Differential Regulation of Ca_V2.1 Channels by Calcium-Binding Protein 1 and Visinin-Like Protein-2 Requires N-Terminal Myristoylation

Effect of myristoylation of CaBP1 on voltage-dependent activation of Ca_V2.1 channels

To investigate the functional role of myristoylation of CaBP1, we used a mutant, CaBP1/G2A, in which the glycine that is normally myristoylated has been mutated to alanine, a mutation that is known to prevent N-terminal myristoylation of proteins (Rocque et al., 1993; Olshevskaya et al., 1997). We began by characterizing the voltage dependence of Ca_V2.1 activation in cells cotransfected with wild-type or myristoyl-deficient CaBP1. Cotransfection of CaBP1 with Ca_V2.1 causes a positive shift and a reduction in the slope of the voltage dependence of activation (Lee et al., 2002). The voltage of half-activation (V_1/2) of Ca²⁺ currents (I_Ca; V_1/2 = 18.6 ± 2.2 mV), measured from the normalized tail current-voltage curve in cells cotransfected with Ca_V2.1 and CaBP1, was shifted by +8.0 mV compared with cells transfected with Ca_V2.1 alone (V_1/2 = 10.6 ± 1.8 mV). The V_1/2 for activation in cells cotransfected with Ca_V2.1 and CaBP1/G2A (V_1/2 = 7.7 ± 1.0 mV) was indistinguishable from that of cells transfected with Ca_V2.1 alone. (Fig. 1A,B, left). The slope of the activation curve was reduced in cells cotransfected with Ca_V2.1 and CaBP1 (slope factor k = 8.1 ± 0.4 mV per e-fold change in current) compared with cells transfected with Ca_V2.1 alone (k = 6.0 ± 0.4 mV). In contrast, there was no significant difference between the slope of the voltage dependence of activation between cells cotransfected with Ca_V2.1 plus CaBP1/G2A (k = 5.7 ± 0.2 mV) versus cells transfected with Ca_V2.1 alone (Fig. 1A,B, right). These data indicate that myristoylation of CaBP1 is required for the positive shift and the reduction of the slope of voltage-dependent activation of Ca_V2.1 channels.

Regulation of Ca_V2.1 by CaBP1 is Ca²⁺ independent (Lee et al., 2002). To test the importance of myristoylation in the absence of Ca²⁺, we characterized the voltage dependence of activation of Ca_V2.1 channels with the replacement of Ca²⁺ with Ba²⁺ as the charge carrier. In cells cotransfected with Ca_V2.1 and CaBP1, the activation curve for Ba²⁺ current (I_Ba) was shifted to more positive potentials (V_1/2 = 10.6 ± 2.7 mV) compared with cells transfected with Ca_V2.1 alone (V_1/2 = 4.6 ± 2.2 mV). When CaBP1/G2A was cotransfected with Ca_V2.1, the voltage dependence of activation (V_1/2 = 0.1 ± 1.8 mV) was indistinguishable from that for cells transfected with only Ca_V2.1 and was significantly different from cells cotransfected with Ca_V2.1 and wild-type CaBP1 (Fig. 1C,D, left). The slopes of the activation curves for I_Ba are not significantly different in any of the three conditions (Fig. 1C,D, right). These results demonstrate a requirement for myristoylation of CaBP1 to shift the voltage dependence of activation. Because CaBP1/G2A retains the ability to bind to Ca_V2.1 channels in transfected tsA-201 cells (Figs. 4C, 5), these results indicate that myristoylation is required for this functional effect of CaBP1.

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Figure 3.

CaBP1/G2A allows Ca²⁺-dependent facilitation of Ca_V2.1 channels. A, Voltage protocol used to measure paired-pulse facilitation. B-D, Voltage dependence of Ca_V2.1 tail currents (I_Ca) evoked before (p1, •) or after (p2, ○) a 50 ms depolarization to +10 mV to allow Ca²⁺ entry.B, Cells transfected with Ca_V2.1, β_2a, and α₂δ (n = 10). C, Cells cotransfected with Ca_V2.1, β_2a, α₂δ and CaBP1 (n = 9). D, Cells cotransfected with Ca_V2.1, β_2a, α₂δ, and CaBP1/G2A (n = 14). E, Paired-pulse ratios of tail currents from data in B-D elicited by an initial depolarization from -80 to +80 mV (^*p < 0.05; ^**p < 0.01). F, Repetitive depolarizations (2 ms)from -80 to +20 mV in Ca²⁺ were given at a frequency of 166 Hz. The first current elicited in the train was normalized to 1.0, with each subsequent current expressed as a fraction of the first and plotted against time. I_Ca from cells transfected with Ca_V2.1, β_2a, α₂δ (•, n = 8), and CaBP1 (, n = 5) or CaBP1/G2A (□, n = 9). The data shown are averages ± SEM, with every 10th data point and every 20th error bar plotted for clarity (^*p < 0.05; ^**p < 0.01). The peak amplitude of I_Ca was substantially reduced by coexpression of CaBP1 (Fig. 6). Therefore, in F, we compared results from cells with peak I_Ca < 1.6 nA to avoid artifacts caused by greater Ca²⁺ entry in the control cells.

