Interdomain Interactions Control Ca2+-Dependent Potentiation in the Cation Channel TRPV4

Several Ca2+-permeable channels, including the non-selective cation channel TRPV4, are subject to Ca2+-dependent facilitation. Although it has been clearly demonstrated in functional experiments that calmodulin (CaM) binding to intracellular domains of TRP channels is involved in this process, the molecular mechanism remains elusive. In this study, we provide experimental evidence for a comprehensive molecular model that explains Ca2+-dependent facilitation of TRPV4. In the resting state, an intracellular domain from the channel N terminus forms an autoinhibitory complex with a C-terminal domain that includes a high-affinity CaM binding site. CaM binding, secondary to rises in intracellular Ca2+, displaces the N-terminal domain which may then form a homologous interaction with an identical domain from a second subunit. This represents a novel potentiation mechanism that may also be relevant in other Ca2+-permeable channels.


Introduction
Ca 2+ -dependent regulation by calmodulin (CaM) has been demonstrated for a large number of intracellular proteins including Ca 2+ -permeable plasma membrane cation channels. Often, this provides a negative feedback mechanism that either prevents excessive, potentially deleterious Ca 2+ influx into the cell, or defines the time course of channel activity. Indeed, CaMdependent inhibition is seen in most of the non-selective, Ca 2+permeable cation channels of the TRP group [1,2,3,4,5]. Within the TRPV subfamily, CaM-dependent regulation has been shown for the capsaicin and temperature-sensitive TRPV1 [3,6,7], temperature-sensitive TRPV3 [8], the highly Ca 2+ -selective epithelial Ca 2+ channels TRPV5 and TRPV6 [4,9,10], and TRPV4 [11].
In a previous study, we identified a CaM binding domain in the C terminus of TRPV4 as the structural basis of the Ca 2+dependent potentiation process [11]. Although CaM binding is a prerequisite for potentiation, the molecular mechanism that couples CaM binding to enhanced channel activity has not been resolved. Using protein interaction experiments and functional assays, we show that the molecular correlate of Ca 2+ -dependent current potentiation is disruption of an interdomain interaction within TRPV4 resulting from CaM binding to a C-terminal site. We provide experimental evidence for an underlying mechanism in which an N-terminal autoinhibitory domain forms a molecular switch that controls facilitation of TRPV4.

