Competitive and Non-competitive Regulation of Calcium-dependent Inactivation in CaV1.2 L-type Ca2+ Channels by Calmodulin and Ca2+-binding Protein 1*

Background: Calmodulin and calcium-binding protein 1 (CaBP1) oppositely regulate inactivation of CaV1.2 channels. Results: Quantitative titration of purified proteins in intact cells suggests competition between CaM and CaBP1 for the CaV1.2 C terminus and an additional non-competitive action of CaBP1. Conclusion: CaBP1 counteracts CaM actions by a dual mechanism. Significance: Our approach provides new insights into mechanisms of Ca2+ channel inactivation. CaV1.2 interacts with the Ca2+ sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, α1C. Ca2+/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca2+ ions (Ca2+-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the α1C- subunit using Förster resonance energy transfer (FRET) and recording of Cav1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of α1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for α1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaM altered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15–20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated α1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that the NT is not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in α1C.

In contrast to CaM, CaBP1 eliminates CDI but accelerates VDI of Ca v 1.2 (42)(43)(44). The mechanism by which CaBP1 counteracts the CDI-promoting action of CaM is unclear. Like CaM, CaBP1 interacts with the IQ domain and the pre-IQ domain of the ␣ 1C subunit (42). Mutations in IQ reduce the apparent affinity of both CaM and CaBP1, and direct competition of CaBP1 and CaM for IQ has been demonstrated (37,42,45). Therefore, it has been proposed that at least part of the functional CaBP1 action occurs through a competition mechanism whereby CaBP1 displaces CaM at the overlapping pCT binding site(s) in the ␣ 1C subunit (42,45).
Furthermore, both CaM and CaBP1 interact with the NT of ␣ 1C . CaM binds at the N-terminal spatial Ca 2ϩ transforming element in the presence of Ca 2ϩ (28,46,47). In contrast, the binding of CaBP1 to the NT of ␣ 1C is Ca 2ϩ -independent (43) and upstream of the CaM binding site (44). Deletion of the first half of the NT of ␣ 1C , which removes the CaM binding site but spares the CaBP1 site, weakened the regulation of CDI by CaBP1 in Ca V 1.2 expressed in a mammalian cell line (43), but the structural mechanism behind this effect is not known. Because the ␣ 1C NT binding sites for CaM and CaBP1 are not overlapping, regulation of CDI by CaBP1 via the NT, which does not involve competition with CaM, is plausible. In all, it is not well understood whether competition of CaBP1 and CaM at the CT is a major mechanism of suppression of CDI by CaBP1 and what the relative roles of CaBP1 and CaM binding sites in the N and C termini of ␣ 1C are.
In this paper we show that the effect of CaBP1 on CDI in Ca V 1.2 expressed in Xenopus oocytes is largely determined by a competition with CaM, probably at the pCT binding site(s). Förster resonance energy transfer (FRET) shows that CaM can displace CaBP1 from pCT but not the NT of ␣ 1C . Titrated injection of recombinant purified CaM and CaBP1 proteins into the oocytes reveals a functional competition that determines the extent of CDI and does not require the NT of ␣ 1C . A residual 15-20% effect of CaBP1 cannot be reversed by excess CaM and may rely on a mechanism that does not involve competition with CaM.
DNA Constructs-cDNA constructs for the labeled proteins were obtained using standard PCR procedures. cDNA constructs for oocyte expression were inserted into the pGEMHJ vector. Enhanced yellow fluorescent protein (YFP) and cerulean (CFP) were as in Ref. 49. Both carried the A206K mutation to avoid dimerization, and YFP carried the Q69M mutation reducing its environmental sensitivity. To create Myr-pCT-CFP, the coding sequence of the myristoylation signal derived from the Src protein (ATGGGGAGTAGCAAGAGCAAGC-CTAAGGACCCCAGCCAGCGCCGG) was inserted between SmaI and EcoRI of pGEM-HJ followed by the coding sequence of pCT, which was inserted between EcoRI and XbaI, and CFP (with stop codon), which was inserted between XbaI and HindIII. To create YFP-NT-CAAX, the coding sequence of YFP was inserted between BamHI and EcoRI of pGEM-HJ followed by the NT (amino acids 1-154), which was inserted between EcoRI and XbaI. The CAAX box was inserted after an adenine to avoid a frameshift, and a three-glycine linker was inserted between HindIII and BstEII. To create CaM-xFP (where xFP stands for either CFP or YFP), the coding sequence of CaM was inserted between BamHI and XbaI (the stop codon was canceled) followed by YFP or CFP with a stop codon between XbaI and HindIII. To create CaBP1-xFP, the coding sequence of CaBP1 was inserted into a EcoRI site (the stop codon was canceled) followed by YFP or CFP with a stop codon between EcoRI and HindIII.
