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J. Biol. Chem., Vol. 275, Issue 50, 39786-39792, December 15, 2000

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Determinants for Calmodulin Binding on Voltage-dependent Ca²⁺ Channels^*

Patricia Pate, Javier Mochca-Morales, Yuejin Wu^§, Jia-Zheng Zhang, George G. Rodney, Irina I. Serysheva, Barbara Y. Williams, Mark E. Anderson^§¶, and Susan L. Hamilton

From the  Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030 and ^§ Division of Cardiology, Department of Medicine, and ^¶ Department of Pharmacology, School of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6300

Received for publication, August 7, 2000, and in revised form, September 12, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Calmodulin, bound to the ₁ subunit of the cardiac L-type calcium channel, is required for calcium-dependent inactivation of this channel. Several laboratories have suggested that the site of interaction of calmodulin with the channel is an IQ-like motif in the carboxyl-terminal region of the ₁ subunit. Mutations in this IQ motif are linked to L-type Ca²⁺ current (I_Ca) facilitation and inactivation. IQ peptides from L, P/Q, N, and R channels all bind Ca²⁺calmodulin but not Ca²⁺-free calmodulin. Another peptide representing a carboxyl-terminal sequence found only in L-type channels (designated the CB domain) binds Ca²⁺calmodulin and enhances Ca²⁺-dependent I_Ca facilitation in cardiac myocytes, suggesting the CB domain is functionally important. Calmodulin blocks the binding of an antibody specific for the CB sequence to the skeletal muscle L-type Ca²⁺ channel, suggesting that this is a calmodulin binding site on the intact protein. The binding of the IQ and CB peptides to calmodulin appears to be competitive, signifying that the two sequences represent either independent or alternative binding sites for calmodulin rather than both sequences contributing to a single binding site.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Calcium dynamically regulates L-type Ca²⁺ current (I_Ca) through opposing processes of facilitation (1) and inactivation (2). Although the mechanisms for these processes remain incompletely understood, critical molecular determinants for Ca²⁺-dependent facilitation and inactivation of the L-type calcium channel have been attributed to the cytoplasmic carboxyl-terminal tail of the ₁ subunit, which contains a putative Ca²⁺ binding EF hand motif (3) and an "IQ-like" motif (4-7). The latter resembles the IQ domains that bind Ca²⁺-free calmodulin (apoCaM)¹ (8), and consistent with this, Peterson et al. (5) and Qin et al. (9) determined that calmodulin (CaM) was necessary for calcium-dependent inactivation of the cardiac channel. P/Q channels have also been shown to be modulated by calmodulin (10).

The IQ-like domain in the L-type channels appears to bind primarily Ca²⁺-CaM rather than apoCaM (5). An isoleucine to alanine mutation in this motif results in loss of Ca²⁺-dependent inactivation and unmasks a strong facilitation by CaM (11, 12). If the isoleucine is changed to a glutamate, the effects of CaM (inactivation and facilitation) are lost (11). A double mutation of I1624A/Q1625A (Swiss Prot Q0815) produced an even more pronounced facilitation (12). These investigators concluded that Ca²⁺-dependent inactivation requires strong binding of CaM to the IQ motif. Peterson et al. (5) find that a mutant CaM that cannot bind Ca²⁺ at any of the four Ca²⁺ binding sites blocks the effects of Ca²⁺-CaM on the L-type Ca²⁺ channel, suggesting that both the Ca²⁺-free and Ca²⁺-bound forms of CaM bind to this channel. However, only the Ca²⁺-bound form can induce inactivation (5). In particular, it appears that Ca²⁺ binding to sites 3 and 4 of CaM are required for Ca²⁺-dependent inactivation (5).

