Calretinin Regulates Ca²⁺-dependent Inactivation and Facilitation of Ca_v2.1 Ca²⁺ Channels through a Direct Interaction with the α₁2.1 Subunit

Background: Ca²⁺-dependent inactivation and facilitation of Ca_v2.1 Ca²⁺ channels are major determinants of neuronal excitability and synaptic plasticity.

Results: The Ca²⁺-binding protein calretinin interacts with Ca_v2.1 and inhibits Ca²⁺-dependent inactivation and enhances facilitation of Ca_v2.1.

Conclusion: In addition to its role as a diffusible Ca²⁺ buffer, calretinin can interact with targets such as Ca_v2.1 and modulate their function.

Significance: Calretinin-Ca_v2.1 interactions may shape Ca²⁺ signaling dynamics in neurons.

Abstract

Voltage-gated Ca_v2.1 Ca²⁺ channels undergo dual modulation by Ca²⁺, Ca²⁺-dependent inactivation (CDI), and Ca²⁺-dependent facilitation (CDF), which can influence synaptic plasticity in the nervous system. Although the molecular determinants controlling CDI and CDF have been the focus of intense research, little is known about the factors regulating these processes in neurons. Here, we show that calretinin (CR), a Ca²⁺-binding protein highly expressed in subpopulations of neurons in the brain, inhibits CDI and enhances CDF by binding directly to α₁2.1. Screening of a phage display library with CR as bait revealed a highly basic CR-binding domain (CRB) present in multiple copies in the cytoplasmic linker between domains II and III of α₁2.1. In pulldown assays, CR binding to fusion proteins containing these CRBs was largely Ca²⁺-dependent. α₁2.1 coimmunoprecipitated with CR antibodies from transfected cells and mouse cerebellum, which confirmed the existence of CR-Ca_v2.1 complexes in vitro and in vivo. In HEK293T cells, CR significantly decreased Ca_v2.1 CDI and increased CDF. CR binding to α₁2.1 was required for these effects, because they were not observed upon substitution of the II-III linker of α₁2.1 with that from the Ca_v1.2 α₁ subunit (α₁1.2), which lacks the CRBs. In addition, coexpression of a protein containing the CRBs blocked the modulatory action of CR, most likely by competing with CR for interactions with α₁2.1. Our findings highlight an unexpected role for CR in directly modulating effectors such as Ca_v2.1, which may have major consequences for Ca²⁺ signaling and neuronal excitability.

Introduction

Voltage-gated Ca_v2.1 channels mediate Ca²⁺ influx that triggers neurotransmitter release from presynaptic nerve terminals at most synapses in the central nervous system (1). Because the amount of neurotransmitter released is proportional to the third or fourth power of the presynaptic Ca²⁺ concentration (2, 3), modulation of Ca_v2.1 properties can greatly alter synaptic output and contribute to mechanisms of synaptic plasticity (4).

Like other Ca_v channels, Ca_v2.1 channels undergo a prominent feedback regulation by incoming Ca²⁺ ions, which depends on calmodulin (CaM)⁴ (5, 6). However, unlike other Ca_v channels, CaM binding to the cytoplasmic C-terminal domain of α₁2.1 mediates both a negative and positive regulation by incoming Ca²⁺ ions, Ca²⁺-dependent inactivation (CDI), and Ca²⁺-dependent facilitation (CDF) (7). Both CDI and CDF have been described for presynaptic Ca_v2.1 channels at central synapses (8–10) and can cause short term depression and facilitation, respectively, of neurotransmitter release (11).

Ca²⁺-dependent modulation of native Ca_v2.1 channels is heterogeneous in neurons (12), which can be due to alternative splicing of sequences encoding the α₁2.1 C-terminal domain (13, 14) or differences in auxiliary Ca_vβ subunit expression (15). In addition, neuron-specific Ca²⁺-binding proteins related to CaM can influence the extent of Ca_v2.1 CDF and CDI: CaBP1 inhibits CDF and enhances CDI (16), whereas VILIP-2 increases CDF and inhibits CDI (17). CaBP1 and VILIP-2 bind to the same sites as CaM in the C-terminal domain of α₁2.1, but structural differences from CaM underlie their distinct modulation of Ca_v2.1 (17, 18).

Within the EF-hand superfamily of Ca²⁺-binding proteins, CaM, CaBP1, and VILIP-2 are considered Ca²⁺ sensors, so were defined by their abilities to undergo Ca²⁺-dependent conformational change and interact with effectors. In contrast, Ca²⁺-binding proteins such as parvalbumin and calbindin D-28k generally act as diffusible Ca²⁺ buffers that modulate cytoplasmic Ca²⁺ signals (19). Like Ca²⁺ chelation by EGTA (7), parvalbumin and calbindin D-28k can significantly alter CDI of Ca_v2.1 channels in transfected HEK293T cells (20). However, because parvalbumin and calbindin D-28k were not reported to associate directly with α₁2.1, their actions on Ca_v2.1 CDI are likely to be indirect and are primarily due to their actions as diffusible Ca²⁺ buffers (20).