Purified Recombinant Protein Injection-All recombinant proteins were expressed in Escherichia coli Tuner (DE3) Codon Plus cells grown in standard media at 37°C (CaM, CaM 1234 ) or 16°C (CaBP1) after induction with isopropyl 1-thio-␤-D-galactopyranoside. For CaM purification, the procedure described by Hayashi et al. (50) was used with slight modifications. After lysis by a microfluidizer (Microfluidics), cell debris was removed by a 1-h centrifugation at 38,700 ϫ g. The supernatant was heated to 90°C for 5 min and then centrifuged for an additional 30 min before loading on a phenyl-Sepharose column. Subsequently, an additional Q-Sepharose chromatography step was added where the protein was eluted by a linear salt gradient (0 -0.5 M NaCl). Protein was dialyzed against deionized water and lyophilized. For CaM 1234 purification, cells were suspended in lysis buffer (100 mM NaCl, 50 mm Tris, pH 7.5, 1 mM phenylmethylsulfonyl fluoride). After lysis, the soluble fraction was heated to 70°C for 5 min and then centrifuged. Soluble fraction was diluted with 1 volume of 50 mM Tris, pH 7.5, 10% glycerol before loading onto a Q-Sepharose (GE Healthcare) column. The protein was eluted by a linear salt gradient (50 -1000 mM NaCl). Selected fractions were loaded onto a hydroxyapatite column pre-equilibrated with 50 mM Tris, pH 7.5, 200 mM NaCl. The protein was eluted by a linear phosphate gradient to 500 mM potassium phosphate, pH 7.5, 100 mM NaCl. Relevant fractions were then loaded onto a HiLoad 16/60 Superdex 75 gel filtration column (GE Healthcare) in 20 mM Tris, pH 7.5, 200 mM NaCl. Finally, CaM 1234 was desalted by a HiTrap 26/10 desalting column (GE Healthcare) in water, lyophilized, and stored at Ϫ20°C.
For CaBP1 purification, all steps were performed on ice or at 4°C. Cell paste was suspended in PBS with a ratio of 10 ml of buffer to 1 g of paste. Triton X-100 (0.1%), 1000 units of DNase, 10 mg of lysozyme, and 1 mM PMSF were added to the suspension. The cells were homogenized and lysed by microfluidizer (Microfluidic). Lysate was centrifuged for 1 h to pellet. The supernatant was loaded onto a glutathione column pre-equilibrated with PBS buffer. After the cell extract was loaded, the column was extensively washed with PBS buffer. Protein was eluted with buffer containing 50 mM Tris, pH 8.0, 100 mM NaCl, and 10 mM glutathione. The eluted protein was digested overnight with thrombin at a ratio of 1 mg of protease to 30 mg of protein. After thrombin digestion, the protein solution was diluted 2-fold in 50 mM MES (pH 6.0), 10% glycerol. Buffer solution was subsequently loaded onto a Q-Sepharose (Amersham Biosciences) column pre-equilibrated with 25 mM MES (pH 6.0), 50 mM NaCl. The protein was eluted by a gradient of 50 -600 mM NaCl (10 column volumes). The final step used a Superdex 200 Hi-prep gel-filtration column pre-equilibrated with 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM DTT. The fractions containing the protein were collected and concentrated to 15 mg/ml using a Vivaspin concentrator with a 5000-Da cutoff. Final protein concentration was determined by absorbance spectroscopy at 280 nm. The protein was aliquoted, flash-frozen in liquid nitrogen, and stored at Ϫ80°C. Circular dichroism spectroscopy showed that purified recombinant CaM, CaM 1234 , and CaBP1 displayed spectra characteristic of ␣-helical proteins and CaM. All proteins showed essentially 100% monodispersity as shown with size exclusion chromatographymulti-angle light scattering, and the molecular masses were completely consistent with calculated masses based on their sequences (data not shown).
For injection into oocytes, proteins were diluted in water (rCaM and rCaM 1234 stock solutions, 12 mM; rCaBP1 stock solution, 700 M), aliquoted, and stored at Ϫ80°C. BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid) was kept in 200 mM stock solution in 0.3 N sodium bicarbonate at Ϫ20°C. BAPTA mixed with H 2 O or with the purified proteins was injected in a 50-nl drop into each oocyte 0.5-1 h before measurement. Calculations of final concentration in the oocyte of BAPTA and the purified proteins have been done assuming a homogenous distribution within an oocyte volume of 1 l.