Other studies suggest that the IQ motif may not be the only determinant necessary for Ca²⁺-dependent inactivation of the channel (7, 13, 14). Adams and Tanabe (15) replaced the I-II loop of the cardiac channel with the corresponding loop of the skeletal channel, which slowed Ca²⁺-dependent inactivation. An effect of replacing the II-III loop was also observed. The role of the EF hands of the carboxyl-terminal region of the ₁ subunit of the L-type channel in Ca²⁺-dependent inactivation has been controversial (6, 7, 14, 16). Bernatchez et al. (16) found that mutations in the EF hand altered Ca²⁺-dependent inactivation, but Zhou et al. (6) found that mutant channels containing a triple mutation that disrupted Ca²⁺ coordinating activity was still inhibited by Ca²⁺. Recently, Peterson et al. (14) demonstrated that replacing four amino acids (VVTL) in the F helix of the putative EF hand of _1C with those of the _1E (MYEM) channel completely abolished Ca²⁺-dependent inactivation. In contrast, mutating the residues presumably involved in coordinating Ca²⁺ reduced the inactivation only about 2-fold. These authors suggested that the EF hand plays a role in transducing the signal generated by Ca²⁺ binding to CaM into channel inactivation. Other regions of the carboxyl tail are also likely to be involved in Ca²⁺-dependent inactivation. Soldatov et al. (13) found that mutating regions between the EF hand and the IQ motif (IKTEG and LLDQV) eliminated Ca²⁺-dependent inactivation. They suggested that a cooperative interaction between these two regions contributed to Ca²⁺-dependent inactivation. Zuhlke and Reuter (7) used deletions to show that three different domains (the putative EF hand, the IQ domain, and the two-amino acid motif, NE, at amino acids 1630 and 1631 of the cardiac L-type channel) are important for the inactivation process. Sequences within the region between amino acids 1572 and 1651 have also been suggested to be important for regulating targeting, conductance, and open probability of the channel (17).

Peterson et al. (5) compared the binding of Ca²⁺-CaM to the IQ-like motifs from N-, P/Q-, and R-type calcium channel  subunits. In their study, the P/Q-type (_1A), R-type (_1E), and N-type (_1B) calcium channels bound Ca²⁺-CaM, but _1B had a much lower affinity. P/Q-type calcium channels display a small amount of calcium-dependent inactivation, whereas R and N do not. If Ca²⁺-CaM binding to the _1C is responsible for Ca²⁺-dependent inactivation, this result raises the question of the functional role of CaM when bound to R- and N-type calcium channels, since they do not undergo Ca²⁺-dependent inactivation.

In this study we show that the IQ domain of the L-type binds partially Ca²⁺-saturated calmodulin, whereas the IQ domains of P/Q, R, and N have a much lower apparent affinity for the partially Ca²⁺-saturated calmodulin. We also show that another sequence (CB) found in the carboxyl-terminal region of L-type channels binds partially and fully Ca²⁺-saturated CaM and enhances I_Ca facilitation in cardiac myocytes, suggesting that CB can participate in Ca²⁺-dependent modulation of I_Ca. This sequence is found between amino acids 1484 and 1509 in skeletal L-type (_1S) and amino acids 1627-1652 in the cardiac L-type channel (_1C). This region has recently been shown to play a role in membrane targeting of the L-type channel (18), suggesting the possibility of a role for calmodulin in this process. The corresponding regions of _1E (amino acids 1828-1853), _1A (1917-1942), and _1B (1815-1840) do not bind calmodulin. This region in L-type channels has been shown by Soldatov et al. (13) to be important for Ca²⁺-dependent inactivation. The finding that two domains of cardiac L-type channels bind CaM and each of these binds partially Ca²⁺-saturated CaM suggests the possibility that both of these domains may contribute to the Ca²⁺-dependent modulation of L-type Ca²⁺ channels.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents-- Bovine brain calmodulin (95% pure) was purchased from Sigma, solubilized in 10 mM MOPS, 1 mM EGTA, 0.02% NaN₃, pH 7.4, and quantified by absorption from 320 to 277 nm to obtain stock solutions of about 300 µM. Peptides were synthesized at the protein lab facility at Baylor College of Medicine and diluted into 200 mM MOPS, pH 7.4, for assays. The antipeptide antibody was prepared by immunization of rabbits with a peptide representing amino acids 1484-1509 of the skeletal L-type channel coupled to keyhole limpet hemocyanin. All electrophoresis reagents were analytical grade from Bio-Rad.

Electrophoretic Mobility Shift Assays-- Calmodulin electrophoretic mobility was evaluated by non-denaturing polyacrylamide gel electrophoresis under discontinuous conditions as a modified technique described by Laemmli (19). Calmodulin in 200 µM CaCl₂ was incubated with the peptides in molar ratios of peptide to calmodulin of 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 5:1, 10:1. Gels evaluating the ability of mutant calmodulins to bind peptides were in molar ratios of peptide to calmodulin of 0.1:1, 1:1, 2:1, 5:1, 10:1 in 200 µM CaCl₂. The extent of the interaction was quantified by densitometer analysis of the absorbance of the uncomplexed CaM at each peptide to CaM molar ratio. Values were normalized to the absorbance in the absence of peptide.

