CaBP1 Regulates Voltage-dependent Inactivation and Activation of CaV1.2 (L-type) Calcium Channels*

CaBP1 is a Ca2+-binding protein that regulates the gating of voltage-gated (CaV) Ca2+ channels. In the CaV1.2 channel α1-subunit (α1C), CaBP1 interacts with cytosolic N- and C-terminal domains and blunts Ca2+-dependent inactivation. To clarify the role of the α1C N-terminal domain in CaBP1 regulation, we compared the effects of CaBP1 on two alternatively spliced variants of α1C containing a long or short N-terminal domain. In both isoforms, CaBP1 inhibited Ca2+-dependent inactivation but also caused a depolarizing shift in voltage-dependent activation and enhanced voltage-dependent inactivation (VDI). In binding assays, CaBP1 interacted with the distal third of the N-terminal domain in a Ca2+-independent manner. This segment is distinct from the previously identified calmodulin-binding site in the N terminus. However, deletion of a segment in the proximal N-terminal domain of both α1C isoforms, which spared the CaBP1-binding site, inhibited the effect of CaBP1 on VDI. This result suggests a modular organization of the α1C N-terminal domain, with separate determinants for CaBP1 binding and transduction of the effect on VDI. Our findings expand the diversity and mechanisms of CaV channel regulation by CaBP1 and define a novel modulatory function for the initial segment of the N terminus of α1C.

CaBP1 is a Ca 2ϩ -binding protein enriched in the brain and retina (2). It has emerged as a prominent regulator of Ca V channel CDI (4). The molecular determinants underlying CaBP1 regulation have been most well characterized for Ca V 1.2 (␣ 1C ) channels, which play crucial roles in Ca 2ϩ signaling and excitability in the nervous and cardiovascular systems (14). In contrast to Ca 2ϩ /CaM, which stabilizes the inactivated state of Ca V 1.2 (24 -26), CaBP1 fully eliminates CDI and supports Ca 2ϩ -dependent facilitation (6,27). CaBP1 binds to the C-terminal CaM-binding ␣ 1C sites but also interacts with the ␣ 1C N-terminal domain, the deletion of which blunts the effect of CaBP1 on CDI (28). The N terminus of ␣ 1C regulates several aspects of channel gating as well as modulation by protein kinase C (29 -32). Within the N terminus, two modular segments are involved in CaM binding (33,34) and regulation of P o (35). A "long N-terminal" alternatively spliced ␣ 1C isoform possesses a 20-amino acid (aa) initial segment (N-terminal inhibitory (NTI) module), which controls the channel's maximal P o (30,35). A second module in the central part of the N-terminal domain is a CaM-binding site, the N-terminal spatial Ca 2ϩ -transforming element (NSCaTE), which regulates CDI in Ca V 1.3 (33), but its role in Ca V 1.2 is less clear (34). The exact location of the CaBP1-binding site within the N terminus of ␣ 1C has not been determined, and whether CaBP1 influences parameters of Ca V 1.2 function other than CDI is unknown.
Here, we report that CaBP1 expression in Xenopus oocytes regulates activation gating and accelerates VDI of Ca V 1.2. Although CaBP1 binds to a site in the distal N-terminal domain that is distinct from the CaM-binding site, CaBP1 regulation of VDI requires an intact proximal domain in the N terminus of ␣ 1C . Deletion of the entire N-terminal domain inhibits but does not fully abolish effects of CaBP1 on VDI, which indicates the presence of additional relevant molecular determinants. These results suggest a new modulatory role for the ␣ 1C N terminus, in which CaBP1 binding and transduction of the modulation of VDI are encoded by distinct N-terminal domains.