Calretinin (CR) is a EF-hand Ca²⁺-binding protein that is highly expressed in cerebellar granule neurons where Ca_v2.1 channels are also expressed (19, 21, 22). Like parvalbumin and calbindin D-28k, CR is considered mainly as a Ca²⁺ signal modulator in neurons. Based on its Ca²⁺ chelating properties, CR can significantly shape the spatiotemporal properties of Ca²⁺ signals generated by plasma membrane and intracellular Ca²⁺ channels (23), yet CR undergoes large conformational changes upon binding to Ca²⁺ (24, 25), which may mediate Ca²⁺-dependent interactions with integral membrane proteins (26). While investigating the potential targets of CR, we discovered a consensus CR-binding motif in α₁2.1. Ca²⁺-dependent binding of CR to this region nullifies CDI and enhances CDF in transfected HEK293T cells, which suggests that local tethering of CR to α₁2.1 is required for channel modulation. Our findings provide the first evidence that CR can directly alter Ca²⁺ signaling through interactions with effectors, thus raising new possibilities for how CR may modulate neuronal and network excitability (27, 28).

EXPERIMENTAL PROCEDURES

cDNAs and Molecular Biology

The following Ca_v2.1 subunit cDNAs were used: α₁2.1 (rbA isoform (29)), β_2A (30), and α₂δ (31). GST-α₁2.1 fusion proteins containing one or more CR-binding (CRB) motifs were generated by PCR and subcloning of the corresponding sequence in EcoRI/XhoI sites of pGEX-4T1 (GE Healthcare). For the chimeric Ca²⁺ channel α₁2.1–1.2 subunit, amino acids 864–983 from rat brain α₁2.1 were replaced by amino acids 807- 923 of rat brain α₁1.2 (rbCII (32)). The corresponding DNA sequence of α₁1.2 was amplified by PCR and cloned into AscI/SgrAI sites of the α₁2.1-containing expression plasmid. GFP-CR was generated by cloning a PCR fragment containing the cDNA coding for human CR (836 bp) in the plasmid pEGFP-C1 (Invitrogen) using CR-specific primers comprising EcoRI and BamHI sites; the sequence of the insert was verified prior to use in experiments. For the expression construct containing the first three CRB motifs of α₁2.1 (mcherry-CRB1–3), the corresponding sequence (amino acids 899–953) and Kozak sequence was amplified from rat brain α₁2.1 by PCR and subcloned into XhoI and HindIII restriction sites of pEGFPN1-mcherry (a kind gift from S. England). All of the cDNA constructs were subject to sequencing prior to use in experiments.

Cell Culture and Transfection

HEK293 cells transformed with SV40 T-antigen (HEK293T) were maintained in DMEM (Invitrogen) with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO₂. The cells were grown to 70- 80% confluence and transfected with Gene Porter reagent (Genlantis, San Diego, CA) or FuGENE 6 (Promega) according to the manufacturer's protocols. For electrophysiology, the cells were plated in 35-mm dishes and transfected with Ca_v2.1 subunit cDNAs: α₁2.1 (2 μg), β_2A (1 μg), and α₂δ (1 μg). pEGFPN1 or GFP-CR was cotransfected (1 μg), which facilitated identification of transfected cells by fluorescence. For pulldown assays, the cells were plated in 60-mm dishes and transfected with GFP-CR (6 μg). For coimmunoprecipitation experiments, the cells were plated in 60-mm dishes and transfected with Ca_v2.1 subunits GFP-α₁2.1 (3.2 μg), β_2A (1.6 μg), and α₂δ (1.6 μg) and pEGFPN1 (1.6 μg) or GFP-CR (1.6 μg).

Phage Display and Binding Assays

Purified human recombinant CR-coated 7.5-cm² plastic plates and the Ph.D.^TM-7 phage display peptide library kit (New England Biolabs Inc., Biocencept, Allschwil, Switzerland) were used according to the manufacturer's protocol with three rounds of panning. The cDNA coding for the surface-exposed heptapeptides (part of the pIII coat protein) was extracted from Escherichia coli strain ER2738, amplified by PCR, and subject to DNA sequencing (Microsynth, GmbH, Balgach, Switzerland).

GST-α₁2.1 fusion proteins containing one or more CRB motifs were expressed in E. coli and purified on glutathione-Sepharose according to standard protocols. For experiments with fluorescently tagged purified CR, Alexa 488-labeled CR (CR*; 0–20 μm) was incubated with GST-α₁2.1 fusion proteins or GST control (10 μm) in binding buffer (140 mm potassium glutamate, 2 mm ATP, 1.25 mm MgCl₂, 20 mm PIPES, 0.4 mm EGTA, pH 7.2, adjusted with KOH). Ca²⁺-containing solutions included 1.6 mm CaCl₂ (Ca_free²⁺ = ∼300 μm). The reactions were carried out at room temperature for 1 h. Sample mixtures were applied to GST MultiTrap 4B (GE Healthcare) 96-well filter plates prepacked with glutathione-Sepharose. Bound CR*-GST-α₁2.1 complexes were eluted with elution buffer (10 mm glutathione, 50 mm Tris-HCl, pH 8.0), and fluorescence was measured with a Victor X3 plate reader (PerkinElmer Life Sciences).