Fluorescence Studies-- Peptide (730 pmol, 2.9 µM) in 100 mM MOPS, pH 7.4, was added to 730 pmol of brain calmodulin in buffers containing Ca²⁺ ranging from 1 nM to 100 µM. The solution was excited at 280 nm, and emission was detected at 330 nm.

Enzyme-linked Immunoabsorbance Assay for Antibody Binding-- Microtiter plates were coated with CHAPS-solubilized sarcoplasmic reticulum membranes (100 µg of protein/well). The plates were then blocked with 3% bovine serum albumin and incubated in the presence of 2 µM CaM. The antipeptide antibody (serial dilutions 1:100 to 1:200,000) was added, and the samples were incubated overnight at 4 °C. The secondary goat anti-rabbit (dilution 1:3000) coupled to alkaline phosphatase was added, and the incubation was continued for 2 h at room temperature. After washing, the plates were developed with the alkaline phosphatase substrate, p-nitrophenyl phosphate disodium salt.

Preparation of Membranes and Purified Skeletal Muscle L-type Calcium Channel (the Dihydropyridine Receptor, DHPR)-- T-tubule membranes and purified DHPR were prepared as we have previously described (20).

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting-- SDS-polyacrylamide gel electrophoresis and Western blotting were performed as described previously (19, 21). Samples (1-2 µg of protein), corresponding to the purified L-type calcium channel were applied to a 7.5% SDS-polyacrylamide gel. One gel was stained with Coomassie Brilliant Blue, and another gel was transferred for Western blotting (21). The primary antibody used was a sequence-specific antibody to amino acids 1484-1509 of skeletal L-type channel, and the second antibody was a goat anti-rabbit antibody coupled to horseradish peroxidase. The blots were developed using the SuperSignal chemiluminescence substrate (Pierce). For competition experiments, CaM (2 µM) was incubated with the blot for 6-8 h at 4 °C before the addition of the first antibody.

Calcium Currents in Cardiac Myocytes-- Voltage clamp experiments were performed in whole cell mode (22) with freshly isolated rabbit ventricular myocytes, (23). L-type Ca²⁺ current (I_Ca) was isolated by adding Cs⁺ and tetraethylammonium chloride and reducing Na⁺ and K⁺ in the pipette and bath solutions. Elimination of the residual current by nifedipine (10 µM) confirmed that the identity of active current was I_Ca (not shown). I_Ca was activated (0.5 Hz) by voltage command pulses from 80 to +10 mV for 300 ms at 24 °C. Total charge movement was determined by integrating inward I_Ca during the command step using pClamp 6.2 (Axon Instruments) and expressed as a ratio of the nth to the 1st stimulated "beat." The pipette (intracellular) solution was 120.0 mM CsCl, 10.0 mM EGTA, 10.0 mM HEPES, 10.0 mM tetraethylammonium chloride, 5.0 mM phosphocreatine, 3.0 mM CaCl₂, 1.0 mM MgATP, 1.0 mM NaGTP, and pH was adjusted to 7.2 with 1.0 N CsOH. The calculated resting free [Ca²⁺] was ~100 nM (24) in the pipette solution. In some experiments, the CaM-binding peptide CB-L-B (100 µM) was included in the pipette solution, and all cells were dialyzed for 5 min before initiating experiments. The bath (extracellular) solution was N-methyl-D-glucamine 137.0 mM, 25.0 mM CsCl, 10.0 mM HEPES, 10.0 mM glucose, 1.8 mM CaCl₂, 0.5 nM MgCl₂, and the pH was adjusted to 7.4 with 12 N HCl. For experiments designed to eliminate CaM binding, Ca²⁺ was omitted, EGTA was substituted with BAPTA in the pipette solution, and Ba²⁺ was substituted for Ca²⁺ in the bath solution. The null hypothesis was rejected for p < 0.05 using the unpaired Student's t test or analysis of variance as appropriate, and data were expressed as means ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Binding of CaM to Peptides from the Different Voltage-dependent Ca²⁺ Channels-- Partial sequences for the carboxyl-terminal tails of the different voltage-dependent Ca²⁺ channels are shown in Fig. 1. IQ peptides corresponding to the IQ motif of the different voltage-dependent channels are underlined and italicized in this figure. The cardiac IQ motif has been shown to contribute to CaM binding and Ca²⁺-dependent inactivation (5, 7, 9, 11). However, other sequences in the carboxyl-terminal region of _1C have also been shown to play important roles in Ca²⁺-dependent inactivation. Soldatov et al. (13) indicated the amino acids IKTEG and LLDQV, whereas Zuhlke et al. (7) suggested that the Asn and Glu at positions 1630 and 1631 were crucial for inactivation. This region of the cardiac L-type channel is highly homologous to that of the skeletal L-type channel, with only two conservative changes (Table I, italicized letters). To address the question of how this region of the L-type channels contributes to CaM binding, we synthesized a peptide with the sequence containing these elements. The primary interest of our current studies is the skeletal muscle protein, and therefore, we prepared a peptide matching the skeletal sequence (designated CB-L-A, Table I). Other shorter peptides synthesized (see below) had identical sequences in the cardiac and skeletal proteins.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   The aligned sequences of the amino-terminal parts of carboxyl-terminal tails of the 1 subunits of the Lsk (skeletal), Lc (cardiac), P/Q-, N-, and the R-type calcium channels. The sequences were obtained from the Swiss-Prot data base (accession numbers: CCAA (brain P/Q-type, 1A) O00555; CCAB (brain N-type, 1B) Q00975; CCAC (cardiac L-type, 1C) Q13936; CCAE (brain R-type, 1E) Q15878; CCAS (skeletal, L-type, 1S) Q13698. The CB sequences are underlined, and the IQ sequences are underlined and italicized.