Results and Discussion
The C-terminal CaM binding site in TRPV4 binds to the CaM C lobe Because CaM interaction with a C-terminal binding site in TRPV4 has been shown to be an essential step in the Ca 2+dependent potentiation of TRPV4 [11], we attempted to obtain initial clues about the underlying molecular mechanism by studying the CaM binding geometry. We analyzed the properties of the complex between CaM and C-terminal fragments of the human TRPV4 ortholog, and investigated the structural determinants within the CaM molecule that are involved in binding.
Fluorescence polarization experiments with the carboxyfluorescein-labeled CaM binding peptide, P5, as the tracer molecule (Fig. 1A, Table S1), showed that CaM binding was Ca 2+dependent and half-maximal at 3.2 mM Ca 2+ (Fig. 1C). Thus, in contrast to some other TRP channels, where CaM binding is present at nanomolar Ca 2+ concentrations [4,29], CaM binding to TRPV4 depends on Ca 2+ concentrations above those in resting cells. The Ca 2+ concentration at which CaM binding to P5 was half-maximal is higher than the IC 50 for steady-state inhibition of TRPV4 by intracellular Ca 2+ in electrophysiological experiments (around 600 nM, [25]). However, during the dynamic response to an agonist, potentiation via this CaM binding site clearly precedes the inactivation process [11]. A possible explanation for the apparent discrepancy in the Ca 2+ dependencies is that CaM binding to the full-length channel may occur at lower concentrations than to the short fragment used here. Alternatively, inactivation may be more sensitive to Ca 2+ , but a slower process than potentiation, or, in the case e.g. of Ca 2+ entry through the channel, components of the potentiatory process may be exposed to a higher local Ca 2+ concentration than those involved in inactivation.
A characteristic feature of the CaM molecule is its symmetry with respect to its domain composition. The homologous N-and C-terminal lobes that both comprise two Ca 2+ -binding EF hand motifs (Fig. 1B), each expose a hydrophobic interaction domain on the molecular surface secondary to Ca 2+ binding and may thus contribute independently to target binding. To quantify the individual affinities of the isolated CaM lobes to P5, we performed P5 fluorescence polarization experiments at different lobe concentrations in 100 mM Ca 2+ (Fig. 1D). The Kd values were 400 nM, 700 nM and 25 mM for CaM and the CaM C-terminal (CaM-C) and N-terminal (CaM-N) lobes, respectively, indicating a 36-fold higher affinity of the CaM C lobe over the N lobe. In an independent experimental approach, we subjected a longer Cterminal TRPV4 fragment, C2 (Fig. 1A), to a CaM interaction assay that is based on the AlphaScreen proximity assay technology (Fig. 2, Fig. S1, Methods S1). Using different CaM constructs, we analyzed the relative binding affinities of the two CaM lobes alone or CaM mutants that are Ca 2+ binding-deficient in the N-lobe (CaM 12 ), C-lobe (CaM 34 ) or both lobes (CaM 1234 ) at 100 mM Ca 2+ (Fig. 1E). Again, the interaction signal was found to be highly dependent on the presence of the functional C-lobe ( To assess whether this finding is functionally relevant, we tested the effect of overexpression of CaM and CaM mutants on TRPV4 activity. In nominally Ca 2+ -free solutions, activation of TRPV4 by the phorbol ester 4a-PMA, a partial TRPV4 agonist [30]that we used in a previous study [11], is weak and slow. Addition of Ca 2+ after activation by 4a-PMA results in facilitation followed by inactivation of the channel [11]. We measured the current increase on adding Ca 2+ (2 mM) in HEK293 cells expressing YFP (not shown) or TRPV4 alone (control), or TRPV4 together with CaM or CaM mutants (Fig. 1F). CaM or the CaM mutants had no effect on spontaneous currents, or currents activated by 4a-PMA in a nominally Ca 2+ -free solution (not shown). A current increase upon addition of Ca 2+ was observed in all cells except those expressing YFP alone. In the latter, currents were not significantly changed at +100 mV (difference: 0.13360.438 pA/ pF, n = 8, p = 0.7688), but significantly decreased at -100 mV (difference-1.09760.436 pA/pF, n = 8, p = 0.04), probably due to a decrease in leakage current. CaM slightly increased the TRPV4 response to Ca 2+ , but the increase is not statistically significant, indicating that in HEK cells, the CaM concentration is not a limiting factor in TRPV4 potentiation. CaM mutants with a dysfunctional C lobe (CaM 34 , CaM 1234 ) had no effect (Fig. 1F), probably because these mutants do not interact with the TRPV4 C-terminal domain and do not functionally compete with endogenous CaM. In contrast, current increases were significantly smaller with the mutant CaM 12 that binds to TRPV4. This mutant may bind and displace endogenous CaM, but, owing to the N-terminal mutation, cannot fully substitute for the wild type molecule. This suggests that, in spite of its lower affinity to the Cterminal CaM binding site in TRPV4, the CaM N lobe contributes functionally to TRPV4 potentiation by a mechanism which remains unclear.
These results with CaM mutants differ from those on Ca 2+dependent potentiation in other TRP channels. Coexpression of CaM 1234 abolished TRPC6 activation secondary to G q proteincoupled receptor activation or diacylglycerol application [31,32] and strongly decreased activation of the adenosine diphosphor-ibose-sensitive channel TRPM2 [33,34]. For TRPM2, coimmunoprecipitation experiments demonstrated CaM 1234 binding to the channel, indicating that Ca 2+ -independent CaM binding contributes to the facilitation mechanism. In contrast to these findings, TRPV4 neither binds to CaM in Ca 2+ -free buffers, nor to CaM 1234 , and potentiation of its activity directly depends on CaM interaction triggered by a rise in Ca 2+ .
How does CaM binding facilitate Ca 2+ entry through TRPV4 at the molecular level? Two general molecular switching mechanisms seemed conceivable. The first comprises a ternary interaction in which Ca 2+ -loaded CaM binds to two different sites within the TRPV4 protein thus bringing two regulatory domains in proximity to each other. Indeed, asymmetrical CaM binding has been demonstrated for the pore-forming subunits of the CaV1/CaV2 [35,36] and SK [37,38] channel families, and a similar mechanism has been proposed for the Ca 2+ -dependent desensitization of TRPV1 [6,7]. Using CaM overlay experiments on a peptide library representing the cytosolic termini of TRPV4 and fluorescence polarization experiments we identified three additional CaM binding sites in TRPV4 that may serve as anchoring domains for the CaM N lobe (Fig. S3). However, in fluorescence polarization experiments, their CaM affinities were in the micromolar range ( Fig. S4) and none bound to CaM-N with higher affinity than to CaM-C (Table S2).