Electrophysiology and Data Analysis-Xenopus oocytes were injected with BAPTA to a final concentration of 1.5-2.5 mM, and Ca 2ϩ currents were measured at 22-25°C essentially as described (44). In brief, the two-electrode voltage clamp technique was used to measure whole cell currents using Gene-Clamp 500 amplifier (Axon Instruments). The external solutions contained 40 mM Ca(OH) 2 , 50 mM NaOH, and 2 mM KOH titrated to pH 7.5 with methanesulfonic acid. All current protocols were routinely repeated in the presence of 200 M Cd 2ϩ . Net Ca 2ϩ current was obtained by subtracting the currents recorded in Cd 2ϩ -containing solution from the currents in control Ca 2ϩ solution to eliminate endogenous non-Ca 2ϩ and leak currents. All recordings were obtained by a 400-ms step pulse from Ϫ80 mV (holding potential) to several test pulses from Ϫ30 mV up to 60 mV in a 10-mV step. Maximal current amplitude was usually reached at 20 mV. Data acquisition and analysis were done using the pCLAMP10 software (Axon Instruments). Graph design and fit were done by SigmaPlot 11 software. r 400 and r 50 denote the fractional residual current left after 400 and 50 ms, respectively, after the peak of I Ca divided by the maximal current. r 400 and r 50 are numbers between 0 and 1, corresponding to full and no inactivation, respectively.
Dose-effect relationships for rCaM 1234 (see Fig. 4C) and rCaBP1 (see Fig. 5B) were fit to a one-site saturation isotherm, or the Hill equation where E is the fractional effect of CaBP1 or CaM 1234 , C is the concentration of the injected protein, K d,app is the apparent dissociation constant, and n is the Hill coefficient (51). The curves describing the dose-dependent attenuation of CaBP1 effect by the injected rCaM were obtained by fitting the data to a one-site competition model, or a one-site model with non-competing fraction where K d stands for the apparent dissociation constant of rCaM, and f r is the CaM-resistant fraction of CaBP1 regulation of CDI.
Imaging and FRET assays in oocytes were performed essentially as described (49,52). Oocytes were imaged in ND96 solution in a 0.7-mm glass-bottom dish. Fluorescence emissions from CFP and YFP-tagged proteins were collected from the animal hemisphere of the oocyte with a Zeiss inverted confocal microscope (Zeiss Axiovert LSM 510META) using a 20 ϫ 0.75 NA air objective and laser excitations of 405 and 514 nm, respectively. To construct FRET titration curves using data from different experiments, molar ratios of donor and acceptor have been calculated in each oocyte as described (52). In brief, we imaged a double-labeled protein expressing both CFP and YFP at a 1:1 stoichiometry (YFP-IRK1-CFP, DL-IRK1) in each experiment and converted the fluorescence of CFP and YFP into their molar ratio by calculating the slope of the CFP to YFP fluorescence of DL-IRK1. Then this slope was used as a reference to convert CFP and YFP fluorescence in all other experimental groups into a molar ratio. To measure FRET, we used a spectrum-based method (53). Briefly, two emission spectra were collected from each oocyte, one with 405-nm excitation and the other with 514-nm excitation. A scaled CFP spectrum, collected from control oocytes expressing CFP-tagged proteins only, was used to normalize the CFP emissions from the spectrum taken from oocytes expressing both fluorophores at 405-nm excitation. This procedure allows one to dissect the YFP emission spectrum, termed F 405 . F 405 has two components: one due to direct excitation of YFP (F 405 direct ) and one due to FRET (F 405 FRET ). F 405 is normalized to the total YFP emission with 514-nm excitation at the same oocyte, F 514 . The resulting ratio, termed Ratio A, can be expressed as The direct excitation component in the calculated Ratio A, termed Ratio A 0 , is experimentally determined from a large population of oocytes expressing only YFP-tagged proteins. This allows an accurate calculation of the bleed-through of direct excitation of YFP by the 405-nm laser. The difference between Ratio A and Ratio A 0 (Ratio A Ϫ Ratio A 0 ) is directly proportional to FRET efficiency, The apparent FRET efficiency in an individual cell, E app , can be calculated as (54), where ⑀ D and ⑀ A are molar extinction coefficients for the donor and acceptor, respectively, at the donor excitation wavelength (55).
FRET data in Fig. 1, C-E, were binned by grouping E app of all cells within consecutive donor/acceptor ratio ranges of 0.5, except for the last bin where there were too few points, and all data in this variable bin were pooled together. Both S.E. for both donor/acceptor ratio (horizontal S.E. bars) and for E app (vertical S.E. bars) are shown for this point in all plots.

CaBP1 and CaM Interactions with the NT and the CT of ␣ 1C -
We first addressed the interactions of CaM and CaBP1 with NT and CT separately. It has been proposed that in the presence of Ca 2ϩ , CaM may "bridge" between NT and CT in L-type VGCCs (28,46) forming a triple complex, which could complicate the interpretation of the results. However, a later study demonstrated that no such bridge is formed in the ␣ 1C subunit of Ca V 1.2 (47), supporting the approach taken here.