To assess the interaction of the peptide with CaM, we examined its ability to bind to CaM on nondenaturing gels. The interaction was analyzed by measuring the absorbance of the CaM band at increasing peptide to CaM ratios in the presence of 200 µM Ca²⁺. As shown in Fig. 2, CB-L-A binds CaM in the presence of Ca²⁺. A representative Coomassie-stained gel is shown in Fig. 2A, top panel, and the absorbance of the CaM band with increasing peptide for three independent experiments is shown in Fig. 2B. To determine which amino acids in this sequence are important for the interaction with CaM, we synthesized several other peptides (CB-L-B, CB-L-C, CB-L-D). The sequences of these peptides are shown in Table I. All of the peptides were able to interact with Ca²⁺-CaM (Fig. 2), suggesting that only the sequence LRAIIKKIWKRTSMKLL is required for CaM binding. The two shorter peptides (CB-L-C and CB-L-D) both produced multiple bands in the presence of CaM. The reason for this is not clear. It may be that, under these conditions, more than one peptide can bind to the CaM molecule, possibly by binding at each of the two CaM lobes. We chose CB-L-B, whose sequence is the same in the cardiac and skeletal muscle L-type channels and has similar affinity for CaM as CB-L-A, for subsequent studies because of its simpler gel pattern and because it was shorter and hence less expensive to prepare than CB-L-A.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   The interaction of the L-type CB region with CaM. CaM was incubated with peptide in increasing peptide to CaM molar ratios before electrophoresis on 20% nondenaturing gels (200 µM Ca2+). The gels were stained with Coomassie Blue. Panel A: representative Coomassie Blue-stained 20% non-denaturing gels of samples containing CaM and increasing molar ratios of CB-L-A, CB-L-B, CB-L-C, and CB-L-D. Solid arrows denote calmodulin. Dashed arrows denote the peptide-CaM complex. The first lane in each panel contains CaM only. Ratios of peptide to CaM (beginning in lane 2) are 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 5:1, and 10:1. Panel B, the intensity of the Coomassie-stained CaM band on the nondenaturing gels in the presence of increasing peptide was determined by densitometry and then normalized to the intensity of the CaM band in the absence of peptide. This is plotted as % control. The data represent three independent determinations. Solid circles, CB-L-A; solid squares, CB-L-B; solid triangles, CB-L-C; solid diamonds, CB-L-D.