For experiments with GFP-CR expressed in HEK293T cells, transfected cells were harvested and homogenized in 1 ml of ice-cold cell lysis buffer (50 mm Tris-HCl, 150 mm NaCl, 1% Triton X-100, 0.25% (w/v) sodium deoxycholate, 1 mm EDTA, pH 7.4, and protease inhibitors) containing either 1.6 mm CaCl₂ or 1 mm EGTA. The homogenate was rotated at 4 °C for 1 h to solubilize membrane proteins, and the insoluble material was separated by centrifugation at 16,100 × g (30 min). The supernatant (300 μl) was incubated with 40 μl of a 50% slurry of immobilized GST-CRB1–3 brought to 1 ml with the lysis buffer at 4 °C overnight. The beads were washed three times with 1 ml of ice-cold lysis buffer, and the bound proteins were eluted with SDS-containing sample buffer, subjected to SDS-PAGE, and transferred to nitrocellulose. Mouse monoclonal antibodies anti-GFP (1:3000; Santa Cruz Biotechnology, Santa Cruz, CA) were used to detect bound GFP-CR by Western blot.

Coimmunoprecipitation

For coimmunoprecipitation from transfected HEK293T cells, transfected cells were harvested 48 h after transfection. The cell lysates were prepared as described in binding assays and incubated with 5 μg of CR antibodies (Swant) and 40 μl of protein A-Sepharose (50% slurry) overnight, rotating at 4 °C. After three washes with 1 ml of cell lysis buffer, the proteins were eluted and analyzed by SDS-PAGE. Coimmunoprecipitated proteins were detected by Western blotting with anti-α₁2.1 antibodies (1:300; Alomone Labs, Jerusalem, Israel) and anti-GFP antibodies (1:3000; Santa Cruz Biotechnology).

For coimmunoprecipitation from mouse brain, the cerebellum was homogenized in 1 ml of 250STMDPS buffer (250 mm sucrose, 50 mm Tris-HCl, 5 mm MgCl₂, 1 mm DTT, pH 7.4, and protease inhibitors). The nuclear fraction was removed by centrifugation at 800 × g for 15 min. The membrane fraction was separated from the cytosolic fraction by ultracentrifugation at 100,000 × g for 1 h. The membrane pellet was solubilized in 1 ml of solubilization buffer (Tris-buffered saline (50 mm Tris-HCl, 150 mm NaCl, pH 7.5), 1% Triton X-100, and protease inhibitors) at 4 °C for 30 min, and insoluble material was removed by ultracentrifugation at 100,000 × g for 1 h. Either 5 μg of rabbit IgG (Invitrogen) or anti-CR antibodies (Swant, Marly, Switzerland) were added to the solubilized membrane proteins along with 50 μl of protein A-Sepharose (50% slurry; Sigma-Aldrich). The reactions were continued overnight with end over end rotation at 4 °C. The resin was collected by centrifugation and rinsed three times with 1 ml of solubilization buffer. Bound proteins were eluted and resolved by 4–12% SDS-polyacrylamide gel and Western blotting with anti-CR antibodies (1:5000; Swant) and anti-α₁2.1 antibodies as described above.

Electrophysiology of Transfected HEK293T Cells

Whole cell patch clamp recordings were acquired 36–60 h of post-transfection with a HEKA (Lambrecht/Pfalz, Rheinland-Pfalz, Germany) EPC-9 patch clamp amplifier. External recording solution contained 150 mm Tris, 1 mm MgCl₂, and 10 mm CaCl₂ or BaCl₂. Internal solution contained 140 mm N-methyl-d-glucamine, 10 mm HEPES, 2 mm MgCl₂, 2 mm Mg-ATP, and 0.5 mm EGTA. The pH of both solutions was adjusted to 7.3 with methanesulfonic acid. Electrode resistances were 1–2 MΩ in the bath solution, and series resistance was ∼2–4 MΩ, compensated 60–80%. The membrane potential was held at −60 mV prior to action potential waveform protocols or to −80 mV prior to all other experiments. The acquired data were analyzed using Igor Pro software (Wavemetrics), and statistics were performed using SigmaPlot (Systat Software). All of the averaged data are presented as the means ± S.E. In the figures, numbers of cells (n) are indicated in parentheses.

