Abstract

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In neurons, entry of extracellular calcium (Ca²⁺) into synaptic terminals through Ca_v2.1 (P/Q-type) Ca²⁺ channels is the driving force for exocytosis of neurotransmitter-containing synaptic vesicles. This class of Ca²⁺ channel is, therefore, pivotal during normal neurotransmission in higher organisms. In response to channel opening and Ca²⁺ influx, specific Ca²⁺-binding proteins associate with cytoplasmic regulatory domains of the P/Q channel to modulate subsequent channel opening. Channel modulation in this way influences synaptic plasticity with consequences for higher-level processes such as learning and memory acquisition. The ubiquitous Ca²⁺-sensing protein calmodulin (CaM) regulates the activity of all types of mammalian voltage-gated Ca²⁺ channels, including the P/Q class, by direct binding to specific regulatory motifs. More recently, experimental evidence has highlighted a role for additional Ca²⁺-binding proteins, particularly of the CaBP and NCS families in the regulation of P/Q channels. NCS-1 is a protein found from yeast to humans and that regulates a diverse number of cellular functions. Physiological and genetic evidence indicates that NCS-1 regulates P/Q channel activity, including calcium-dependent facilitation, although a direct physical association between the proteins has yet to be demonstrated. In this study, we aimed to determine if there is a direct interaction between NCS-1 and the C-terminal cytoplasmic tail of the Ca_v2.1 α-subunit. Using distinct but complementary approaches, including in vitro binding of bacterially expressed recombinant proteins, fluorescence spectrophotometry, isothermal titration calorimetry, nuclear magnetic resonance, and expression of fluorescently tagged proteins in mammalian cells, we show direct binding and demonstrate that CaM can compete for it. We speculate about how NCS-1/Ca_v2.1 association might add to the complexity of calcium channel regulation mediated by other known calcium-sensing proteins and how this might help to fine-tune neurotransmission in the mammalian central nervous system.

Experimental Procedures

Results

The aim of this study was to test, using a range of complementary methods, the possibility that the small calcium-sensing protein NCS-1 directly interacts with the Ca_v2.1 P/Q-type Ca²⁺ channel as has been indirectly suggested in prior functional studies.²⁰⁻²³ With the knowledge that other known small EF-hand Ca²⁺-binding proteins interact with the α-subunit of the P/Q channel predominantly through motifs located in the C-terminal tail, we reasoned that the same domains represent the most likely sites for an NCS-1 interaction. In initial experiments, we generated a recombinant, GST-tagged, form of the P/Q α₁ subunit C-terminal tail domain corresponding to residues 1898–2035 (Figure ​(Figure1,1, GST-PQ). This fragment encompasses both the IQ motif (IM in the P/Q-type channel) and the CaM-binding domain (CBD).¹⁰ Two further constructs derived from this precursor were the CBD alone [residues 1950–2035 (Figure ​(Figure1,1, GST-CBD)] and an IQ motif mutant in which the P/Q channel IM residues at positions 1913 and 1914 were mutated to glutamic acid to generate an IM → EE double mutant (Figure ​(Figure1,1, GST^IM→EE). This mutation has been previously shown to abolish CaM-dependent CDF and CDI of the human P/Q-type channel.⁷ We used these constructs to investigate whether recombinant NCS-1 could associate with P/Q-type C-terminal regulatory domains in vitro. NCS-1 exhibited Ca²⁺-dependent binding to GST-PQ but was not observed to bind to either GST-CBD or GST^IM→EE under any tested conditions (Figure ​(Figure1A),1A), suggesting a specific interaction with residues in the IQ region of the channel. Averaged densitometry data from three independent binding experiments are listed in Table 1.

