Molecular Determinants of Modulation of Ca_V2.1 Channels by Visinin-like Protein 2

Background: Regulation of calcium channels by calcium sensor proteins mediates short-term synaptic plasticity.

Results: Visinin-like protein-2 (VILIP-2) increases facilitation of calcium channels through its N terminus, interlobe linker, and EF-hands 3 and 4.

Conclusion: Specific domains of VILIP-2 are responsible for regulation, including adjacent EF-hands that bind calcium.

Significance: These results reveal the molecular code used by calcium-sensor proteins to differentially regulate short-term synaptic plasticity.

Abstract

Ca_V2.1 channels, which conduct P/Q-type Ca²⁺ currents, initiate synaptic transmission at most synapses in the central nervous system. Ca²⁺/calmodulin-dependent facilitation and inactivation of these channels contributes to short-term facilitation and depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin (CaM) from its binding site, differentially regulate Ca_V2.1 channels, and contribute to the diversity of short-term synaptic plasticity. The neuronal calcium sensor protein visinin-like protein 2 (VILIP-2) inhibits inactivation and enhances facilitation of Ca_V2.1 channels. Here we examine the molecular determinants for differential regulation of Ca_V2.1 channels by VILIP-2 and CaM by construction and functional analysis of chimeras in which the functional domains of VILIP-2 are substituted in CaM. Our results show that the N-terminal domain, including its myristoylation site, the central α-helix, and the C-terminal lobe containing EF-hands 3 and 4 of VILIP-2 are sufficient to transfer its regulatory properties to CaM. This regulation by VILIP-2 requires binding to the IQ-like domain of Ca_V2.1 channels. Our results identify the essential molecular determinants of differential regulation of Ca_V2.1 channels by VILIP-2 and define the molecular code that these proteins use to control short-term synaptic plasticity.

Introduction

P/Q-type Ca²⁺ currents conducted by Ca_V2.1 channels initiate neurotransmitter release at most synapses in the central nervous system (1, 2). Short-term synaptic plasticity modulates neurotransmitter release in response to trains of repetitive action potentials, which often evoke facilitation followed by depression of the postsynaptic response (3–5). This regulatory mechanism encodes information in the frequency and pattern of action potentials and transmits it to the postsynaptic cell as a change in synaptic strength (3–5). Ca_V2.1 channels are required for synaptic facilitation at the Calyx of Held, a large, rapidly transmitting synapse in the auditory system (6). At that synapse, a component of short-term synaptic facilitation has been correlated with facilitation of P/Q-type calcium currents (6, 7), and the rapid phase of synaptic depression has been correlated with Ca²⁺-dependent inactivation of P/Q-type calcium currents (8, 9).

In non-neuronal cells, Ca²⁺ influx through transfected Ca_V2.1 channels acts as a feedback regulator and causes facilitation followed by inactivation of the P/Q-type Ca²⁺ current in trains of repetitive depolarizations (10–14). Ca²⁺-dependent facilitation and inactivation are mediated by binding of Ca²⁺/CaM to a bipartite site in the C-terminal domain of Ca_V2.1 channels composed of an IQ-like motif (IM)² and a CaM-binding domain (CBD) (12, 14). Mutation of the IQ-like motif (IM-AA) abolishes Ca²⁺-dependent facilitation, whereas deletion of the CBD (ΔCBD) reduces inactivation and has a lesser effect on facilitation (12, 14). Superior cervical ganglion neurons transfected with Ca_V2.1 channels show short-term synaptic facilitation and depression, and mutations of the IQ-like motif and CBD block facilitation and depression, respectively (15).

Short-term synaptic plasticity is diverse, with facilitation, depression, or facilitation followed by depression observed at different synapses in the central nervous system (3). As CaM is ubiquitously expressed, other proteins must determine the diversity of short-term synaptic plasticity. CaM is the founding member of a large family of CaS proteins, which are differentially expressed in neurons (16–18). Calcium-binding protein 1 (CaBP1) and visinin-like protein 2 (VILIP-2) differentially regulate Ca_V2.1 channels (19, 20). CaBP1 reduces facilitation and increases inactivation (19), whereas VILIP-2 slows the rate of inactivation and enhances facilitation (20). Mutation of the IQ-like motif (IM-AA) combined with the deletion of the CBD prevent VILIP-2 binding to Ca_V2.1 channels (20).

Both CaM and CaS proteins have four EF-hands connected by a central α-helix (16–18). VILIP-2 and CaBP1 have large N-terminal domains with a myristoyl lipid anchor (16–18). All four EF-hands of CaM bind Ca²⁺, whereas EF-hand 1 of VILIP-2 and EF-hand 2 of CaBP1 have changes in amino acid sequence that prevent high-affinity Ca²⁺ binding (16–18). In CaM regulation of Ca_V2.1 channels, local increases in Ca²⁺ lead to binding to the C-terminal lobe (EF-hands 3 and 4) and initiate facilitation by interaction with the IQ-like domain (11, 12, 14), whereas global increases in Ca²⁺ bind to the N-terminal lobe of CaM and mediate inactivation by interaction with the CBD (11, 12, 14). The domains of VILIP-2 responsible for differential regulation of Ca_V2.1 channels are unknown. In this study, we have constructed and analyzed chimeras of VILIP-2 and CaM to define the molecular basis for the differential regulation of Ca_V2.1 channels by VILIP-2. Our results show that the N-terminal domain up to the first EF-hand plus the C-terminal lobe of VILIP-2, together with its interlobe α-helix, are both necessary and sufficient to confer VILIP-2-like regulatory properties on CaM. These results further define the molecular code that is used for CaS protein-dependent modulation of short-term synaptic plasticity.

