1Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas; and 2Department of Molecular and Cellular Biochemistry, Ohio State University, Columbus, Ohio
Submitted 16 April 2004 ; accepted in final form 6 October 2004
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ABSTRACT |
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Ca2+-dependent inactivation; Ca2+-dependent facilitation; apocalmodulin
Calmodulin binding to an "IQ-like" motif in the COOH-terminal region of the 1C-subunit of the CaV1.2 has been shown to be crucial for both CDI and CDF (14), raising questions as to how a single Ca2+ sensor, binding at a single site, could produce opposite effects on channel activity. A mutant CaM (E1234Q) that cannot bind Ca2+ at any of the four Ca2+ binding sites (EF hands) competitively inhibits the interaction of Ca2+ CaM with the cardiac L-type Ca2+ channel (11), suggesting that both the Ca2+-free and Ca2+-bound forms of CaM bind to this channel. However, only the Ca2+-bound form can produce inactivation (11). It has been proposed that CaM in the Ca2+-free state (apoCaM) is anchored to the Ca2+ channels in the region of the IQ motif (8). Our laboratory (12) has shown that a peptide representing amino acids 16271685 (human sequence) of the L-type Ca2+ channel binds apoCaM.
The variable lobe dependence and differential response to added Ca2+ buffers among the voltage-dependent Ca2+ channels (8) imply that the N and C lobes of CaM are likely to have very different Ca2+ binding properties when bound to the different channels. We have examined the Ca2+ binding properties of CaM bound to peptides matching the sequences of the IQ motifs of the Lc (cardiac), P/Q, R, and N-type voltage-dependent Ca2+ channels.
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MATERIALS AND METHODS |
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Bovine brain CaM (95% pure) was purchased from Sigma (St. Louis, MO), solubilized in 10 mM MOPS (pH 7.4), 1 mM EGTA, 0.02% NaN3, and quantified by absorption from 320 to 277 nm to obtain stock solutions of 300 µM (2). All peptides were synthesized at the protein laboratory facility at Baylor College of Medicine and were diluted into 200 mM MOPS (pH 7.4) for assays. F19W and F92W were purified as described by Black et al. (2).
Methods
Determination of affinity of F19W and F92W for LcIQ peptide. LcIQ peptide has a predicted isoelectric point >10, requiring the need to silanize the negatively charged quartz cuvettes and glassware to prevent loss of peptide. The cuvettes and glassware were dipped in a solution of 1% N-trimethoxysilylpropyl-N,N,N-triethylammonium (United Chemical Technologies, Bristol, PA) in methanol, rinsed in 100% methanol, then baked at 115°C for several hours. A range of LcIQ concentrations (from 0 to 1,500 nM) were prepared in solutions containing 200 nM CaM (F19W, F92W, or control), 30 mM MOPS, 100 mM KCl, 1 mM EGTA, and 2 mM CaCl2. The buffer was titrated to pH 7.2 after all the ingredients were added. After 45 min of incubation, the fluorescence spectra were collected on a fluorometer (model PC-1; ISS, Urbana-Champaign, IL) by tryptophan excitation at 295 nm using a 0.5-mm slit (4 nm band pass) and collecting the emission spectrum from 310 to 400 nm. A solar blind excitation filter and a 295-nm cut-on emission filter (Oriel, Stamford, CT) were used to reduce interference.
Because peptide binding shifts both the peak emission and the intensity of emission from F19W CaM and F92W CaM, the extent of binding is not simply proportional to intensity changes at any particular wavelength or to the shift of peak emission. To determine the extent of peptide binding from the spectral data, spectra at various peptide concentrations were assumed to be linear combinations of the spectra for unbound and fully bound CaM. Matrix methods were then used to extract the relative contribution of each end point spectrum to the spectra at intermediate concentrations as follows: S = B*W, where S is the matrix of the spectral data at all concentrations of peptide; B is a basis-set matrix of the two end point spectra (zero and maximal peptide concentration); and W is the matrix containing the relative contributions of the two end point spectra to the spectra at all peptide concentrations. The values of W essentially represent the extent of titration; here, W contains the titration data of interest. To solve for W, we used the following equation: W = B1*S. Because the B matrix is generally rectangular and cannot be inverted, the matrix pseudo-inverse was applied to determine B1 and then calculate W, as implemented in MatLab (version 13; MathWorks, Natick, MA). The values in W were then plotted against peptide concentration to determine the titration curves.
