Differential modulation of Ca_v2.1 channels by calmodulin and Ca²⁺-binding protein 1

CaBP1 interacts with the CBD of α₁2.1

Although CaBP1 is a neuron-specific Ca²⁺-binding protein that shares nearly 56% amino acid sequence identity with CaM²⁵, it differs in having a consensus site for N-terminal myristoylation, an alternatively spliced region, inactivating amino acid substitutions in the second of the four EF-hand motifs, and an extra turn in the helical domain that links the N- and C-terminal lobes (Fig. 1a). To determine whether CaBP1 can substitute for CaM in interactions with Ca_v2.1 channels, we tested the ability of CaBP1 to interact with various intracellular domains of the α₁2.1 subunit. In yeast two-hybrid assays, CaBP1 activated transcription of HIS3 and lacZ reporter genes only in yeast that had been cotransformed with α₁2.1 constructs that included the CBD (Fig. 1b and c). CaBP1 did not interact with the IQ domain or with a control plasmid that lacked the CaBP1 coding region. These results indicated that CaBP1 may modulate Ca_v2.1 channel function through interactions with the CBD.

To confirm that CaBP1 associated with the CBD in the intact channel, we tested whether CaBP1 co-immunoprecipitated with Ca_v2.1 channels from cotransfected tsA-201 cells. In this assay, CaM co-immunoprecipitates with α₁2.1 in a Ca²⁺-dependent manner only when the cells are exposed to Ca²⁺ ionophore⁹. Under these conditions CaBP1 also co-immunoprecipitated with α₁2.1. When Ca²⁺ was buffered with 10 mM EGTA, however, the association of CaBP1 with the channel was not affected (Fig. 2a). This co-immunoprecipitation of CaBP1 was specific because CaBP1 was not immunoprecipitated with control IgG or with α₁2.1-specific antibodies in cells transfected with CaBP1 alone, and CaBP1 did not co-immunoprecipitate with α₁2.1 subunits that lacked the CBD. Thus, despite its Ca²⁺ independence, the interaction between CaBP1 and Ca_v2.1 channels requires the same intracellular domain of Ca_v2.1 that binds CaM.

CaBP1 associates with neuronal Ca_v2.1 channels

To determine whether CaBP1 associated with endogenous Ca_v2.1 Ca²⁺ channels, co-immunoprecipitation experiments were done with extracts from rat cerebellum, which contains high concentrations of α₁2.1 and CaBP1 mRNA^25,26. Immunoblots of CaBP1 showed two proteins (28 and 36 kDa) that specifically co-immunoprecipitated with α₁2.1 (Fig. 2b, left) but not with control IgG (Fig. 2b, middle). The 36-kDa species might represent caldendrin, a larger isoform of CaBP1 that is produced from alternative splicing^25,27. The 28-kDa species was consistent in size with the predicted molecular mass of the long CaBP1 isoform that we used in transfected cells (Fig. 2b, right), in support of a physiological interaction between neuronal Ca_v2.1 channels and CaBP1.

To identify the potential cellular sites of interaction between CaBP1 and α₁2.1, we immunostained rat brain sections with antibodies specific for both proteins. Compared with the immunostaining of α₁2.1, the immunostaining for CaBP1 was generally far more restricted within the brain and more commonly associated with somatodendritic regions than with nerve terminals. However, CaBP1 and α₁2.1 showed similar patterns of punctate staining in the CA1 region of the hippocampus and in the molecular layer of the cerebellum (Fig. 3a–f). As a large proportion of punctate labeling of α₁2.1 in the cerebellum colocalizes with that of syntaxin²⁸, it is likely that CaBP1 and Ca_v2.1 channels coexist in at least some presynaptic nerve terminals. Immunostaining for CaBP1 and for α₁2.1 also overlapped in clusters along the dendrites of cerebellar Purkinje neurons and in structures that resembled dendritic spines (data not shown), however, which suggests that CaBP1 may associate with Ca_v2.1 channels in the post- as well as in the presynaptic membrane.