Effect of myristoylation of CaBP1 on inactivation of Ca_V2.1 channels

To further examine whether myristoylation of CaBP1 was required for acceleration of inactivation of Ca_V2.1 channels, we compared the effects of wild-type and CaBP1/G2A on inactivation of I_Ca (Fig. 2A,B) and quantitated the results as the ratio of the current remaining 200 ms after the initiation of a 1 s depolarization to the peak current during the depolarization (I₂₀₀/I_pk) (Fig. 2C). The rate of inactivation of I_Ca is faster in cells in which Ca_V2.1 is coexpressed with CaBP1 (I₂₀₀/I_pk = 0.65 ± 0.06) (Fig. 2A, light gray trace) compared with cells in which Ca_V2.1 is modulated by endogenous CaM (I₂₀₀/I_pk = 0.83 ± 0.03) (Fig. 2A, black trace; C), as described previously (Lee et al., 2002). I_Ca inactivates more slowly in cells coexpressing Ca_V2.1 and CaBP1/G2A (I₂₀₀/I_pk = 0.84 ± 0.02) (Fig. 2B, dark gray trace) than in cells coexpressing Ca_V2.1 channels and wild-type CaBP1 (Fig. 2B, light gray trace; C) and is not different from inactivation of Ca_V2.1 when channels are modulated by endogenous CaM (Fig. 2C). These data indicate that myristoylation of CaBP1 is required to accelerate the rate of inactivation of I_Ca conducted by Ca_V2.1 channels.

To examine the effect of Ca²⁺, we performed experiments in which Ba²⁺ was used as the charge carrier instead of Ca²⁺. Wild-type CaBP1 accelerates the inactivation of I_Ba, (Fig, 2D, light gray trace). In contrast, CaBP1/G2A does not accelerate inactivation of Ca_V2.1 compared with wild-type CaBP1 (Fig. 2E, dark gray trace) [I₂₀₀/I_pk = 0.92 ± 0.02 for CaBP1/G2A compared with 0.7 ± 0.07 for CaBP1 (Fig. 2F)]. Similar to experiments measuring the rate of I_Ca inactivation, the rate of Ca_V2.1 inactivation of I_Ba is not significantly different when channels are expressed alone (I₂₀₀/I_pk = 0.92 ± 0.01) or coexpressed with CaBP1/G2A (I₂₀₀/I_pk = 0.92 ± 0.02) (Fig. 2F).

Ca²⁺-dependent facilitation of Ca_V2.1 channels coexpressed with CaBP1/G2A

Ca²⁺/CaM binding to the IQ-like domain in the C terminus of Ca_V2.1 channels produces Ca²⁺-dependent facilitation in response to paired pulses or trains of action potential-like stimuli (Lee et al., 2000, 2003), whereas coexpression of CaBP1 blocks facilitation (Lee et al., 2002). We assessed whether CaBP1/G2A could also block Ca²⁺-dependent facilitation of Ca_V2.1 channels. In cells expressing Ca_V2.1 alone, where channels are modulated by endogenous CaM, Ca²⁺ influx during a short prepulse produced a significant increase in the tail current elicited by a subsequent test pulse across a wide range of test pulse potentials (Fig. 3A,B,E). In cells expressing both Ca_V2.1 and CaBP1, the amplitude of the tail current elicited after the prepulse was not increased (Fig. 3C,E). In contrast, facilitation was observed with the same voltage protocol when CaBP1/G2A was coexpressed with Ca_V2.1 channels (Fig. 3D,E), indicating that myristoylation of CaBP1 is required to block Ca²⁺/CaM-induced facilitation of Ca_V2.1 channels. These data support the conclusion that myristoylation of CaBP1 is required to produce functional modulation of Ca_V2.1 channels that is distinct from modulation by CaM.

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Figure 4.