CaM binding disrupts an interdomain interaction
The alternative scenario for CaM-dependent current facilitation in TRPV4 involves a disinhibition process in which CaM binding displaces a regulatory domain that is attached to TRPV4 in the resting state. Prototypical mechanisms involving the displacement of autoinhibitory domains from a functional center are found in the CaM-dependent activation of CaMK II [39] and the plasma membrane Ca 2+ pump [40,41]. Multiple lines of evidence indicate that this is indeed the mechanism of the CaM-dependent potentiation in TRPV4.
First, we did a complementation experiment to test whether a CaM molecule that binds to the C2 fragment of TRPV4 through its C-lobe can form an additional interaction with another CaM binding domain thus forming a ternary complex. We focused on the N-terminal P2 peptide which with its accumulation of positively charged amino acids around a central tryptophan residue resembles the CaM-binding M13 peptide from the skeletal muscle myosin light chain kinase, a prototypical CaM binding motif. We used an AlphaScreen-based proximity assay (Fig. 2, Methods S1) in which the acceptor beads were coated with the Cterminal fragment C2-Flag, and the donor beads either with the N-terminal CaM binding peptide P2-biotin, or, as a negative control, with C2-biotin. The interaction between the proteins was measured in the presence of increasing CaM concentrations in 100 mM Ca 2+ or 1 mM EGTA (Fig. 3A). Surprisingly, instead of CaM-dependent complex formation, a strong constitutive interaction between the N-and C-terminal TRPV4 fragments was observed in the absence of CaM (Fig. 3A). CaM competitively reduced this interaction in a Ca 2+ -dependent fashion with an IC 50 of 166 nM in 100 mM Ca 2+ , a value that is in agreement with the Kd observed for its binding to C2 (see Fig. 1D). The control reaction in which both acceptor and donor beads were coated with the C2 protein did not show interaction in the absence or presence of CaM (Fig. 3A).
To determine the Ca 2+ -dependence of the complex between P2 and C2, an AlphaScreen experiment was performed in the absence of CaM but with Ca 2+ concentrations between 10 nM and 10 mM (Fig. 3B). The interaction was already present at 0 Ca 2+ and essentially Ca 2+ -insensitive over a wide range of Ca 2+ Figure 2. AlphaScreen-based CaM interaction assay. Optical stimulation of phthalocyanine compounds contained in the AlphaScreen donor beads excites ambient oxygen molecules into the metastable singlet state. The energy conveyed by the molecule is converted into light emission by the acceptor bead. Owing to the limited lifespan of singlet oxygen in solution, the AlphaScreen signal emission is highly dependent on the distance between the two beads. In this study, AlphaScreen donor and acceptor beads were modified for the detection of CaM/TRPV4 or interdomain interactions. Streptavidincoated donor and anti-Flag acceptor beads were used to attach the proteins to the bead surface. doi:10.1371/journal.pone.0010580.g002 concentrations. Very high Ca 2+ concentrations above 100 mM, however, inhibited binding of the fragments, a process that is probably secondary to unspecific Ca 2+ binding and has been observed for other protein interactions including CaM binding to TRPV4 [11].
To more finely map the interaction site, we then performed overlay experiments with a partial peptide library from the TRPV4 N terminus (Fig. S3A). Probing with the C2 fragment resulted in strong binding to a site that overlaps with the P2 peptide. In a similar fashion, a peptide array from the TRPV4 C terminus was probed with the N-terminal N3 fragment (Fig. 1A) that includes P2 (Fig. S3B). Consistent with the finding that CaM inhibits the interaction, strong interaction was found with a region that corresponds to the CaM binding peptide P5.
The interdomain interaction could also be shown using the longer N-terminal TRPV4 fragments N2 (wt in Fig. 3C) and N3 (data not shown) instead of P2. In an AlphaScreen interaction experiment, donor and acceptor beads were incubated with 10 nM C2-biotin and N2-Flag at concentrations ranging from 30 pM to 1.5 mM, respectively (Fig. 3C). Increasing N2-Flag concentrations resulted in increasing C2 interaction signals with a maximum at the bead capacity of approximately 10 nM bound protein. Further increases in the N2-Flag concentration led to signal reduction owing to a hook effect of the assay system, demonstrating specific interaction of the fragments (see Methods S1).
Secondly, N2 mutants in which key basic amino acids within the N-terminal binding motif were changed into alanine showed significantly weaker binding to C2. We used site-directed mutagenesis to create N2 mutants that lack either the hydrophobic (WIA) or the positively charged residues (KRA) in the N-terminal interaction domain (Fig. 3D). In AlphaScreen-based C2 interaction assays, both constructs, WIA and KRA, had a significantly lower binding affinity to the C2 fragment (Fig. 3C,E) with respective AlphaScreen signals of 48.465.5% and 14.862.1% of the wild type at the given component concentrations.
The same was found when CFP-and YFP-fusion protein of the full-length N and C-termini, respectively, (N1, C1, Fig. 1A) were tested for interaction in fluorescence resonance energy transfer (FRET) experiments after coexpression in HEK293 cells (Fig. 4). This is in agreement with a recent study [42] that showed high FRET efficiency in TRPV4 for both homologous N-terminal and N-terminal to C-terminal interactions. Interestingly, the interaction site represented by the P2 peptide is close to a proline-rich domain that has been shown to interact with PACSIN 3, a regulatory protein that controls basal TRPV4 activity and stimulus specificity [43,44].
To investigate the interaction in the full-length channel protein, we used a TRPV4 expression construct in which CFP and YFP were fused to the N-and C termini, respectively (Fig. 5). Fluorescence resonance energy transfer (FRET) experiments in unstimulated HEK293 cells expressing the construct revealed a significantly stronger FRET efficiency between the fluorophores than in cells cotransfected with a TRPV4-CFP fusion construct and YFP (Fig. 5A,B). The same was found in cells cotransfected with CFP-TRPV4 and TRPV4-YFP (Fig. 5B) suggesting that interactions can occur between subunits in the homotetrameric channel. All constructs resulted in functional channels (data not shown). Interestingly, both stimulation of CFP-TRPV4-YFP by decreases in the extracellular osmolarity (Fig. 5C) or application of 4a-PMA (not shown) in Ca 2+ -containing buffer, and intracellular Ca 2+ release after stimulation of the endogenous M3 muscarinic receptor resulted in transient decreases in the FRET-and concomitant increases in the donor channel fluorescences, indicating a decrease in FRET efficiency (Fig. 5C).