EXPERIMENTAL PROCEDURES

Construction of Chimeras

We named the chimeras with a combination of two letters and four numbers. The letters are: N for the N-terminal domain including the myristoyl moiety and H for the interlobe helix. The four numbers correspond to the four EF-hands from N- to C-terminal. The VILIP-2 functional domains are highlighted in bold and underlined, whereas the CaM domains are in normal font. To exchange the functional domains of CaM and VILIP-2, overlapping PCR was used to swap the functional domains of VILIP-2 with those of CaM and inserted into the pcDNA3.1 vector at the 5′-XhoI and 3′-XbaI sites. The Pfam database (21) was used to determine the boundaries of CaM and VILIP-2 functional domains. The N12H34, N12H34, N12H34, and N12H34 chimeras were generated by using overlapping PCR to fuse either the N terminus (a.a. 1–23), the N terminus to the end of EF-hand 1 (a.a. 1–55), EF-2 hand (a.a. 1–92), or helix (a.a. 1–96) of VILIP-2 and replaced the corresponding regions of CaM in pcDNA3.1. The N12H34 chimera was used to generate N12H34 and N12H34. Using overlapping PCR, either the EF-hand 3 and EF-hand 4 (a.a. 97–end) or the helix, EF-hand 3, and EF-hand 4 (a.a. 93–end) of VILIP-2 replaced the corresponding segments of N12H34 and in pcDNA3.1.

Cell Culture and Transfection

Human embryonic kidney tsA-201 cells in DMEM/Ham's F12 with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 100 units/ml penicillin and streptomycin (Invitrogen) were grown to ∼80% confluence at 37 °C and 10% CO₂. Cells in 20-mm wells were transfected with cDNAs encoding Ca²⁺ channel subunits α₁2.1 (2 μg), β_2a (1.5 μg), α₂δ (1 μg), and VILIP-2 (1 μg) using TransIT-LT1 (Mirus Bio LLC, Madison, WI). cDNA encoding enhanced GFP was co-transfected to visualize the transfected cells (19). After 24 h, the cells were washed in serum-containing medium and subcultured on sterile 8-mm coverslips. Finally, 24–48 h after transfection, the cells were used for electrophysiological recordings.

Electrophysiological Recordings and Data Analysis

Whole-cell voltage clamp recordings were acquired from transfected tsA-201 cells at room temperature. The recordings were done in an extracellular solution containing (in mm) 10 CaCl₂ or 10 BaCl₂, 150 Tris, 1 MgCl₂ (305 mosm) and with an intracellular solution consisting of (in mm) 120 N-methyl-d-glucamine, 60 Hepes, 1 MgCl₂, 2 Mg-ATP, and 0.5 EGTA (295 mosm). The pH of both solutions was adjusted to pH 7.3 with methanesulfonic acid. Recordings were made with a HEKA EPC 10 patch clamp amplifier using PULSE software (HEKA Elektronik, Lambrecht, Germany) and filtered at 3 kHz. Leak and capacitive transients were subtracted using a P/−4 protocol. Voltage protocols were adjusted to compensate for the more negative voltage dependence of activation in extracellular Ba²⁺ solution. Data analysis was performed using IGOR (WaveMetrics, Lake Oswego, OR). Activation curves were fit to determine values for the voltage of half-activation (V½) and the slope (k) using the following Boltzmann equation: y = (y_max − y_min)/(1 + exp(V½ − V)/k) + y_min. All average data represent the mean ± S.E. Statistical significance was determined using Student's unpaired t test.

Binding Assays

MBP-VILIP-2 or MBP alone was immobilized on amylose beads (New England Biolabs, Beverly, MA) that were extensively washed with PBS buffer. The MBP proteins were incubated with 4 μg of CBD-GST, IM-GST, or GST alone proteins for 1 h at 4 °C. The binding buffer contained (in mm): 200 NaCl, 1 MgCl₂, 20 Tris-HCl, and 1% Triton X-100. Ca²⁺ and EGTA were added as described under “Results.” After extensive washing, bound proteins were eluted at 97 °C with sample buffer (4×) and separated on a NuPAGE gel (Invitrogen). Immunoblotting was performed with monoclonal anti-GST (Sigma) or anti-MBP (New England Biolabs) antibodies. Blots were extensively washed with Tris-buffered saline with Tween-20. Analysis of the blots was done using the National Institutes of Health ImageJ program, and relative binding was normalized to control GST or MBP.

RESULTS

Modulation of Ca_V2.1 Channels by CaM and VILIP-2

CaM and CaS proteins are modular, composed of four EF-hand motifs separated by a central α-helix (Fig. 1A). All four EF-hands of CaM are active in binding Ca²⁺, whereas EF-hand 1 of VILIP-2 is inactive (16–18). In addition, VILIP-2 has an extended N-terminal domain with a myristoyl lipid anchor (16–18). These molecular differences in VILIP-2 must be responsible for its unique regulatory properties, but the molecular determinants of differential regulation remain unknown. Chimeras of VILIP-2 and CaM were constructed in which the structural domains of VILIP-2 were substituted in CaM individually or in combinations (Fig. 1A). In this experimental strategy, the transferred domains from VILIP-2 confer VILIP-2-like regulatory properties on the chimeras, and overexpression of the chimeras displaces endogenous CaM and induces VILIP-2-like regulation of Ca_V2.1 channels. Overexpression of CaM itself does not have any functional effect, suggesting that it is already at a saturating concentration (19). Therefore, distinctive regulatory effects conferred by expression of the chimeras can be ascribed to molecular differences in the chimeras and not to additive effects of CaM plus the chimera. All of the chimeras we constructed were functionally expressed because each of them had a clearly detectable effect on regulation of Ca_V2.1 channels (supplemental Tables 1 and 2). However, only one chimera was able to fully confer the regulatory properties of VILIP-2 as described below.