RESULTS

CR Interacts with Ca_v2.1

While screening a heptapeptide phage display library for CR-interacting proteins, we identified a consensus CR-binding sequence, H(R/K)HRRR(E/D), consisting of five or six basic residues (His/Arg) flanked by an acidic (Glu/Asp) residue (Fig. 1A). Bioinformatic analysis revealed that this CRB sequence was repeated five times in the cytoplasmic loop between domains 2 and 3 of α₁2.1 (Fig. 1B). The CRBs are C-terminal to the synaptic protein interaction site (“synprint”) (33) and are highly conserved in α₁2.1 between species. The CRB-containing region is also present in the Ca_v2.2 α₁ subunit (α₁2.2; supplemental Fig. S1) but absent in Ca_v1 α₁ subunits. To test whether the CRB(s) directly interact with CR, pulldown assays were performed with GST fusion proteins containing the first three CRBs in α₁2.1 (GST-CRB1–3). CR bound to GST-CRB1–3, but not to control GST, in the presence of Ca²⁺ (1.6 mm). Binding of CR was Ca²⁺-dependent because it was prevented when free Ca²⁺ was chelated with EGTA (1 mm; Fig. 1C). To estimate the affinity of CR binding to α₁2.1, GST-CRB1–3 was incubated with varying amounts of fluorescently tagged CR. Binding was relatively low affinity (EC₅₀ = ∼10 μm) and was stronger with than without Ca²⁺ (Fig. 1D). Compared with CR binding to a GST fusion protein containing only the most N-terminal CRB (CRB1), CR binding in the linear range of the binding curve was increased by 19.6 ± 7.9% and 32.6 ± 13.6% for GST proteins containing two (CRB1,2) and three (CRB1–3) motifs, respectively (data not shown). These results confirm that CR binds in a Ca²⁺-dependent manner to the CRBs in α₁2.1.

FIGURE 1.

CR binds to α₁2.1. A, top panel, schematic of CR with functional (EF1–5, white) and nonfunctional (EF6, gray) Ca²⁺-binding domains indicated. Bottom panel, consensus sequence for CR binding. B, schematic of Ca_v2.1 subunit, α₁2.1, with CRB domains in the II-III linker indicated (bold, underlined). Italics mark the synprint region. The sequence present in the GST- α₁2.1 fusion protein used for binding assays is shaded gray. C, pulldown assay of GFP-CR and GST-CRB1–3. GST (third and sixth lanes) or GST-CRB1–3 (second and fourth lanes) were immobilized on glutathione-agarose beads and incubated with GFP-CR transfected HEK293T cell lysate in the presence of 1.6 mm CaCl₂ or 1 mm EGTA. In represents ∼10% of the lysate used in the assay. Top panel, Western blot with GFP antibody. Bottom panel, integrity and levels of GST fusion proteins were shown by Ponceau S staining. D, in vitro assay for CR binding to GST- CRB1–3. GST or GST-CRB1–3 was tested for binding to Alexa 488-labeled CR in the absence (−) or presence (+) of Ca²⁺ (∼300 μm). Fluorescence values were normalized to that obtained with 20 μm CR in the presence of Ca²⁺ (% maximal binding). The data are from three independent experiments done in duplicate. E, coimmunoprecipitation of Ca_v2.1 and CR from HEK293T cells. Cells transfected with Ca_v2.1 subunits alone or cotransfected with GFP-CR were subjected to lysis and immunoprecipitation (IP) with rabbit CR antibodies. Immunoprecipitated proteins were detected by Western blotting with α₁2.1 (top panel) or GFP (bottom panel) antibodies. F, coimmunprecipitation of Ca_v2.1 and CR from mouse cerebellum. Mouse cerebellum lysates were incubated with rabbit antibodies against CR or control rabbit IgG. Immunoprecipitated proteins were detected by Western blotting with α₁2.1 (top panel) or calretinin (bottom panel) antibodies. The results shown in C and F are representative of three independent experiments; E is representative of two independent experiments.

If CR is an interacting partner of Ca_v2.1, it should also associate with the intact channel. To test this prediction, we coexpressed CR and Ca_v2.1 in HEK293T cells and used CR antibodies in coimmunoprecipitation experiments. CR antibodies brought down α₁2.1 in cells cotransfected with Ca_v2.1+ CR. Some α₁2.1 was brought down nonspecifically by CR antibodies because a small amount of α₁2.1 was detected in immunoprecipitations from cells transfected with Ca_v2.1 alone (Fig. 1E). However, a larger signal corresponding to α₁2.1 was consistently detected when Ca_v2.1 was coexpressed with CR, indicating some specific association of CR with α₁2.1. To verify these results in the native context, we performed coimmunoprecipitation experiments with extracts from mouse cerebellum given that CR and Ca_v2.1 are both expressed highly in cerebellar granule neurons (19, 21, 22). In these experiments, CR antibodies but not control IgG coimmunoprecipitated α₁2.1 (Fig. 1F). Together, these results demonstrate that CR associates with recombinant and native Ca_v2.1 channels in the brain.