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Figure 1

Interactions of NCS-1 and CaM with rat PQ α₁ subunit C-terminal binding motifs. (A) Recombinant NCS-1 was incubated with GST-PQ (residues 1898–2035 of the rat PQ α₁ subunit, encompassing both the IQ and CBD motifs), GST^IM→EE (equivalent to the IQ motif present in Ca_v1.x channels), and GST-CBD (residues 1950–2035 of the rat PQ α₁ subunit) in the presence or absence of 1 μM free Ca²⁺. Bound NCS-1 was detected by Western blotting with a specific antibody. This blot is a representative result, and averaged data from three independent binding assays are listed in Table 1. (B) Densitometry quantification of an NCS-1/GST-PQ binding Ca²⁺ dose response. Recombinant NCS-1 (1 μM) was incubated with 1 μM GST-PQ in the presence of increasing concentrations of free Ca²⁺ and bound protein detected by Western blotting with an anti-NCS-1 specific antibody. Western blot signal intensities were quantified by densitometry and plotted as a function of free Ca²⁺ concentration present in the binding reaction mixture. (C) Recombinant CaM was incubated with GST-PQ, GST^IM→EE, and GST-CBD in the presence or absence of 1 μM free Ca²⁺. Bound CaM was detected by Western blotting with a specific antibody.

A Ca²⁺ titration of binding of NCS-1 to GST-PQ confirmed the Ca²⁺ dependency of the interaction and allowed us to derive a K_d of 0.7 μM based on densitometry quantification of Western blot data (Figure ​(Figure1B).1B). For comparison, we performed the same binding analysis with CaM (Figure ​(Figure1C).1C). Similar to our results with NCS-1, CaM displayed Ca²⁺-dependent binding to GST-PQ but no detectable binding under any conditions to either GST-CBD or GST^IM→EE.

Our initial data suggested that both NCS-1 and CaM could interact with the same IQ motif region of the rat P/Q-type channel, and therefore, we extended our binding studies to evaluate the potential existence of a common, overlapping, binding site for both proteins. Competitive association of both proteins with GST-PQ was assessed by binding of NCS-1 to GST-PQ in the presence of increasing concentrations of recombinant CaM (Figure ​(Figure2).2). Western blot results from these experiments highlighted a clear displacement of NCS-1 from GST-PQ that was first apparent at a CaM concentration between 0.3 and 1 μM (Figure ​(Figure2A).2A). Densitometry analysis of the Western blot data confirmed a competitive association between NCS-1 and CaM for GST-PQ (Figure ​(Figure2B).2B). Our initial experiments indicated that the IQ-like motif of the P/Q-type channel is important for interaction with both NCS-1 and CaM, and therefore, in subsequent NMR and ITC analyses, we focused on only this region of the channel.

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Figure 2

Competitive interaction between NCS-1 and CaM for binding to GST-PQ. (A) NCS-1 (5 μM) was incubated with 5 μM GST-PQ in the presence of varying concentrations of CaM. Bound NCS-1 and CaM were visualized by Western blotting with specific antibodies. (B) Data from panel A were quantified by densitometry, and NCS-1/CaM bound to GST-PQ was plotted as a function of the CaM concentration present in the binding assay.

Further testing of the ability of NCS-1 to bind the P/Q-type channel and to delineate the NCS-1- and CaM-binding regions of the P/Q-type channel was performed using NMR experiments. A sample of ¹⁵N-labeled P/Q spanning residues 1909–2035 (encompassing much of the IQ motif and the CBD) was prepared. Addition of CaM shows chemical shift perturbations of glycine and tryptophan residues, whereas NCS-1 has no effect on either group of residues (Figure ​(Figure3).3). Glycine and tryptophan residues are located in the CBD region but not in or around the IQ segment. Hence, it is clear that NCS-1 does not bind to the CBD of the P/Q channel, although there is an indication that there is weak binding of CaM to this site.