The affinity of CaBP1/G2A for Ca_V2.1 channels is reduced. Lysates from cells (transfected as indicated) were collected in medium with 10 mm EGTA or 2 mm Ca²⁺ and were immunoprecipitated with an antibody recognizing the α₁ subunit of Ca_V2.1 or IgG. The immunoprecipitate was analyzed by immunoblotting for the presence of CaBP1 or CaBP1/G2A. Top panels show immunoblots of immunoprecipitates, and bottom panels show immunoblots of lysates. A, Cells were analyzed without cross-linking. B, Cells were treated with the bifunctional cross-linker DSP, as described in Materials and Methods, before lysis. C, The extent of coimmunoprecipitation in B was estimated from densitometric scans of CaBP1 and CaBP1/G2A in the coimmunoprecipitate and normalized for expression of the corresponding bands in the lysate. The value for wild-type CaBP1 was set to 1, and the fraction of CaBP1/G2A binding was normalized by the same value for side-by-side experiments. The data shown are mean ± SEM (n = 8 in EGTA; n = 7 in 2 mm Ca²⁺).

To further test whether myristoylation of CaBP1/G2A is required for modulation of Ca_V2.1 channels, we measured facilitation in response to trains of 2 ms depolarizations from -80 to +20 mV. These brief repetitive depolarizations maximize Ca²⁺/CaM-dependent facilitation by preventing Ca²⁺-dependent inactivation (Lee et al., 2000) and are similar to trains of action potentials and therefore represent more physiologically relevant stimuli. I_Ca modulated by endogenous CaM in cells expressing Ca_V2.1 alone showed increasing facilitation as the number of stimuli increased (Fig. 3F, black circles). Cells coexpressing CaBP1 and Ca_V2.1 had much reduced facilitation, which became insignificant by 800 ms of stimulation (Fig. 3F, gray circles). I_Ca in cells coexpressing CaBP1/G2A and Ca_V2.1 channels exhibited an intermediate effect (Fig. 3F, open squares). Initially, I_Ca showed facilitation that was not different from the facilitation induced by CaM. However, as the number of stimuli increased, facilitation of I_Ca decreased, and, by the end of the train of 250 stimuli, facilitation of I_Ca remaining in cells coexpressing CaBP1/G2A was significantly less than in cells expressing only endogenous CaM. Our working model for the effects of nCaBPs on Ca_V2.1 channels (see Discussion) is that CaBP1 blocks facilitation by inducing such rapid, voltage-dependent inactivation during each depolarizing stimulus that facilitation is occluded. Our results with CaBP1/G2A show that this effect is not detectable in paired-pulse experiments and in the initial few depolarizations during trains of stimuli. In contrast, CaBP1/G2A does accelerate pulse-wise inactivation and thereby reduces facilitation later in trains of stimuli, as illustrated in Figure 3F. These data suggest that CaBP1/G2A retains some ability to enhance inactivation, which builds up from pulse to pulse in repetitive pulse experiments. A <1% increase in cumulative inactivation of I_Ca in each depolarization/repolarization cycle in the 250-pulse train would be sufficient to reduce the degree of facilitation by the extent observed in Figure 3F.

Binding of CaBP1/G2A to Ca_V2.1 channels

Modulation of Ca_V2.1 channels by CaBP1/G2A was indistinguishable from modulation of channels by CaM in every functional test we performed except one, the modulation in response to trains of action potential-like stimuli. Because myristoylation is known to be important for membrane targeting of many nCaBPs, it was possible that CaBP1/G2A was not localized at the plasma membrane, where it could exert functional effects on the channel. Alternatively, CaBP1/G2A may bind to Ca_V2.1 channels but may not be capable of modulating their function normally. To distinguish between these possibilities, we assessed the ability of CaBP1/G2A to bind to Ca_V2.1 channels in transfected cells. CaBP1 coimmunoprecipitated with Ca_V2.1 in cotransfected tsA-201 cells when cells were treated with the Ca²⁺ ionophore ionomycin to increase intracellular Ca²⁺ or when Ca²⁺ was buffered with EGTA (Fig. 4A), consistent with previous work (Lee et al., 2002). In coimmunoprecipitations performed side by side with wild-type CaBP1, CaBP1/G2A did not coimmunoprecipitate with the channel as effectively as wild type in the presence or absence of Ca²⁺ (Fig. 4A), indicating that the CaBP1/G2A does not bind to the channel as stably as CaBP1. However, the appearance of a faint band corresponding in size to CaBP1/G2A in the coimmunoprecipitate without Ca²⁺ suggests that CaBP1/G2A can bind to Ca_V2.1 channels stably enough to be detected after extraction and immunoprecipitation.