To study the possible competition between CaM and CaBP1 in an intact cell, we first used the FRET two-hybrid method (34) in Xenopus oocytes (Fig. 1B). We constructed RNAs corresponding to the following fluorescently labeled proteins: cytosolic NT domain (amino acids 1-154), the proximal part of the CT from the boundary with the plasma membrane up to the end of IQ domain, pCT (amino acids 1505-1671) (Fig. 1A), CaBP1, and CaM. An important modification of the FRET twohybrid method was the introduction of a plasma membrane anchor in the NT and pCT in order to imitate their proximity to the membrane in the full channel (Figs. 1, C and D, upper panel schematic). To accomplish this we added a prenylation signal element to the carboxyl end of the NT (CAAX box originating from K-Ras) (56). For pCT, we added a myristoylation signal from Src (57) at the N-terminal end. The fluorophores in these constructs are located distally to the membrane anchor, YFP in the NT (YFP-NT) and CFP in the pCT (pCT-CFP). To assess actual molar ratios of CFP and YFP, in a separate group of oocytes we expressed a double-labeled protein containing both CFP and YFP at a 1:1 stoichiometry (DL-IRK1; see "Experimental Procedures"). FRET was quantitated as apparent FRET efficiency (E app ) for a range of donor/acceptor molar ratios to verify saturation of the signal and to avoid underestimation of FRET at low donor/acceptor ratios (52,58). The value of E app at saturation reflects proximity among fluorophores within a FRET pair (53).
We observed FRET between fluorescently labeled CaBP1 and both YFP-NT and pCT-CFP (Fig. 1C). The FRET signal saturated at high donor/acceptor ratios, which indicates a specific interaction. We also observed FRET between CaM-YFP and pCT-CFP, but not between CaM-CFP and YFP-NT (Fig. 1D). The latter is in line with the known Ca 2ϩ dependence of this interaction (see "Discussion"). Our FRET experiments also show that there is no FRET between CaM-CFP and CaBP1-YFP or between CaM-YFP and CaM-CFP (Fig. 1E).
If CaM and CaBP1 could simultaneously interact with separate sites in the channel, the coexpression of the channel with CaM and CaBP1 should strengthen the FRET signal between CaM and CaBP1. However, there was no difference in the FRET signal in oocytes expressing CaM and CaBP1 and those expressing ␣ 1C , CaM, and CaBP1 (Fig. 1E). This result suggests that CaM and CaBP1 do not simultaneously bind to ␣ 1C . However, because of the limitations of FRET methodology, which only applies to distances less than 10 nm (59), the possibility of such a simultaneous binding cannot be ruled out.
CaM Reduces FRET of CaBP1 with CT but Not with NT-If CaBP1 inhibits CDI by competing with CaM for a shared binding site in ␣ 1C , then CaM should antagonize the interaction of CaBP1 with the channel and reverse the effect of CaBP1 on CDI. To examine whether excess CaM reduces the interaction between CaBP1 with NT or pCT, we injected purified recombinant CaM protein produced in E. coli (rCaM) to an approximate final concentration of 350 M in the oocyte and monitored changes in FRET between YFP-NT and CaBP1-CFP or between pCT-CFP and CaBP1-YFP. FRET was monitored in individual oocytes before and after the injection of rCaM. rCaM did not change the FRET between YFP-NT and CaBP1-CFP ( Fig. 2A), but it significantly reduced the FRET between the pCT-CFP and CaBP1-YFP (Fig. 2B). Thus, CaM interferes with the interaction of CaBP1 with the pCT, but not with the NT, under the same experimental conditions.
Overexpression of CaM Attenuates the Functional Effect but Also the Expression Level of CaBP1-Next, we coexpressed Ca V 1.2 with CaBP1-CFP (Fig. 3A) and observed the typical slowing of current decay, manifested in the increase in r 400 value (% of current remaining after 400 ms pulse (24)) ( Fig. 3, B and C), which is due to inhibition of CDI. Coexpression of increasing the amounts of CaM-YFP RNA while maintaining constant RNA amounts of CaBP1-CFP and Ca V 1.2 and caused a reduction in r 400 value in an RNA dose-dependent manner (Fig.   3, A-C). This result appears compatible with a competition between CaM and CaBP1. However, careful examination of expression levels revealed that coexpression of CaM-YFP caused a decrease in the level of expressed CaBP1-CFP protein (Fig. 3, D and E). We did not succeed in preventing the reduction in CaBP1-CFP levels by widely varying RNA doses of both proteins and by switching the fluorophores (using CaM-CFP and CaBP1-YFP; data not shown). The decrease in the amount of CaBP1 protein might well underlie the apparent disappearance of the effect of CaBP1 on CDI. Such a decrease may reflect saturation of the protein synthesis machinery (e.g. Ref. 60) or a process of mutual regulation of biogenesis of these proteins, which may deserve further study. Furthermore, we could not achieve a molar excess of expressed CaM protein over CaBP1. Even with a 20:1 ratio of RNA, the actual amount of expressed CaM did not exceed that of CaBP1 ( Fig. 3E and data not shown). We conclude that the overexpression strategy is inadequate to assess competition between CaBP1 and CaM both because of insufficient expression of CaM and because of a decrease in CaBP1 levels after coexpression of CaM.
