Phosphatidylinositol 4,5-Bisphosphate Increases Ca²⁺ Affinity of Synaptotagmin-1 by 40-fold

Background: Synaptotagmin-1, a Ca²⁺ sensor of neuronal exocytosis, interacts with the anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂).

Results: Microscale thermophoresis shows that PIP₂ binding to the polybasic patch of synaptotagmin-1 increases the Ca²⁺ affinity by >40-fold.

Conclusion: PIP₂ and Ca²⁺ binding to synaptotagmin-1 is strongly cooperative.

Significance: Understanding the interplay between Ca²⁺, synaptotagmin-1, and PIP₂ is crucial for our understanding of neurotransmitter release.

Abstract

Synaptotagmin-1 is the main Ca²⁺ sensor of neuronal exocytosis. It binds to both Ca²⁺ and the anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), but the precise cooperativity of this binding is still poorly understood. Here, we used microscale thermophoresis to quantify the cooperative binding of PIP₂ and Ca²⁺ to synaptotagmin-1. We found that PIP₂ bound to the well conserved polybasic patch of the C2B domain with an apparent dissociation constant of ∼20 μm. PIP₂ binding reduced the apparent dissociation constant for Ca²⁺ from ∼250 to <5 μm. Thus, our data show that PIP₂ makes synaptotagmin-1 >40-fold more sensitive to Ca²⁺. This interplay between Ca²⁺, synaptotagmin-1, and PIP₂ is crucial for neurotransmitter release.

Introduction

In the synaptic terminal, neurotransmitter release is mediated by fusion of synaptic vesicles with the plasma membrane. Fusion is triggered by a sudden increase in the cytoplasmic Ca²⁺ concentration in response to membrane depolarization. The protein synaptotagmin-1 (together with synaptotagmin-2 and synaptotagmin-9) is the main Ca²⁺ sensor of the fast phase of neuronal exocytosis (reviewed in Ref. 1). Synaptotagmin-1 contains a single transmembrane domain close to the N terminus, which anchors the protein to synaptic vesicles. The transmembrane domain is connected by a 61-residue unstructured linker to two C2 domains, C2A and C2B. The mechanism by which synaptotagmin-1 triggers membrane fusion is still debated, but structural rearrangements of the plasma membrane and/or interactions with SNARE proteins have been implicated (1).

Ca²⁺ binding to synaptotagmin-1, originally demonstrated by equilibrium dialysis using native protein (2), has been characterized by isothermal titration calorimetry (3) and NMR (4–6) using a soluble fragment containing both C2 domains (C2AB fragment, residues 97–421). The C2A domain binds to three Ca²⁺ ions with affinities ranging from 50 μm to 10 mm. The C2B domain binds two Ca²⁺ ions, both with ∼200 μm affinity.

In the presence of Ca²⁺, the C2 domains of synaptotagmin-1 also bind to membranes containing anionic phospholipids, with little specificity for the phospholipid species (3, 6–14). Interestingly, binding already occurs at Ca²⁺ concentrations well below the Ca²⁺ affinity of free synaptotagmin-1. Here, anionic phospholipid headgroups complement the Ca²⁺-binding sites, increasing the affinity of C2AB for Ca²⁺ to ∼5–100 μm (3, 6–8, 11, 13). In the absence of Ca²⁺, a conserved polybasic lysine patch located on the C2B domain can also bind to anionic lipids, and this binding is strongly preferential for the polyanionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂)⁴ (3, 9–14). Binding of PIP₂ to the polybasic patch might increase the Ca²⁺ affinity (12), although this is still controversial (3) and has hitherto not been characterized in detail.

Experimentally, measuring synaptotagmin-1 binding to PIP₂ and/or Ca²⁺ is not trivial. Isothermal titration calorimetry and NMR require high (100 μm to 1 mm) concentrations of protein (3–5). Therefore, high affinities well below these concentrations cannot be accurately determined with these approaches. Binding of synaptotagmin to PIP₂ is often inferred from binding of the C2 domains to artificial membranes containing a defined fraction of PIP₂ (e.g. by FRET (3), pulldown assays (11, 13), or density flotations (3, 12)). However, it is difficult to quantitatively distinguish Ca²⁺ from PIP₂ binding with these approaches. We have recently shown (10) that Ca²⁺ binding to synaptotagmin-1 can be directly measured with a new technique called microscale thermophoresis (MST) (15, 16). MST is based on the principle that molecules move along a temperature gradient in a capillary (the Soret effect). Upon binding to Ca²⁺ or PIP₂, the surface properties of synaptotagmin-1 change, resulting in an altered thermophoretic behavior. In this study, we applied MST to study PIP₂ and Ca²⁺ cooperative binding to synaptotagmin-1.