A Comparison of the IQ and CB Peptides for Binding CaM-- We next synthesized two sets of peptides: 1) peptides matching the IQ motifs from cardiac L-, skeletal L- and the P/Q-, N-, and R-type voltage-dependent calcium channels and 2) peptides matching the regions that most closely align with the CB region of these same channels. The sequences of these peptides are shown in Table I. We assessed and compared the interaction of these peptides (both IQ and CB) with CaM on nondenaturing gels. These data for the IQ and CB peptides are summarized in Fig. 3, A and B, respectively. We found, in agreement with Peterson et al. (5), that the IQ domains from all of the different channels bound CaM in the presence of Ca²⁺. Of the CB peptides, only those corresponding to L-type channels were able to bind CaM with high affinity in the presence of Ca²⁺. The CB-R, however, showed a small amount of binding at high molar ratios. All of the IQ peptides had a strong affinity for CaM with a relative order at high Ca²⁺ of IQ-Lc = IQ-R > IQ-P/Q > IQ-L-sk > IQ-N. In contrast, only the CB peptide from the L-type Ca²⁺ channels had a significant affinity for CaM.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Comparison of the Interaction of the IQ and CB peptides with CaM. CaM was incubated with peptide in increasing peptide to CaM molar ratios before electrophoresis on 20% nondenaturing gels (200 µM Ca2+). The gels were stained with Coomassie Blue, and the absorbance of the CaM band was measured. The data represent three independent determinations, and the values are normalized to the absorbance of CaM in the absence of peptide. Panel A: solid circles, IQ-Lc; open circles, IQ-Lsk; solid squares, P/Q-IQ; solid triangles, IQ-N; solid diamonds, IQ-R. Ratios of CaM to peptide are similar to those in Fig. 2. Panel B, the intensity of the Coomassie-stained CaM band on the nondenaturing gels in the presence of increasing peptide concentration was determined by densitometry and then normalized to the intensity of the CaM band in the absence of peptide. This is plotted in this figure as % control. The data represent three independent determinations. Solid circles, CB-L-B; solid squares, CB-P/Q; solid triangles, CB-N; solid diamonds, CB-R.

Ca²⁺ Dependence of the Interaction of the Peptides with CaM-- Previous studies suggest that the L-type channel (cardiac) can bind CaM at low Ca²⁺ (5). None of the peptides used in our study were able to bind to CaM if the gels were electrophoresed in the presence of 1 mM EGTA with no added Ca²⁺ (data not shown). This binding site, therefore, does not represent a binding site for apoCaM. We next examined the ability of the various peptides to bind to CaMs that are mutated in the first two (B12), the second two (B34), or all four Ca²⁺ binding sites (B1234) on CaM. All mutations in CaM involve Glu to Gln substitutions at the z positions for coordinating Ca²⁺, resulting in a greatly decreased affinity of the EF hand for Ca²⁺ (25). All gels were electrophoresed in the presence of 200 µM Ca²⁺. Consistent with the findings with apoCaM, none of the peptides could bind to the B1234 mutant (data not shown). However, as can be seen in Fig. 4, both the IQ peptide from the cardiac L-type channel and CB-L-B peptide were able to bind B12 with an affinity similar to that seen with the wild type CaM. The IQ-P/Q and IQ-R peptides could also bind B12, but to a lesser extent. The cardiac IQ peptide was also able to bind B34. None of the other peptides bound B34 as well as IQ-Lc. The mutation of either the amino- or carboxyl-terminal Ca²⁺ binding sites in CaM greatly reduced the affinity of the IQ domains of the skeletal L-type, R-type, and N-type to bind CaM, suggesting that these sites prefer fully Ca²⁺ saturated CaM. The other CB-L peptides (A, C, and D) bound the mutant CaMs similar to CB-L-B (data not shown).

View larger version (68K): [in this window] [in a new window]   Fig. 4.   Interaction of CaMs with mutations in Ca2+ binding sites with the IQ and CB peptides. Panel A, peptides at increasing molar ratios (beginning in lane 2: 0.1:1, 1:1, 2:1, 5:1, and 10:1) were incubated with the B12 CaM and then electrophoresed on nondenaturing gels as described previously. Panel B, peptides (beginning in lane 2: 0.1:1, 1:1, 2:1, 5:1 and 10:1) were incubated with the B34 CaM at increasing molar ratios and then electrophoresed on nondenaturing gels as described previously.

To quantify the Ca²⁺ dependence of the cardiac L-type CB peptide-CaM interaction we examined the change in tryptophan fluorescence of the CB peptide upon binding CaM. The emission spectra for the peptide and CaM, alone and in combination, are shown in Fig. 5A. The binding of CaM to the peptide increases the emission and results in a shift of the peak to shorter wavelengths. The Ca²⁺ dependence of this interaction is shown in Fig. 5B. The EC₅₀ for the Ca²⁺-dependent enhancement of the CB-L-B peptide-CaM interaction is 105 ± 5 nM (n = 3). The absence of a tryptophan in the IQ peptides prevented this type of analysis of their Ca²⁺ dependence.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of Ca2+ on the interaction of CB-L-B with CaM. CB-L-B has a tryptophan at amino acid 15 (corresponding to amino acid 1498 in the intact skeletal L-type 1 subunit), allowing its interaction with CaM (2.9 µM) to be assessed by changes in fluorescence. Excitation was at 280 nm, and emission in relative fluorescence units (RFU) was detected between 300-410 nm. Panel A, the emission spectrum of CB-L-B at high Ca2+ in the presence and absence of CaM. Solid triangles, buffer alone; open circles, CaM alone; closed circles, CB-L-B alone; solid squares, CaM + CB-L-B. Panel B, changes in the fluorescence of a 1:1 mixture of CaM (2.9 µM) to peptide at increasing Ca2+ concentrations. Triangles, CaM + CB-L-B; solid circles, peptide alone; solid squares, CaM alone.