CR Modulates CDI and CDF of Ca_v2.1

To investigate the functional consequences of CR interacting with α₁2.1, we compared the properties of Ca_v2.1 transfected alone or cotransfected with CR in HEK293T cells. Because Ca_v2.1 channels are strongly modulated by Ca²⁺, we analyzed both Ca²⁺ and Ba²⁺ currents (I_Ca and I_Ba, respectively). In initial experiments, we found that CR had no significant impact on I_Ca or I_Ba current density or on voltage-dependent activation of I_Ba, although it caused a modest positive shift in the current-voltage (I-V) relation for I_Ca (V½ = 2.7 mV ± 0.7 mV for Ca_v2.1 alone versus 7.3 ± 0.4 mV for Ca_v2.1+CR, p < 0.001; k = −5.4 ± 0.2 for Ca_v2.1 alone versus −4.8 ± 0.1 for Ca_v2.1+CR, p = 0.03, both by t test; Fig. 2).

FIGURE 2.

CR modestly inhibits voltage-dependent activation of Ca_v2.1 I_Ca. Voltage protocol for current-voltage (I-V) relations and representative I_Ca (A) and I_Ba (B) current traces are shown for cells transfected with Ca_v2.1 alone or cotransfected with CR (+CR) with I-V curves shown below.

Ca²⁺-dependent binding of CR to the channel could dynamically alter Ca²⁺-dependent modulation of Ca_v2.1. Because of a reliance on global elevations in Ca²⁺, CDI can be suppressed by Ca²⁺ buffers such as EGTA (5–10 mm). In contrast, CDF, which depends on rapid, local Ca²⁺ elevations, is not blunted by high concentrations of EGTA (6, 7). If CR acted as a Ca²⁺ buffer when bound to the channel, it could suppress CDI of Ca_v2.1. To test this, we performed experiments dissecting voltage- from Ca²⁺-dependent inactivation (VDI and CDI, respectively). Voltage protocols consisted of two test pulses (P1 and P2) separated by a conditioning prepulse (Fig. 3A, pre) to varying voltages. With both Ca²⁺ and Ba²⁺ as the charge carrier, the second test current is smaller than the first because of inactivation induced by the prepulse. The ratio of the P2:P1 test current amplitudes can therefore be used as a metric for inactivation (Fig. 3B). For Ca_v2.1 I_Ca, inactivation is significantly stronger than for I_Ba (P2/P1 = 0.31 ± 0.07 for I_Ca versus 0.68 ± 0.04 for I_Ba, p < 0.001). In addition, the P2:P1 ratio exhibits U-shaped dependence on prepulse voltage with a minimum at the prepulse voltage evoking the maximal inward I_Ca (Fig. 3B). In contrast, I_Ba shows a more modest and monotic increase in inactivation with prepulse voltage caused by VDI (Fig. 3, A and B). Because Ca_v2.1 current density positively affects CDI (14, 20), we restricted analysis to cells exhibiting maximal current densities between 13 and 19 pA/pF.

FIGURE 3.

CR inhibits CDI and enhances Ca_v2.1 CDF. A, voltage protocol and representative traces for I_Ca and I_Ba are shown. P1 (black) and P2 (gray) test currents are overlaid for comparison. B, the ratio of the amplitude of the P2 and P1 current (P2/P1) was plotted against prepulse voltage for I_Ca and I_Ba for cells transfected with Ca_v2.1 alone or Ca_v2.1+CR. C, left panel, P2/P1 obtained with 0-mV prepulse voltage for Ca_v2.1 and Ca_v2.1+CR. #, p < 0.001 compared with I_Ca; *, p < 0.001 by t test. Right panel, CDI or CDF obtained with 0-mV prepulse represents P2/P1 ratio for I_Ca minus the mean P2/P1 ratio for I_Ba. *, p < 0.001, t test.

Compared with the strong CDI in cells transfected with Ca_v2.1 alone, CDI was virtually undetectable across the full range of prepulse voltages in cells cotransfected with CR (Fig. 3, A and B). At prepulse voltages between −20 and +10 mV, I_Ca actually underwent facilitation (P2/P1 > 1). Facilitation was Ca²⁺-dependent in that there was no effect of CR on inactivation of I_Ba (Fig. 3C). Consistent with protocols that induce CDF (6), a prepulse to 0 mV induced a significant acceleration in activation kinetics of the P2 compared with the P1 current for both Ca_v2.1 alone (τ_P1 = 6 ± 1.3 ms versus τ_P2 = 2 ± 0.3 ms, n = 6, p = 0.02) and Ca_v2.1+CR (τ_P1 = 12.4 ± 1 ms, versus τ_P2 = 1.9 ± 0.2 ms, n = 9, p < 0.001). However, the prepulse-induced speeding of the P2 activation kinetics was significantly greater for cells cotransfected with CR compared with Ca_v2.1 alone (∼125%, p = 0.001). To quantitate the Ca²⁺-dependent modulation seen with a 0-mV prepulse, the P2/P1 ratio of I_Ba was subtracted from that for I_Ca. This analysis clearly indicated that unlike Ca_v2.1 alone, which shows significant CDI, CR caused overt CDF (Fig. 3C).