Electrophysiological studies of HEK293T cells involved transient transfection of HEK293T cells with Ca V 1.2 with or without CaBP1. In some experiments, we utilized T-REx-293 cells (Invitrogen) stably transfected with GFP-tagged CaBP1 under tetracycline-inducible expression. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C under 5% CO 2 . Cells plated in 35-mm tissue culture dishes were grown to 65-80% confluency and transfected with FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's protocol. Cells were transfected with 2 g of DNA coding for the rabbit LNT␣ 1C or rat rbcII␣ 1C variant or their truncated forms, 1 g of ␤ 2a , 1 g of ␣ 2 ␦, and 0.05 g of enhanced GFP with or without 0.05-0.1 g of CaBP1, all in the pcDNA3 vector. For stably transfected T-REx-293 cells, cells were transfected only with Ca V 1.2 subunit cDNAs and 0.05 g of mCherry plasmid to mark transfected cells. Tetracycline (1 g/ml) was added to the culture medium 6 -8 h before recording to induce CaBP1 expression. Patch-clamp recordings of all cells were made at least 24 h after transfection. There were no significant differences between channel properties or regulation by CaBP1 in the transiently transfected and stably transfected cells, so the data from both systems were combined for analysis.
Electrophysiology and Data Analysis-In Xenopus oocytes, the procedures were essentially as described (35). In brief, whole cell currents were recorded with the GeneClamp 500 amplifier Molecular Devices using a two-electrode voltage clamp. 25-30 nl of 50 mM BAPTA (Ca 2ϩ chelator) was routinely injected into the oocytes 0.5-3 h before the measurement of currents. This procedure usually blocked the endogenous Ca 2ϩ -dependent Cl Ϫ currents; cells with residual Cl Ϫ currents (distinguished by long-lasting inward tails at Ϫ80 mV) were excluded from analysis. The extracellular solutions contained 40 mM Ba(OH) 2 or Ca(OH) 2 , 50 mM NaOH, 2 mM KOH, and 5 mM HEPES, titrated to pH 7.5 with methanesulfonic acid. In each oocyte, the net current was obtained by subtraction of the residual currents recorded with the same protocols after applying 200 M CdCl 2 to the same solution (supplemental Fig. S1A) (35). Stimulation, data acquisition, and analysis were per-formed using pCLAMP 10.2 software (Molecular Devices). All experiments were performed at 20 -22°C. Currents were measured by 400-ms, 1000-ms, or 10-s pulses from a resting potential of Ϫ80 mV to different potentials, with 60-s intervals for the 10-s pulses and with 10-s intervals for shorter pulses. A currentvoltage (I-V) curve was fitted to the Boltzmann equation in the form I ϭ G max (V m Ϫ V rev )/(1 ϩ exp(Ϫ(V m Ϫ V a )/K a )), where K a is the slope factor, V a is the half-maximal activation voltage, G max is the maximal macroscopic conductance, and V rev is the reversal potential of the current. The parameters obtained for G max and V rev were then used to calculate fractional conductance at each V m using the equation The waveform of decay of the Ba 2ϩ current (I Ba ) was fitted, using a Levenberg-Marquardt algorithm, to a two-exponential equation in the form f(t) ϭ A fast e Ϫt/fast ϩ A slow e Ϫt/slow ϩ C, where A is the contribution of each kinetic component ( fast or slow ) of the decay of I Ba , and C is the non-inactivating component (supplemental Fig. S1B and Table S2).
In transfected HEK293T cells, Ba 2ϩ currents were recorded in whole cell patch-clamp configuration at room temperature. Extracellular recording solutions contained 150 mM Tris, 2 mM MgCl 2 , and 10 mM BaCl 2 . Intracellular recording solutions contained 130 mM N-methyl-D-glucamine, 60 mM HEPES, 1 mM MgCl 2 , 2 mM MgATP, and 5 mM EGTA. The pH of the extracellular and intracellular recording solutions was adjusted to 7.3 with methanesulfonic acid. Reagents used for electrophysiological recordings were obtained from Sigma. Currents were recorded with an EPC 9 patch-clamp amplifier driven by PULSE software (HEKA Electronics, Lambrecht/Pfalz, Germany). Leak and capacitive transients were subtracted using a P4 protocol. To measure VDI, I Ba was evoked by 1-s depolarizations to ϩ10 mV from Ϫ80 mV. r 400 , r 1000 , and r 2000 are defined as the residual current (I res ) left after 400, 1000, and 2000 ms, respectively, divided by the maximal current, r x ϭ I res /I peak . Data were analyzed using pCLAMP (Molecular Devices) or routines written in IGOR Pro software (WaveMetrics, Lake Oswego, OR). Averaged data are presented as means Ϯ S.E. Statistical differences between two groups were determined by Student's t test, and multiple group comparisons were done by one-way analysis of variance followed by Dunnett's or Bonferroni tests. Graphs and statistical analysis were done with SigmaPlot (SPSS, Inc., Chicago, IL).
