Ca2+ and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] are key agents in membrane-associated signalling events. Their temporal and spatial regulation is crucial for activation or recruitment of proteins in the plasma membrane. In fact, the interaction of several signalling proteins with PI(4,5)P2 has been shown to be tightly regulated and dependent on the presence of Ca2+, with co-operative binding in some cases. In these proteins, PI(4,5)P2 and Ca2+ binding typically occurs at different binding sites. In addition, several PI(4,5)P2-binding proteins are known targets of calmodulin (CaM), which, depending on the presence of calcium, can compete with PI(4,5)P2 for protein interaction, translating Ca2+ transient microdomains into variations of PI(4,5)P2 lateral organization in time and space. The present review highlights different examples of calcium-dependent PI(4,5)P2-binding proteins and discusses the possible impact of this dual regulation on fine-tuning of protein activity by triggering target membrane binding in the presence of subtle changes in the levels of calcium or PI(4,5)P2.
Living cells have the ability to adapt and respond efficiently to environmental stimuli, mostly due to the presence of messengers whose concentration varies in time and space. In mammalian cells, Ca2+ and phosphate ions (e.g. as adenosine phosphates or phospholipids) play a key role in many cell signalling pathways, mainly as a result of localized changes in protein electrostatics. Ca2+ binding, as well as phosphorylation, is able to change protein conformation and charge, ultimately affecting their potential interactions and assuring signal transduction . In this context, it is of great importance to better understand the concerted action of Ca2+ and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2].
PI(4,5)P2 is the major polyphosphoinositide present in the inner leaflet of the plasma membrane of mammalian cells. It represents approximately 1 mol% of total plasma membrane phospholipid content, a steady-state level sustained by consecutive phosphorylations and dephosphorylations.
Much of PI(4,5)P2's functional multiplicity is associated with its capacity to bind several protein domains, targeting proteins to the plasma membrane and controlling their activity in time and space. PI(4,5)P2-binding domains include pleckstrin homology (PH) domains, Tubby domains and C2 domains, among others [2,3]. Furthermore, electrostatic interactions with PI(4,5)P2 are also likely to occur through the polybasic region of some proteins . All together, these lipid–protein interactions play a key role in the regulation of numerous vital cell functions, from actin cytoskeleton attachment and reorganization to membrane trafficking. For example, the interaction of PI(4,5)P2 with ezrin/radixin/moesin (ERM proteins), vinculin and talin, is responsible for the anchoring of actin filaments to the plasma membrane and assembly of focal adhesions . This phospholipid is also involved in membrane ruffle formation and cell motility, through its interaction with the GMC proteins myristoylated alanine-rich C kinase substrate (MARCKS), growth-associated protein of 43 kDa (GAP-43) and cortical/cytoskeleton-associated protein of 23 kDa (CAP-23) [6,7]. PI(4,5)P2 has also been associated with early stages of endocytosis, interacting with clathrin adaptor proteins such as AP180/clathrin assembly lymphoid myeloid leukaemia protein (CALM) during the recruitment of the clathrin coat . Later on in the mechanism, the interaction of PI(4,5)P2 with dynamin and synaptojanin-1 is related with the fission of endocytic pits  and clathrin uncoating  respectively.
As well as in endocytosis, PI(4,5)P2 plays different roles throughout exocytosis, interacting with distinct proteins during vesicle docking, priming or fusion. Some of the more studied examples include synaptotagmin  (associated with Ca2+-triggered exocytosis), rabphilin [the effector of rab3 proteins responsible for controlling the soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) complex formation]  and the SNARE protein syntaxin-1 .
Several PI(4,5)P2-binding proteins have been shown to be sensitive to calcium fluctuations, and in several cases PI(4,5)P2 and calcium binding was shown to be co-operative [11,14]. In the present review, we briefly summarize different examples of calcium-dependent changes in the affinity of PI(4,5)P2-binding proteins to membranes. The effect of Ca2+ on PI(4,5)P2 membrane distribution and its consequences on PI(4,5)P2–protein interactions are also addressed.
Role of calcium in membrane binding by C2 domains
C2 domains are important protein modules for eukaryotic membrane signalling, being involved in signal transduction and membrane trafficking . These domains interact with PI(4,5)P2 and are composed of approximately 130 amino acid residues . The overall structure of the C2 domain fold is represented by a Greek key motif composed of a pair of four-stranded β-sheets with flexible surface loops and helices.