FIGURE 1.

Modulation of Ca_V2.1 channels by CaM and VILIP-2. A, schematic drawing of CaM/VILIP-2 chimeras. EF-hands are indicated by rectangles, and the central α-helix is indicated by a spiral. Filled rectangles, CaM EF-hands; striped rectangles, VILIP-2 EF-hands that bind Ca²⁺ with high affinity; open rectangles, VILIP-2 EF-hands that do not bind Ca²⁺ with high affinity. B, mean normalized I_Ca (n = 20) and I_Ba (n = 20) evoked by a 1-s depolarizing test pulse from −80 to +20 or +10 mV, respectively. C, mean ratio of residual current amplitude measured at the end of the 1-s pulse (I_Res) over peak current amplitude (I_Peak) from Ca_V2.1-transfected cells in Ca²⁺ (black) and Ba²⁺(white). D, mean normalized I_Ca (n = 10) and I_Ba (n = 9) from cells transfected with Ca_V2.1 and VILIP-2. E, mean ratio of I_Res over I_Peak from Ca_V2.1 and VILIP-2-transfected cells in Ca²⁺ (black) and Ba²⁺ (white). F, mean normalized I_Ca (n = 19) and I_Ba (n = 12) during 100-Hz train of 5-ms pulses from −80 to +20 or +10 mV, respectively. G, paired pulse ratios (PPR) from tail currents evoked by test pulses from −80 to +30 mV for I_Ca (black; n = 25) or +20 mV for I_Ba (white; n = 13) without a prepulse (P1) or with a 50-ms prepulse to +10 mV (P2). H, mean normalized I_Ca + 20 mV (n = 10) and I_Ba + 10 mV (n = 10) during 100-Hz trains of pulses from Ca_V2.1 and VILIP-2-transfected cells. I, PPR from tail currents of I_Ca (black; n = 21) or I_Ba (white; n = 19) without a prepulse (P1) or with a 50-ms prepulse to +10 mV (P2) in cells transfected with Ca_V2.1 and VILIP-2. The data shown are averages ± S.E. Asterisks indicate a significant difference (***, p < 0.001).

Ca²⁺/CaM-dependent inactivation of Ca_V2.1 channels was examined in transfected tsA-201 cells by application of a 1-s test pulse from −80 to 20 mV. Evoked Ca²⁺ currents inactivated rapidly when compared with currents recorded after replacing Ca²⁺ with Ba²⁺ as the charge carrier (Fig. 1, B and C). This Ca²⁺-dependent component of inactivation is caused by Ca²⁺/CaM interaction with the CBD in the C-terminal domain of Ca_V2.1 channels (10–12, 14). Co-expression of VILIP-2 with Ca_V2.1 prevented the Ca²⁺-dependent component of inactivation of Ca_V2.1 channels (Fig. 1, D and E).

To analyze Ca²⁺/CaM regulation in response to more physiological stimuli, Ca²⁺ and Ba²⁺ currents were recorded during a train of repetitive depolarizations to 20 mV for 5 ms at 100 Hz. Ca²⁺ currents were facilitated by the first depolarizations and then progressively inactivated (Fig. 1F). Both Ca²⁺-dependent facilitation and Ca²⁺-dependent inactivation were prevented when Ba²⁺ was the charge carrier (Fig. 1F). Ca²⁺-dependent facilitation was also observed when Ca_V2.1 tail currents were measured to assess conductance of Ca_V2.1 channels following paired depolarizations to 30 mV for 8 ms with or without a prepulse to 10 mV for 50 ms (Fig. 1G). In the presence of VILIP-2, repetitive depolarizations resulted in enhanced Ca²⁺-dependent facilitation and slowed Ca²⁺-dependent inactivation in cells expressing Ca_V2.1 channels (Fig. 1H) when compared with endogenous CaM (Fig. 1F). VILIP-2 induced Ca²⁺-dependent facilitation of the Ca_V2.1 tail current in a paired-pulse protocol (Fig. 1I), but the magnitude of Ca²⁺-dependent facilitation was not significantly different from CaM (Fig. 1, G and I). These effects of VILIP-2 on Ca²⁺-dependent facilitation and inactivation of Ca_V2.1 channels are consistent with a model in which VILIP-2 displaces CaM and regulates Ca_V2.1 channels by slowing Ca²⁺-dependent inactivation and enhancing Ca²⁺-dependent facilitation (20).

Role of N-terminal Lobe of VILIP-2 in Ca²⁺-dependent Facilitation and Inactivation

CaM/VILIP-2 chimeras (Fig. 1A) were analyzed to examine which part of VILIP-2 mediates Ca²⁺-dependent facilitation. We included the N-terminal domain of VILIP-2 in all chimeras tested because it has been shown that deletion of the myristoylation site from the N terminus of VILIP-2 prevents its differential regulation of Ca_V2.1 channels (22). We tested three chimeras of the N-terminal lobe of VILIP-2. The first one included the N-terminal domain with its myristoyl moiety plus EF-hand 1 (N12H34), the second one added EF-hand 2 (N12H34), and the third one included the interlobe helix (N12H34) (Fig. 1A). Similar to endogenous CaM (Fig. 1B), co-expression of N12H34 with Ca_V2.1 channels supported normal Ca²⁺-dependent inactivation (Fig. 2, A and G). In contrast, co-expression of either N12H34 or N12H34 with Ca_V2.1 channels caused significantly faster inactivation of both I_Ca and I_Ba (Fig. 2, C, E, and G). Moreover, substitution of Ca²⁺ with Ba²⁺ did not slow inactivation for N12H34 (Fig. 2, C and G). Thus, the N-terminal lobe of VILIP-2 increases the rate of inactivation when substituted in CaM, opposite to the effect of intact VILIP-2.