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Figure 3

Delineation of binding of NCS-1 to PQ 1909–2035. ¹H–¹⁵N HSQC spectra of ¹⁵N-labeled PQ1909–2035 in the presence (A) and absence of CaM (B) and of NCS-1 (C), showing the glycine amide resonance, and (D) tryptophan N^εH resonances. Panel A shows the full ¹⁵N–¹H spectrum; expanded regions show resonances from only glycine amide (B and C) and tryptophan indole (D) groups. (E) The amino acid sequence of PQ 1909–2035 is shown with glycine and tryptophan residues colored blue. The likely CaM- and NCS-1-binding region is indicated.¹⁵

Knowing from the experiments described above that the major site of binding of NCS-1 on the P/Q C-terminal domain was likely to be the IQ motif, we used a synthetic PQIQ peptide in subsequent experiments. The intrinsic tryptophan fluorescence of NCS-1 was used to measure interactions with the PQIQ peptide (Figure ​(Figure4),4), using an approach similar to that described for the interactions with the D2 dopamine receptor peptide.²⁷ NCS-1 has two tryptophan residues; addition of the PQIQ peptide resulted in a decrease in fluorescence and allowed titration of the peptide over a range of concentrations (Figure ​(Figure2A).2A). The data for the change in fluorescence versus concentration were analyzed using a logistic fit. The dose response upon titration with the PQIQ indicated half-maximal binding at 1.036 ± 0.07 μM with a Hill coefficient of 1.7. Hill coefficients greater than 1 would be consistent with a 2:1 stoichiometry of peptide binding to NCS-1 because NCS-1 itself was monomeric.²⁷ ITC shows that the interaction is entropically driven. Because of the tendency of the protein complex to aggregate at the high peptide (100 μM) and protein concentrations (1 mM) required to observe binding by this method, it was not possible to obtain reliable equilibrium binding constants using ITC. The affinity obtained for the interaction of NCS-1 with the PQIQ peptide is almost 1 order of magnitude lower than the reported affinities involving the calmodulin lobes. From ITC experiments, K_d values of 51 ± 20 and 4.32 ± 0.39 nM were obtained for binding of the Ca²⁺/N and Ca²⁺/C lobes, respectively, to the IQ domain of the PQ channel.³¹ The two lobes in NCS-1 are intimately linked, and attempts to express the two NCS-1 lobes as independently folded domains soluble at micomolar concentrations have met with little success so far. Therefore, experiments that aimed to disentangle the relative affinities of the two NCS-1 domains for the PQIQ peptide have not been possible.

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Figure 4

Binding of the PQIQ peptide to NCS-1 monitored using tryptophan fluorescence. Changes in tryptophan fluorescence following sequential additions of the PQIQ peptide to the final indicated concentrations. Recombinant NCS-1 at a concentration of 1 μM in a buffer [50 mM Tris (pH 7.5), 50 mM NaCl, and 5 mM CaCl₂] was titrated with PQIQ at 50 μM to 1 mM under the same buffer conditions. The data were fit using a logistic equation and nonlinear curve fitting. Data are plotted as means ± SEM for three independent experiments.

To determine which region of the PQIQ peptide forms the NCS-1-binding site, we again used NMR. In these experiments, [¹³C,¹⁵N]NCS-1 was titrated into a sample of the unlabeled PQIQ peptide and ¹³C- and ¹⁵N-filtered TOCSY and NOESY data were acquired. Analysis of the side chain resonances, in particular the methyl and tyrosine resonances, reveals selective line broadening for 14 residues, YAAMMIMEYYRQSK (Figure ​(Figure5A).5A). Hence, a subset of the 27 amino acids in the polypeptide sequence forms the main interaction site for NCS-1 binding, with the residues beyond this core peptide region further enhancing the interaction. The length of this motif correlates well with the recently reported structure of NCS-1 in complex with the C-terminal peptide from the human D2 dopamine receptor (Protein Data Bank entry 2YOU). The 14-amino acid motif is short enough for it to bind NCS-1 with a stoichiometry of 2:1 (peptide:NCS-1) and supports the fluorescence binding data.

