2 with different strengths or otherwise differ in their PIP2 sensitivity (Huang et al. 1998; Zhang et al. 1999; Li et al. 2005). We use the term ‘apparent affinity’ to describe the concentration dependence of the sensitivity of a given ion channel or transporter to PIP2. Channels with a low apparent PIP2 affinity require high levels of PIP2 in the membrane to activate, and those with a high apparent PIP2 affinity require much less. Thus, the tonic activity of a PIP2-sensitive protein depends on the apparent affinity of the protein for PIP2 in relation to tonic PIP2 abundance, and the possible modulatory effect of altered [PIP2] depends on whether the altered PIP2 abundance would be expected to change the fraction of target proteins bound by PIP2. The lower the apparent affinity of a given ion channel for PIP2, the higher sensitivity it will display towards PIP2 depletion, and on the contrary, channels with high apparent PIP2 affinity may never respond to physiological PIP2 depletion because the PIP2 abundance in the membrane may never fall below their threshold level for unbinding (Rohacs et al. 2003; Li et al. 2005; Chen et al. 2006).

Let us consider more carefully the family of PIP2-sensitive M-type (KCNQ or Kv7) K+ channels. All five KCNQ subunits have been shown to be inhibited by PIP2 depletion (Suh & Hille, 2002; Zhang et al. 2003; Loussouarn et al. 2003; Li et al. 2005); however, as shown by direct application of soluble PIP2 to inside-out patches, their apparent PIP2 affinities are highly divergent. Thus, the most sensitive KCNQ2 and KCNQ4 subunits have an approximately 100-fold lower apparent affinity for PIP2 than that of the least sensitive KCNQ3 (Li et al. 2005). As expected if PIP2 directly governs the maximal open probability (Po) of KCNQ channels, KCNQ subunits with a low apparent affinity for PIP2 also display in cell-attached patches a very low maximal Po (governed by tonic PIP2 abundance), whereas KCNQ3 has a maximal Po near unity (Selyanko et al. 2001; Li et al. 2004, 2005). Accordingly, KCNQ openers that act by increasing channel Po (such as N-ethylmaleimide or H2O2) are virtually ineffective against KCNQ3 (Li et al. 2004; Gamper et al. 2006). Likewise, an increase in tonic plasma membrane PIP2 abundance produced by over-expression of PI(4)5-kinase had little effect on macroscopic KCNQ3 currents, but dramatically increased KCNQ2 currents (Li et al. 2005). The, as yet untested, prediction is that among homomeric KCNQ channels, KCNQ3 should be the least sensitive to modulation by PLC-coupled receptors which act by depleting [PIP2]. A similar relationship between channel Po and apparent PIP2 affinity is predicted for Kir channels. Constitutively active Kir1 and Kir2 are thought to have a very high apparent affinity for PIP2, and the G protein-activated Kir3 channels a much lower apparent affinity, such that at tonic membrane PIP2 abundance, the former are already maximally activated, whereas the latter require Gβγ binding to activate, perhaps by increasing the channel affinity for the lipid. For the case of Ca2+–CaM action on M channels, the possible mechanism is again a dynamic alteration in the affinity of the channels for PIP2, this time decreasing it, such that tonic [PIP2] is insufficient to maintain binding to the channels. Further and more quantitative work is required to confirm these ideas.

Technical considerations

  1. Top of page
  2. Abstract
  3. Specificity of PIP2 signalling
  4. PIP2 affinity of membrane proteins
  5. Technical considerations
  6. Perspectives
  7. References
  8. Appendix