C2 domains can interact with lipids using two separate binding sites, one containing Ca2+-binding loops in a lysine-rich cluster  and another with a cationic β-groove, which selectively binds to phosphoinositides in a Ca2+-independent matter .
Several signalling proteins containing these C2 domains, such as rabphilin-3A , protein kinase C (PKC)  or synaptotagmin , have specificity for inositol rings in the membrane, especially to PI(4,5)P2 in a Ca2+-dependent manner, as shown in Figure 1. The C2A domain of rabphilin-3A was shown to bind the PI(4,5)P2 headgroup with 16-fold higher affinity in the presence of saturating concentrations of Ca2+, whereas its C2B domain binds the PI(4,5)P2 headgroup in a Ca2+-independent fashion with low affinity . On the other hand, the interaction of the C2A domain of rabphilin-3A with the PI(4,5)P2 headgroup increased its affinity for Ca2+, revealing co-operativity between Ca2+ and PI(4,5)P2 binding.
Representative structures of the C2A domain of rabphilin-3A (PDB code 4LT7) , the C2 domain of PKCα (PDB code 3GPE) , the C2B domain of synaptotagmin-1 (PDB code 1TJX)  and annexin II (PDB code 2HYW) 
Furthermore, binding of the C2 domain of PKCα  and the C2B domain of synaptotagmin-1  to PI(4,5)P2 was also shown to be Ca2+-dependent (Figure 1). Synaptotagmin-1, a calcium sensor involved in synaptic vesicle fusion, contains a single transmembrane domain close to the N-terminus, which anchors the protein to synaptic vesicles . This domain is connected by a 61-residue unstructured linker to two C2 domains, C2A and C2B. The C2A domain binds three Ca2+ ions, with affinities ranging from 50 μM to 10 mM, whereas the C2B domain binds two Ca2+ ions, both with ~200 μM affinity . As observed for other C2 domains, synaptotagmin-1 C2 domains can bind to anionic phospholipids with low specificity by completing the Ca2+-binding sites with the anionic phospholipid headgroups, whereas interaction with PI(4,5)P2 occurs through the conserved cationic β-groove , as represented in Figure 1. The interaction between PI(4,5)P2 and the C2B domain of synaptotagmin was shown to increase the affinity of the C2B domain for Ca2+ more than 40-fold. Conversely, Ca2+-binding to the C2B domain increases the affinity for PI(4,5)P2 more than 10-fold , revealing similar co-operativity between Ca2+ and PI(4,5)P2 binding as the one observed for rabphilin-3A.
The mechanism responsible for this co-operativity is not the co-ordination of calcium and PI(4,5)P2 in the binding sites, since the respective binding sites within the C2 domains are located at some distance, but are probably related to protein conformational changes .
The dependence on both calcium and PI(4,5)P2 for binding of PKCα has been defined as a target-activated messenger affinity (TAMA) mechanism . According to this, both the messenger and the target lipid must be present in sufficient concentrations for effective protein binding to the membrane, allowing for protein targeting to PI(4,5)P2-enriched domains during calcium signalling.
The same mechanism must be operating for rabphilin-3A and synaptotagmin-1, and it is likely to allow for fine-tuning of protein activity in response to subtle changes in the levels of calcium or PI(4,5)P2.
Ca2+-dependent PI(4,5)P2 binding by annexins
Annexins are a conserved family of proteins that share the ability to reversibly bind anionic phospholipids in a Ca2+-dependent fashion . They have been implicated in several vital cell functions, including membrane–cytoskeleton interactions, membrane trafficking, endocytosis, exocytosis, regulation of membrane protein activity, signal transduction and also Ca2+ channel activity [22–24].
Each member of the annexin family presents two distinct domains: a highly conserved C-terminal core and a variable N-terminal extension. The annexin core domain is composed of four (eight in annexin A6) homologous α-helical do-mains of approximately 70 amino acid residues, presenting an overall shape of a slightly curved disc surrounding a central hydrophilic pore. Both Ca2+- and phospholipid-binding sites are located on the convex side of this core domain, whereas the concave side interacts with the N-terminal domain. This N-terminus varies in length, amino acid sequence and post-translational modifications along the annexin family, influencing the stability and specific functions of each individual protein .