We further analyzed the effects of CR on Ca_v2.1 CDF using action potential (AP) waveforms. During a train of 200-Hz AP stimuli, CDF is evident as a progressive increase in the amplitude of I_Ca; I_Ba undergoes less facilitation, which is voltage-dependent (Fig. 4, A and B). Consistent with our data obtained with conditioning prepulses (Fig. 3), CR caused a significantly larger increase in I_Ca but not I_Ba by the end of the train (Fig. 4C). We measured CDF as the difference in the amplitude of I_Ca and I_Ba at the end (0.5 s) of the train. By this metric, CDF for Ca_v2.1 was significantly greater with CR (0.22 ± 0.01, n = 10) than without (0.13 ± 0.03, n = 8; p < 0.01, t test). These results confirm that CR enhances CDF of Ca_v2.1.

FIGURE 4.

CR enhances Ca_v2.1 CDF during AP trains. A, AP waveform and representative I_Ca evoked by 200-Hz train. The dashed line indicates initial current amplitude. Representative traces from the first (black) and 100th (gray) AP were overlaid for comparison for I_Ca and I_Ba in cells transfected with Ca_v2.1 alone or Ca_v2.1+CR. B, fractional current represents test current amplitudes normalized to the first in the train and plotted against time. C, the maximal fractional current at 0.5 s was compared for I_Ca and I_Ba. #, p < 0.001 compared with I_Ca. *, p < 0.03 by t test.

These effects of CR on Ca_v2.1 CDI and CDF could be due to its actions as a freely diffusible Ca²⁺ buffer. Alternatively, CR binding to the α₁2.1 II-III linker may directly suppress CDI and enhances CDF. To distinguish between these possibilities, we took advantage of the fact that the CRBs present in α₁2.1 are not conserved in the Ca_v1.2 α₁ subunit (α₁1.2). If CR binding to α₁2.1 is necessary for Ca_v2.1 modulation, then chimeric channels in which the 2–3 linker of α₁1.2 is substituted for that in α₁2.1 (Ca_v2.1–1.2) should prevent effects of CR on CDI and CDF. Consistent with this prediction, Ca_v2.1–1.2 channels underwent CDI that was not affected by CR (Fig. 5). In addition, there was no significant difference in CDF during AP trains in cells transfected with Ca_v2.1–1.2 alone and cells cotransfected with CR (p = 0.57; Fig. 6). These results show that the CRBs are required for CR modulation of Ca_v2.1 CDI and CDF.

FIGURE 5.

Ca_v2. 1 channels lacking the CRBs are insensitive to CR modulation of CDI. A–C, same as in Fig. 3 except for Ca_v2.1–1.2 alone and Ca_v2.1–1.2+CR.

FIGURE 6.

Ca_v2. 1 channels lacking the CRBs are insensitive to CR modulation of CDF. A and B, same as in Fig. 4, A and B, except for Ca_v2.1–1.2 alone and Ca_v2.1–1.2+CR. C, CDF shown for wild-type Ca_v2.1 or Ca_v2.1–1.2 ± CR. *, p < 0.005.

To verify the importance of CR binding to the CRBs for Ca_v2.1 modulation, we tested the impact of a peptide, which should competitively displace CR from binding sites in the α₁2.1 II-III linker. Because our biochemical experiments indicated that CR binds to a peptide sequence including the first three CRBs (Fig. 1), we generated a mcherry-tagged peptide containing this region (CRB1–3). Because CR had no effect on I_Ba either in inactivation or facilitation protocols (Figs. 3 and 4), we restricted analysis to I_Ca and assumed effects on I_Ca inactivation and facilitation reflected effects on CDI and CDF, respectively. When cotransfected with Ca_v2.1+CR, CRB1–3 significantly opposed the decrease in CDI caused by CR (for a 0-mV prepulse, P2/P1 = 1.0 ± 0.05 for Ca_v2.1+CR versus 0.58 ± 0.11 for +CR+CRB1–3; p < 0.01; Fig. 7A). Coexpression of CRB1–3 also significantly inhibited the effect of CR on CDF by the end of a 1-s AP train (p < 0.05; Fig. 7B). In contrast, there was no difference in CDI (p = 0.43 for a 0-mV prepulse) or CDF (p = 0.93 at the end of the 1-s train) in cells transfected with Ca_v2.1 alone and those cotransfected with Ca_v2.1+CRB1–3 (Fig. 7, C and D), which argued against nonspecific inhibitory effects of CRB1–3 on Ca_v2.1 that were independent of CR modulation. Taken together, our results support a mechanism in which CR binding to the α₁2.1 II-III linker directly inhibits Ca_v2.1 CDI and enables enhanced CDF of Ca_v2.1 during repetitive stimuli.