Pulldown Assays with GST-fused Proteins-Procedures were essentially as described (29). In brief, [ 35 S]Met/Cys-labeled CaBP1 or CaM was translated on the template of an in vitro synthesized RNA using a rabbit reticulocyte translation kit (Promega). DNAs of ␣ 1C segments designed to create GST fusion proteins were cloned into the pGEX-4T-1 vector. GSTfused proteins were produced in Escherichia coli (strain BL21-RIL) transfected with the cDNAs (see Fig. 3A) and grown in standard medium at 37 or 18°C after induction with isopropyl ␤-D-thiogalactopyranoside. The GST fusion proteins were extracted from E. coli using an Amersham Biosciences kit. The protein concentration was estimated using a Bio-Rad protein assay kit. Purified GST fusion proteins (5-10 g) or purified GST (ϳ10 g) was incubated with 5 l of the lysate containing the 35 S-labeled proteins in PBS containing 0.05% Tween 20 with 1 mM CaCl 2 or EGTA. The final reaction volume was 300 l. GST fusion protein was immobilized on glutathione-Sepharose beads (Amersham Biosciences) for 30 min at 4°C and washed. Following washing, GST fusion proteins were eluted with 20 mM reduced glutathione in 120 mM NaCl and 100 mM Tris-HCl (pH 8) and analyzed by SDS-PAGE. Gels were stained to detect proportional protein concentration with Coomassie Brilliant Blue R-250 (Bio-Rad). The labeled products were identified by autoradiography using a PhosphorImager (Molecular Dynamics).

RESULTS
CaBP1 Accelerates VDI of Ca V 1.2-CaBP1 inhibition of CDI in Ca V 1.2 has been described for a SNT␣ 1C isoform from rat brain, rbcII (6,28). However, a LNT␣ 1C isoform is also expressed in the brain (37). The LNT␣ 1C isoform includes a 46-aa initial segment encoded by exon 1a (38), which contains the NTI module (aa 1-20) (see Fig. 3A and supplemental Fig. S4 for sequence) that regulates the channel's P o (35). The SNT␣ 1C isoform (39 -41) has an initial N-terminal segment encoded by the alternative exon 1 (42,43). This 16-aa segment is partly homologous to aa 6 -20 of the long N terminus (see Fig. 3A and supplemental Fig. S4) but does not retain functionality of the NTI module in terms of modulation of P o of Ca V 1.2 (35). Considering the importance of the rbcII␣ 1C N terminus in CaBP1 regulation of CDI (28), we examined whether CaBP1 might differentially regulate the LNT␣ 1C and SNT␣ 1C isoforms. We employed two-electrode voltage-clamp recordings in Xenopus oocytes injected with RNAs encoding LNT␣ 1C or SNT␣ 1C together with the auxiliary ␣ 2 ␦-1 and Ca V ␤ 2b -subunits. CDI and VDI were studied with Ca 2ϩ and Ba 2ϩ as the permeant ions, respectively. Ba 2ϩ (I Ba ) or Ca 2ϩ (I Ca ) currents were isolated by subtracting Cd 2ϩ -insensitive currents from the total current (supplemental Fig. S1A) (35). Ba 2ϩ currents via Ca V channels are widely considered as inactivating essentially by the VDI mechanism, and their decay provides a reliable measure of VDI kinetics (e.g. Ref. 25).