Inhibition of the interaction between N and C termini renders TRPV4 permanently potentiated
As a third line of evidence, the full-length WIA and KRA mutants displayed significantly enhanced currents during activation in nominally Ca 2+ -free media (Fig. 6). In whole-cell patchclamp experiments, TRPV4 wild type showed an initial decrease in spontaneous activity after establishment of the whole cell configuration (Fig. 6A). Addition of 4a-PMA thereafter in the nominal absence of Ca 2+ led to a slow, small increase in currents which after reaching a plateau were then strongly, but transiently potentiated on readdition of Ca 2+ . Potentiation was rapidly followed by Ca 2+ -dependent inhibition. Both mutants showed clear differences to wild type TRPV4. Significantly larger currents were activated by 4a-PMA in the nominally Ca 2+ -free medium and no potentiation, only inhibition was observed on readdition of Ca 2+ (Fig. 6B, C). Interestingly, the current densities attained by the mutants in 4a-PMA were similar to those for wild type TRPV4 after potentiation by Ca 2+ . Thus, channels with the mutations that prevent or reduce interactions between the N and C termini do not require increases in intracellular Ca 2+ for strong current activation.
All data provide strong evidence for an interaction in TRPV4 between an N-terminal domain represented by the P2 peptide and a site in the C terminus, P5, that also binds to CaM. Our functional data show that disruption of the interaction by mutagenesis of key amino acids leads to constitutive current potentiation when the channel is stimulated in the absence of extracellular Ca 2+ . This is in good agreement with a previous study [11] that demonstrated that TRPV4 mutants in which CaM binding to the C terminus is abolished result in channels not potentiated by Ca 2+ addition.
A similar interdomain interaction mechanism has also been found in cyclic nucleotide-gated channels [45]. Here, an Nterminal domain is capable of interacting with both the cyclic nucleotide binding region in the C terminus of the protein and Ca 2+ /CaM. In contrast to TRPV4, however, this is functionally coupled to autoregulatory channel inactivation. The same group demonstrated using GFP-FRET experiments that the interaction occurs between adjacent subunits of the tetrameric protein rather than within the same subunit [46].