EXPERIMENTAL PROCEDURES

The C2AB fragment of synaptotagmin-1 (rat sequence, residues 97–421) was expressed in Escherichia coli and purified as described (3, 10). The single cysteine mutant (C278S/S342C) was labeled with Alexa Fluor 488-maleimide (Invitrogen) as described (3, 10). Liposomes were prepared by extrusion of rehydrated lipid films through 100-nm pores (polycarbonate membranes, Avestin) (17). All lipids were from Avanti Polar Lipids. MST was measured with ∼50 nm fluorescently labeled C2AB in 20 mm HEPES, 150 mm KCl, and 2.5 mg/ml BSA at pH 7.4. The samples were added to hydrophobic capillaries (NanoTemper Technologies), and MST was measured with a NanoTemper Monolith NT.015 system (25% light-emitting diode, 40% IR laser power). The label-free (tryptophan) experiments were performed with 1 μm wild-type C2AB, no BSA, and the NanoTemper Monolith NT.LabelFree instrument (80% UV light-emitting diode, 40% IR laser power). The MST curves were fitted with simple Michaelis-Menten kinetics to obtain the apparent dissociation constant for Ca²⁺ (K_Ca) or PIP₂ (K_PIP2). For Ca²⁺ binding, T = A − B/(K_Ca + [Ca²⁺]), where T is the percentage of fluorescence after heating, [Ca²⁺] is the total calcium concentration in the capillary, and A and B are conversion factors for the thermophoresis.

RESULTS

We performed MST measurements on the Alexa Fluor 488-labeled C2AB fragment of synaptotagmin-1 (residues 97–421). With this technique, a glass capillary is filled with a dilute protein solution (50 nm). Fluorescence is then measured at a spot in the capillary that is heated with a focused IR laser beam. Heating (by ∼5 °C) results in the generation of a temperature gradient along the axis of the capillary (Fig. 1, A and B). The C2AB fragment thermodiffuses out of this heated spot (measured by fluorescence recording), resulting in a protein gradient that is reversed when the IR laser is switched off. The amount of fluorescence decrease at the heated spot (the MST signal) was changed in the presence of Ca²⁺, thus providing a direct readout of Ca²⁺ binding to the C2AB fragment. Evidently, Ca²⁺ binding alters the thermophoretic (i.e. surface, charge) properties and thereby the thermodiffusion of synaptotagmin (10). Varying the calcium concentration in the capillary thus allowed us to obtain a binding curve (Fig. 1C).

FIGURE 1.

Ca²⁺ binding to C2AB measured by MST. A, principle of MST. A capillary containing 50 nm Alexa Fluor 488-labeled C2AB is locally heated by a focused IR laser (IR on). C2AB thermodiffuses away from the heated spot, causing a local depletion and a drop in fluorescence. Ca²⁺ binding changes the thermophoretic properties of C2AB, resulting in a decreased thermodiffusion. B, MST time traces of 16 different Ca²⁺ concentrations (ranging from 0 to 5 mm). Note that thermodiffusion is reduced at high Ca²⁺ concentrations. C, dependence of the MST signal on the Ca²⁺ concentration (measured 30 s after turning on heating; data from B). The solid line is a fit with Michaelis-Menten kinetics, yielding an apparent dissociation constant of K_Ca = 221 μm. No change in the MST signal was observed in the presence of Mg²⁺ or when a mutant impaired in Ca²⁺ binding was used (D178A/D230A/D232A/D309A/D363A/D365A, called C2a*b* (a*b*)). D, same as C but using unlabeled C2AB. MST was measured using intrinsic tryptophan fluorescence and fitted, yielding K_Ca = 206 μm. Error bars indicate the range of data points obtained from at least two measurements.

We fitted the binding curves with simple Michaelis-Menten kinetics assuming a single binding site (see “Experimental Procedures”). This model does not take into account binding of multiple Ca²⁺ ions (or PIP₂ molecules; see below), and for some curves, this simplification may affect the quality of the fit. However, the overall quality of the data did not warrant fitting with a more sophisticated binding model. Thus, we could not differentiate between the different calcium-binding sites, and we report only the apparent dissociation constant (K_Ca).