FIGURE 2.

Modulation of Ca_V2.1 channels by chimeras of CaM and VILIP-2 N-terminal lobe. A, mean normalized I_Ca (n = 17) and I_Ba (n = 15) in cells transfected with Ca_V2.1 together with N12H34 chimera. B, mean normalized I_Ca (n = 16) and I_Ba (n = 18) during 100-Hz train of pulses from cells transfected with Ca_V2.1 and N12H34 chimera. C, mean normalized I_Ca (n = 12) and I_Ba (n = 10) from cells transfected with Ca_V2.1 together with N12H34 chimera. D, mean normalized I_Ca (n = 16) and I_Ba (n = 12) during 100-Hz train of pulses from cells transfected with Ca_V2.1 and N12H34 chimera. E, mean normalized I_Ca (n = 11) and I_Ba (n = 11) from cells transfected with Ca_V2.1 together with N12H34 chimera. F, mean normalized I_Ca (n = 8) and I_Ba (n = 10) during 100-Hz train of pulses from cells transfected with Ca_V2.1 and N12H34 chimera. G, mean ratio of I_Res over I_Peak from currents in A, C, and E in Ca²⁺ and Ba²⁺. Horizontal solid or dashed lines correspond to average Ca²⁺ or Ba²⁺ currents from cells transfected with Ca_V2.1 and VILIP-2, respectively. H, PPR from tail currents of I_Ca or I_Ba without a prepulse (P1) or with a 50-ms prepulse to +10 mV (P2) in cells transfected with Ca_V2.1 and CaM/VILIP-2 N-terminal lobe chimeras as in A, C, and E. The data shown are averages ± S.E. Asterisks indicate a significant difference (***, p < 0.001; **, p < 0.01; *, p < 0.05).

A similar pattern of effects on inactivation was observed in trains of depolarizations (Fig. 2, B, D, and F). The N12H34 chimera reduced facilitation when compared with CaM but induced similar Ca²⁺-dependent inactivation of I_Ca during a train of depolarizations (Figs. 1F and 2B). In contrast, the N12H34 and N12H34 chimeras prevented facilitation in a paired-pulse protocol and during a train of depolarizations, as well as accelerated inactivation during trains of depolarizations, similar to their effects during single pulses (Fig. 2, D, F, and H). Taken together, these results suggest that the N-terminal lobe of VILIP-2 is not responsible for its increase in facilitation or its slowing of inactivation because transfer of its EF-hands to CaM has the opposite effect on channel regulation.

Role of C-terminal Lobe of VILIP-2 in Ca²⁺-dependent Facilitation and Inactivation

To test whether facilitation is mediated by interaction of the C-terminal lobe of VILIP-2 with Ca_V2.1 channels, we constructed a CaM/VILIP-2 C-terminal lobe chimera (N12H34; Fig. 1A). Co-expression of N12H34 with Ca_V2.1 produced Ca²⁺-dependent inactivation similar to endogenous CaM during single depolarizations (Figs. 1, B and C, and 3, A and B). However, in contrast to CaM (Fig. 1, F and G), the N12H34 chimera blocked facilitation in a paired-pulse protocol and during trains of depolarizations, leaving only Ca²⁺-dependent inactivation (Fig. 4, A and B). Thus, the VILIP-2 C-terminal lobe alone does not induce facilitation of Ca_V2.1 channels when substituted in CaM chimeras.

FIGURE 3.

Modulation of Ca_V2.1 by chimeras of CaM and VILIP-2 C-terminal lobe. A, mean normalized I_Ca (n = 10) and I_Ba (n = 24) from cells transfected with Ca_V2.1 together with N12H34. B, mean ratio of I_Res over I_Peak from Ca_V2.1 and N12H34 chimera in Ca²⁺ and Ba²⁺. C, mean normalized I_Ca (n = 12) and I_Ba (n = 15) from cells transfected with Ca_V2.1 together with N12H34 chimera. D, mean ratio of I_Res over I_Peak from Ca_V2.1 and N12H34 chimera in Ca²⁺ and Ba²⁺. E, mean normalized I_Ca from cells transfected with Ca_V2.1 together with VILIP-2 (n = 10) or N12H34 chimera (n = 12). F, mean ratio of I_Res over I_Peak for I_Ca from cells transfected with Ca_V2.1and VILIP-2 or N12H34 chimera. The data shown are averages ± S.E. Asterisks indicate a significant difference (***, p < 0.001).

FIGURE 4.

Regulation of Ca²⁺-dependent facilitation by chimeras of CaM and VILIP-2 C-terminal lobe. A, mean normalized I_Ca (n = 14) and I_Ba (n = 24) during 100-Hz train of pulses from cells transfected with Ca_V2.1 together with N12H34 chimera. B, PPR from tail currents of I_Ca (n = 8) or I_Ba (n = 8) without a prepulse (P1) or with a 50-ms prepulse to +10 mV (P2) in cells transfected with Ca_V2.1 and N12H34 chimera. C, mean normalized I_Ca (n = 10) and I_Ba (n = 10) during 100-Hz train of pulses from cells transfected with Ca_V2.1 together with N12H34 chimera. D, PPR from tail currents of I_Ca (n = 12) or I_Ba (n = 10) without a prepulse (P1) or with a prepulse (P2) in cells transfected with Ca_V2.1 and N12H34 chimera. E, mean normalized I_Ca during 100-Hz train of pulses from cells with Ca_V2.1 together with VILIP-2 (n = 10) or N12H34 chimera (n = 10). F, voltage dependence of activation of Ca_V2.1 channels alone (circles; n = 10), with VILIP-2 (squares; n = 10) or with N12H34 chimera (triangles; n = 10). The data shown are averages ± S.E. Asterisks indicate a significant difference (***, p < 0.001).