Ca2+ usually plays a dual role in the regulation of annexin function. In one hand, Ca2+ ions are the mediators of membrane binding, simultaneously co-ordinating carbonyl and carboxy groups of the protein and phosphoryl moieties of phosphatidylserine (PS) . On the other hand, as shown for annexin A1, Ca2+ binding can result in a change in conformation that leads to the exposure of the N-terminus and its interaction with other cytosolic proteins .
Despite the similarity of the core domain, annexins present different lipid specificity and are sensitive to different Ca2+ concentrations , allowing for cytosol-to-membrane translocation to specific sites in the membrane as a response to different Ca2+ concentrations. For example, the free Ca2+ concentration required for binding to liposomes containing PS may vary from 20 μM to 100 nM for annexin A5 and annexin A2–S100A10 (A2t) heterotetrameric complex respectively [21,27].
Ca2+-dependent PI(4,5)P2 binding has been demonstrated for different annexins (A2, A4 and A8) [28–30], the more extensively studied being annexin A2 (AnxA2). A2t binds PI(4,5)P2 with high affinity, whereas monomeric AnxA2 exhibits significantly less affinity for inositol binding . The binding site for PI(4,5)P2 within AnxA2 in the heterotetrameric form was shown to involve Lys279 and Lys281 . As illustrated in Figure 1, these residues are located within a calcium binding loop, and the carbonyl groups of several residues adjacent to the PI(4,5)P2-binding residues participate in calcium binding.
In the presence of Ca2+, the interaction of PI(4,5)P2 with A2t results in the formation of micrometre-size PI(4,5)P2 clusters whose formation and stability depend on the presence of cholesterol . These PI(4,5)P2-enriched raft-like domains coincide with the accumulation of the AnxA2-binding partner filamentous actin (F-actin), suggesting that AnxA2 may act as an actin nucleator at PI(4,5)P2 membrane patches, promoting the reorganization of the actin cytoskeleton . Moreover, the formation of these microdomains suggests a wider involvement of AnxA2 in PI(4,5)P2-related processes that are dependent on its lateral clustering, as already demonstrated for Ca2+-triggered granule exocytosis in chromaffin cells .
Ca2+ modulation of CaM/PI(4,5)P2-binding proteins
Calmodulin (CaM) is a small ubiquitous adaptor protein that functions as the major transducer of small and transient Ca2+ signals inside cells. Structurally, Ca2+-free CaM (apoCaM) presents a dumbbell-like shape composed of a flexible α-helix flanked by two globular domains, each one consisting of two helix–loop–helix motifs (EF hands) connected by a short antiparallel β-sheet [33,34]. Each domain can bind two Ca2+ ions, the C-terminal lobe presenting higher affinity for Ca2+ (Kd ≈ 1 μM) than the N-terminal one (Kd ≈ 12 μM) . Upon Ca2+ binding (Ca2+/CaM), CaM becomes more elongated and hydrophobic residues in each domain are exposed . These hydrophobic patches can bind to a variety of target proteins, activating its Ca2+ sensor activity and triggering its ability to relieve protein autoinhibition, remodel active sites, and dimerize proteins [33,36].
In cells, there are hundreds of proteins that contain CaM recruitment sites and consequently bind CaM. Their heterogeneity in structure and function can be explained by the structural flexibility of the calcium sensor. CaM targets differ in their affinity for apoCaM and Ca2+/CaM, but can also modulate CaM sensitivity for Ca2+. Taking into account the low level of free CaM inside cells (i.e. ~1% in cardiac myocytes) , these differences in affinity usually dictate which proteins are activated at any given time, especially after Ca2+ stimulation .
Among the CaM protein targets, a few were also shown to bind PI(4,5)P2 and, for that reason, make the bridge between Ca2+ signalling and PI(4,5)P2-regulated cellular functions. These CaM/PI(4,5)P2-binding proteins include not only the GMC proteins, also called ‘pipmodulins’ , but also the SNARE proteins synaptobrevin-2 and syntaxin-1 , among others. Structurally, these proteins present an unstructured cluster of positively charged amino acid residues (effector domain) capable of electrostatically sequestering PI(4,5)P2 to localized patches in the membrane (Figure 2). Since CaM is also negatively charged, it can compete with PI(4,5)P2 as a response to Ca2+ concentration fluctuations, depending on the relative affinity of the protein for apoCaM and Ca2+/CaM [1,40].