It should be pointed out that the uncertainties in PIP2-mediated signalling discussed here arise mainly not from the lack of rigor of investigators, but rather from the inherent technological difficulties associated with the physical/chemical properties of phosphoinositides in biological systems. PIP2 is a polar phospholipid with a variable net charge (subject to pH and membrane interactions; McLaughlin et al. 2002), that forms micelles in aqueous solution. These circumstances make it very hard to quantitatively deliver PIP2 to the inner leaflet of the plasma membrane in experiments on living cells, so one often uses short side-chain analogues with lesser known properties. Although widely used to indicate involvement of PIP2 in signalling pathways, PIP2 antibodies or chelators (such as heparin or polylysine) usually are not very specific. An early approach to study PIP2 sensitivity of transport proteins was to focus on the recovery from receptor-induced inhibition by using pharmacological blockade of PI4-kinase with wortmannin to block PIP2 re-synthesis after putative receptor-induced depletion by PLC (Suh & Hille, 2002; Zhang et al. 2003; Gamper et al. 2004; Zaika et al. 2006). However, wortmannin is more specific for PI3-kinases (Balla, 2001), requiring high concentrations (∼50 μm) to block PI4-kinases, increasing the possibility of side-effects. In addition, not all isoforms of PI4-kinase are wortmannin sensitive, suggesting the existence of wortmannin-insensitive pools of PIP2 (Heilmeyer et al. 2003). Localization of PIP2 and its quantification within the plasma membrane is another difficult task. Popular approaches to track phosphoinositide localization and abundance in the membrane involve the use of optical PIP2 reporters (such as green fluorescent protein (GFP)-tagged plekstrin homology domains) or detergent-based membrane fractionation; both methods suffer from the fundamental artefact of changing the object of the measurement by the very measurement itself. Thus, introduction into the plasma membrane of labelled PIP2-binding proteins may create microdomains that otherwise would not form (Janmey & Lindberg, 2004); likewise, treatment of membranes with detergents (such as Triton X-100) may induce PIP2 clustering that is not present in untreated membranes (van Rheenen et al. 2005).

There are several complications with the measurement of PIP2 levels in living cells. Firstly, receptor-triggered PIP2 hydrolysis is usually accompanied by the release of its by-products, DAG and IP3, with each triggering its own downstream signalling pathways. Therefore, to study PIP2-specific signals, one must block the other signals using pharmacological or genetic tools (blocking IP3 receptors, chelating cytosolic Ca2+, knocking-out PKC, etc.). Secondly, the hitherto most popular method of monitoring PIP2 hydrolysis is based on the translocation of an GFP-tagged PIP2-binding plekstrin homology (PH) domain of PLCδ (PLC-PH) that binds to both PIP2 and IP3. In unstimulated cells, tonic [IP3] is very low, so this construct is highly localized to the membrane. Upon activation of PLC and hydrolysis of PIP2, the PLC-PH probe translocates to the IP3 accumulating in the cytosol, which can be easily optically monitored (Stauffer et al. 1998; Raucher et al. 2000; Gamper et al. 2004; Suh et al. 2004; Winks et al. 2005). Although this probe was initially thought to be a simple monitor of membrane [PIP2] (and so a handy read-out for channel modulation studies), it has a 10- to 20-fold higher affinity for IP3 over PIP2 (Varnai & Balla, 1998; Hirose et al. 1999; Nash et al. 2001); thus, its translocation does not prove an actual drop in membrane [PIP2] (the signal we are interested in) but often rather the release of IP3. Accordingly, translocation of PLC-PH in response to PLC activation is absent when cytosolic IP3 accumulation is prevented by over expression of an IP3 5-phosphatase (Hirose et al. 1999; Horowitz et al. 2005; Suh et al. 2006). Thus, the quantification of PIP2 with this probe requires careful calibration with fixed levels of cytoplasmic IP3 (Winks et al. 2005). Recently, another PIP2 probe has been used that appears to have little affinity for IP3. It is based on the PIP2 binding domain within the carboxy terminus of the transcription factor called ‘tubby’, a domain that is apparently insensitive to any signals downstream of PIP2 hydrolysis, such as IP3, PKC or Ca2+ (Santagata et al. 2001). With one residue mutated (R332H) which slightly weakens its PIP2 affinity, the yellow fluorescent protein (YFP)-tagged R332H-tubby construct translocated from the membrane to cytosol upon muscarinic stimulation in sympathetic neurons, reflecting the depletion of PIP2 in the membrane (Hughes et al. 2007). This tubby-based probe may become widely used as an optical indicator of [PIP2].