Injection of Purified CaM Protein Removes the Effect of CaM1234 on CDI-We next attempted to use injection of purified proteins as an assay for functional competition between CaM and CaBP1. First, we tested whether injection of the purified rCaM will alleviate the effect of CaM 1234 . CaM 1234 is a CaM mutant devoid of Ca 2ϩ binding to its four EF-hands (61), which mimics apoCaM. CaM 1234 binds to pCT of ␣ 1C but cannot bind Ca 2ϩ . Heterologously expressed CaM 1234 suppresses CDI, presumably by replacing the endogenous apoCaM anchored to the pCT and thus preventing CDI when Ca 2ϩ enters the cell via the channel (24,27,41,62). Because both CaM 1234 and CaM bind to the same site(s) in the CT at low basal levels of Ca 2ϩ (29), we expected that injected rCaM protein will compete for the binding site and replace the rCaM 1234 .
Injection of purified rCaM 1234 to an approximate final concentration of 450 M in the oocyte (see "Experimental Procedures") resulted in a robust inhibition of CDI; r 400 changed from 0.18 Ϯ 0.02 to 0.84 Ϯ 0.03 (Fig. 4, A and B). rCaM alone did not affect CDI in the same concentration range (see Fig. 5A). For the following calculations, the net change in r 400 caused by CaM 1234 (⌬r 400 ϭ 0.84 -0.18 ϭ 0.66) was taken as 100% effect of CaM 1234 (Fig. 4B) . Injection of rCaM 1234 to 26 and 75 M produced 74 and 86% of maximum effect, respectively (Fig. 4, B and C). Data were fit to the Hill equation (Equation 2, "Experimental Procedures"), yielding an apparent dissociation constant of rCaM 1234 protein, K d,app , of about 8.8 M (Fig. 4C, black curve). The Hill coefficient was about 0.93, indicating a probable occupancy of one site. A one-site binding isotherm fit (Fig.  4C, gray curve), which can be viewed as a specific case of the Hill equation with a Hill coefficient equal to one (Equation 1, "Experimental Procedures"), gave a K d,app of 9.7 M.
Note that at 26 M, CaM 1234 protein produces ϳ74% of its maximal effect, placing this concentration below saturation and within the dynamic range where a reduction of its binding by a competitor will result in a visible reduction in functional effect. Injection of 350 M rCaM protein together with 75 or 26 M rCaM 1234 protein abolished 86 and 99% of the CaM 1234 effect on CDI, respectively (Fig. 4B). We assume that the final concentration of CaM after injection might be slightly higher than 350 M because of the presence of freely diffusible unbound endogenous CaM in the oocyte, although the latter is not expected to be substantial as most of CaM is probably restricted by interactions with its intracellular targets (63). Fig. 5A shows that although injection of rCaM did not affect CDI, the injection of the purified protein rCaBP1 decreased CDI. Using the same methodology as with rCaM 1234 , we injected increasing amounts of rCaBP1 into oocytes expressing Ca V 1.2. rCaBP1 caused a dose-dependent attenuation of CDI (Fig. 5Ba) with a K d,app of 0.78 M when fit to a standard one-site binding isotherm (Fig. 5Bb, purple line). Interestingly, fitting to the Hill equation gave a better fit with a K d,app of 0.81 M and a Hill coefficient of 1.9 (Fig. 5Bb, black  line), implying an interaction of CaBP1 with two or more binding sites in Ca v 1.2. Injection of 26 M rCaBP1 into oocytes expressing Ca V 1.2 maximally increased the r 400 value to 0.72 Ϯ 0.16, whereas 2.6 M produced ϳ90% of the maximal effect (Fig. 5B). Notably, the purified CaBP1 was produced in E. coli with no endogenous myristoyl transferase activity and thus lacked N-terminal myristoylation (64). This indicates that myristoylation of CaBP1 is not essential for the regulation of CDI in Ca V 1.2 under our experimental conditions. Next, we examined whether rCaM abolishes the effect of rCaBP1. Like with CaM 1234 , we chose to titrate rCaM in the presence of a concentration of CaBP1, which is ϳ3-fold the K d,app (2.6 M for CaBP1, 26 M for CaM 1234 ) and produces less than a maximal effect, which should be within the dynamic range suitable to study a competition mechanism. We mixed increasing amounts of rCaM with 2.6 M rCaBP1 protein and injected the protein mixtures into oocytes expressing Ca V 1.2. rCaM attenuated the effect of 2.6 M rCaBP1 (measured as r 400 ) in a dose-dependent manner (Fig. 5C). However, rCaM did not fully abolish the effect of rCaBP1. The dose-dependent inhibition by rCaM of the effect of rCaBP1 on CDI was not adequately fit to a simple competition model (Fig. 5D, red line). Rather, there was a fraction (ϳ15%) of the CaBP1 effect that could not be eliminated even with a 154-fold molar excess (400 M) of rCaM over rCaBP1 (Fig. 5, C and D, black line). This finding is in contrast to the effect of CaM 1234 , which was fully abolished by a 13-fold excess of rCaM. This result indicates that the effect of rCaBP1 is largely (ϳ85%) due to competition with CaM, but a residual action of CaBP1 is mediated by another mechanism. The K d,app for rCaM, calculated for the competitive fraction of its inhibition of rCaBP1 effect, was 6.3 M, very close to the 8.8 M estimated for CaM 1234 from the experiments of Fig. 4, A-C. This latter result supports the notion that, in its competition with CaBP1, CaM binds to the same site as apoCaM (CaM 1234 ).