It is possible that the binding of CaBP1/G2A to the channel was reduced because of incorrect targeting of CaBP1/G2A to the plasma membrane or because of a reduction in the stability of the complex of CaBP1/G2A and Ca_V2.1, which allowed dissociation during extraction and immunoprecipitation. To address the latter possibility, we performed coimmunoprecipitations after treating intact cells with a bifunctional cross-linker (DSP) to covalently link complexes of Ca_V2.1 and CaBP1/G2A in situ in intact cells. Under these conditions, CaBP1/G2A did coimmunoprecipitate with Ca_V2.1 channels both when Ca²⁺ was elevated by treating cells with Ca²⁺ ionophore before cross-linking and when cell lysates were buffered with EGTA. However, the amount of CaBP1/G2A that coimmunoprecipitated with the channel appeared to be reduced compared with wild-type CaBP1 in some experiments (Fig. 4B). Densitometric scans of the immunoprecipitated bands and normalization to the level of expression in the corresponding lysate samples indicated a significant decrease in coimmunoprecipitation of CaBP1/G2A compared with wild type in the absence of Ca²⁺ (Fig. 4C) (0.47 ± 0.09, SEM; n = 8; p < 0.01) but no decrease in the coimmunoprecipitation of CaBP1/G2A compared with wild type in the presence of Ca²⁺ (0.84 ± 0.15, SEM; n = 7; p > 0.05). Thus, CaBP1/G2A does bind to Ca_V2.1 channels effectively in transfected tsA-201 cells, although its binding may be reduced compared with wild-type CaBP1 in the absence of Ca²⁺.

Colocalization of CaBP1/G2A with Ca_V2.1 channels

We performed immunocytochemical experiments to further assess the ability of CaBP1/G2A to bind to Ca_V2.1 in intact cells. First, we examined the subcellular localization of CaBP1 or CaBP1/G2A expressed alone in tsA-201 cells. In cells transfected with CaBP1 only, immunolabeling was seen at or near the plasma membrane with exclusion from the nucleus (Fig. 5A). A similar pattern of immunolabeling was observed in cells that were treated with ionomycin to increase intracellular Ca²⁺ (Fig. 5B), indicating that CaBP1 does not undergo a Ca²⁺-dependent membrane translocation, similar to other nCaBPs such as NCS-1 and K⁺ channel interacting protein 1 (KChip1) (O'Callaghan and Burgoyne, 2003, 2004). In contrast, in cells transfected with CaBP1/G2A, immunolabeling of CaBP1/G2A was more diffuse, without exclusion from the nucleus (Fig. 5C), consistent with an important role of myristoylation in targeting of CaBP1 (Haynes et al., 2004). The pattern of immunolabeling was similar when cells were treated with ionomycin (Fig. 5D). No labeling was detected in control experiments in which transfected cells were labeled with a secondary antibody only (data not shown). In addition, untransfected cells treated with both primary and secondary antibodies showed no labeling (data not shown). These data indicate that myristoylation of CaBP1 is necessary for targeting at or near the plasma membrane in tsA-201 cells without Ca²⁺ channels.

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Figure 5.

Myristoyl-deficient CaBP1 colocalizes with Ca_V2.1 channels in tsA-201 cells. A, B, Confocal images of tsA-201 cells transfected with CaBP1 alone and fixed at resting Ca²⁺ levels (A) or after treatment with ionomycin (B). C, D, Confocal images of cells transfected with CaBP1/G2A alone and fixed at resting Ca²⁺ levels (C) or after treatment with ionomycin (D). E, F, I, J, M, N, Confocal images of cells transfected with Ca_V2.1, β_2a, α₂δ, and CaBP1 fixed at resting Ca²⁺ levels (E, I, M) or after treatment with ionomycin (F, J, N). G, H, K, L, O, P, Confocal images of cells transfected with Ca_V2.1, β_2a, α₂δ, and CaBP1/G2A fixed at resting Ca²⁺ levels (G, K, O) or after treatment with ionomycin (H, L, P). Scale bar, 50 μm. Q, Line scans of cells transfected with CaBP1/G2A alone, showing fluorescence intensity as a function of position across the cell diameter after fixation at resting Ca²⁺ levels (n = 8). R, Line scans of cells transfected with Ca_V2.1, β_2a, α₂δ, and CaBP1/G2A, fixed at resting Ca²⁺ levels (n = 10). S, Line scans of cells transfected with CaBP1/G2A alone, fixed after treatment with ionomycin (n = 4). T, Line scans of cells transfected with Ca_V2.1, β_2a, α₂δ, and CaBP1/G2A, fixed after treatment with ionomycin (n = 7). Red traces in Q-T correspond to line scans of the cells in images in C, G, D, and H, respectively, whereas black traces were obtained from other similarly treated cells.