To examine whether the addition of the interlobe helix of VILIP-2 is required for Ca²⁺-dependent facilitation of Ca_V2.1 channels, we tested a chimera in which the interlobe helix was added to the C-terminal lobe of VILIP-2 (N12H34; Fig. 1A). N12H34 prevented Ca²⁺-dependent inactivation so that I_Ca and I_Ba had an identical, slow rate of inactivation (Fig. 3, C and D). The slow rate of inactivation in the presence of N12H34 was identical to that induced by VILIP-2 (Fig. 3, E and F). In addition, N12H34 caused Ca²⁺-dependent facilitation in a paired-pulse protocol and during trains of depolarizations (Fig. 4, C and D), which was similar to VILIP-2 (Fig. 4E). Co-expression of VILIP-2 or the N12H34 chimera with Ca_V2.1 channels did not affect the voltage dependence of activation in response to different depolarizing membrane potentials (Fig. 4F). Taken together, the results from these chimeras indicate that the key elements required for the slowed Ca²⁺-dependent inactivation and increased Ca²⁺-dependent facilitation induced by VILIP-2 reside in the C-terminal lobe and the interlobe helix.

Role of IQ-like Motif and CBD of Ca_V2.1 Channels in Regulation by VILIP-2

The IQ-like motif and the downstream CBD in the C-terminal domain of Ca_V2.1 channels form the CaS protein regulatory site that is required for modulation by Ca²⁺ binding to endogenous CaM (14). Alanine substitution of the first two residues in the IQ-like motif of Ca_V2.1 channels (IM-AA) abolishes Ca²⁺/CaM-dependent facilitation during trains of depolarizations (12, 14). Ca²⁺ currents were evoked by single depolarizations or repetitive depolarizations at 100 Hz to assess the effects of VILIP-2 on Ca_V2.1/IM-AA channels. I_Ca conducted by Ca_V2.1/IM-AA channels had a rapid rate of inactivation, consistent with an intact Ca²⁺-dependent inactivation process as described previously (Fig. 5, A and B) (12, 14). Co-expression of VILIP-2 or N12H34 blocked Ca²⁺-dependent inactivation and slowed the overall inactivation rate to the level observed with Ba²⁺ as current carrier (Fig. 5, A, B, D, and E). These results indicate that the IM-AA mutation is not sufficient to prevent the effect of VILIP-2 to inhibit Ca²⁺-dependent inactivation. In contrast, both VILIP-2 and N12H34 failed to induce facilitation of the Ca_V2.1/IM-AA channels (Fig. 5, C and F). These results demonstrate that binding of VILIP-2 to the IQ-like domain of Ca_V2.1 channels is a primary requirement for Ca²⁺-dependent facilitation but not for Ca²⁺-dependent inactivation.

FIGURE 5.

Functions of the IQ-like motif in modulation of Ca_V2.1 channels by VILIP-2. A, mean normalized I_Ca from cells transfected with Ca_V2.1 and VILIP-2 (closed circles, n = 10), Ca_V2.1/IM-AA (open triangles; n = 9), and Ca_V2.1/IM-AA together with VILIP-2 (open circles; n = 10). B, mean ratio of I_Res over I_Peak from Ca_V2.1 and VILIP-2 (black), Ca_V2.1/IM-AA together with VILIP-2 (white), and Ca_V2.1/IM-AA (diagonal lines). C, mean normalized I_Ca during 100-Hz train of pulses from cells transfected with Ca_V2.1 and VILIP-2 (closed circles; n = 10) or Ca_V2.1/IM-AA together with VILIP-2 (open circles; n = 9). D, mean normalized I_Ca from cells transfected with Ca_V2.1/IM-AA alone (open triangles; n = 10), Ca_V2.1/IM-AA together with VILIP-2 (closed circles; n = 10); and Ca_V2.1/IM-AA together with N12H34 chimera (open circles; n = 10). E, mean ratio of I_Res over I_Peak from Ca_V2.1/IM-AA alone (diagonal lines), Ca_V2.1/IM-AA together with VILIP-2 (black) or Ca_V2.1/IM-AA together with N12H34 chimera (white). F, mean normalized I_Ca during 100-Hz train of pulses from cells transfected with Ca_V2.1/IM-AA with VILIP-2 (closed circles; n = 10) and Ca_V2.1/IM-AA together with N12H34 chimera (open circles; n = 10). The data shown are averages ± S.E. Asterisks indicate a significant difference (***, p < 0.001).

Deletion of the CBD has been shown to reduce Ca²⁺-dependent inactivation of Ca_V2.1 channels mediated by endogenous CaM (14). Unfortunately, this effect prevents testing the role of this site in VILIP-2 action because the effects of VILIP-2 would be occluded by the effects of the mutation. Thus, as expected, co-expression of VILIP-2 and N12H34 with Ca_V2.1 channels lacking the CBD domain (ΔCBD) had no effect on inactivation (Fig. 6, A, B, D, and E). To investigate the effects of VILIP-2 on facilitation of Ca_V2.1 channels lacking the CBD domain, we depolarized tsA-201 cells to +20 mV for 5 ms at a frequency of 100 Hz and recorded I_Ca. Ca_V2.1ΔCBD channels showed reduced facilitation and slower Ca²⁺-dependent inactivation than WT Ca_V2.1 channels (Figs. 1F and 6C). Ca_V2.1ΔCBD channels co-expressed with VILIP-2 or N12H34 chimera showed increased facilitation and slowed inactivation of I_Ca (Fig. 6, C and F). These results show that the CBD is not required for enhancement of facilitation of Ca_V2.1 channels by VILIP-2, consistent with previous results showing that it is not required for facilitation by endogenous CaM (14).