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Figure 5

¹H TOCSY and HSQC spectra showing PQIQ–NCS-1 interactions. (A) The TOCSY spectrum of the PQIQ peptide delineates the NCS-1-binding region. Line broadening was observed for selected resonances in the presence of a 2-fold excess of NCS-1. Line-broadened resolved resonances from Y4, A7, A8, I11, and R16 are highlighted: aromatic resonances (left) and aliphatic resonances (middle and right). (B) ¹H–¹⁵N HSQC spectra of [¹⁵N]NCS-1 (0.8 mM, black) in the presence of PQIQ (final concentration of 0.4 mM, red) in 50 mM Tris buffer (pH 6.5), 50 mM NaCl, and 5 mM CaCl₂ at 300 K.

NMR ¹H–¹⁵N HSQC of NCS-1 in the presence of a 2-fold excess of PQIQ shows resonances that are universally broadened although still discernible to confirm that the protein remains folded (Figure ​(Figure5B).5B). Addition of equimolar amounts of unlabeled CaM/Ca²⁺ to the preformed ¹⁵N-labeled NCS-1/PQIQ complex led to a complete reversion of the ¹H–¹⁵N HSQC C spectrum to that of free NCS-1. This is further confirmation that NCS-1 and CaM bind to similar sites on the PQ channel, with, as expected, CaM binding the peptide sufficiently strongly to displace it from NCS-1.

The experiments described above show that a direct interaction of NCS-1 with the P/Q C-terminus could be detected using three distinct in vitro biochemical approaches. To provide evidence of the interaction of NCS-1 with the P/Q channel regulatory domain in a more physiological setting, we studied the localization of these proteins ex vivo in cultured HeLa cells. For these experiments, we returned to the use of a longer recombinant construct encompassing both the IQ and CBD domains of the P/Q C-terminus. We first examined the cellular localization of both NCS-1-mCherry¹⁸ and a C-terminally YFP-tagged variant of P/Q 1898–2035 to which an N-terminal myristoylation consensus sequence had been attached (^myrPQ-YFP). In previous studies, this approach has been used to successfully direct isolated soluble domains of plasma membrane proteins to the plasma membrane for functional analyses.²⁵ NCS-1-mCherry exhibited a predominantly perinuclear localization consistent with its association with the TGN in addition to some plasma membrane localization (Figure ​(Figure66A).^32myrPQ-YFP was observed to target the plasma membrane and/or large cytosolic puncta (Figure ​(Figure6A).6A). Plasma membrane association of ^myrPQ-YFP suggested that myristoylation of the peptide was accurately targeting a fraction of PQ to the expected location. Interestingly, in cells exhibiting ^myrPQ-YFP puncta, co-expressed NCS-1-mCherry failed to localize to the TGN or plasma membrane and instead colocalized with ^myrPQ-YFP on the enlarged punctate structures (Figure ​(Figure6B).6B). We quantified these phenomena and were able to demonstrate that in 39.5 ± 9.2% of cells cotransfected with NCS-1-mCherry and ^myrPQ-YFP puncta were observed and that in 100% of this subpopulation NCS-1 protein was associated with large ^myrPQ-YFP positive punctate structures (Figure ​(Figure6C,6C, NCS-1-mCh + ^myrPQ-YFP). This was similar to the number of cells exhibiting puncta in ^myrPQ-YFP/mCherry controls [43.6 ± 3% (Figure ​(Figure6C,6C, ^myrPQ-YFP + mCherry)]. In contrast, control cells expressing NCS-1-mCherry along with EYFP protein exhibited no observable formation of puncta (Figure ​(Figure6C,6C, NCS-1-mCh + EYFP). It should be noted that colocalization of NCS-1-mCherry with ^myrPQ-YFP in punctate structures was specific for NCS-1 as no colocalization was observed with the soluble mCherry protein in control cotransfections (data not shown). These observations are consistent with a direct interaction of NCS-1-mCherry with ^myrPQ-YFP in a cellular context.