Subsequently, we examined the ability of CaBP1 and CaBP1/G2A to colocalize with Ca_V2.1 channels in intact cells. When tsA-201 cells were cotransfected with CaBP1 and Ca_V2.1 and fixed at resting Ca²⁺ levels, immunolabeling showed colocalization of CaBP1 and Ca_V2.1 at or near the plasma membrane (Fig. 5E,I,M), consistent with the binding of CaBP1 to Ca_V2.1 observed in coimmunoprecipitation studies. When cells were treated with ionomycin to increase intracellular Ca²⁺ levels, the pattern of immunolabeling (Fig. 5F,J,N) was not different from labeling of cells at resting Ca²⁺ levels, consistent with the finding that regulation of Ca_V2.1 channels by CaBP1 is Ca²⁺ independent (Lee et al., 2002). Cells cotransfected with CaBP1/G2A and Ca_V2.1 also showed colocalization of CaBP1/G2A and Ca_V2.1 at or near the plasma membrane at both resting Ca²⁺ levels (Fig. 5G,K,O) and elevated intracellular Ca²⁺ levels (Fig. 5H,L,P). Thus, coexpression of Ca_V2.1 channels alters the distribution of CaBP1/G2A from general distribution throughout the cell when expressed alone (Fig. 5C,D) to colocalization with Ca_V2.1 channels at or near the plasma membrane when expressed together (Fig. 5G,H,O,P). These results indicate that CaBP1/G2A binds to Ca_V2.1 channels in intact tsA-201 cells, consistent with our coimmunoprecipitation results on cross-linked complexes of CaBP1/G2A and Ca_V2.1 channels.

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Figure 6.

CaBP1 and VILIP-2 differentially modulate Ca_V2.1 expression. A, Ca²⁺ tail current-voltage curves from cells cotransfected with Ca_V2.1, β_2a, α₂δ (•, n = 15), and CaBP1 (○, n = 13), or CaBP1/G2A (□, n = 18) show myristoylation of CaBP1 is required to decrease Ca_V2.1 current density. The data shown are averages ± SEM (^*p < 0.05; ^**p < 0.01). B, Top, Immunoblots for the α₁2.1 subunit or heat shock protein 90 (Hsp90) from lysates of cells transfected with Ca_V2.1, β_2a, α₂δ (left), plus CaBP1 (middle), or VILIP-2 (right). Bottom, Lysates from cells transfected with Ca_V2.1, β_2a, α₂δ (n = 9), and CaBP1 (n = 6), or VILIP-2 (n = 5) were analyzed by immunoblotting for α₁2.1, Hsp90, and CaBP1 or VILIP-2. Both isoforms of α₁2.1 (Sakurai et al., 1996) were included in the densitometric scan. α₁2.1 expression was normalized to Hsp90 intensity to control for equal loading. Error bars represent SEM.

Additional evidence to support binding of CaBP1/G2A to Ca_V2.1 channels in intact cells derives from comparison of line scans of the subcellular localization of CaBP1/G2A when expressed alone with the localization of CaBP1/G2A when coexpressed with Ca_V2.1 channels. Line scans across the diameter of the cell through the center of the nucleus show a plateau of fluorescence across the cell at both resting and elevated intracellular Ca²⁺ levels for cells transfected with CaBP1/G2A alone (Fig. 5Q,S). In contrast, cells coexpressing CaBP1/G2A and Ca_V2.1 show substantial peaks of fluorescence at or near the plasma membrane (Fig. 5R,T). This quantitative change in the subcellular localization of CaBP1/G2A caused by overexpression of a target for binding indicates that CaBP1/G2A binds to Ca_V2.1 channels in intact cells.

Differential modulation of Ca_V2.1 expression by CaBP1 and VILIP-2

In addition to the previously observed effects of CaBP1 on the function of Ca_V2.1 channels (Lee et al., 2002), we characterized a new modulatory effect of CaBP1 on expression of Ca_V2.1 channels. In cells cotransfected with CaBP1 and Ca_V2.1 channels, the amplitude of the Ca²⁺ tail current (Fig. 6A, open circles) was smaller than that for cells transfected with Ca_V2.1 alone (Fig. 6A, filled circles) over the entire range of voltages tested. This effect is Ca²⁺ independent, because amplitudes of I_Ba in cells transfected with CaBP1 and Ca_V2.1 are also smaller than in cells transfected with Ca_V2.1 channels only (data not shown). These data indicate that CaBP1 substantially decreases peak currents conducted by Ca_V2.1 channels. In cells cotransfected with CaBP1/G2A and Ca_V2.1 channels, the peak Ca²⁺ tail current (Fig. 6A, open squares) is not different from cells transfected with Ca_V2.1 channels only (Fig. 6A, filled circles). A similar effect of CaBP1/G2A is observed for I_Ba (data not shown). Therefore, consistent with its other modulatory effects, myristoylation of CaBP1 is required to decrease expression of functional Ca_V2.1 channels.