Example of a PI(4,5)P2/CaM switching mechanism
This PI(4,5)P2/CaM switching mechanism, represented in Figure 2, has been well demonstrated for the ‘pipmodulin’ MARCKS, a natively unstructured protein anchored to the plasma membrane through an N-terminal myristate. Because it presents a conserved basic effector domain, it also interacts electrostatically with negatively charged phospholipids . Several experiments have shown that 13 basic residues are responsible for laterally sequestering 3 PI(4,5)P2 molecules in a reversible manner [4,41,42]. MARCKS presents high affinity for Ca2+/CaM (~4 nM) . As a consequence, when the local Ca2+ concentration increases, Ca2+/CaM competes with PI(4,5)P2 for the polybasic region of the protein. Because the myristate anchor is not enough to hold the protein in the membrane, MARCKS translocates to the cytosol, releasing the sequestered PI(4,5)P2 molecules [43,44] and consequently increasing its free concentration in the plasma membrane. In summary, CaM translates the Ca2+ transient microdomains into variations of PI(4,5)P2 lateral organization, contributing for the tight regulation of PI(4,5)P2-dependent cellular processes.
Direct PI(4,5)P2–Ca2+ interaction
So far we have discussed the effect of Ca2+ on PI(4,5)P2–protein interactions. However, Ca2+ may have a direct impact on PI(4,5)P2 lateral distribution, facilitating protein sorting to the plasma membrane at sites already enriched in PI(4,5)P2. At non-physiological concentrations of both Ca2+ (≥1 mM) and/or PI(4,5)P2 (5–50%), Ca2+ was already shown to promote macroscopic clustering of PI(4,5)P2 in membrane model systems, in lipid monolayers [45,46], large unilamellar vesicles (LUVs)  and giant unilamellar vesicles (GUVs) . Recently, our group and others have demonstrated that Ca2+-induced PI(4,5)P2 clustering occurs even at physiological concentrations of PI(4,5)P2 and Ca2+, in supported lipid monolayers  and free-standing bilayers .
Moreover, in artificial membranes containing cholesterol, Ca2+ seems to favour partition of PI(4,5)P2 molecules to raft-like domains [45,49]. This suggests that Ca2+ may even potentiate protein anchoring in lipid rafts, by increasing PI(4,5)P2 density in these microdomains.
Altogether, these recent findings seem to indicate that PI(4,5)P2, besides interacting with Ca2+ sensors such as C2-contaning proteins, annexins and CaM, may act as a Ca2+ sensor itself, responsive to physiological Ca2+ concentrations.
The Ca2+-dependent binding of several signalling proteins to PI(4,5)P2 is well established. This dependence is not based on the simultaneous co-ordination of Ca2+ ions with acidic residues from the protein and phosphate groups from phospholipids as observed with many PS-binding domains, but typically involves two different sets of protein residues (or binding sites). In addition, several CaM/PI(4,5)P2-binding proteins exhibit Ca2+-dependent binding of PI(4,5)P2 due to competition of CaM with the phosphoinositol for protein binding. In such conditions, these PI(4,5)P2-binding proteins operate as integrators of two different signals: Ca2+ and PI(4,5)P2 concentrations. The physiological relevance of these mechanisms is likely to be associated with the possibility of fine-tuning protein activity by triggering target membrane binding in the presence of subtle changes in the levels of calcium or PI(4,5)P2.
Membrane Morphology and Function: A Biochemical Society Focused Meeting held at Hotel del Camerlengo, Fara San Martino, Abruzzo, Italy, 5–8 May 2014. Organized and Edited by Banafshé Larijani [IKERBASQUE, Basque Foundation for Science and Unidad de Biofísica (CSIC-UPV/EHU), University of the Basque Country, Spain] and Marco Falasca (Barts and The London School of Medicine and Dentistry, U.K.)
annexin 2–S100A10 heterotetrameric complex
myristoylated alanine-rich C kinase substrate
protein kinase C
soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor
M.J.S. and F.F. are recipients of fellowships from Fundação para a Ciência e a Tecnologia (FCT) [grant numbers SFRH/BD/80575/2011 and SFRH/BPD/64320/2009 respectively]. We acknowledge funding by FCT [project references PTDC/QUI-BIQ/112067/2009 and RECI/CTM-POL/0342/2012].