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Figure 6

Expression of NCS-1 and PQ in HeLa cells. (A) HeLa cells were cotransfected with NCS-1-mCherry (red) and EYFP (green) or mCherry (red) or (B) NCS-1-mCherry (red) and ^myrPQ-YFP (green). Regions of colocalization are colored yellow in overlay images. Scale bars are 10 μm. (C) Quantification of data from panels A and B. Cells were counted and scored for the presence of puncta {three independent experiments from each control condition [NCS-1-mCh + EYFP and ^myrPQ-YFP + mCherry (A)]} or four independent experiments [NCS-1-mCh + ^myrPQ-YFP (B)]. The total number of cells counted for all experiments and for each condition is shown above the corresponding bar on the histogram. All data are plotted as means ± SEM.

Discussion

In this paper, we set out to examine whether NCS-1 is able to interact directly with the C-terminus of the α₁ subunit of the P/Q-type channel. To avoid a potentially misleading indication of a positive interaction, we tested this using three separate in vitro biochemical/biophysical assays using either a long construct from the P/Q C-terminus or a synthetic IQ domain peptide as well as a test through cellular colocalization. The use of multiple techniques gives a more rigorous approach to addressing the question of whether a significant direct interaction does exist. In all cases, we were able to detect a direct interaction between NCS-1 and P/Q-type Ca²⁺ channels.

P/Q-type channel regulation by small Ca²⁺-binding proteins is essential in the mammalian CNS for the processes of CDI and CDF that drive short-term presynaptic plasticity.⁶ Alterations in synaptic plasticity of this form in turn influence information processing and neural network behavior that underpin the normal functioning of the CNS.³³ A large body of experimental evidence that describes at the molecular level how CaM is able to regulate all Ca_v-type channels, including those of the P/Q subtype, has accumulated. CaM is constitutively associated with the P/Q α subunit and is considered an auxiliary subunit of the channel. Apo-CaM is believed to bind to the P/Q α subunit at a site outside of, but in the proximity of, the Ca²⁺-dependent IQ-binding site, as determined using a Förster resonance energy transfer approach in cultured mammalian cells.³⁴ It has been suggested that this arrangement locates CaM within striking distance of the regulatory IQ motif to ensure fast responses to Ca²⁺ influx on channel opening.³⁴ Ca²⁺-dependent binding of CaM to the IQ motif is then able to modulate both CDI and CDF depending on the precise nature of the Ca²⁺ signal. Mechanistically, this system has been resolved in elegant experiments showing that Ca²⁺ entry through individual P/Qα channels selectively activates the CaM C-lobe to mediate CDF. Larger Ca²⁺ signals emanating from multiple local channel opening events conversely activate the CaM N-lobe to drive CDI.^3,7,35 Our data confirming a Ca²⁺-dependent interaction of CaM with the P/Q containing the IQ motif are consistent with previous biochemical⁷ and structural analyses.³¹ A second CaM-binding site, the CaM-binding domain (CBD), has been reported in related studies of P/Q–channel interactions.^5,13 In this study, although we have been unable to replicate binding of CaM to this region of the rat α_1A CBD using pull-down experiments, the NMR data suggest that CaM, but not NCS-1, binds to the proposed CBD, albeit rather weakly. The weak nature of this interaction might explain why there are discrepancies in the literature regarding the significance of the CBD. Pull-down-type experiments select for high-affinity interactions, whereas NMR approaches are able to detect lower-affinity transient binding events consistent with these results. Our data help reconcile apparently conflicting experimental data as some studies do observe a CaM–CBD interaction^5,13 whereas others do not.⁷