FIGURE 6.

Functions of the CBD in modulation of Ca_V2.1 channels by VILIP-2. A, mean normalized I_Ca from cells transfected with Ca_V2.1 and VILIP-2 (closed circles; n = 10) or Ca_V2.1-ΔCBD together with VILIP-2 (open circles; n = 10). B, mean ratio of I_Res over I_Peak from Ca_V2.1 and VILIP-2 (black) or Ca_V2.1-ΔCBD together with VILIP-2 (white). C, mean normalized I_Ca during 100-Hz train of pulses from cells transfected with Ca_V2.1 and VILIP-2 (closed circles; n = 10), Ca_V2.1-ΔCBD together with VILIP-2 (open circles; n = 6), and Ca_V2.1-ΔCBD alone (open triangles; n = 10). D, mean normalized I_Ca from cells transfected with Ca_V2.1-ΔCBD together with VILIP-2 (closed circles; n = 10) and Ca_V2.1-ΔCBD together with N12H34 chimera (open circles; n = 10). E, mean ratio of I_Res over I_Peak from Ca_V2.1-ΔCBD and VILIP-2 (black) or Ca_V2.1-ΔCBD together with N12H34 chimera (white). F, mean normalized I_Ca during 100-Hz train of pulses from cells transfected with Ca_V2.1-ΔCBD together with VILIP-2 (closed circles; n = 10) and Ca_V2.1-ΔCBD together with N12H34 chimera (open circles; n = 10). The data shown are averages ± S.E.

We further tested the site of VILIP-2 action in Ca²⁺-dependent facilitation using the double mutant Ca_V2.1ΔCBD/IM-AA. As expected, co-expression of VILIP-2 or the N12H34 chimera with Ca_V2.1ΔCBD/IM-AA had no effect on the slow rate of inactivation of I_Ca characteristic of mutants with the CBD deleted (Fig. 7, A, B, D, and E). However, this double mutation completely blocked facilitation induced by VILIP-2 during trains of depolarizations (Fig. 7, C and F), as expected from our results on Ca_V2.1/IM-AA channels. These results confirm that the IM-AA mutation effectively blocks enhanced facilitation by VILIP-2. Taken together, the results from our studies of mutations of the IQ-like motif and CBD indicate that the IM residues in the IQ-like motif are required for Ca²⁺-dependent facilitation but not for Ca²⁺-dependent inactivation of Ca_V2.1 channels and that the CBD is not required for Ca²⁺-dependent facilitation. These conclusions are consistent with previous findings on CaM regulation of Ca_V2.1 channels (14).

FIGURE 7.

Modulation of Ca_V2.1-ΔCBD/IM-AA channels by VILIP-2. A, mean normalized I_Ca from cells transfected with Ca_V2.1 and VILIP-2 (closed circles; n = 10) or Ca_V2.1-ΔCBD/IM-AA together with VILIP-2 (open circles; n = 12). B, mean ratio of I_Res over I_Peak from Ca_V2.1 and VILIP-2 (black) or Ca_V2.1-ΔCBD/IM-AA together with VILIP-2 (white). C, mean normalized I_Ca during 100-Hz train of pulses from cells transfected with Ca_V2.1 and VILIP-2 (closed circles; n = 10) or Ca_V2.1-ΔCBD/IM-AA together with VILIP-2 (open circles; n = 12). D, mean normalized I_Ca from cells transfected with Ca_V2.1-ΔCBD/IM-AA together with VILIP-2 (closed circles; n = 10) and Ca_V2.1-ΔCBD/IM-AA together with N12H34 chimera (open circles; n = 10). E, mean ratio of I_Res over I_Peak from Ca_V2.1-ΔCBD/IM-AA and VILIP-2 (black) or Ca_V2.1-ΔCBD/IM-AA together with N12H34 chimera (white). F, mean normalized I_Ca during 100-Hz train of pulses from cells transfected with Ca_V2.1-ΔCBD/IM-AA together with VILIP-2 (closed circles; n = 10) and Ca_V2.1-ΔCBD/IM-AA together with N12H34 chimera (open circles; n = 10). The data shown are averages ± S.E.

Binding of VILIP-2 to IQ-like Motif and CBD in Vitro

To test direct binding of VILIP-2 to the IQ-like motif, we used MBP-tagged VILIP-2 immobilized on amylose beads (Fig. 8A, Bait) and GST-tagged IM (Fig. 8A, Prey). We observed Ca²⁺-independent binding of the GST-tagged IM domain (a.a. 1848–1964) to VILIP-2-MBP, suggesting stable interaction of these two proteins at basal Ca²⁺ levels (Fig. 8, A and B). These results indicate that the IQ-like motif should be dominant in VILIP-2 binding at low Ca²⁺ levels in resting cells. Surprisingly, the IM-AA mutation did not prevent VILIP-2 binding to the IQ-like motif in vitro (Fig. 8, C and D). These results explain how VILIP-2 can retain its inhibition of Ca²⁺-dependent inactivation in the Ca_V1.2/IM-AA mutant because it should displace CaM, whose binding is blocked by the IM-AA mutation (14), and thereby prevent Ca²⁺-dependent inactivation as observed in Fig. 5, A and D. However, binding of VILIP-2 to Ca_V2.1 channels with the IM-AA mutation is not able to induce facilitation during trains of stimuli (Fig. 5, C and F).