Another recent technical advance makes it possible to manipulate PIP2 levels in individual living cells on the fast time scale of a patch-clamp experiment. It exploits the binding of intracellular immunophilin proteins to a phosphoinositide 3-kinase-related kinase involved in cell proliferation called mammalian target of rapamycin (mTOR) induced by the immunosuppressive compound rapamycin and its analogues. In particular, rapamycin induces the dimerization of FK506 binding protein (FKBP, it also binds the immunosuppressant FK506) and the FKBP-rapamycin binding (FRB) domain of mTOR, this being the immunosuppressant mechanism of action of these drugs (Tamaoki et al. 1986). Two groups have cleverly harnessed this chemically induced dimerization (CID) of FKBP and the FRB domains by fusing one (the FRB domain) with a membrane-localization tag from Lyn kinase, and the other (FKBP) to both a fluorescent protein such as CFP and a PI kinase or PIP2 phosphatase (Suh et al. 2006; Varnai et al. 2006). Upon addition of rapamycin or an analogue to the bathing solution, CID occurs, bringing the kinase or phosphatase to the membrane, and membrane PIP2 levels are thus either rapidly depressed or elevated. Since the cellular ionic current or transporter activity can be simultaneously monitored (as well as single-cell imaging performed), this is a powerful method for assessing the role of PIP2 in the function of membrane proteins, and we expect it to quickly gain wide use. The results from this technique are discussed at length elsewhere in this special issue of The Journal of Physiology (Suh & Hille, 2007; Balla, 2007).

Perspectives

  1. Top of page
  2. Abstract
  3. Specificity of PIP2 signalling
  4. PIP2 affinity of membrane proteins
  5. Technical considerations
  6. Perspectives
  7. References
  8. Appendix

In summary, the biophysical and biochemical properties of phosphoinositides make it difficult to quantitatively and specifically monitor, localize, or manipulate PIP2 in living cells. This requires the use of a broad spectrum of approaches to accumulate sufficient evidence to conclude whether a given ion channel or transporter is PIP2 sensitive, if the sensitivity is physiologically relevant, and if PIP2 abundance is involved in signalling pathways linking receptor activation and regulation of the target membrane protein.

We make an analogy with the current level of our understanding of PIP2 signalling to that in the early years of Ca2+ signalling when many Ca2+-dependent processes were discovered, but little was yet known about localized Ca2+ signalling, [Ca2+]i microdomains, Ca2+-binding proteins and other sophisticated mechanisms supplying clockwork precision to signalling machinery. With increasing numbers of PIP2-sensitive processes discovered every year, the necessity for a comprehensive conceptual framework for target-specific phosphoinositide signalling appears increasingly apparent.