C2AB bound to Ca²⁺ with K_Ca = 221 ± 23 μm (n = 3). Control experiments with Mg²⁺ or a mutant with disrupted Ca²⁺ binding (D178A/D230A/D232A/D309A/D363A/D365A, called C2a*b*) (3, 10) showed that the change in the MST signal was indeed due to binding of Ca²⁺ ions to the established binding sites in the C2 domains. Furthermore, the MST measurements were not affected by the presence of the dye because a similar binding constant of K_Ca = 206 ± 40 μm was obtained with the unlabeled C2AB fragment using the intrinsic tryptophan fluorescence as the readout (C2AB has three tryptophans) (Fig. 1D). We then set out to study the cooperativity of Ca²⁺ and PIP₂ binding.

No apparent change in the Ca²⁺-dependent thermophoretic behavior of C2AB was observed in the presence of liposomes composed of pure 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (2.5 mm total lipid concentration; K_Ca = 226.7 ± 50.7 μm) (Fig. 2A). In contrast, the apparent affinity for Ca²⁺ increased by ∼5-fold when only 10% of these liposomes were replaced with a liposome population composed of 95% 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine and 5% PIP₂ (K_Ca = 46.0 ± 5.9 μm). Accordingly, the addition of 50 μm Ca²⁺ (well below the K_Ca of C2AB) resulted in ∼4-fold stronger binding to PIP₂-containing liposomes (from K_PIP2 = 45.3 ± 9.25 μm to 13.3 ± 2.9 μm total PIP₂ concentration) (Fig. 2B). 50 μm Ca²⁺ also increased C2AB binding to liposomes containing a more physiological lipid composition (phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine/cholesterol at a molar ratio of 5:2:1:1) but only if 1 mol % PIP₂ was present (Fig. 2C). Thus, synaptotagmin-1 binds to anionic membranes and Ca²⁺ in a cooperative manner, as reported previously (3, 6–13). We performed a set of experiments with water-solubilized PIP₂ to further characterize this cooperativity.

FIGURE 2.

Ca²⁺ dependence of MST signal of C2AB in presence of PIP₂-containing liposomes. A, Ca²⁺ binding of the C2AB fragment in the presence of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (PC)-containing liposomes (2.5 mm total lipid concentration) yielded an apparent dissociation constant of K_Ca = 226.7 ± 50.7 μm (black). However, when 10% of the 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine-containing liposomes contained 5 mol % PIP₂, the affinity increased by ∼5-fold to K_Ca = 46.0 ± 5.9 μm (red). B, liposome binding as a function of the fraction of PIP₂-containing liposomes. In all cases, the total lipid concentration was 2.5 mm, but the fraction of liposomes containing 5 mol % PIP₂ varied. In the absence of Ca²⁺, C2AB bound to the PIP₂ membranes with K_PIP2 = 36.2 ± 7.4% (or 45.3 μm PIP₂; cyan). In the presence of 50 μm Ca²⁺, the affinity increased by 4-fold to K_PIP2 = 10.6 ± 2.3% (or 13.3 μm PIP₂; green). C, binding of C2AB to liposomes composed of a 5:2:1:1 molar ratio of brain isolated phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol. C2AB did not bind to liposomes lacking PIP₂ regardless of the presence (green) or absence (blue) of 50 μm Ca²⁺. In contrast, C2AB bound to liposomes containing 1 mol % PIP₂ already in the absence of Ca²⁺ (red). Similar to B, 50 μm Ca²⁺ increased the binding affinity (K_PIP2 = 50.9 ± 20.0 μm total lipid concentration; black). D, Ca²⁺ binding curve of C2AB in the presence (K_Ca = 17.7 ± 0.7 μm; pink) or absence (K_Ca = 265.2 ± 27.4 μm; blue) of 20 μm PIP₂ in solution. 1 mm Mg²⁺ was present to suppress potentially unspecific Ca²⁺-PIP₂ interactions. Error bars indicate the range of data points obtained from at least two measurements.

One of the main advantages of MST compared with alternative techniques for measuring Ca²⁺ binding is the low concentration of protein that is required: measurements could be carried out with C2AB concentrations as low as 50 nm, which is 3–4 orders of magnitude below that reported for isothermal titration calorimetry (3) or NMR (4–6). This low concentration allowed us to measure PIP₂ binding by adding PIP₂ directly to the capillary (Fig. 2D). Even PIP₂ isolated from porcine brain with long fatty acid acyl chains (dominant species C18:0 and C20:4) is water-soluble at concentrations up to ∼9 mm and does poorly form micelles because of its high anionic charge (18).