CaBP1 enhances inactivation of Ca_v2.1 channels

To elucidate the functional consequences of the interaction between CaBP1 and Ca_v2.1 channels, we determined the effect of CaBP1 on Ca²⁺ currents (I_Ca) in whole-cell patch-clamp recordings of transfected tsA-201 cells. We first compared the effects of transfected CaBP1 and endogenous CaM on inactivation of I_Ca. We have shown previously that Ca^2+Ca/CaM enhances the inactivation of I_Ca during step depolarizations when intracellular recording solutions contain 0.5 mM EGTA^9,10. Here, inactivation of I_Ca caused by Ca²⁺/CaM proceeded with a single exponential time course (τ = 852.3 ± 63.7 ms at +20 mV, n = 18) that was relatively insensitive to the test voltage (Fig. 4a and b). By contrast, CaBP1 caused I_Ca to decay significantly faster than in cells that were transfected with only Ca_v2.1.

In almost all of the cells that were cotransfected with CaBP1, the decay of I_Ca evoked by +20- and +30-mV pulses was best fit by a double exponential function, with a slow component similar to control and a fast component comprising 30–40% of the peak current (Fig. 4c). With a +10-mV test pulse, however, biphasic inactivation was detected in only 11 out of 20 cells that had been cotransfected with CaBP1. At this test voltage, which elicits the peak inward I_Ca, Ca²⁺/CaM-dependent inactivation is maximal¹⁰. Therefore, the absence of a fast phase of inactivation in some cells cotransfected with CaBP1 might have resulted from more effective competition by Ca²⁺/CaM.

Competition between CaM and CaBP1 for Ca_v2.1 channels was supported further by the observation of a marked reduction in Ca²⁺-dependent inactivation in cells cotransfected with CaBP1 (Fig. 5a and b). Enhanced inactivation caused by CaM results in a significant reduction in the residual current at the end of a 1-second depolarizing test pulse normalized to the peak current (I_res/I_pk) for I_Ca as compared with I_Ba (refs. ^{9, 10}). By contrast, in cells cotransfected with CaBP1, I_res/I_pk was already reduced when Ba²⁺ was the charge carrier and was not significantly different for I_Ca and I_Ba (Fig. 5a and b). Ca²⁺-dependent inactivation was not affected in the same way by cotransfection with CaM instead of CaBP1, which indicated that the faster, Ca²⁺-independent inactivation was a specific consequence of the modulation of Ca_v2.1 channels by CaBP1.

To clarify the effects of CaBP1 on fast inactivation of Ca_v2.1 channels, we measured the amplitude of I_Ca at the 200-ms time point during a 1-s test pulse and normalized this to the peak current (I₂₀₀/I_pk, Fig. 5c and d). We used more positive test voltages to limit Ca²⁺ entry, thus minimizing the contribution of endogenous CaM in these experiments. With 0.5 mM EGTA, faster inactivation of I_Ca in cells with CaBP1 caused a significant decrease in I₂₀₀/I_pk (0.45 ± 0.06 for CaBP1 versus 0.79 ± 0.03 for control, p < 0.01). CaBP1 significantly enhanced fast inactivation of I_Ba (I₂₀₀/I_pk of 0.43 ± 0.09 for CaBP1 versus 0.74 ± 0.07 for control, p < 0.05) and also of I_Ca with 10 mM of the intracellular calcium chelator BAPTA (I₂₀₀/I_pk of 0.56 ± 0.08 for CaBP1 versus 0.85 ± 0.03 for control, p < 0.02). The CBD was essential for these effects on inactivation, because CaBP1 had no effect on channels in which this domain had been deleted (Fig. 5c and d, Ca_v2.1_ΔCBD; p > 0.3). Together with biochemical analyses, these results support a Ca²⁺-independent association of CaBP1 with the CBD, which mediates a strong acceleration of Ca_v2.1 channel inactivation that does not require Ca²⁺ influx or intracellular accumulation of Ca²⁺.

CaBP1 shifts voltage dependence of Ca_v2.1 activation

In cells cotransfected with CaBP1 and Ca_v2.1, the normalized tail current–voltage curve was shifted positively and was shallower than in cells transfected with Ca_v2.1 alone (Fig. 6a). CaBP1 caused significant increases in the half-activation voltage, V_1/2 (12.8 ± 1.3 mV for CaBP1 versus 4.5 ± 0.9 mV for control, p < 0.01), and slope factor of the tail current–voltage curve (−8. 7 ± 0.5 mV for CaBP1 versus −5.2 ± 0.6 mV for control, p < 0.01). Similar to the other actions of CaBP1 on Ca_v2.1 channels, these effects on I_Ca activation were essentially reproduced with intracellular BAPTA and extracellular Ba²⁺ but were not observed with Ca_v2.1_ΔCBD channels (Fig. 6b–d), which indicates that the Ca²⁺-independent association of CaBP1 with the CBD results in a newly identified, multifaceted regulation of Ca_v2.1 channels.