References

  1. Top of page
  2. Abstract
  3. Specificity of PIP2 signalling
  4. PIP2 affinity of membrane proteins
  5. Technical considerations
  6. Perspectives
  7. References
  8. Appendix
  • Aderem A (1992). The MARCKS brothers: a family of protein kinase C substrates. Cell 71, 713716.
  • Arbuzova A, Murray D & McLaughlin S (1998). MARCKS, membranes, and calmodulin: kinetics of their interaction. Biochim Biophys Acta 1376, 369379.
  • Balla T (2001). Pharmacology of phosphoinositides, regulators of multiple cellular functions. Curr Pharm Des 7, 475507.
  • Balla T (2007). Imaging and manipulating phosphoinositides in living cells. J Physiol 582, 927938.
  • Bernheim L, Beech DJ & Hille B (1991). A diffusible second messenger mediates one of the pathways coupling receptors to calcium channels in rat sympathetic neurons. Neuron 6, 859867.
  • Berridge MJ (1984). Inositol trisphosphate and diacylglycerol as second messengers. Biochem J 220, 345360.
  • Bofill-Cardona E, Vartian N, Nanoff C, Freissmuth M & Boehm S (2000). Two different signaling mechanisms involved in the excitation of rat sympathetic neurons by uridine nucleotides. Mol Pharmacol 57, 11651172.
  • Chen X, Talley EM, Patel N, Gomis A, McIntire WE, Dong B, Viana F, Garrison JC & Bayliss DA (2006). Inhibition of a background potassium channel by Gq protein alpha-subunits. Proc Natl Acad Sci U S A 103, 34223427.
  • Cho H, Kim YA & Ho WK (2006). Phosphate number and acyl chain length determine the subcellular location and lateral mobility of phosphoinositides. Mol Cells 22, 97103.
  • Cho H, Kim YA, Yoon JY, Lee D, Kim JH, Lee SH & Ho WK (2005a). Low mobility of phosphatidylinositol 4,5-bisphosphate underlies receptor specificity of Gq-mediated ion channel regulation in atrial myocytes. Proc Natl Acad Sci U S A 102, 1524115246.
  • Cho H, Lee D, Lee SH & Ho WK (2005b). Receptor-induced depletion of phosphatidylinositol 4,5-bisphosphate inhibits inwardly rectifying K+ channels in a receptor-specific manner. Proc Natl Acad Sci U S A 102, 46434648.
  • Coppolino MG, Dierckman R, Loijens J, Collins RF, Pouladi M, Jongstra-Bilen J, Schreiber AD, Trimble WS, Anderson R & Grinstein S (2002). Inhibition of phosphatidylinositol-4-phosphate 5-kinase Ia impairs localized actin remodeling and suppresses phagocytosis. J Biol Chem 277, 4384943857.
  • Cruzblanca H, Koh DS & Hille B (1998). Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons. Proc Natl Acad Sci U S A 95, 71517156.
  • Delmas P & Brown DA (2002). Junctional signaling microdomains: bridging the gap between the neuronal cell surface and Ca2+ stores. Neuron 36, 787790.
  • Delmas P & Brown DA (2005). Pathways modulating neural KCNQ/M (Kv7) potassium channels. Nat Rev Neurosci 6, 850862.
  • Delmas P, Coste B, Gamper N & Shapiro MS (2005). Phosphoinositide lipid second messengers: new paradigms for calcium channel modulation. Neuron 47, 179182.
  • Delmas P, Wanaverbecq N, Abogadie FC, Mistry M & Brown DA (2002). Signaling microdomains define the specificity of receptor-mediated InsP3 pathways in neurons. Neuron 34, 209220.
  • Fernandes F, Loura LM, Fedorov A & Prieto M (2006). Absence of clustering of phosphatidylinositol-(4,5)-bisphosphate in fluid phosphatidylcholine. J Lipid Res 47, 15211525.
  • Ford CP, Stemkowski PL, Light PE & Smith PA (2003). Experiments to test the role of phosphatidylinositol 4,5-bisphosphate in neurotransmitter-induced M-channel closure in bullfrog sympathetic neurons. J Neurosci 23, 49314941.
  • Gambhir A, Hangyas-Mihalyne G, Zaitseva I, Cafiso DS, Wang J, Murray D, Pentyala SN, Smith SO & McLaughlin S (2004). Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins. Biophys J 86, 21882207.
  • Gamper N, Li Y & Shapiro MS (2005). Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+/calmodulin. Mol Biol Cell 16, 35383551.
  • Gamper N, Reznikov V, Yamada Y, Yang J & Shapiro MS (2004). Phosphotidylinositol 4,5-bisphosphate signals underlie receptor-specific Gq/11-mediated modulation of N-type Ca2+ channels. J Neurosci 24, 1098010992.
  • Gamper N & Shapiro MS (2003). Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels. J Gen Physiol 122, 1731.
  • Gamper N, Zaika O, Li Y, Martin P, Hernandez CC, Perez MR, Wang AY, Jaffe DB & Shapiro MS (2006). Oxidative modification of M-type K+ channels as a mechanism of cytoprotective neuronal silencing. EMBO J 25, 49965004.