Strikingly, the affinity for Ca²⁺ binding increased by 15-fold in the presence of 20 μm PIP₂ (from K_Ca = 265.2 ± 27.4 μm to 17.7 ± 0.7 μm) (Fig. 2D). In this experiment, an excess of 1 mm Mg²⁺ was present to suppress potential nonspecific interactions of Ca²⁺ with PIP₂ or C2AB. At higher PIP₂ concentrations, the Ca²⁺ affinity increased even further (to >40-fold; K_Ca = 3.3 ± 1.3 μm at 40–80 μm PIP₂ compared with 221 ± 23 μm without PIP₂) (Fig. 3, A–C). Accordingly, the addition of Ca²⁺ progressively increased the binding affinity of C2AB for PIP₂ (from K_PIP2 = 20 ± 5 μm without Ca²⁺ to <2 μm at >20 μm Ca²⁺). This cooperativity is not specific for PIP₂ or the length of the acyl chains because another phosphoinositide (20 μm phosphatidylinositol 3,5-bisphosphate) or short-chain PIP₂ (20 μm 1,2-dioctanoyl-sn-glycero-3-phosphatidylinositol 4′,5′-bisphosphate; C8:0) also increased the apparent Ca²⁺ affinity (K_Ca = 11 ± 5 and 8 ± 5 μm, respectively).

FIGURE 3.

Ca²⁺ and PIP₂ binding to C2AB measured by MST. A, MST as a function of both Ca²⁺ and PIP₂. Each x and y curve (thus with the same Ca²⁺ or PIP₂ concentrations) was fitted with Michaelis-Menten kinetics to obtain the apparent dissociation constants (K_Ca and K_PIP2; see Fig. 4). B, two Ca²⁺ binding curves from A and their corresponding fits in the absence (K_Ca = 221 μm; black) or presence (K_Ca = 4.6 μm; red) of 80 μm PIP₂. C, two PIP₂ binding curves from A in the absence (K_PIP2 = 20 μm; blue) or presence (KPIP2 < 5 μm; green) of 2.5 mm Ca²⁺. D–F, same as A–C but for the KAKA mutant (K326A/K327A) (12). Compared with the wild type, the amplitude of the fluorescence changes of the KAKA mutant was reduced due to the altered thermophoretic properties that resulted from the substitution of charged residues. In E, the solid (no PIP₂) and dashed (80 μm PIP₂) lines are fits with K_Ca = 195 and 61 μm, respectively. Note that for the KAKA mutant, PIP₂ binding was dramatically reduced compared with the wild type. Each experiment was repeated at least twice; error bars show the range of data points.

PIP₂ binding required the well conserved polybasic patch that is located on the C2B domain because removal of two lysines from this patch (K326A/K327A, the so-called KAKA mutant (12)) (Fig. 3, D–F, and Fig. 4, A and B) almost completely abolished PIP₂-dependent MST changes, even at very high Ca²⁺ concentrations. Accordingly, the apparent affinity for Ca²⁺ was increased by only ∼3-fold in the presence of 80 μm PIP₂ (from K_Ca = 195 ± 35 μm to 61 ± 11 μm). Thus, we could detect only PIP₂ binding to the polybasic patch and did not observe PIP₂ binding via the Ca²⁺-binding sites on the C2A and C2B domains, in contrast to previous observations by us and others (3, 10–12, 14). It is likely that, for the interaction of the Ca²⁺-binding pockets with the membrane, hydrophobic residues surrounding these pockets must insert into the membrane (6–8, 11, 12, 14), although we cannot exclude that PIP₂ binding to the Ca²⁺ sites is silent (i.e. does not change the MST signal). Nevertheless, the Ca²⁺-binding pocket of the C2B domain does affect PIP₂ binding to the polybasic patch because disruption of Ca²⁺ binding to the C2B domain (D309A/D363A/D365A, called C2Ab*) reduced the affinity for PIP₂ by ∼4-fold (from K_PIP2 = 20.4 ± 5.2 μm to 70 ± 24 μm) (Fig. 4B).

FIGURE 4.

Cooperative Ca²⁺ and PIP₂ binding to C2AB. The apparent dissociation constants for Ca²⁺ binding (K_Ca; A) and PIP₂ binding (K_PIP2; B) were determined by MST. Wild-type C2AB (see Fig. 3, A–C) and various mutants were tested: KAKA (K326A/K327A; see Fig. 3, D–F), C2a*B (a*B; D178A/D230A/D232A), C2Ab* (Ab*; D309A/D363A/D365A), C2a*b* (a*b*; D178A/D230A/D232A/D309A/D363A/D365A), and KAKA/C2a*B (KAKA a*B). The KAKA/C2Ab* and KAKA/C2a*b* mutants are not shown in the figure because PIP₂ and Ca²⁺ binding could not be detected with MST (see Fig. 1C). Error bars show the range of data points obtained from at least two measurements. C, conservation of the PIP₂-binding sites. The crystal structure of the C2B domain (purple; Protein Data Bank code 1TJX (26)) was overlapped with that of the PIP₂-bound PKCα C2 domain (green; code 3GPE (25)). D, all residues that stabilize the PIP₂ headgroup (orange) are conserved in the C2B domain (see also Ref. 25).