Nondenaturing gels in which the CB-L-B peptide was added at increasing concentrations to a 2:1 mixture of IQ-Lsk (Fig. 6A) and CaM, respectively, showed that the CB-L-B peptide could apparently displace the Lsk- IQ peptide from its complex with CaM, indicating a competitive interaction of these two peptides. In contrast, a 1:1 mixture of IQ-Lc (Fig. 6B) with CaM showed very little displacement by CB-L-B. This is likely due to the higher affinity of IQ-Lc compared with IQ-Lsk for CaM. There is no band corresponding to CaM complexed simultaneously to both peptides. These data clearly show that L-IQ and CB-L-B cannot bind simultaneously to CaM. Our data support a model in which the CB and IQ regions represent either distinct or alternative binding sites rather than both sequences contributing to a single binding site.

View larger version (71K): [in this window] [in a new window]   Fig. 6.   Competition between IQ and CB peptides for binding to CaM. CaM was incubated with IQ-L in the presence of increasing molar ratios of CB-L-B. The samples were then electrophoresed on 20% non-denaturing gels and stained with Coomassie Blue. Panel A is with IQ-Lsk; molar ratios of IQ:CaM:CB-L-B are 0:1:20 (lane 1), 0:1:0 (lane 2), 2:1:0 (lane 3), 2:1:0.5 (lane 4), 2:1:1 (lane 5), 2:1:3 (lane 6), 2:1:5 (lane 7), 2:1:10 (lane 8), and 2:1:20 (lane 9). Panel B is with IQ-Lc; molar ratios of IQ:CaM:CB-L-B are 0:1:20 (lane 1), 0:1:0 (lane 2), 1:1:0 (lane 3), 1:1:0.5 (lane 4), 1:1:1 (lane 5), 1:1:3 (lane 6), 1:1:5 (lane 7), 1:1:10 (lane 8), and 1:1:20 (lane 9). Left dashed arrow, CaM-CB-L-B; right small solid arrow, CaM alone; right large solid arrow, CaM -IQ-L complex.

Demonstration of the Ability of the L-type Ca²⁺ Channel to Bind Calmodulin Close to the CB Site-- To demonstrate that the full-length _1S subunit can bind calmodulin at a site close to the CB sequence, we prepared an anti-peptide antibody to peptide CB-L-B and examined the ability of calmodulin to block the binding of the antibody to the ₁ subunit of the DHPR on Western blots (Fig. 7A). The skeletal protein was used for these studies because it is more abundant than the cardiac channel, and its sequence in the CB region is identical to that of the cardiac channel. Pre-incubation of the blots with calmodulin before the addition of the antibody blocked the labeling of this subunit by antibody, suggesting that the denatured _1S protein bound calmodulin at a site close to the antibody binding site. To assess the effects of calmodulin on the binding of the antibody to a channel in the presence of a nondenaturing detergent, we partially solubilized membranes in CHAPS and examined the ability of calmodulin to block the interaction with the antibody in an enzyme-linked immunoabsorbance assay (Fig. 7B). Again, calmodulin blocked the interaction of the antibody with the DHPR.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   CaM binds close to the CB sequence in the intact DHPR. Purified DHPR was electrophoresed on 7.5% SDS gels. The gels were transferred and Western-blotted with anti-CB-L-B peptide antibody as described under "Materials and Methods." A, lane 1 is the Coomassie-stained molecular weight markers. Lane 2 contains the purified DHPR (2 µg). Lanes 3 and 4 are the ECL-developed Western blot of DHPR incubated without (lane 3) and with (lane 4) 2 µM CaM before the addition of first antibody. B, skeletal muscle t-tubule membranes (100 µg/well) in 200 µl of 0.1 M sodium carbonate, pH 9.6, 0.1% CHAPS were bound to the wells of a microtiter plate. Previous studies have shown that this treatment does not destroy the ability of this protein to bind [3H]PN200-110 J.-Z. Zhang and S. L. Hamilton, unpublished observation). After an overnight incubation at 4 °C, the plates were blocked with 3% bovine serum albumin. CaM (2 µM) was added to half the plates. After incubating for 30 min, anti-CB-L-B antibody (dilutions from 1:100 to 1:200,000) was added and incubated for 2 h at room temperature. Bound antibody was detected as described under "Materials and Methods." Closed circles, control; open circles, antibody added in the presence of CaM.