FIGURE 8.

Binding of VILIP-2 to the IQ-like motif. A, Ca²⁺ independent binding of GST-tagged IM domain (Prey) (a.a. 1848–1964) with MBP-tagged VILIP-2 immobilized on amylose resin beads (Bait) in the presence of 2 μm Ca²⁺, 100 μm Ca²⁺, or 10 mm EGTA detected by immunoblot with anti-GST antibodies. B, normalized relative binding of the IM domain with VILIP-2. C, identical experiment to panel A but with the IM-AA mutant. D, normalized relative binding of the IM-AA domain with VILIP-2. The data shown are averages ± S.E. Asterisks indicate a significant difference (***, p < 0.001; **, p < 0.01).

Finally, we used MBP-tagged VILIP-2 immobilized on amylose beads (Fig. 9A, Bait) and GST-tagged CBD (Fig. 9A, Prey) to measure direct binding of VILIP-2 to the CBD. We observed Ca²⁺-dependent binding of the GST-tagged CBD (a.a. 1959–2035) to VILIP-2-MBP, but only at 100 μm Ca²⁺ (Fig. 9, A and B). These results indicate that interaction of VILIP-2 with the CBD is likely to contribute to its overall binding affinity only during trains of stimuli that raise the concentration of free Ca²⁺ to this level near active zones where Ca_V2.1 channels are clustered (4, 5).

FIGURE 9.

Binding of VILIP-2 to the CBD. A, Ca²⁺-dependent binding of GST-tagged CBD (Prey) (a.a. 1959–2035) with MBP-tagged VILIP-2 immobilized on amylose resin beads (Bait) in the presence of 2 μm Ca²⁺, 100 μm Ca²⁺, or 10 mm EGTA detected by immunoblot with anti-MBP antibodies. B, normalized relative binding of the CBD with VILIP-2. The data shown are averages ± S.E. Asterisks indicate a significant difference (***, p < 0.001).

DISCUSSION

N-terminal Lobes of CaBP1 and VILIP-2 Enhance Ca²⁺-dependent Inactivation

CaM and neuronal CaS proteins contain four EF-hand motifs separated into two lobes by a central α-helical domain. CaS proteins differ from CaM in that they have an extended N-terminal domain, including an N-terminal myristoyl lipid anchor, and at least one EF-hand that cannot bind Ca²⁺. For example, EF-hand 1 of VILIP-2 and EF-hand 2 of CaBP1 do not bind Ca²⁺ (17, 23). The N-terminal lobe of CaM containing EF-hands 1 and 2 is primarily responsible for Ca²⁺-dependent inactivation (12, 14). The N-terminal domain and EF-hand 2 of CaBP1 are sufficient to enhance inactivation in chimeras with CaM, suggesting that CaBP1 modulation of Ca_V2.1 channels is mediated by its unique EF-hand that is inactive in Ca²⁺ binding (24). Our results presented here show that transfer of the N-terminal lobe of VILIP-2 containing its EF-hands 1 and 2 increases inactivation of Ca_V2.1 channels in both Ca²⁺ and Ba²⁺ extracellular solutions (Figs. 2, C and G, and Fig. 10A). These results suggest that the N-terminal lobe containing the EF-hands that are inactive in Ca²⁺ binding (EF-hand 2 of CaBP1 and EF-hand 1 of VILIP-2) may actually be constitutively active in enhancing inactivation independent of Ca²⁺ in the context of the complete N-terminal lobes of these CaS proteins. The chimeric CaS proteins may be dominant over endogenous CaM in this respect. The ability of the N-terminal lobe to enhance inactivation is apparently suppressed in intact VILIP-2 by the presence of the C-terminal lobe of the protein plus the interlobe helix.

FIGURE 10.

Regulation of Ca_V2.1 channels by proposed functional domains of VILIP-2. A, schematic drawing of VILIP-2. The grayed area denotes its functional domains responsible for enhanced inactivation. B, schematic drawing of VILIP-2. The grayed area denotes its functional domains responsible for enhanced facilitation. C, schematic drawing of the interaction of CaM with the IM and CBD domains of the Ca_V2.1 channel. D, schematic drawing of the interaction of VILIP-2 with the IM and CBD domains of the Ca_V2.1 channel after displacement of CaM. The solid line with arrow indicates the dominant interaction of VILIP-2 with the IM domain.

C-terminal Lobe of VILIP-2 Enhances Ca²⁺-dependent Facilitation

CaM induces Ca²⁺-dependent facilitation through interaction of its C-terminal lobe containing EF-hands 3 and 4 with the IQ-like motif of Ca_V2.1 channels (14). VILIP-2 enhances Ca²⁺-dependent facilitation of Ca_V2.1 channels (20). Our results show that the functional domain of VILIP-2 responsible for enhanced facilitation is the C-terminal lobe together with the interlobe helix (Fig. 10B). Thus, Ca²⁺-dependent facilitation is controlled by the C-terminal lobes of both CaM and VILIP-2. Chelation of Ca²⁺ by 10 mm EGTA blocks Ca²⁺-dependent inactivation but not Ca²⁺-dependent facilitation, consistent with activation of the facilitation process in response to brief, local increases in Ca²⁺ that cannot be intercepted by EGTA (11). These results suggest that Ca²⁺ influx through Ca_V2.1 channels generates a local increase in Ca²⁺, which promotes Ca²⁺ binding to the C-terminal lobe of preassociated CaM and initiates Ca²⁺-dependent facilitation by interaction with the IQ-like motif (Fig. 10C). In contrast, VILIP-2 binds to the IQ-like domain itself at low levels of Ca²⁺, prevents CaM binding, and enhances Ca²⁺-dependent facilitation when local Ca²⁺ levels rise (Fig. 10D). More global Ca²⁺ increases result in Ca²⁺ binding to the N-terminal lobe of CaM and initiation of Ca²⁺-dependent inactivation (Fig. 10C), which is prevented in the presence of VILIP-2 by its ability to bind at low levels of Ca²⁺ and displace CaM binding (Fig. 10D).