NCS-1, a multifunctional Ca²⁺ sensor conserved from yeast to humans, has documented roles in the regulation of membrane trafficking,^36,37 ion channel activity,^38,39 dopamine receptor signaling,^27,40 long-term depression,⁴¹ autism,^18,42 and memory acquisition.^43,44 NCS-1 exhibits enriched expression in mammalian brain tissue,⁴⁵ and many of the aforementioned functions are related to neuronal activity. NCS-1 interacts with a number of targets in common with CaM,²⁴ and a previous functional study identified links between NCS-1 and CDF of P/Q-type channel activity in rat primary neurons.²⁰ Other studies have additionally indicated a cellular connection between NCS-1 and P/Q-type channel activity in bovine adrenal chromaffin cells^21,22 and Drosophila.²³ With this information in mind, we have tested the possibility that NCS-1 can directly bind to P/Q regulatory elements in common with CaM and other EF-hand-containing Ca²⁺ sensors.^10,14 Our data show that NCS-1 can associate with the IQ motif of rat P/Qα_1A in a Ca²⁺-dependent manner and that this binding site must overlap to some extent with that of CaM because CaM can compete for binding to the same region leading to NCS-1 displacement. This is the first example of a CaM-related Ca²⁺-sensing protein behaving in a manner almost identical to that of CaM with respect to IQ binding. Both CaBP1¹⁰ and VILIP-2¹⁴ interact with the CBD or require this domain for functional activity. CaM binds predominantly to the IQ domain with a possible weak interaction with the CBD, and NCS-1 binds to only the IQ domain in the presence of Ca²⁺. The weak CaM–CBD interaction that we have observed in this study might also help to explain why CaBP1 and VILIP-2 are able to effectively compete with CaM at this site.^10,14

The precise details of the NCS-1 interaction are still to be elucidated. In rat primary neurons, NCS-1 appears to play a role during CDF,²⁰ and in Drosophila, loss of the NCS-1 orthologue Frequenin leads to a reduced level of entry of Ca²⁺ into neurons and defective synaptic transmission.²³ These data suggest that at least one function of NCS-1 is in positively regulating P/Q channel opening. Our data now provide an attractive biochemical explanation for these cellular studies and act as a platform for further cellular and structural investigations studying the interplay between CaM and NCS-1 during regulation of P/Q channel activity. CaM is expressed in all neurons, and one possibility is that specific neuronal populations that also express NCS-1 are able to utilize this Ca²⁺ sensor as an additional modulator of P/Q activity. NCS-1 has a Ca²⁺ affinity higher than that of CaM^15,46 and therefore could potentially interact with the IQ motif over a different range of local Ca²⁺ concentrations to modulate activity in a manner independent of CaM. As Ca²⁺ concentrations increase, CaM could then displace NCS-1 to exert its documented roles in CDF and CDI. The N- and C-lobes of CaM, as described, exert distinct modes of P/Q channel regulation. NCS-1, unlike CaM, does not have distinct lobes but rather a compact globular structure.⁴⁷ The orientations of the N- and C-lobes with respect to one another differ in the two proteins, giving rise to different conformations of the ligand-binding site. Residues from both lobes in NCS-1 together form a large, contiguous solvent-exposed hydrophobic ligand-binding crevice, resembling the palm of a partially open hand. In the Ca²⁺/CaM–PQIQ complex, the hydrophobic binding pocket is more enclosed, resembling a closed hand that envelops the PQIQ peptide. It is, therefore, not possible to model a structure of NCS-1 in complex with the PQIQ peptide based on the Ca²⁺/CaM complex structure. It is likely that binding the PQIQ peptide would require a significant conformational change to the overall structure of NCS-1 to accommodate the peptide.

Using a combination of in vitro biochemical, NMR and ITC biophysical, and cellular approaches, we provide the first evidence of a direct interaction of NCS-1 with P/Q-type voltage-gated Ca²⁺ channels. Voltage-gated Ca²⁺ channel modulation has direct implications for synaptic plasticity and complex neural processing in higher organisms. Our work expands the number of small Ca²⁺-sensing proteins that can interact with the essential P/Q-type neuronal channel and provides further insights into the complexity of the regulation of voltage-gated Ca²⁺ channels in the mammalian CNS. Extensive further work will be required to determine the full structural basis for the interaction of NCS-1 with the P/Q IQ domain.

Glossary

Notes

This work was supported by Wellcome Trust Prize Ph.D. studentships awarded to V.M.M. and P.A.C.T. and a University of Liverpool Studentship awarded to S.R.P.

Notes

The authors declare no competing financial interest.

References