CB-L-B Alters Calcium Currents in Cardiac Myocytes-- The L-type Ca²⁺ channel IQ domain can powerfully direct I_Ca facilitation (11, 12) and inactivation (5, 11, 12) by an unknown molecular mechanism(s). To test for possible L-type Ca²⁺ channel regulatory actions of CB, I_Ca was measured in cardiomyocytes dialyzed with CB-L-B under conditions favorable or adverse to Ca²⁺-CaM-dependent binding. CB-L-B enhanced I_Ca facilitation compared with control cells in the presence of a physiologic intracellular Ca²⁺ concentration (Fig. 8, a, b, and e), but CB-L-B was without effect on I_Ca during increased intracellular Ca²⁺ buffering when extracellular Ca²⁺ was substituted for Ba²⁺ (Fig. 8, c, d, and e). These data support the hypothesis that CB helps to determine Ca²⁺-CaM-dependent regulation of L-type Ca²⁺ currents.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   CB-L-B enhances Ca2+ channel facilitation. Enhanced ICa facilitation by CB-L-B peptide requires conditions favorable to CaM binding. The addition of CB-L-B peptide to the pipette solution increases ICa facilitation (panel b) compared with control cells (panel a). Both ICa facilitation and the effect of CB-L-B peptide is eliminated by substituting Ba2+ for Ca2+ (panels c and d). The ICa tracings are superimposed for comparison for the 1st (B1) and 10th (B10) beats; the calibration bars indicate 100 ms (horizontal) and 1000 pA (vertical) for panels a-d. Panel e shows the summary data for the ratio of integrated ICa for each stimulated beat divided by integrated ICa from the first stimulated beat. ICa ratios were significantly (p < 0.05) greater for cells dialyzed with CB-L-B peptide compared with control cells for beats 8-20 only in the presence of Ca2+.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

L-type Ca²⁺ channels show Ca²⁺-dependent inactivation that requires CaM. Recent work has centered on the IQ-like domain in the carboxyl terminus of the  subunit in Ca²⁺ channels as representing the CaM binding site. However, this is an incomplete picture since voltage-dependent Ca²⁺ channels that do not show Ca²⁺-dependent inactivation bind CaM at their corresponding IQ-like sequences in the carboxyl terminus. This raises the question of why the binding of Ca²⁺-CaM to the IQ domain on the L-type channel leads to Ca²⁺-dependent inactivation, whereas the binding to IQ domains of the other channels does not. If the IQ domain is the primary binding site for CaM on these channels, the binding to the IQ domain of the L-type channel must lead to a secondary change that does not occur in the other channels. Recently Soldatov et al. (13), Zuhlke and Reuter (7), and Peterson et al. (14) found that determinants outside of the IQ motif are necessary for Ca²⁺-dependent inactivation. Soldatov et al. (13) showed that the sequences IKTEG and LLDQV are required, and Zuhlke and Reuter (7) demonstrated the requirement for the IQ domain and residues 1630 and 1631 for Ca²⁺-dependent inactivation (7). These sequences immediately bracket the CB sequence in L-type channels that we have shown binds CaM. Petersen et al. (14) demonstrated that the exchange of the sequence VVTL in the F helix of the putative EF hand with the corresponding sequence of the R channel abolished Ca²⁺-dependent inactivation. The widely spaced regions of the carboxyl tail involved in Ca²⁺-dependent inactivation suggest that some of these regions are involved in the CaM binding, whereas others may contribute to the regulation of the channel that occurs after CaM binds. Other portions might be crucial for the communication between these two domains.