I Ca demonstrated typical strong CDI, which was substantially slowed by coexpression of CaBP1 in both SNT␣ 1C and LNT␣ 1C isoforms (I Ca ) (Fig. 1A). Thus, Xenopus oocytes recapitulate the effect of CaBP1 on CDI described in HEK293T cells (6,27). However, a novel observation was the acceleration of VDI seen with Ba 2ϩ as the permeant ion (I Ba ) (Fig. 1A). The extent of inactivation was measured as r 400 (24), i.e. the residual current remaining at the end of a 400-ms test pulse normalized to the peak current amplitude. For both ␣ 1C isoforms, CaBP1 significantly accelerated VDI (decreased r 400 ) at all positive voltages (Fig. 1, B and C). CaBP1 Enhances VDI Mainly by Changing the Fraction of Fast and Slow Components of Inactivation-Given the relatively slow time course of Ca V 1.2 VDI, we next used 10-s depolarizing pulses to characterize the effects of CaBP1 on the kinetics of VDI (Fig. 2). For LNT␣ 1C and SNT␣ 1C alone, I Ba showed faster decay kinetics at more positive membrane voltages, a hallmark of VDI (Fig. 2, A-C, and supplemental Fig. S3). The decay of I Ba was best fitted with a double-exponential function ( Fig. 2A and supplemental Fig. S1, B and C), giving time constants of fast ϳ 0.6 s and slow ϳ 3 s at ϩ10 mV (Fig. 2C, Table   1, and supplemental Fig. S1B and Table S1), supporting the involvement of at least two processes in VDI of Ca V 1.2 (21). CaBP1 accelerated I Ba decay kinetics at all voltages in both LNT␣ 1C and SNT␣ 1C isoforms ( Fig. 2B and supplemental Fig.  S3A). Acceleration of VDI by CaBP1 was dose-dependent and maximal with 5-10 ng of CaBP1 RNA/oocyte in both LNT␣ 1C and SNT␣ 1C isoforms (supplemental Fig. S2A). The kinetic analysis of current decay showed that, at less depolarized voltages, CaBP1 decreased both fast and slow . At more depolarized voltages, CaBP1 significantly increased the fast inactivating fraction (A fast ) with a concomitant decrease in the slowly inactivating fraction (A slow ) (Fig. 2C). Together, these changes account for the acceleration of VDI by CaBP1 at all voltages.
CaBP1-binding Site in the N Terminus, and No Overlap with the NTI or NSCaTE Module-The N-terminal domain of ␣ 1C binds both CaM and CaBP1 (28,29). The binding of CaM is Ca 2ϩ -dependent and has been mapped to aa 60 -100, with crucial binding determinants between aa 80 and 92 (33, 34) conserved in all isoforms of ␣ 1C (supplemental Fig. S4A), as well as in Ca V 1.3␣ 1 (␣ 1D ). We tested if CaBP1 and CaM share similar molecular determinants for binding in the ␣ 1C N-terminal domain in GST pulldown assays. GST fusion proteins that cover the complete long N terminus of ␣ 1C , as shown schematically in Fig. 3A, were used to pull down in vitro synthesized radiolabeled CaBP1. CaBP1 bound to aa 95-154 and to a shorter segment (aa 95-140) but not to segments located N-terminally to aa 94 (Fig. 3B). Importantly, CaBP1 did not bind to the segment at aa 60 -100, which has been shown to strongly bind CaM in the same assay (supplemental Fig. S4B) (34). These results locate the CaBP1-binding site to aa 95-140, a segment fully conserved in all ␣ 1C isoforms; the crucial binding determinants are probably between aa 120 and 140. Thus, unlike the C terminus (6), in the N terminus, there is no overlap between CaM-and CaBP1-binding sites (Fig. 3A). Consistent with previous findings (28), CaBP1 binding to the N-terminal fragments was Ca 2ϩ -independent in that binding was similar in the presence of either Ca 2ϩ or EGTA (Fig. 3B). We estimate that the Ca 2ϩ -dependent binding of CaBP1 to the N terminus is weaker than that of CaM because the ratio of bound to loaded protein ("input," i.e. initial amount of protein in the binding reaction) was always smaller for CaBP1 than for CaM (compare Fig. 3B and supplemental Fig. S4B). These results further underscore differences in the physical interactions of CaBP1 and CaM with Ca V 1.2 that could contribute to their distinct modulation of channel function.