We then performed MST experiments with mutants disrupted in Ca²⁺ binding to the C2A domain (D178A/D230A/D232A, called C2a*B). Surprisingly, only a small and insignificant PIP₂- or Ca²⁺-dependent change in the MST signal of C2a*B was observed compared with the wild type (Fig. 4, A and B). Accordingly, the combination of C2a*B with the KAKA mutation did not markedly differ from the KAKA mutant with all Ca²⁺-binding sites intact. Apparently, Ca²⁺ binding to the C2A domain does not result in a detectable change in the thermophoretic properties of the C2AB fragment. In contrast, Ca²⁺ binding could no longer be detected by MST upon disruption of the C2B domain. Thus, only Ca²⁺ binding to the C2B domain seems to change the thermophoretic properties of the C2AB fragment, indicating that the calcium-dependent changes reported above are exclusively mediated by the C2B domain. Perhaps this selectivity is related to the thermodynamically divergent modes of Ca²⁺ binding of synaptotagmin-1: Ca²⁺ binding to the C2A domain is endothermic, and that to the C2B domain is exothermic (3). Finally, Ca²⁺ concentrations above 100 μm increased the apparent PIP₂ affinity of synaptotagmin-1 even when both Ca²⁺-binding sites were disrupted (double mutant C2a*b*) (Fig. 4B). This indicates that Ca²⁺ was still able to bind to the double mutant at very high Ca²⁺ concentrations in the presence of PIP₂, perhaps by binding directly to PIP₂ (19, 20).

DISCUSSION

In this work, we have shown that PIP₂ binds to the polybasic patch of the C2B domain of synaptotagmin-1, in agreement with earlier studies (10–14, 21). PIP₂ binding to the polybasic patch increases the apparent affinity of the C2B domain for Ca²⁺ by >40-fold. Conversely, Ca²⁺ binding to the C2B domain increases the affinity for PIP₂ by >10-fold. Cooperative PIP₂ and Ca²⁺ binding to synaptotagmin-1 has been observed previously (12). This cooperativity is probably not caused by complementation of the Ca²⁺-binding sites, as suggested earlier by us and others (3, 6–8), because the polybasic patch and the Ca²⁺-binding sites are located quite far apart (Fig. 4C). Instead, PIP₂ may interact in a structurally less defined manner with the polybasic patch and other solvent-exposed basic residues (9, 12), and this may increase the Ca²⁺ affinity simply by charge screening. Alternatively, the polybasic patch may form a structurally defined complex with PIP₂ similar to the C2 domains of rabphilin-3A and PKCα (22–25). In fact, cooperative PIP₂ and Ca²⁺ binding has been observed for these C2 domains (22–24), very similar to our observations for the C2B domain. Moreover, the crystal structure of the C2B domain (26) can be superimposed with that of the PIP₂-bound C2 domain of PKCα (25), rendering it likely that PIP₂ binds to the C2AB fragment of synaptotagmin-1 in a similar manner (Fig. 4, C and D). Thus, it is conceivable that such PIP₂ binding increases the Ca²⁺ affinity via a conformational change. However, how PIP₂ and Ca²⁺ precisely bind in a cooperative manner to synaptotagmin-1 remains to be elucidated.

Together, we conclude that PIP₂ binding to the polybasic patch of synaptotagmin-1 dramatically increases the Ca²⁺ sensitivity. As discussed previously (12), this explains the reduced release probability of the KAKA mutant in hippocampal neurons (12, 27) and in Drosophila (28). It also explains why in vivo already 10 μm Ca²⁺ is sufficient for physiological release of neurotransmitters in the calyx of Held (29). PIP₂ modulation of synaptotagmin-1 may well be of major physiological relevance when considering that PIP₂ is the predominant phospholipid species at the sites of docked vesicles in PC12 cells (30).

Finally, our work demonstrates the value of MST for measuring molecular interactions. Although we were unable to detect Ca²⁺ binding to the C2A domain under our conditions, MST can be extremely sensitive and allows for monitoring medium and high affinity interactions with only picomoles of material. MST has the potential to complement the limited set of techniques available to measure Ca²⁺ and PIP₂ binding to proteins under equilibrium conditions such as isothermal titration calorimetry and NMR.