Injection of rCaM Protein Reduces but Does Not Abolish the Effect of CaBP1-
CDI usually shows two kinetic components, a fast phase and a slower phase, with time constants in the 10s and 100s-ms ranges, respectively (5,65). An effect of CaBP1 on the faster component may be overlooked when using only the r 400 parameter. In the current traces of Fig. 5A, rCaM successfully eliminates the inhibitory effect of rCaBP1 on the fast component of CDI, and the effect of CaBP1 that is not suppressed by excess rCaM appears only in the slower component. To analyze this quantitatively, we measured changes in inactivation at the 50 ms time point, r 50 . As shown in Fig. 5E, 200 -400 M rCaM fully restored the fast component of CDI to a value seen in the absence of CaBP1.
139 N-terminal Amino Acids of ␣ 1C Are Not Involved in the Effect of CaBP1 on CDI-Previous reports showed the importance of the NT in the CaBP1-induced slowing of CDI; partial deletion of NT attenuated the action of CaBP1 on CDI in HEK 293 cells (37,43). We injected rCaBP1 protein into oocytes expressing Ca V 1.2 containing an ␣ 1C mutant, ⌬139-␣ 1C , which lacks the first 139 amino acids and thus most of the CaBP1 binding site in the NT (44). rCaBP1 significantly reduced the CDI of ⌬139-␣ 1C , as in the wild-type channel (Fig. 6A). Moreover, coinjection of rCaM protein with 2.6 M rCaBP1 showed a similar dose-dependent effect of rCaM as for the wild-type channel, with a K d,app of 7.9 M and a residual CaBP1 effect on r 400 of 20%, which remained even at 400 M rCaM (Fig. 6, B and C, black line). As with WT-␣ 1C , the dose-dependent inhibition by rCaM was not adequately fit to a simple competition model (Fig. 6C, red line). Analysis of the CaBP1 effect after 50 ms showed a complete removal of the CaBP1 effect by 200 -400 M rCaM (Fig. 6D). These data indicate that in Xenopus oocytes the ␣ 1C NT is dispensable for CaBP1 regulation of CDI and that competition with CaM likely takes place at another site(s) in ␣ 1C .

DISCUSSION
To date full-length Ca 2ϩ channels are not yet available for quantitative in vitro studies. Only partial information on the structural and thermodynamic aspects of interactions of segments of the pCT with CaM and CaBP1 is available for VGCCs (35, 45, 66 -69). Accordingly, heterologous expression has been the leading strategy to study the molecular regulation of VGCCs by CaM-and Ca 2ϩ -binding proteins. In this work we have exploited the advantages of the oocyte expression system for a quantitative analysis of the opposing regulation of Ca V 1.2 by CaBP1 and CaM in a living cell.