CaBP1 Regulates Voltage-dependent Inactivation of Ca V 1.2
CaBP1 Depolarizes Voltage-dependent Activation of Ca V 1.2-Because CaBP1 reduces the voltage sensitivity of activation in P/Q-type (Ca V 2.1) channels (5, 11), we also examined the possibility of such regulation in Ca V 1.2 channels expressed in Xenopus oocytes. CaBP1 caused a 10 -12.5-mV depolarizing shift in the I-V curves and the corresponding conductancevoltage (G-V) curves of the LNT␣ 1C and SNT␣ 1C isoforms ( Fig. 4; see Table 2 for Boltzmann fit parameters). CaBP1 inhibited Ca V 1.2 activation in a dose-dependent manner with the maximal effect achieved with 5-10 ng of CaBP1 RNA/oocyte (supplemental Fig. S2B). CaBP1 specifically altered the midpoint value of the G-V curve, the V a . All other parameters of the Boltzmann equation did not change significantly (Table 2).
To assess whether the N terminus of ␣ 1C is involved in CaBP1-induced changes in activation, we used several mutants. ⌬139-LNT␣ 1C lacks most of the cytosolic N terminus of ␣ 1C , including both the NSCaTE module and the CaBP1-binding site. ⌬46-LNT␣ 1C lacks the initial segment of the N terminus (46 aa encoded by exon 1a in LNT␣ 1C or 16 aa encoded by exon 1 in SNT␣ 1C ) but retains the CaM-binding site (NSCaTE module) and the CaBP1-binding segment. The shift in the G-V curve was preserved in both ⌬46-LNT␣ 1C and ⌬139-LNT␣ 1C (Fig. 4). CaBP1 similarly affected the voltage dependence of channels with other N-terminal deletions and mutations: ⌬20-LNT␣ 1C , ⌬20-LNT␣ 1C , the WIR mutant (a triple mutation that abolishes CaM binding to the N terminus (33,34)), and channels in which the ␤ 2b -subunit was replaced by ␤ 2a or ␤ 3 . In all cases, a 6 -10-mV shift in V a was observed upon coexpression of CaBP1 (Table 2). In all, the effect of CaBP1 on voltage-dependent activation appears to be independent of the N terminus of ␣ 1C .
Effect of CaBP1 on VDI Is Regulated by the First 46 aa of the ␣ 1C N Terminus-We next used the N-terminal mutants to examine whether the N terminus of ␣ 1C is involved in CaBP1 regulation of VDI. To quantitate the extent of VDI, in addition to r 400 , we also used r 2000 (the fraction of residual current after 2000 ms), which might better reflect changes in slower components of VDI compared with r 400 . We used ⌬46-LNT␣ 1C , ⌬139-LNT␣ 1C , and two additional mutants in which we deleted either the proximal 20 aa (⌬20-LNT␣ 1C ) or the distal 26 aa (⌬20 -46-LNT␣ 1C ) from the 46-aa-long initial segment (Fig.  3A) (35). Except for ⌬20 -46-LNT␣ 1C , which contains the NTI   Table 2.

CaBP1
Regulates Voltage-dependent Inactivation of Ca V 1.2 APRIL 22, 2011 • VOLUME 286 • NUMBER 16 module and has the low P o characteristic of LNT␣ 1C (35), the biophysical properties of these four deletion constructs are generally similar to those of the SNT␣ 1C isoform.
Our previous work suggested that VDI was accelerated in the ⌬139-LNT␣ 1C mutant (29). In agreement with this, the kinetics of VDI in ⌬139-LNT␣ 1C and, to a smaller extent, in SNT␣ 1C and ⌬46-LNT␣ 1C are faster than those in wild-type LNT␣ 1C (Fig. 5, Table 1, and supplemental Fig. S5A). Changes in VDI in ⌬20-LNT␣ 1C and ⌬20 -46-LNT␣ 1C were less pronounced and not statistically significant (supplemental Fig. S5A). Together, these results implicate the N terminus of ␣ 1C in the regulation of VDI.