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Figure 7.

Myristoylation of VILIP-2 is required for distinct modulation of Ca_V2.1 channels. A, B, I_Ca elicited by a 1 s depolarization from -80 to +10 mV in cells transfected with Ca_V2.1, β_2a, α₂δ (A, black trace, n = 17), and VILIP-2 (A, B, light gray trace, n = 12), or VILIP-2/G2A (B, dark gray trace, n = 7). C, Amplitudes of the residual Ca²⁺ currents at the ends of the depolarizations (I_res) were normalized to the peak (I_pk) and plotted for the data in A and B. The data are shown as smoothed averages ± SEM with every 100th error bar plotted for clarity (^*p < 0.05; ^**p < 0.01).

To examine the mechanism by which CaBP1 decreases peak currents, we measured the level of Ca_V2.1 protein in lysates of transfected cells compared with the constitutively expressed housekeeping protein Hsp90 (heat shock protein 90). Immunoblots of lysates from tsA-201 cells transfected with CaBP1 and Ca_V2.1 show a decrease in the intensity of staining for α₁2.1 compared with cells transfected with Ca_V2.1 alone (Fig. 6B). To determine whether this decrease in Ca_V2.1 expression was an artifact of cotransfection, we cotransfected a different nCaBP that modulates Ca_V2.1 channels, VILIP-2 (Lautermilch et al., 2005). Lysates from cells expressing Ca_V2.1 and VILIP-2 did not show a decrease in staining for α₁2.1 (Fig. 6B), indicating that the reduction of expression is a specific effect of CaBP1.

Effect of myristoylation on modulation of Ca_V2.1 channels by VILIP-2

Coexpression of VILIP-2 has the opposite effect on the rate of inactivation of Ca_V2.1 channels as CaBP1 (Lautermilch et al., 2005). To determine whether myristoylation, which is a conserved feature of the nCaBP family, is also required for the distinct modulation of Ca_V2.1 channels by VILIP-2, we used a myristoyl-deficient mutant of VILIP-2, VILIP-2/G2A. I_Ca in cells coexpressing Ca_V2.1 and VILIP-2 inactivates more slowly [I_res/I_pk = 0.65 ± 0.04 (Fig. 7A, light gray trace)] than I_Ca in cells expressing only Ca_V2.1 channels modulated by endogenous CaM (I_res/I_pk = 0.40 ± 0.04) (Fig. 7A, black trace), consistent with previous findings (Lautermilch et al., 2005). However, I_Ca in cells coexpressing VILIP-2/G2A and Ca_V2.1 channels inactivates more rapidly [I_res/I_pk = 0.44 ± 0.07 (Fig. 7B, dark gray trace)] than I_Ca in cells coexpressing VILIP-2 and Ca_V2.1 (Fig. 7B, light gray trace) and is not significantly different from modulation of Ca_V2.1 inactivation by endogenous CaM (Fig. 7C). These data indicate that myristoylation of VILIP-2 is required to slow the rate of Ca_V2.1 inactivation and suggest that myristoylation, a conserved feature of nCaBPs, may be essential to allow these proteins to differentially regulate Ca_V2.1 channels.

Myristoylation of CaBP1 and VILIP-2 is required for their distinct modulation of inactivation

Our results with the myristoylation-deficient mutants CaBP1/G2A and VILIP-2/G2A show that myristoylation of CaBP1 and VILIP-2 is required to produce their characteristic regulation of inactivation of Ca_V2.1 channels, which is distinct from regulation by CaM. CaBP1 increases inactivation of Ca_V2.1 channels whether Ca²⁺ or Ba²⁺ is the permeant ion, whereas VILIP-2 decreases inactivation whether Ca²⁺ or Ba²⁺ is the permeantion. In contrast, coexpression of the myristoylation-deficient mutants with Ca_V2.1 channels has no effect on Ca²⁺-dependent inactivation. Thus, myristoylation is required for the characteristic effects of these nCaBPs on Ca_V2.1 channels, although they have opposite effects on inactivation.