  • Haugh JM, Codazzi F, Teruel M & Meyer T (2000). Spatial sensing in fibroblasts mediated by 3′ phosphoinositides. J Cell Biol 151, 12691280.
  • Heilmeyer LM Jr, Vereb G Jr, Vereb G, Kakuk A & Szivak I (2003). Mammalian phosphatidylinositol 4-kinases. IUBMB Life 55, 5965.
  • Hinchliffe KA, Ciruela A & Irvine RF (1998). PIPkins1, their substrates and their products: new functions for old enzymes. Biochim Biophys Acta 1436, 87104.
  • Hirose K, Kadowaki S, Tanabe M, Takeshima H & Iino M (1999). Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns. Science 284, 15271530.
  • Horowitz LF, Hirdes W, Suh BC, Hilgemann DW, Mackie K & Hille B (2005). Phospholipase C in living cells: activation, inhibition, Ca2+ requirement, and regulation of M current. J Gen Physiol 126, 243262.
  • Huang CL, Feng S & Hilgemann DW (1998). Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gbg. Nature 391, 803806.
  • Hughes S, Marsh S, Tinker A & Brown D (2007). PIP2-dependent inhibition of M-type (Kv7.2/7.3) potassium channels: direct on-line assessment of PIP2 depletion by Gq-coupled receptors in single living neurons. Pflugers Arch in press.
  • Itoh T, Koshiba S, Kigawa T, Kikuchi A, Yokoyama S & Takenawa T (2001). Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis. Science 291, 10471051.
  • Janmey PA & Lindberg U (2004). Cytoskeletal regulation: rich in lipids. Nat Rev Mol Cell Biol 5, 658666.
  • Koizumi S, Rosa P, Willars GB, Challiss RA, Taverna E, Francolini M, Bootman MD, Lipp P, Inoue K, Roder J & Jeromin A (2002). Mechanisms underlying the neuronal calcium sensor-1-evoked enhancement of exocytosis in PC12 cells. J Biol Chem 277, 3031530324.
  • Laux T, Fukami K, Thelen M, Golub T, Frey D & Caroni P (2000). GAP43, MARCKS, and CAP23 modulate PI(4,5)P2 at plasmalemmal rafts, and regulate cell cortex actin dynamics through a common mechanism. J Cell Biol 149, 14551472.
  • Lechner SG, Hussl S, Schicker KW, Drobny H & Boehm S (2005). Presynaptic inhibition via a phospholipase C- and phosphatidylinositol bisphosphate-dependent regulation of neuronal Ca2+ channels. Mol Pharmacol 68, 13871396.
  • Li Y, Gamper N, Hilgemann DW & Shapiro MS (2005). Regulation of Kv7 (KCNQ) K+ channel open probability by phosphatidylinositol 4,5-bisphosphate. J Neurosci 25, 98259835.
  • Li Y, Gamper N & Shapiro MS (2004). Single-channel analysis of KCNQ K+ channels reveals the mechanism of augmentation by a cysteine-modifying reagent. J Neurosci 24, 50795090.
  • Ling K, Doughman RL, Firestone AJ, Bunce MW & Anderson RA (2002). Type Ig phosphatidylinositol phosphate kinase targets and regulates focal adhesions. Nature 420, 8993.
  • Loussouarn G, Park KH, Bellocq C, Baro I, Charpentier F & Escande D (2003). Phosphatidylinositol-4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K+ channels. EMBO J 22, 54125421.
  • McLaughlin S & Murray D (2005). Plasma membrane phosphoinositide organization by protein electrostatics. Nature 438, 605611.
  • McLaughlin S, Wang J, Gambhir A & Murray D (2002). PIP2 and proteins: interactions, organization, and information flow. Annu Rev Biophys Biomol Struct 31, 151175.
  • Nash MS, Young KW, Willars GB, Challiss RA & Nahorski SR (2001). Single-cell imaging of graded Ins(1,4,5)P3 production following G-protein-coupled-receptor activation. Biochem J 356, 137142.
  • Pike LJ & Casey L (1996). Localization and turnover of phosphatidylinositol 4,5-bisphosphate in caveolin-enriched membrane domains. J Biol Chem 271, 2645326456.
  • Pike LJ & Miller JM (1998). Cholesterol depletion delocalizes phosphatidylinositol bisphosphate and inhibits hormone-stimulated phosphatidylinositol turnover. J Biol Chem 273, 2229822304.
  • Raucher D, Stauffer T, Chen W, Shen K, Guo S, York JD, Sheetz MP & Meyer T (2000). Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion. Cell 100, 221228.
  • Redfern DA & Gericke A (2005). pH-dependent domain formation in phosphatidylinositol polyphosphate/phosphatidylcholine mixed vesicles. J Lipid Res 46, 504515.
  • Rohacs T (2007). Regulation of TRP channels by PIP2. Pflugers Arch 453, 753762.
  • Rohacs T, Lopes CM, Jin T, Ramdya PP, Molnar Z & Logothetis DE (2003). Specificity of activation by phosphoinositides determines lipid regulation of Kir channels. Proc Natl Acad Sci U S A 100, 745750.