Ca²⁺-dependent facilitation is not supported by CaBP1

Activity-dependent increases in intracellular Ca²⁺ cause an initial facilitation of I_Ca owing to the interaction of Ca²⁺/CaM with Ca_v2.1 channels (refs ^{10, 15}). This Ca²⁺-dependent facilitation was evident with 0.5 mM intracellular EGTA in paired-pulse protocols, in which Ca²⁺ influx during a short prepulse induced a significant increase in the tail current elicited by a subsequent test pulse (Fig. 7a). With the same voltage protocol, no facilitation of I_Ca was observed in cells cotransfected with CaBP1 (Fig. 7b). Because of the strong voltage-dependent enhancement of I_Ca inactivation caused by CaBP1, it was possible that paired-pulse facilitation in cells cotransfected with CaBP1 might have been obscured by the onset of inactivation during the conditioning prepulse. Alternatively, CaBP1, unlike CaM, might not support Ca²⁺-dependent facilitation of I_Ca.

To distinguish between these possibilities, we analyzed the properties of I_Ca during trains of short (5-ms) repetitive depolarizations, which should initially minimize the impact of voltage-dependent inactivation and reveal facilitation of I_Ca early in the train. With 0.5 mM intracellular EGTA, Ca_v2.1 Ca²⁺ currents undergo a sustained facilitation and gradually inactivate below initial current amplitudes after 800 ms of repetitive pulses (Fig. 7c), an effect that depends on Ca²⁺/CaM¹⁰. In cells cotransfected with CaBP1, facilitation of I_Ca was reduced markedly, with current amplitudes rapidly inactivating below initial values only 200 ms into the train (Fig. 7c).

The maximum facilitated I_Ca amplitude at 50 ms in cells cotransfected with CaBP1 (1.04 ± 0.02, n = 13) was not significantly different from the Ca²⁺-independent facilitation of Ba²⁺ currents in cells transfected with Ca_v2.1 alone (1.04 ± 0.03, n =5, p = 0.80) or cotransfected with CaBP1 (1.02 ± 0.02, n = 11, p = 0.52; Fig. 7d), which indicated that CaBP1 does not support Ca²⁺-dependent facilitation of Ca_v2.1 channels. Together with the enhanced inactivation and positive shifts in activation caused by CaBP1, the absence of Ca²⁺-dependent facilitation would strongly limit voltage-dependent Ca²⁺ entry through Ca_v2.1 channels. These results highlight further the different modulation of these Ca²⁺ channels by CaBP1 and CaM.

Ca²⁺-independent binding and modulation by CaBP1

The Ca²⁺ independence of the interaction between CaBP1 and Ca_v2.1 was unexpected given the previously observed Ca²⁺-dependent association of CaBP1 with other CaM targets²⁵. It is possible that very local rises in Ca²⁺ might have escaped buffering by BAPTA in our experiments, which could have been sufficient for binding to CaBP1 and for causing Ca²⁺-dependent modulation of I_Ca. This possibility seems unlikely, however, because the modulation by CaBP1 did not change appreciably when Ba²⁺ was the permeant ion; Ba²⁺ ions bind to EF-hand motifs with relatively low affinity and so should not reproduce Ca²⁺-dependent regulation of target molecules²⁹.

Calcium-free CaM can associate with and regulate several targets, including the ryanodine receptor RyR1, cyclic GMP kinase and a CaM-dependent adenylyl cyclase from Bordetella pertussis^30-32. In addition, GCAPs—photoreceptor Ca²⁺-binding proteins—activate guanylyl cyclases in their Ca²⁺-free forms^33,34. Thus, CaBP1 might have a similar flexibility and interact with and regulate some effectors without binding Ca²⁺. Although we cannot exclude the possibility that CaBP1 may modulate some aspects of Ca²⁺ channel function in a Ca²⁺-dependent manner, we found no evidence to support a requirement for Ca²⁺ in the effects of CaBP1 on the activation and inactivation of Ca_v2.1 channels. Thus, despite the Ca²⁺-sensing capability of CaBP1, we propose that CaBP1 itself does not mediate Ca²⁺-dependent regulation of Ca_v2.1 channels, but might indirectly influence feedback regulation by Ca²⁺ by competing with CaM. Thus, CaBP1 may act more like auxiliary Ca²⁺ channel β-subunits by altering the intrinsic properties of Ca_v2.1 channels to fine-tune voltage-gated Ca²⁺ entry in specific classes of neurons.