Myristoylation of CaBP1 is required for block of paired-pulse facilitation and for modulation of expression and activation

CaBP1 blocks Ca²⁺/CaM-dependent facilitation of Ca_V2.1 channels, but VILIP-2 does not (Lee et al., 2002; Lautermilch et al., 2005). As for inactivation, block of paired-pulse facilitation of Ca_V2.1 channels by CaBP1 requires myristoylation, although partial block of facilitation by CaBP1/G2A is observed late in trains of stimuli. Similarly, CaBP1 positively shifts voltage-dependent activation and reduces functional expression of Ca_V2.1 channels, whereas VILIP-2 does not. These effects of CaBP1 also require myristoylation. Thus, myristoylation is required for the characteristic effects of CaBP1 on expression, activation, inactivation, and paired-pulse facilitation of Ca_V2.1 channels and for the normal level of block of facilitation in trains of stimuli.

Although the effects of CaBP1 on Ca_V2.1 channel function were described previously, the decrease in the amplitude of peak Ca_V2.1 currents because of reduced expression of the α₁2.1 subunit is a new effect. Our results do not distinguish whether this reduction in protein expression is caused by decreased transcription and/or translation, decreased posttranslational processing and targeting, or increased degradation. However, effects on transcription and translation are less likely, because these processes are driven by the promoter and translation start signals in the cDNA vector rather than those specified by the Ca_V2.1 gene. Although VILIP-2 does not change the expression of Ca_V2.1, the related nCaBP, NCS-1, reduces expression of the α₁ subunits of Ca_V1.2, Ca_V2.1, and Ca_V2.2 channels when expressed with β₁, β₂, and β₄ subunits but not with β₃ subunits (Rousset et al., 2003). This suggests that regulation of Ca_V2.1 peak current amplitude by CaBP1 may also require specific β subunit(s). However, we found that CaBP1 reduces peak Ca_V2.1 currents when coexpressed with β_1b (data not shown) as well as with β_2a.

Myristoylation enhances, but is not required for, binding of CaBP1

CaBP1 and CaM bind to the CaM-binding domain (CBD) in the C terminus of Ca_V2.1 channels (Lee et al., 2002). Coimmunoprecipitation experiments with in situ cross-linking indicate that myristoyl-deficient CaBP1 binds to Ca_V2.1 channels but with reduced affinity and/or stability in the absence of Ca²⁺. Colocalization studies indicate that Ca_V2.1 channels bind myristoyl-deficient CaBP1 in intact cells and redirect its distribution to or near the plasma membrane. The failure of CaBP1/G2A to alter activation, inactivation, and paired-pulse facilitation of Ca_V2.1 channels, although it binds to them in intact cells, indicates that myristoyl-deficient CaBP1 acts functionally like CaM when bound to their common binding site.

In previous work (Lee et al., 2002) and in these experiments, we found that CaBP1 binding and action is independent of Ca²⁺ from the resting level in tsA-201 cells to the high level achieved in the presence of Ca²⁺ ionophore. In contrast, Haynes et al. (2004) have shown that Ca_V2.1 channels in brain extracts interact with immobilized glutathione S-transferase/CaBP1 in a Ca²⁺-dependent manner. One explanation for this apparent discrepancy may be that binding of CaBP1 occurs in intact cells at 37°C and resting intracellular Ca²⁺ (∼100 nm) in our experiments, whereas binding occurs in vitro at 4°C and nominally zero Ca²⁺ in the experiments by Haynes et al. (2004).

Control of CaBP1 localization by N-terminal myristoylation

Myristoylation has been shown to control membrane localization of several nCaBPs, including NCS-1, KChip1, hippocalcin, VILIP-1, and recoverin (Dizhoor et al., 1993; Kobayashi et al., 1993; Spilker et al., 1997; O'Callaghan and Burgoyne, 2003; O'Callaghan et al., 2003). Although myristoylation of CaBP1 is necessary for localization at or near the plasma membrane when it is expressed alone [Haynes et al. (2004) and the present study], we conclude that myristoylation of CaBP1 is not essential for association with a membrane-bound target protein like Ca_V2.1 channels. Overexpression of other target proteins changes the localization of nCaBPs (O'Callaghan et al., 2003). For example, overexpression of K_v4.2 localizes KChip1 to the plasma membrane, whereas KChip1 is located in punctate intracellular structures when expressed alone (O'Callaghan et al., 2003).