The N-terminal interaction domain is capable of dimerization
When the chromatographically purified N2 protein was subjected to SDS-PAGE, a strong band of the expected size of 34.8 kD was detected (Fig. 7A). In addition, a second band was visible with a molecular weight compatible with a dimeric form of N2 (Fig. 7A). In gel filtration experiments under non-denaturing conditions, a single peak was detected at a size that corresponds to the dimeric form (Fig. S5). We then used an AlphaScreen-based assay to investigate the interaction of the N-terminal P2 peptide with the wild type N2 fragment or the N2 mutants WIA and KRA in nominally Ca 2+ -free buffers (Fig. 7B). P2 bound strongly to wild type N2 and the WIA mutant, but not to the KRA mutant. Similar results were obtained using the biotinylated N2 fragment instead of the P2 peptide (data not shown). These data show that the Nterminal domain constituted by the P2 peptide, in addition to its affinity for the C-terminal binding site, has the ability to form homodimeric interactions.
FRET experiments were performed to validate the interaction under intracellular conditions (Fig. 7C). N1-YFP was coexpressed with N1-CFP or CFP alone in HEK293 cells and the donor fluorescence measured during selective YFP photobleaching. The respective maximal relative donor fluorescence increases after acceptor bleaching were 1.2760.03 and 1.0460.01. We conclude that there is significant interaction between two molecules of the full-length TRPV4 N terminus that is dependent on the integrity of the domain formed by the P2 peptide.
It is known that TRPV4 forms homotetrameric complexes [47]. The N termini of adjacent subunits are thus readily accessible for homologous interaction. Indeed, previous studies [42] demonstrated that strong binding between the N-terminal tails exists in TRPV4. Studies in TRPV4 [48], TRPV5 [49] and TRPV6 [50] indicated that specific N-terminal ankyrin domains are crucial structures for channel self assembly. However, the N2 fragment that includes the P2 peptide is located N-terminal to the ankyrin domains (Fig. 1A). The dual affinity of this domain to both a homologous domain of a second subunit and the C-terminal CaM binding site suggests that it may contribute to transient rather than static interactions in the functional channel.

Dual role of the N-terminal interaction domain
To investigate which of the alternative interactions of the P2 peptide is relevant in the absence and presence of CaM binding to the TRPV4 C terminus, we performed a series of competition experiments (Fig. 7D,E). AlphaScreen acceptor and donor beads were coated with C2-Flag and either P2-biotin (Fig. 7D) or CaMbiotin at 100 mM Ca 2+ (Fig. 7E) and incubated with untagged C2 or N2 competitor proteins at the indicated concentrations. In the P2/C2 complex (Fig. 7D), the IC 50 values for C2 and N2 were 47 nM and 166 nM, respectively, indicating a 3.5-fold higher apparent affinity of the C2 fragment. A similar finding was observed when the CaM/C2 interaction was subjected to competition by C2 or N2 (Fig. 7E). The IC 50 values for C2 and N2 were 38 nM and 522 nM. Taken together, the data indicate a higher affinity of the N-terminal binding site towards C2 than to a second N-terminal fragment, suggesting that in the resting state of the channel when CaM is not bound to the C-terminal binding site, the interaction between the N and C termini prevails.
In summary, this study provides a comprehensive model for the Ca 2+ -dependent potentiation of TRPV4 currents. Using different experimental strategies, we have demonstrated that an interaction between an N-terminal and a C-terminal site in TRPV4 is present at nanomolar Ca 2+ concentrations (Fig. 8A), but becomes disrupted secondary to CaM binding to the TRPV4 C terminus at micromolar Ca 2+ concentrations (Fig. 8B). In in vitro FRET experiments, the interaction was shown to occur between channel subunits, presumably in the channel homotetramer. However, from the experimental data, we cannot exclude the possibility that the N and C termini of a single channel subunit can interact.
Evidence for an interaction between N and C termini was also obtained in functional experiments. While TRPV4 mutants that are CaM binding-deficient result in currents that are small and do not potentiate secondary to rises in the intracellular Ca 2+ concentration [11], mutants in which the interaction is absent result in large currents in the absence of Ca 2+ (Fig. 6). A second, homologous interaction between the N termini of adjacent TRPV4 subunits that is present when CaM is bound to the Cterminal site may contribute to conformational changes that lead to current facilitation (Fig. 8B), but we currently have no functional evidence in support of this. This model represents a new Ca 2+ -dependent regulatory mechanism that may also be found in other CaM-regulated cation channels.