Considering that the approximately ϩ10-mV shift in activation caused by CaBP1 may alter VDI kinetics, we compared VDI of the various channel constructs at ϩ10 mV in the absence of CaBP1 and at ϩ20 mV in the presence of CaBP1 (Fig. 5, A and  B). This comparison also revealed a strong attenuation of the effect of CaBP1 on ⌬46-LNT␣ 1C and ⌬139-LNT␣ 1C channels. The small residual effect of CaBP1 on r 2000 seen in both mutants (correlated with residual changes in A fast and A slow ) (supplemental Fig. S6) was significantly smaller than that seen in the wild-type channels (p Ͻ 0.001 compared with LNT␣ 1C , one-way analysis of variance) (Fig. 5C). We conclude that the initial segments of both LNT␣ 1C and SNT␣ 1C isoforms play a major role in transducing the CaBP1-induced acceleration of VDI.
We confirmed these observations in HEK293T cells expressing either the rat SNT␣ 1C isoform (rbcII) or the rabbit LNT␣ 1C isoform and several of the N-terminal deletion mutants (supplemental Fig. S5B). Inactivation was measured as r 1000 in HEK293T cells as described previously (6). In the wild-type channels, CaBP1 significantly accelerated VDI, decreasing r 1000 by 31% in rbcII and by 27% in rabbit LNT␣ 1C . Deletion of the first 64 aa in rbcII␣ 1C or of 46 aa in LNT␣ 1C completely abolished the CaBP1-induced change in r 1000 of I Ba .
To further define the determinants within the initial 46 aa required for CaBP1 modulation of VDI, we analyzed the ⌬20-LNT␣ 1C and ⌬20 -46-LNT␣ 1C deletion mutants. However, both ⌬20-LNT␣ 1C and ⌬20 -46-LNT␣ 1C manifested enhancement of VDI by CaBP1 similar to the wild-type channels ( Fig. 5 and supplemental Fig. S7), suggesting that either the first or last half of this proximal N-terminal region is sufficient for modulation of VDI by CaBP1.

DISCUSSION
Here, we have demonstrated that the neuronal Ca 2ϩ -binding protein CaBP1 profoundly regulates multiple aspects of gating of the Ca V 1.2 channel. In addition to the previously described regulation of CDI, we have shown that CaBP1 also regulates activation gating and VDI. The N terminus of ␣ 1C is important for the regulation of VDI by CaBP1. CaBP1 binds to the distal third of the N-terminal tail, adjacent to the first transmembrane domain of the channel, yet deletion mutagenesis showed that the functionally important part of N terminus is its initial segment, which does not bind CaBP1 in a direct biochemical assay. Importantly, the initial segments of both LNT␣ 1C and SNT␣ 1C isoforms, encoded by either one of the alternative first exons of the ␣ 1C gene CACNA1C (exon 1a or 1, respectively), function as regulators of the effect of CaBP1 on VDI. This demonstrates, for the first time, a novel modulatory function for the initial segment of the N terminus of the short isoform of ␣ 1C .
Role of the N Terminus of ␣ 1C and Mechanism of Action-Our results indicate that the initial segment of ␣ 1C plays a crucial role in CaBP1 regulation of VDI. The channel construct lacking the initial segment (⌬46-LNT␣ 1C ), which starts essentially with the protein sequence encoded by the obligatory exon 2 (44,45), retains only a residual small regulation of VDI by CaBP1. Importantly, the effect of CaBP1 is equally restored by the addition of either the short 16-aa initial segment encoded by exon 1 or the long 46-aa segment encoded by the alternative exon 1a. Thus, regulation by CaBP1 is observed in both isoforms of ␣ 1C . (A third N-terminal isoform of ␣ 1C starting with a 9-aa segment encoded by the alternative exon 1c is expressed in rat smooth muscle, but no such isoform was identified in humans (46).) The ability of the 16 aa-initial segment of the SNT␣ 1C isoform to regulate CaBP1 modulation contrasts with the inability of this region to regulate the channel's maximal open probability, a function that is unique to the partially homologous 20 aa of the LNT␣ 1C isoform, the NTI module (35). This distinction indicates that the initial segment of the N terminus regulates maximal P o and VDI by distinct molecular mechanisms. This concept is supported by the finding that another Ca 2ϩ -binding protein, KChiP (K V channel-interacting protein), which interacts with the N terminus of ␣ 1C , regulates only P o but not inactivation of the cardiac LNT␣ 1C isoform (10).