Myristoyl groups can impart regulated membrane association to nCaBPs via a Ca²⁺-myristoyl switch. However, the constitutive membrane association of CaBP1 in the absence of Ca²⁺ indicates that either CaBP1 does not have a functional Ca²⁺-myristoyl switch or its Ca²⁺-myristoyl switch is activated at the resting intracellular Ca²⁺. NCS-1/frequenin and KChip1 are also membrane-associated in their Ca²⁺-free and Ca²⁺-bound states (O'Callaghan and Burgoyne, 2003). The NMR structure of frequenin, a Drosophila homolog of NCS-1, shows that the myristoyl group is exposed to an aqueous environment in Ca²⁺-free and Ca²⁺-bound forms (Ames et al., 2000), which provides a structural basis for lack of a Ca²⁺-myristoyl switch. A similar structural mechanism may explain the lack of CaBP1 translocation in response to increases in intracellular Ca²⁺. The presence or absence of a Ca²⁺-myristoyl switch may determine the time scale on which nCaBPs respond to Ca²⁺. Proteins that undergo a Ca²⁺-myristoyl switch may require longer, more widespread elevations in Ca²⁺ to translocate to membranes and regulate their Ca²⁺-dependent targets, whereas nCaBPs that are constitutively bound to their targets may regulate them constitutively or may quickly respond to local Ca²⁺ elevations (O'Callaghan et al., 2002; O'Callaghan and Burgoyne, 2003).

Control of CaBP1 and VILIP-2 function by N-terminal myristoylation

Although myristoylation is not essential for binding of CaBP1 to Ca_V2.1 channels, it is required for normal functional activity. How might myristoylation control the function of CaBP1 and VILIP-2? In addition to their role in targeting to specific membrane compartments, myristoyl groups often regulate the functional activity of nCaBPs. For example, myristoylation of recoverin is not necessary to inhibit rhodopsin kinase (Chen et al., 1995). However, by inducing cooperativity in Ca²⁺ binding to the two functional EF-hands in recoverin, myristoylation increases its inhibition of rhodopsin kinase (Kawamura et al., 1994; Calvert et al., 1995; Senin et al., 1995). Myristoylation also enhances cooperativity of Ca²⁺ binding to neurocalcin δ (Ames et al., 1995; Ladant, 1995). Thus, conformational changes in nCaBPs can be controlled by N-terminal myristoylation.

Our results provide a preliminary indication of how altered conformational changes in myristoylated CaBP1 and VILIP-2 may cause their distinct regulation of Ca_V2.1 channels. EF-hands 1 and 2 in the N-terminal lobe of CaM interact with the CBD in the C terminus of Ca_v1.2 channels to initiate Ca²⁺-dependent inactivation (Lee et al., 2003). EF-hand 2 of CaBP1 is inactive in Ca²⁺ binding, whereas EF-hand 1 of VILIP-2 is inactive. Our results suggest that interaction of these N-terminal EF-hands with Ca_V2.1 channels is altered by N-terminal myristoylation. In the absence of myristoylation, these EF-hands bind to Ca_V2.1 channels and act like CaM, causing Ca²⁺-dependent inactivation, as we observed for CaBP1/G2A and VILIP-2/G2A. We propose that myristoylation of CaBP1 allows enhancement of inactivation at resting intracellular Ca²⁺ mediated by its inactive EF-hand 2, whereas myristoylation of VILIP-2 allows inhibition of inactivation at resting intracellular Ca²⁺ mediated by its inactive EF-hand 1. The active EF-hands 3 and 4 of CaBP1 and VILIP-2 likely cause CaM-like Ca²⁺-dependent facilitation in both wild-type and myristoyl-deficient forms of these proteins. However, this facilitation is immediately reversed by the enhanced inactivation caused by myristoylated CaBP1 leading to apparent block of facilitation (Fig. 3F), whereas it is enhanced by the slowed inactivation caused by myristoylated VILIP-2 (Lautermilch et al., 2005).

Differential regulation of Ca_V2.1 channels by nCaBPs and synaptic plasticity

nCaBPs that undergo large conformational changes after binding Ca²⁺ (Cox et al., 1994; Burgoyne et al., 2004) and bind Ca²⁺ with submicromolar affinities (Burgoyne and Weiss, 2001) are poised to act as potent regulators of synaptic transmission. nCaBPs may modulate synaptic responses by acting as local buffers that regulate residual Ca²⁺ that accumulates with multiple action potentials (Zucker, 2003). In addition, the Ca²⁺-dependent conformational changes that nCaBPs experience may allow these proteins to act as regulators to produce facilitation and depression of neurotransmission (Zucker, 2003). By regulating presynaptic Ca_V2.1 channels, CaBP1, VILIP-2, and NCS-1 can directly shape presynaptic Ca²⁺ transients, which will have profound effects on the time course and amount of neurotransmitter release. Because CaBP1 and VILIP-2 are differentially expressed throughout the nervous system (Braunewell and Gundelfinger, 1999; Haeseleer et al., 2000), they will regulate the output of neurons in a cell-type-specific manner. Myristoylation, by changing the conformation, cooperativity of Ca²⁺-binding, and targeting of nCaBPs, can tune the functional activity of these proteins.