FIGURE 7.

A CR-binding peptide prevents effect of CR on Ca_v2.1 CDI and CDF. A–D, same as Figs. 3, A and B, and 4, A and B, but for I_Ca only and for cells transfected with Ca_v2.1+CR or +CR+CRB1–3 (A and B) or Ca_v2.1 alone or +CRB1–3 (C and D).

DISCUSSION

Our study provides key evidence for a new role for CR as an integral component of Ca_v2.1 complexes that modulates Ca_v2.1 function. First, CR binds in a Ca²⁺-dependent manner to basic motifs in the α₁2.1 II-III linker. Second, CR forms a complex with Ca_v2.1 channels in the brain. Third, the interaction of CR with the α₁2.1 II-III linker inhibits CDI and enhances CDF of Ca_v2.1 channels and enhances CDF in transfected HEK293T cells. Because of the cellular overlap in CR and Ca_v2.1 expression in the brain (22, 34), CR interactions with Ca_v2.1 should be considered in models of how this protein controls Ca²⁺ signaling and excitability in neurons.

Ca²⁺-dependent Binding of CR to Effectors

CR has long been considered a diffusible Ca²⁺ buffer that can alter spatiotemporal aspects of Ca²⁺ signaling (19, 23). However, multiple lines of evidence suggest that CR may not be freely diffusible and may, in fact, interact with proteins in a manner analogous to “Ca²⁺ sensors” such as CaM. First, CR undergoes large conformational changes upon binding to Ca²⁺ (24, 25). Similar Ca²⁺-dependent structural changes have been reported for the related EF-hand proteins calbindin D-28k and secretagogin (35, 36). As in CaM, Ca²⁺ binding to CR may expose hydrophobic regions of the protein, which allow Ca²⁺-dependent interactions with other proteins. Second, in addition to its cytosolic localization, CR is also abundant in the membrane fraction of cerebellar extracts and less so under conditions of low Ca²⁺ (26). The latter findings could be explained by Ca²⁺-dependent interactions of CR with integral membrane or membrane-associated proteins, much like the well established role of CaM in regulating pre- and post-synaptic effectors (37).

Although our in vitro experiments indicate a relatively low binding affinity (∼10 μm) of CR for the α₁2.1 CRB sequences in GST-CRB1–3 (Fig. 1D), we believe this may underestimate the ability of CR to interact with α₁2.1 in vivo. Because we found greater CR binding to GST proteins containing three compared with two or one CRB sequences, it is likely that the five CRBs in the intact channel may increase the avidity of CR binding. In addition, CR is thought to be expressed at rather high concentrations in some neurons, ranging from ∼40 to 80 μm (27, 38). These concentrations of CR would be sufficient to saturate the CRB sequence(s) in α₁2.1 to a large extent, particularly if the affinity between CR and CRBs is even higher within the intact channel complex than in vitro. Although our biochemical analyses cannot allow conclusions regarding the stoichiometry of CR binding to the α₁2.1 II-III linker, binding of CR to the GST-CRB1–3 protein was cooperative (Fig. 1D). Because GST-CRB1–3 contains three of the five CRB sequences in the α₁2.1 II-III linker, it is possible that cooperativity in the binding assay resulted from multiple CR molecules binding to GST-CRB1–3. Alternatively, many EF-hand proteins including CR isoforms show reversible dimerization in vitro (39). The binding of CR dimers to the CRBs could therefore also lead to the apparent cooperativity in binding to GST-CRB1–3. Additional studies will be required to fully resolve the molecular thermodynamic properties of CR interactions with α₁2.1.

Although our findings that CR coimmunoprecipitates with Ca_v2.1 channels in the brain (Fig. 1F) clearly implicate Ca_v2.1 as a CR target, it is important to note that basic amino acid sequences similar to the α₁2.1 CRBs are likely present in other effectors. In particular, the CRBs are conserved in the “synprint” region of the Ca_v2.2 α₁ subunit (α₁2.2; supplemental Fig. S1). Although Ca_v2.2 channels do not undergo CDF (40), synaptic protein interactions with the II-III linker of α₁2.2 regulate voltage-dependent inactivation of Ca_v2.2 and inhibition of these channels by heterotrimeric G-proteins (41). Thus, it is possible that CR binding to the CRBs in α₁2.2 could have important consequences for the modulation of Ca_v2.2 channels in neurons. The characterization of other CR-interacting proteins other than Ca_v2.1 and how they may be modulated by CR are crucial for fully understanding Ca²⁺ signaling dynamics in the neuronal and non-neuronal cell-types in which CR is expressed (42, 43).