Distinct modulation of Ca_v2.1 by CaBP1 and CaM

We have shown that both CaM and CaBP1 interact with the CBD of the α₁2.1 subunit and that this site is essential for full channel regulation by both proteins. Conflicting evidence indicates that the IQ domain—a sequence that is N-terminal to the CBD—is involved in the modulation of Ca_v2.1 channels by Ca²⁺/CaM¹⁵. Our results do not support the importance of the IQ domain in modulation by CaBP1 because, first, CaBP1 interacted with the CBD but not the IQ domain in yeast two-hybrid assays (Fig. 1b and c); second, deleting the CBD prevented the co-immunoprecipitation of CaBP1 with α₁2.1 (Fig. 2a); and third, the fast inactivation and shifts in the voltage dependence of activation caused by CaBP1 were abolished in channels that lacked the CBD (Figs. 5 and 6). Notably, removing the CBD from α₁2.1 eliminated regulation by CaBP1 more completely than it eliminated regulation by CaM¹⁰. Together, our results indicate that the CBD may be the primary determinant for the functional effects of CaBP1 on Ca_v2.1 channels.

If both CaM and CaBP1 interact with the CBD, how is it that CaBP1 causes Ca²⁺-independent fast inactivation and positively shifted activation, whereas CaM causes Ca²⁺-dependent facilitation and inactivation of Ca_v2.1 channels? One possibility is that key structural features that distinguish CaBP1 from CaM, such as its extra-long central helical domain and N-terminal myristoylation (Fig. 1a), may permit Ca²⁺-independent binding of CaBP1 to the CBD, which might then lead to its unique inhibitory modulation of I_Ca. Future experiments that determine how such differences between CaBP1 and CaM contribute to specific forms of Ca_v2.1 regulation may reveal how ion channels and other signaling molecules are differentially modulated by CaM and related Ca²⁺-binding proteins.

Modulation of neuronal Ca_v2.1 channels by CaBP1

Our immunoprecipitation and immunofluorescence studies showed that CaBP1 and α₁2.1 associate physically in extracts of rat cerebellum and that their subcellular distributions overlap in this brain region, which indicates that CaBP1 may have a physiological role in the regulation of Ca_v2.1 channels. Because both CaM and CaBP1 interacted with the same site on the α₁2.1 subunit, an important issue is whether Ca_v2.1 would interact functionally with CaM and/or CaBP1 in neurons in which both Ca²⁺-binding proteins are expressed.

Although we do not know whether CaM and CaBP1 bind simultaneously to Ca_v2.1 channels, our electrophysiological studies suggested that CaM and CaBP1 might competitively regulate the channel. CaBP1 more strongly enhanced inactivation of I_Ca when the influence of Ca²⁺/CaM was suppressed either with extracellular Ba²⁺ or intracellular BAPTA, or with test voltages that elicited submaximal Ca²⁺ influx. These results imply that when intracellular Ca²⁺ concentrations are high Ca_v2.1 channels may be facilitated predominantly by CaM, and that the inactivating effects of CaBP1 become most prominent when cytoplasmic Ca²⁺ concentrations decline. In this way, CaM and CaBP1 may coordinately act as a molecular switch to intensify neuronal Ca²⁺ influx in response to activity-dependent alterations in intracellular concentrations of Ca²⁺.

NCBPs and synaptic transmission

Emerging evidence supports a role for NCBPs in the regulation of synaptic transmission. In particular, neuronal Ca²⁺ sensor-1 (NCS-1), which is more distantly related to CaM than is CaBP1, regulates neurotransmitter release³⁵, synapse formation³⁶ and neuronal circuits that control associative learning³⁷. Notably, NCS-1 has been implicated in the negative regulation of Ca²⁺ channels in chromaffin cells³⁸, which suggests that Ca_v2.1 channels may be modulated by NCBPs in addition to CaBP1.

Given the widespread distribution of Ca_v2.1 channels throughout the nervous system, the cell type–specific modulation of Ca_v2.1 by CaBP1, CaM or other NCBPs may fundamentally determine the nature of presynaptic and postsynaptic Ca²⁺ signals and the functional consequences of synaptic activity.