CaBP1 and CaM Interact with NT and pCT of ␣ 1C but Compete Only for pCT-Overlap of CaBP1 and CaM pCT binding sites in ␣ 1C and their physical competition for these sites have been demonstrated in vitro with purified ␣ 1C protein segments (42,45), but this has not been tested in living cells. We have addressed the possibility of competition using FRET with membrane-attached, xFP-labeled ␣ 1C NT and pCT segments. The use of membrane-attached cytosolic parts of a transmembrane protein for FRET two-hybrid testing ensures proper orientation of NT and CT relative to the plasma membrane and limits the possibility of artifactual interactions with cytosolic proteins such as CaM and CaBP1. The FRET data suggest that under low-Ca 2ϩ conditions of the resting cell, both CaM and CaBP1 interact with the pCT, corroborating the in vitro studies (26,29,30,(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)45). In contrast, the NT interacts only with CaBP1 but not CaM under low-Ca 2ϩ conditions (Fig. 1). This is again in line with in vitro results, which showed that CaBP1 binding to NT is Ca 2ϩ -independent (43,44), whereas binding of CaM requires Ca 2ϩ , with a half-maximal effective dose of above 1 M (28,46). We conclude that in resting cells the pCT of ␣ 1C is a potential site for physical competition between CaBP1 and CaM. Furthermore, our FRET experiments suggest that such competition takes place under resting conditions with low Ca 2ϩ levels. CaM, injected in excess into oocytes in the form of purified protein, greatly reduced FRET between pCT and CaBP1 (Fig. 2). Because there was no direct interaction between CaM and CaBP1, the simplest interpretation is a direct competition between CaM and CaBP1 for a binding site in the pCT. At present, however, an allosteric effect cannot be excluded. The absence of any effect of CaM on FRET between CaBP1 and NT serves as a control and suggests that there is no competition between CaM and CaBP1 for NT in the quiescent cell.
Two Mechanisms of Regulation of CDI by CaBP1-Our results strongly suggest that competition with CaM for a com-mon binding site underlies a major part of the effect of CaBP1 on CDI in Ca V 1.2. Control experiments were done with CaM 1234 , which inhibits CDI, and this effect has been unanimously interpreted as the result of competition between the native CaM and the expressed CaM 1234 and the replacement of the former by the latter at the anchoring site in pCT (8,24,27,70). Our experiments directly tested this theory using purified proteins, for the first time in a living cell. The injection of purified proteins and all electrophysiological measurements were done in the presence of BAPTA. Under these conditions, the global resting concentration of Ca 2ϩ in the cell remains low, and only the local Ca 2ϩ concentration in the nanodomain adjacent to the channel inner mouth rises transiently during the depolarization and channel opening (31). The results corroborate the single-site competition model for the following reasons. First, the injected CaM 1234 dose-dependently attenuated CDI with a Hill coefficient close to 1 (Fig. 4). This is expected if the injected CaM 1234 replaces the endogenous apoCaM anchored at the pCT of ␣ 1C in the absence of Ca 2ϩ . When Ca 2ϩ enters through the channel, the anchored CaM 1234 is unable to confer CDI. Second, an excess of injected Ca 2ϩ -free CaM fully restored the normal CDI, presumably by competing with the injected CaM 1234 .
We then demonstrated that injection of purified CaBP1 strongly suppresses CDI (Fig. 5) and went on to address the mechanism of CaBP1 action by titration of purified CaM and CaBP1. The main new insight suggested by our data is that CaBP1 acts by two mechanisms, probably via two separate sites. The main mechanism is a competition with CaM for a single site. The second mechanism appears to involve CaBP1 binding to a site from which CaBP1 cannot be removed by CaM. The notion of two sites of action is supported by two lines of evidence. First, the dose dependence of CDI suppression by CaBP1 exhibits a Hill coefficient close to 2, which supports more than one site of binding or action (51). Second, titration of CaM in the presence of a constant concentration of CaBP1 showed that up to 85% of the CaBP1 effect on CDI is reversible in the presence of high CaM concentrations. This result was clearly visible and model-independent. The dose dependence of this CaM effect is compatible with a competition with CaBP1 for a common binding site. However, 15-20% of the CaBP1 effect could not be reversed even by a vast molar excess of CaM, supporting a notion of an additional site of action of CaBP1 that is not affected by excess CaM. Partial kinetic analysis of CDI showed that high doses of CaM fully reversed the CaBP1-induced slowing of the first (fast) phase of CDI, but slowing of the second (slow) phase of CDI persisted; the latter actually accounted for the overall remaining effect of CaBP1. It is unlikely that the CaBP1-induced, CaM-resistant slowing of the second phase of CDI reflects a change in a Ca 2ϩ -independent component of inactivation (VDI), because CaBP1 accelerates VDI rather than slows it down (44). A likely possibility is that the CaM-resistant effect on CDI is mediated through binding of CaBP1 to a site in ␣ 1C where it does not compete with CaM. This is in agreement with previous findings that an N-lobe/linker module of CaBP1 exerts an inhibitory effect on CDI that is independent of IQdomain residues required for CaM binding (45). CaBP1 interacts with ␣ 1C at multiple sites in the NT, CT, and L3 (the cytosolic linker between domains III and IV of ␣ 1C ) (42)(43)(44), and at present we can only speculate which interactions underlie the two CaBP1 effects. In our assays the injected CaM protein similarly opposed CaBP1-induced slowing of CDI both in wild-type ␣ 1C and ⌬139-␣ 1C , a mutant lacking a major part of the NT CaBP1-binding site (44), although we cannot fully rule out some binding to the last remaining 15 cytosolic residues preceding the first transmembrane segment (Fig. 1A). Both in wild-type ␣ 1C and ⌬139-␣ 1C , the CaM-reversed and CaM-resistant components and the K d,app for CaM effect were almost identical. This result indicates that neither of the two effects of rCaBP1 is mediated by its binding to the NT site. Although this might seem contradictory to previous findings that a deletion in the NT of the rat brain Ca v 1.2 ␣ 1 subunit (rbcII) inhibited the effect of CaBP1 on CDI (43), it is possible that the NT plays a tethering role for CaBP1 that is important at physiological (low) expression levels of CaBP1. This role could be bypassed by high CaBP1 protein levels accomplished by overexpression with RNA or injection of purified protein. Nevertheless, our current findings indicate that the NT is unlikely to be the site where CaBP1 regulation of CDI cannot be eliminated by CaM, which implicates a potential role for the pCT or the L3 linker in this process.