AlphaScreen protein interaction measurements
Proteins were expressed in E. coli, affinity purified and biotinylated if applicable (for details see Methods S1). For protein interaction measurements, a proximity assay system based on the AlphaScreen technology (PerkinElmer) was used (see Methods S1). Briefly, streptavidin-coated AlphaScreen donor and anti-Flag acceptor beads at a final concentration of 16.7 mg/ml were incubated in a 384-well plate format (OptiPlate-384, PerkinElmer) with C2-GST-biotin or P2-biotin and the respective Flag-tagged interaction protein at concentrations of 10 nM, if not otherwise indicated. Assay buffer conditions were 50 mM Tris, pH 7.4, 100 mM KCl, and 0.1% BSA. After incubation for 90 min at room temperature, the AlphaScreen output signal was recorded in a Fusion alpha FP instrument (PerkinElmer) at 2 s reading time per well. AlphaScreen signals are given as mean6SEM in counts per second (cps) units.

Fluorescence polarization
Tracer peptides were synthesized and coupled to carboxyfluorescein (Dr. Sven Rothemund, Core unit peptide technologies,  IZKF, Leipzig). Protein interaction experiments were performed in buffers containing 50 mM Tris, pH 7.5, 100 mM KCl, 0.1% BSA and calcium at the indicated concentrations. Calcium concentrations below 50 mM were buffered with HEDTA, EGTA or EDTA at a buffer concentration of 10 mM. CaM concentrations were 10 mM or as indicated; the tracer peptide concentration was 100 nM. Fluorescence anisotropy was measured in a 384-well black multititer plate (Fluotrac 200, Greiner) using a Fusion alpha FP instrument (PerkinElmer) at 1 s reading time per well. Fluorescence polarization (FP) was calculated according to: P~I par {I perp I par zI perp with I par and I perp as the fluorescence emission intensities in planes parallel and perpendicular to the excitation plane. FP signals are given as mean6SEM of triplicate measurements in milli-P (mP) units.

GFP-FRET in vivo interaction
Single-cell GFP-FRET was measured in a fluorescence imaging system. Excitation of donor (CFP) and acceptor (YFP) fluorescence was at 430 nm and 520 nm, respectively. After baseline recording, the acceptor fluorescence was selectively photobleached by 30 cycles of 5 s expositions at 520 nm while measuring the donor and acceptor fluorescences. Both channels were normalized to the emission intensity immediately before acceptor bleaching. (See Methods S1 for details).

Electrophysiology
Patch clamp recordings were performed on HEK293 cells in the whole cell configuration. Cells were clamped at a potential of 220 mV, and current-voltage (I-V) relations were obtained from voltage ramps from 2100 mV to +100 mV. The standard extracellular solution contained 140 mM NaCl, 5 mM CsCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose, and 10 mM HEPES (pH 7.4 with NaOH). In nominally Ca 2+ -free solutions, Ca 2+ was omitted. The standard intracellular solution contained 110 mM cesium methanesulfonate, 25 mM CsCl, 2 mM MgCl 2 , 0.362 mM CaCl 2 , 1 mM EGTA, and 30 mM HEPES (pH 7.2 with CsOH) with a calculated [Ca 2+ ] of 100 nM. (See Methods S1 for details).

Supporting Information
Methods S1 Supplementary Methods.  Figure S2 Ca 2+ -dependent binding of C2 to CaM or CaM mutants that are partially Ca 2+ -binding defective. The Ca 2+dependence of C2-biotin binding to wild type CaM-Flag or mutants that are Ca 2+ -binding deficient in the N lobe (CaM 12 ), the C lobe (CaM 34 ) or both lobes (CaM 1234 ) was measured in an AlphaScreen assay at the indicated Ca 2+ concentrations. Half maximal binding was observed at 8.861.0 mM, 10.761.0 mM, 27.061.1 mM Ca 2+ for CaM, CaM 12 and CaM 34 , respectively with a common hill coefficient of 2.0 (n = 3 independent experiments in triplicate). CaM 1234 did not show measurable C2 interaction. To demonstrate the Ca 2+ -independence of the assay system itself, a GST-Flag-biotin fusion protein was incubated with the AlphaScreen beads at 100 mM Ca 2+ or 1 mM EGTA (inset). The AlphaScreen readout was independent of the Ca 2+ concentration.