Our data with the different IQ and CB peptides are summarized in Table I. Consistent with the results of Peterson et al. (5), we found that the IQ peptides matching the sequence from the L, P/Q, N, and R channels all bound Ca²⁺-CaM. However, only cardiac IQ-L was able to bind (in the presence of Ca²⁺) the CaMs mutated in the Ca²⁺ binding sites (B12 and B34) with an affinity comparable with that of wild type CaM. The P/Q and R channel IQ peptides bound B12 CaM and B34 CaM with reduced affinity compared with wild type CaM, and the N and L-sk IQ peptide showed no ability to bind these mutant CaMs. None of the peptides were able to bind to CaM in less than 10 nanomolar Ca²⁺ concentrations or to a CaM mutated at all four Ca²⁺ binding sites. These peptides do not, therefore, represent binding sites for apoCaM. The cardiac L-type channel and, to a lesser extent, the P/Q channels show Ca²⁺-dependent modulation. The other channels do not. The unique ability of the cardiac L-type IQ sequence to bind CaM that is not fully Ca²⁺ saturated may allow CaM to bind to this channel under resting Ca²⁺ conditions. Higher Ca²⁺ concentrations could then produce Ca²⁺-dependent inactivation. This is the first demonstration of a property of the IQ site on the L-type channels that sets it apart from the IQ sites on the other channels.

We also found that a domain (CB) between the EF hand and the IQ domain of L-type channels was able to bind B12 CaM and wild type CaM in the presence of Ca²⁺. Mutations in Ca²⁺ binding sites 3 and 4 on CaM greatly reduced the affinity of CaM for this sequence. The Ca²⁺ dependence of the interaction of CB-L-B with CaM (EC₅₀ = 100 nM) suggests that this interaction could also take place at resting Ca²⁺ levels in the cell, preparing the channel for inactivation when the Ca²⁺ reaches the appropriate levels. Half-maximal inhibition of Ca²⁺ channel activity by Ca²⁺ occurs at about 4 µM Ca²⁺ (26). The CB-L-B peptide also enhanced I_Ca facilitation in cardiac myocytes in a Ca²⁺-dependent manner, suggesting that this sequence may have functional significance for regulating L-type Ca²⁺ channels. The P/Q, R, and N channels did not have comparable sequences that bound CaM. This is the second feature that distinguishes the cardiac L-type channels from the other voltage-dependent channels. The identified CaM binding sequence is within a domain previously suggested to play important roles in regulating Ca²⁺-dependent inactivation (7, 13), targeting (17, 18, 27), conductance (17), and open probability (17) of the channel. These findings suggest a role for calmodulin in regulating other aspects of L-type calcium channel function.

In addition to the peptide data, other workers have found that amino acids within and bracketing this sequence are necessary for Ca²⁺-dependent inactivation of the channel. Our data support these findings. We have shown that calmodulin can block the interaction of an antibody to the CB-L-B sequence with both the SDS-denatured and the CHAPS-solubilized skeletal muscle L-type channel. Although these findings support a model in which the CB sequence is a CaM binding site, we cannot rule out the possibility that CaM binding to the nearby IQ sequence sterically hinders antibody binding to the CB sequence. Mutations of this region coupled to analysis of CaM binding will be necessary to resolve this issue.

The carboxyl-terminal tail of the L-type channels has also been suggested to play a role in membrane targeting of this protein (18). The CB sequence that we demonstrate to bind CaM is within the region that has recently been suggested to play a role in membrane targeting of the L-type channels (18). CaM binding may also contribute to this process.

The IQ and CB regions of the cardiac L-type channel could either each bind a molecule of CaM or they could both contribute to the same CaM binding site. We have demonstrated that the interactions of the L-type IQ and CB peptides with CaM are competitive. Based on our findings, we propose a model in which the IQ and CB domains in the carboxyl-terminal tails of the L-type channels represent either two distinct binding sites for CaM or alternative sites for the interaction with partially and fully saturated CaM. One possibility is that only one CaM can bind to the carboxyl tail, and which site (IQ versus CB) is occupied is controlled by factors regulating the conformation of this region (for example, Ca²⁺ binding to the EF hand).

	FOOTNOTES

^* This work is supported by a grant from the Muscular Dystrophy Association and National Institutes of Health Grant AR44864 (to S. L. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030. Tel.: 713-798-3894; Fax: 713-798-5441; E-mail: susanh@bcm.tmc.edu.

Published, JBC Papers in Press, September 25, 2000, DOI 10.1074/jbc.M007158200

	ABBREVIATIONS

The abbreviations used are: apoCaM apocalmodulin, Ca²⁺-free calmodulin; CaM, calmodulin; Ca²⁺-CaM, Ca²⁺-bound calmodulin; CB peptide, CaM-binding peptide representing amino acids 1484-1509 of the skeletal muscle 1 subunit L-type channel and 1627-1652 of the cardiac L-type channel; CHAPS, 3-[(3-chloamidopropyl)-dimethylammonio]-1-propanesulfonate; DHPR, dihydropyridine receptor; EF hand, Ca²⁺ binding motif; IQ, motif for binding CaM; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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