Our finding that deletion of most of the N terminus does not influence the effect of CaBP1 on voltage dependence of activation highlights the specificity with which the N terminus regulates the VDI modulation by CaBP1. Specificity is also supported by the preservation of CaBP1-dependent regulation of VDI in mutants in which the first or second portion of the 46-aa segment was deleted, ⌬20-LNT␣ 1C and ⌬20 -46-LNT␣ 1C . These two mutants differ by the presence of the NTI module (in ⌬20 -46-LNT␣ 1C ) and the corresponding ϳ7-fold difference in maximal P o (35). Our finding that CaBP1 still modulates VDI in ⌬20-LNT␣ 1C and ⌬20 -46-LNT␣ 1C raises the possibility that the length of the N terminus is the critical parameter. A similar proposal has been made for the N terminus of Ca V ␤ based on the ability of truncations of this region to prevent effects of Ca V ␤ on voltage-dependent inactivation (47,48). However, we cannot rule out that there are two distinct regions that mediate the effect of CaBP1 on VDI, which are present in the first and second halves of the 46-aa initial segment and in the 16 aa of SNT␣ 1C . Interestingly, CaBP1 did not interact directly with the initial N-terminal segment of LNT␣ 1C , although we cannot rule out that low affinity interactions may have evaded our pulldown assays. Because deletion of most of the N terminus including the CaBP1-binding site does not alter VDI regulation by CaBP1 any more than deletion of the initial segment alone, we propose that the initial segment plays a key role in transducing the effect of CaBP1. This effect may require CaBP1 binding to the distal N terminus and/or to the previously characterized site in the C-terminal domain (6). The initial segment of the N terminus may allosterically couple CaBP1 binding to VDI modulation. We proposed a similar mechanism to account for the ability of the Ca V ␤-subunit to inhibit P o via the NTI module despite lack of a direct Ca V ␤-N terminus interaction (30).
Complex Regulation of VDI by the N Terminus of ␣ 1C -The mechanism underlying inactivation of high voltage Ca V channels is not completely understood but may involve "hinged lid" or pore block-type mechanisms (20,49). The N-terminal tail of ␣ 1C has previously been proposed to regulate the inactivation process (29,31), in addition to other well established determinants (the C-terminal tail, cytosolic loop I, and the pore itself in ␣ 1C ; Ca V ␤; and CaM) (18,20,21,50). Our results further suggest a complex regulation of the inactivation process by multiple parts of the N-terminal tail of the Ca V 1.2 ␣ 1C -subunit. Thus, despite the crucial role of the initial segment in regulation of VDI by CaBP1, it only moderately affects the VDI process itself: the overall kinetics of VDI are only slightly accelerated by the deletion of the initial segment (46 or 16 aa, respectively) compared with SNT␣ 1C . Although removal of a part of a protein can distort its conformation and affect functions that are normally not controlled by this part, we note that the ⌬139-LNT␣ 1C mutant shows otherwise standard activation gating, P o , amplitude, and regulation by Ca V ␤ similar to those of the SNT␣ 1C isoform (35).
CaBP1 Regulates VDI in Ca V 1.2, and Possible Physiological Implications-The depolarizing shift in the voltage dependence of activation and the acceleration of VDI are expected to reduce Ca 2ϩ entry in excitable cells. On the other hand, CDI is reduced by CaBP1, which would enhance Ca 2ϩ entry (6). Dual opposite regulation of VDI and CDI resembles the effect of sorcin (8) and may be physiologically relevant. For Ca V 1.2 channels in ventricular cardiomyocytes, VDI may primarily regulate channel function under basal conditions, whereas CDI is dominant upon ␤-adrenergic stimulation (51,52). The importance of VDI as a Ca V 1.2 regulatory mechanism is further underscored by findings that the Timothy syndrome mutation G406R, which leads to autism, cardiac arrhythmia, and developmental abnormalities (53), inhibits VDI but spares CDI (54). CaBP1 is colocalized with L-type Ca 2ϩ channels in somatodendritic areas of neurons in the brain (4) and is well posed to regulate their function. Therefore, CaBP1 regulation of VDI and CDI may contribute significantly toward activity-dependent regulation of neuronal Ca 2ϩ signals.