Ca_v2.1 CDI/CDF Modulation by CR

Significant progress has been made in elucidating the mechanisms underlying Ca_v2.1 CDI and CDF (see Ref. 44 for review). CDI depends on the N-terminal lobe of CaM, which responds to global rather than local Ca²⁺ signals. CDF depends on the C-lobe of CaM, which likely binds local Ca²⁺ ions as they emerge from the channel pore (6, 45). In support of this model, intracellular dialysis with EGTA (10 mm) prevents CDI but not CDF (7). Moreover, CDF but not CDI is observed at the single channel level (46). This latter result and the findings that CDI increases with Ca_v2.1 current density (14, 20) illustrate that CDI depends on Ca²⁺ influx through multiple open channels and so should be sensitive to Ca²⁺ buffering. Based on the ability of CR to rapidly depress presynaptic Ca²⁺ signals (47), it is assumed that CR will act as a Ca²⁺ buffer when faced with a rise in intracellular Ca²⁺. Therefore, it is perhaps not surprising that coexpression of CR with Ca_v2.1 inhibited CDI. The remarkable result is that CR accomplishes this through its association with the α₁2.1 II-III linker (Figs. 6 and 7). Our biochemical and electrophysiological experiments show that disabling CR interactions with the α₁2.1 II-III linker prevents the channel modulation. The tethered CR may rapidly suppress global Ca²⁺ elevations that support CDI, which subsequently enhances CDF during trains of AP waveforms (Figs. 3 and 4). Alternatively, CR binding to the II-III linker could allosterically modulate CDI in the manner of a Ca²⁺ sensor. Despite the presence of molecular determinants for CDI and CDF in the C-terminal domain of Ca_v α₁ subunits, auxiliary Ca_vβ subunit interactions with the I-II linker have been shown to modulate CDI and CDF of Ca_v1.2 channels (48). Experimental dissection of a Ca²⁺ buffering versus Ca²⁺ sensor mechanism is complicated by the possibility that disrupting the Ca²⁺ buffering activity of CR might also negate its ability to interact with the channel but nevertheless is an important goal for future studies.

It is noteworthy that the CRBs in the α₁2.1 II-III linker overlap with the bipartite synprint site in α₁2.1. Interactions between SNAREs and the synprint are thought to promote efficient coupling of Ca_v2 channels and exocytosis in presynaptic nerve terminals based on evidence that peptides containing the synprint site impair neurotransmission (49, 50). Our findings suggest that such peptides may also influence CR regulation of Ca_v2.1 CDI and CDF and so may affect synaptic transmission via multiple mechanisms. In addition, splice variants lacking portions of the CRB/synprint region have been identified in neuroendocrine cells and various brain regions (51). The inability of CR to modulate CDI/CDF of such variants may further diversify Ca_v2.1 Ca²⁺ signaling between neuronal subtypes.

Neurophysiological Significance of CR-Ca_v2.1

Immunohistochemical analyses indicate a number of neuronal cell groups in which CR and Ca_v2.1 colocalize. Ca_v2.1 channels are the major presynaptic Ca²⁺ channels in the nerve terminals forming the Calyx of Held synapse in the auditory brainstem (9, 52). CR is detected presynaptically at these synapses but only at significant levels (>18% in rats) after postnatal day 14 (53). Electrophysiological recordings at the Calyx of Held synapse, usually done in brainstem slices from juvenile rats (postnatal day 8–10), indicate that the presynaptic Ca_v2.1 channels undergo CDI and CDF (8–10, 54). Given our findings that CR inhibits CDI and enhances CDF, the developmental increase in CR would be expected to promote the activity-dependent Ca²⁺ influx that may limit synaptic depression and/or increase reliability in the mature Calyx of Held synapse (55).

As our coimmunoprecipitation of Ca_v2.1 with CR from mouse cerebellum would indicate, CR-Ca_v2.1 complexes may play a role in cerebellar granule cells, the predominant cell types expressing CR in the cerebellum (56, 57). Granule cells provide the major excitatory drive to Purkinje neurons in the form of parallel fibers. Genetic inactivation of CR in mice increases the intrinsic excitability of granule cells and Purkinje cell firing rate in vivo (27, 28, 58). With respect to Ca_v2.1 in granule cells, a loss of CR should inhibit Ca_v2.1 Ca²⁺ influx by enhancing CDI (Fig. 3). Decreased I_Ca may seem at odds with the hyperexcitable phenotype of CR^−/− granule cells, because it would be expected to limit activation of Ca²⁺-activated BK channels and subsequently oppose repolarization following an action potential. However, we have observed compensatory changes in Ca_v2.1 subunit expression in cerebellar Purkinje neurons from mice lacking parvalbumin and calbindin D-28k (15), which could also explain the lack of correlation between our findings and that expected in CR^−/− granule cells. In addition, Ca_v2.1/CR interactions may be more relevant presynaptically, where enhanced CDF may support residual Ca²⁺ in parallel fiber terminals that causes short term synaptic plasticity at the parallel fiber-Purkinje cell synapse (59). In summary, our results implicate CR as a novel modulator of Ca_v2.1 channels, which may foreshadow yet additional roles for CR in actively regulating neuronal excitability and synaptic transmission through direct Ca²⁺-dependent interactions with other effectors.