The concentration range of binding was similar to the concentration of CaM; therefore, a significant amount of peptide was bound at the lower ends of the titration curves. To plot the data in terms of free peptide concentration, the free peptide concentration was calculated from the total concentration added and corrected for the bound peptide. Bound peptide was determined from the extent of the titration, given by the values in the matrix W, and the concentration of CaM and assuming 1:1 stoichiometry of binding: [Bound peptide] = w x [CaM], where w represents the weight for the corresponding peptide concentration taken from W. Then [free peptide] = [added peptide] [bound peptide].
Data were fit to a four-parameter Hill equation using SigmaPlot (SSPS, Chicago, IL)
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Determination of Ca2+ affinity of F19W and F92W: 1 µM F19W or F92W.
CaM was incubated with 5 µM peptide for 1 h at room temperature in standardized Ca2+ buffers prepared from Ca2+ calibration kits available from Molecular Probes (Eugene, OR). Fluorescence data were collected on an ISS PC-1 fluorometer using 1 mm slits (8 nm bandwidth) and a 295-nm cut-on emission filter (Oriel). A 295-nm excitation wavelength was used, and the emission was monitored at 330 nm. Data were plotted and analyzed using SigmaPlot (SSPS) software as described above.
Ca2+ dissociation kinetics.
As previously described by Black et al. (2), an Applied Photophysics (model SX.18MV; Leatherhead, UK) stopped-flow instrument was used to measure rates of Ca2+ dissociation (koff) at 22°C. The apparatus has a dead time of 1.35 ms. A 150-W Xenon arc source was used for excitation. The dissociation rates were extracted using the nonlinear Levenberg-Marquardt algorithm implemented in software provided by P. J. King of Applied Photophysics. Each observed rate represents the concerted release of two Ca2+ from either the NH2 terminal or the COOH terminal Ca2+-binding sites. Each trace represents an average of 58 individual traces fit with either a single or double exponential as needed (variance <4 x 105). All fits of the kinetic traces occurred after premixing was complete. Tryptophan fluorescence was measured after rapid mixing of 50 µl of F19W or F92W CaM (4 µM), peptide (20 µM), and Ca2+ (200 µM) in 10 mM MOPS, 90 mM KCl, pH 7.0, with an equal volume of EGTA (10 mM). A 320-nm cut-on filter (Oriel) was used to minimize contamination from changes in the intrinsic tyrosine fluorescence of CaM and peptides. Each tryptophan fluorescence trace was fit with the following single exponential equation: A*ek + C, where A is the amplitude of the fluorescence change, and kt is the rate at which the change is occurring.
Ca2+ dissociation rates were verified by the rapid mixing of CaM (8 µM), IQ peptide (40 µM), Ca2+ (15 µM) in 10 mM MOPS, and 90 mM KCl, pH 7.0, with an equal volume of the fluorescent Ca2+ chelator Quin-2 (150 µM). A 510-nm broad band-pass filter (Oriel) monitored the emission due to excitation at 330 nm. Each Quin-2 fluorescence trace was fit with the double exponential equation A1*ek1t + A2*ek2t + C, where A1 and A2 are the individual amplitudes of each component of the fluorescence change, and k1 and k2 are the corresponding rates of change. A double exponential was required to fit the Quin-2 signal reporting both the NH2 terminal (fast) and COOH terminal (slow) Ca2+ dissociation rates. The changes in Quin-2 fluorescence were converted into moles of Ca2+ dissociating from CaM, as previously described by Johnson et al. (4). Briefly, monitoring the increase in Quin-2 fluorescence with increasing concentrations of Ca2+ (10, 20, 40, and 80 µM) allowed for the conversion of observed Quin-2 fluorescence to molar [Ca2+]. Quin-2 fluorescence increased linearly as a function of total [Ca2+], allowing the Quin-2 fluorescence increase to be used to calculate the total number of moles of Ca2+ that dissociate from each lobe of CaM.
Calculation of Ca2+ association rate constant. Ca2+ association rates were estimated using the relationship kon = koff/Kd, assuming that koff and Kd represent the concerted release or binding events of Ca2+ ions from the Ca2+-binding sites of the mutant CaM, as described by Wang et al. (13). koff represents the dissociation of both Ca2+ ions from each lobe of CaM due to the inability to distinguish the dissociation events of the individual ions via Quin-2 and/or tryptophan fluorescence. Apparent Kd values represent the concerted binding of both Ca2+ ions assuming that both Ca2+ ions bind indistinguishably to the Ca2+ binding sites of the CaM mutants.