Roles of CBD and IQ-like Motif in Regulation of Ca_V2.1 Channels by VILIP-2

Previous results showed that deletion of the CBD of Ca_V2.1 channels preferentially decreases Ca²⁺/CaM-dependent inactivation and that mutation of the IQ-like motif preferentially reduces Ca²⁺/CaM-dependent facilitation (14). As expected from these results, Ca_V2.1-ΔCBD channels conduct Ca²⁺ currents that lack Ca²⁺-dependent inactivation, and in this respect, they are functionally similar to WT Ca_V2.1 co-expressed with VILIP-2 (20). Co-expression of VILIP-2 with Ca_V2.1-ΔCBD channels has no effect, but this is expected because these channels have no remaining Ca²⁺-dependent inactivation for VILIP-2 to inhibit.

Ca²⁺-dependent facilitation of Ca_V2.1-ΔCBD channels was enhanced by VILIP-2 similarly to WT Ca_V2.1. These results are consistent with previous work showing that the Ca²⁺/CaM-dependent facilitation is blocked by the IM-AA mutation and is less affected by deletion of the CBD (14). Taken together, these results suggest that enhanced facilitation of Ca_V2.1 channels by VILIP-2 is not mediated by its interaction with the CBD but do not allow a definite conclusion on the role of the CBD in the effects of VILIP-2 on Ca²⁺-dependent inactivation.

It has previously been shown that binding of CaM to the IQ-like motif induces Ca²⁺-dependent facilitation (12, 14). Our results indicate that VILIP-2 also binds to the IQ-like domain and that mutation of its first two residues (IM-AA) completely prevents enhancement of facilitation by VILIP-2. These results therefore suggest that the IQ-like domain is necessary for VILIP-2 modulation of Ca²⁺-dependent facilitation. In contrast to these results, we did not observe any effect of the IM-AA mutation on reduction of Ca²⁺-dependent inactivation in the presence of VILIP-2. These results match previous work in which the IM-AA mutation blocked Ca²⁺/CaM-dependent facilitation but did not have a significant effect on Ca²⁺-dependent inactivation (14). Overall, these results suggest that Ca²⁺-dependent facilitation requires high-affinity binding of CaM or VILIP-2 to the IQ-like domain, which induces a conformational change that enhances facilitation in Ca_V2.1 channels but not in the Ca_V2.1/IM-AA mutant. In contrast, Ca²⁺-dependent inactivation does not require binding to the IM domain and is retained in the IM-AA mutant, which therefore can still respond to VILIP-2 with slowed inactivation.

CaS Proteins and Short-term Synaptic Plasticity

Our previous results show that VILIP-2 and CaBP1 bind to the same regulatory site as CaM and differentially regulate Ca_V2.1 channels, favoring facilitation or inactivation, respectively (19, 20). Regulation of Ca_V2.1 channels expressed in synapses of sympathetic ganglion neurons by this mechanism causes synaptic facilitation and a rapid phase of synaptic depression (15). These results demonstrate that binding of Ca²⁺/CaM to the IQ-like motif and CBD of Ca_V2.1 channels is sufficient to induce short-term synaptic plasticity and to determine its direction, i.e. facilitation versus depression. Neuronal calcium sensor-1 (NCS-1), which is closely related to VILIP-2, can enhance P/Q-type Ca²⁺ currents in the Calyx of Held and can facilitate synaptic transmission in the Calyx of Held and in cultured hippocampal neurons (26, 27), but there is no evidence that it acts by directly binding to Ca_V2.1 channels. VILIP-2 and CaBP1 favor facilitation or depression, respectively, at sympathetic neuron synapses transfected with Ca_V2.1 channels and CaS proteins (28). These effects on short-term synaptic plasticity are blocked by mutation of the IQ-like motif and the CBD, demonstrating that direct regulation of Ca_V2.1 channels by binding of these neuronal CaS proteins can regulate short-term synaptic plasticity in a bidirectional manner. Because these CaS proteins can serve as a bidirectional switch and induce facilitation or depression of synaptic transmission, it is of great interest to understand the molecular basis for their differential regulation of Ca_V2.1 channels.

CaM is the most abundant CaS protein and is expressed at high levels in most eukaryotic cells (29, 30). VILIP-2 is mainly expressed in the caudate-putamen, neocortex, hippocampus, and cerebellum (17, 31). Ca_V2.1 is also present in those brain areas and is differentially localized in specific sets of synapses (32–35). Neuronal CaS proteins have higher affinities for Ca²⁺ than CaM (16, 25). Therefore, differential expression of these neurospecific CaS proteins, together with their distinct affinities for binding to Ca_V2.1 channels, would fine-tune the neurotransmitter release and synaptic plasticity initiated by these channels (4). Our results presented here provide the initial insights into the molecular code for the differential regulation of Ca_V2.1 channels and synaptic plasticity by different neuronal CaS proteins. Evidently, domain-specific differences in amino acid sequence in these CaS proteins are both necessary and sufficient to define the regulatory mode of these crucial neuronal signaling proteins in short-term synaptic plasticity.