CaM and CaBP1 have overlapping interaction sites on the pCT of ␣ 1C . CaBP1 and CaM interact with the distal pre-IQ and the IQ domains. The latter binds the C-lobes of CaM and CaBP1, and this is where CaBP1 competes with CaM (42,45). Thus, the IQ domain seems to be a plausible candidate site for mutual competitive effects of CaBP1 and CaM. However, as discussed above, the competition between injected CaM and expressed or injected CaBP1, revealed by our FRET and functional experiments, takes place under low-Ca 2ϩ conditions. This consideration points to the apoCaM anchoring site(s), which may be located mainly in the pre-IQ segment (71), as a plausible site(s) of competition. In sum, further studies are necessary to pinpoint the interactions through which CaBP1 exerts its two effects on CDI.
CaM-CaBP1 Competition in a Living Cell; Quantitative Aspects and Limitations-Calibrated heterologous protein expression was instrumental in studying the interactions between ␣ 1C subdomains and CaM or CaBP1, but it failed to provide the molar excess of one of the competing proteins over the other, essential for any comprehensive analysis of competitive regulation. We have overcome this limitation by injecting purified recombinant proteins, which fully recapitulated the known effects of their heterologous expression. Both protein and overexpression still share the drawback of a possible nonhomogenous distribution within the cell and buffering by endogenous proteins. Nevertheless, for the injected proteins, the strict dose dependence and saturability of the effects of CaM 1234 and CaBP1 indicate proportionality between total injected protein and its concentration in the vicinity of the channel.
We note that analysis of protein interactions in terms of the Hill equation, or a "standard" binding isotherm, is a simplification for two reasons. First, it assumes that molar amount of the "ligand" (e.g. CaM) is significantly higher than that of the "receptor" (␣ 1C ) (51). Reassuringly, with the injected protein strategy this is probably justified given our ability to vary the amount of the injected protein in a very wide range. Second, the initial CDI slowing by the injected CaM 1234 or CaBP1 is a result of competition with the endogenous Ca V 1.2-associated CaM. Under these conditions, the dose of the competitor that will produce a 50% maximal effect (EC 50 ) and, correspondingly, its K d,app , depends on the concentration of endogenous CaM, which is unknown (in general, the effective dose of a competing protein depends on its own K d but also on the concentration and K d of the other competitor). This is illustrated in Figs. 5D and 6C for the case of titration of CaM over a constant amount of CaBP1; compare the black lines, which show a fit to data with 2.6 M CaBP1, and the blue lines, showing simulated data with 26 M CaBP1. Therefore, the calculated K d,app values cannot be taken as estimates of real absolute values of K d .
Notwithstanding these caveats, for a constant level of endogenous CaM, the values of K d,app for different proteins, which compete with the endogenous CaM for the same site, provide a measure of their relative affinity for this site. Our results show that, with respect to the regulation of kinetics of CDI, CaBP1 has a higher affinity to Ca v 1.2 than CaM 1234 in low-Ca 2ϩ conditions (1.5-2.5 mM intracellular BAPTA). The K d,app calculated from the direct dose-effect curves was ϳ0.8 M for CaBP1 and about 9 M for CaM 1234 . Moreover, K d,app calculated for CaM from a different set of experiments (the competition with CaBP1 under low-Ca 2ϩ conditions (the apoCaM case)) was 6 -8 M both for the wild-type ␣ 1C and the ⌬139-␣ 1C mutant (for the CaM-sensitive fraction of CaBP1 effect). The similarity of ␣ 1C K d,app values for apoCaM and the CaM 1234 mutant, obtained from different types of measurement, supports the validity of these estimates. In all, our data suggest that the affinity of CaBP1 for Ca v 1.2 in a living cells is greater than that of apoCaM, enabling regulation of CDI by CaBP1 despite the high cellular levels of CaM and the high affinity of CaM for its binding sites in the pCT (7,66).