  • Rozelle AL, Machesky LM, Yamamoto M, Driessens MH, Insall RH, Roth MG, Luby-Phelps K, Marriott G, Hall A & Yin HL (2000). Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3. Curr Biol 10, 311320.
  • Santagata S, Boggon TJ, Baird CL, Gomez CA, Zhao J, Shan WS, Myszka DG & Shapiro L (2001). G-protein signaling through tubby proteins. Science 292, 20412050.
  • Selyanko AA, Hadley JK & Brown DA (2001). Properties of single M-type KCNQ2/KCNQ3 potassium channels expressed in mammalian cells. J Physiol 534, 1524.
  • Selyanko AA, Stansfeld CE & Brown DA (1992). Closure of potassium M-channels by muscarinic acetylcholine-receptor stimulants requires a diffusible messenger. Proc R Soc Lond B Biol Sci 250, 119125.
  • Shapiro MS, Wollmuth LP & Hille B (1994). Angiotensin II inhibits calcium and M current channels in rat sympathetic neurons via G proteins. Neuron 12, 13191329.
  • Simonsen A, Wurmser AE, Emr SD & Stenmark H (2001). The role of phosphoinositides in membrane transport. Curr Opin Cell Biol 13, 485492.
  • Soejima M & Noma A (1984). Mode of regulation of the ACh-sensitive K-channel by the muscarinic receptor in rabbit atrial cells. Pflugers Arch 400, 424431.
  • Stauffer TP, Ahn S & Meyer T (1998). Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells. Curr Biol 8, 343346.
  • Suh BC & Hille B (2002). Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron 35, 507520.
  • Suh BC & Hille B (2005). Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr Opin Neurobiol 15, 370378.
  • Suh BC & Hille B (2007). Regulation of KCNQ channels by manipulation of phosphoinositides. J Physiol 582, 911916.
  • Suh BC, Horowitz LF, Hirdes W, Mackie K & Hille B (2004). Regulation of KCNQ2/KCNQ3 current by G protein cycling: the kinetics of receptor-mediated signaling by Gq. J Gen Physiol 123, 663683.
  • Suh BC, Inoue T, Meyer T & Hille B (2006). Rapid chemically induced changes of PtdIns(4,5) 2 gate KCNQ ion channels. Science 314, 14541457.
  • Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M & Tomita F (1986). Staurosporine, a potent inhibitor of phospholipid/Ca2+-dependent protein kinase. Biochem Biophys Res Commun 135, 397402.
  • Van Rheenen J, Achame EM, Janssen H, Calafat J & Jalink K (2005). PIP2 signaling in lipid domains: a critical re-evaluation. EMBO J 24, 16641673.
  • Varnai P & Balla T (1998). Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools. J Cell Biol 143, 501510.
  • Varnai P, Thyagarajan B, Rohacs T & Balla T (2006). Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells. J Cell Biol 175, 377382.
  • Wang J, Gambhir A, Hangyas-Mihalyne G, Murray D, Golebiewska U & McLaughlin S (2002). Lateral sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions. J Biol Chem 277, 3440134412.
  • Winks JS, Hughes S, Filippov AK, Tatulian L, Abogadie FC, Brown DA & Marsh SJ (2005). Relationship between membrane phosphatidylinositol-4,5-bisphosphate and receptor-mediated inhibition of native neuronal M channels. J Neurosci 25, 34003413.
  • Xie LH, John SA, Ribalet B & Weiss JN (2006). Activation of inwardly rectifying potassium (Kir) channels by phosphatidylinosital-4,5-bisphosphate (PIP2): Interaction with other regulatory ligands. Prog Biophys Mol Biol 94, 320325.
  • Xu C, Watras J & Loew LM (2003). Kinetic analysis of receptor-activated phosphoinositide turnover. J Cell Biol 161, 779791.
  • Zaika O, Lara L, Gamper N, Hilgemann DW, Jaffe DB & Shapiro MS (2006). Angiotensin II regulates neuronal excitability via PIP2-dependent modulation of Kv7 (M-type) K+ channels. J Physiol 575, 4967.
  • Zaika O, Tolstykh GP, Jaffe DB & Shapiro MS (2007). IP3-mediated Ca2+ signals direct purinergic P2Y-receptor regulation of neuronal ion channels. J Neurosci (in press).
  • Zhang H, Craciun LC, Mirshahi T, Rohacs T, Lopes CM, Jin T & Logothetis DE (2003). PIP2 activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents. Neuron 37, 963975.
  • Zhang H, He C, Yan X, Mirshahi T & Logothetis DE (1999). Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions. Nat Cell Biol 1, 183188.

Appendix

  1. Top of page
  2. Abstract
  3. Specificity of PIP2 signalling
  4. PIP2 affinity of membrane proteins
  5. Technical considerations
  6. Perspectives
  7. References
  8. Appendix

Acknowledgements

The work in the laboratory of N.G. is supported by the Wellcome Trust Grant 080593/Z/06/Z, and in the laboratory of M.S.S. by NIH grant RO1 NS043394.