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RESULTS |
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To determine the effects of the different IQ peptides on Ca2+ binding properties of the N- and C-lobes of CaM, we used CaM mutants with phenylalanine 19 or 92 mutated to tryptophan. Tryptophan substitutions within the paired EF hands of each domain of CaM are sensitive to local Ca2+-dependent conformational changes (2, 5). F19W and F92W CaM are similar to earlier CaM mutants used to assess the site-specific order of Ca2+ binding to CaM and the effect of the chelating residues within the Ca2+ binding loops on Ca2+ affinity (2, 5). The IQ peptides used in this study are listed in Table 1.
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Fluorescence emission (330 nm) of F19W and F92W alone and in the presence of different IQ peptides as a function of Ca2+ concentration are shown in Figs. 2 and 3, respectively. Data for all of the peptides are summarized in Table 2. All of the IQ peptides, except for N-IQ, increased the maximal fluorescence yield of F19W (see Table 2). All of the peptides, except for Lc-IQ, increased the maximal fluorescence yield for F92W. When not bound to a peptide, the C-lobe of CaM has a higher affinity for Ca2+ than the N-lobe, but when bound to any of the IQ peptides, the affinity of the two lobes for Ca2+ is similar. The Lc-IQ peptide produces the greatest increase in the Ca2+ affinities of both lobes of CaM (100x increase in apparent Ca2+ affinity of the N-lobe and
20x increase in apparent Ca2+ affinity of the C-lobe). The N-IQ peptide produces a small effect on the Ca2+ affinity of the N-lobe of CaM (
2x increase in apparent Ca2+ affinity), but does not alter the Ca2+ affinity of the C-lobe.
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Changes in Ca2+ concentrations inside a cell are likely to occur rapidly and, hence, it is important to determine the kinetic constants for Ca2+ binding to CaM bound to the IQ peptides. The increased Ca2+ affinity of CaM bound to the IQ peptides could arise from an increased rate of Ca2+ association and/or decreased rate of Ca2+ dissociation. The determination of which rate constants are altered by the interactions with the IQ peptides could shed light on why intracellular Ca2+ buffers are more likely to alter CDI of P/Q, R, and N channels than that of L-type channels (8). Our first method to determine the rate of Ca2+ dissociation from the lobes of CaM involved stopped flow measurements of changes in tryptophan fluorescence of F19W or F92W (complexed to the peptides) as a function of time after the addition of EGTA to chelate free Ca2+ (eliminating rebinding after dissociation). Dissociation curves are shown in Figs. 5 and 6 for F19W and F92W (in the absence or presence of the indicated peptides). The values for the dissociation rate constants (k1) obtained with F19W and F92W tryptophan fluorescence are presented in Table 3. All of the peptides slowed the rate of dissociation of Ca2+ from both lobes of CaM, as determined by changes in the fluorescence of F19W and F92W, with the exception of the values. The dissociation of Ca2+ from the N-lobe of F19W bound to the N-IQ peptide was significantly faster than when F19W was bound to any of the other peptides.
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Effects of Peptides on Dissociation of Ca2+ Measured by Changes in Quin-2 Fluorescence
To determine whether the F19W or F92W mutations in CaM are altering the Ca2+ binding properties of CaM and to measure Ca2+ dissociation from the lobes of CaM by an alternative method, we used stopped flow measurements of changes in the Quin-2 fluorescence upon binding Ca2+ released from the CaM-IQ complexes (Fig. 7). For these experiments, we used wild-type CaM, F19W, and F92W complexed to the peptides. As expected, two kinetic components were detected in the Quin-2 experiments (dissociation from both the N and C lobes). The dissociation values obtained are summarized in Table 4. The values for the faster component detected with Quin-2 were similar to the dissociation rates calculated from the change in tryptophan fluorescence of F19W for each peptide while the values for the slower component were similar to values obtained from the changes in tryptophan fluorescence of F92W (compare Tables 3 and 4). In addition, the two rates of Ca2+ dissociation from wild-type CaM bound to each of the peptides were similar to that of F19W and F92W. These findings suggest that the tryptophan substitutions themselves did not greatly alter the Ca2+ binding properties of CaM when complexed to the IQ peptides. The close correlation between the 2 kinetic components detected by Quin-2 fluorescence (Table 4) and the rates of decrease in the tryptophan fluorescence as Ca2+ dissociates from the F19W and F92W complexes (Table 3) suggest that these tryptophan mutants accurately report Ca2+ binding events in the N-lobe and C-lobe of CaM. The N-IQ peptide produced the smallest effects on Ca2+ dissociation from the N-lobe.
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While there is a good correlation between the decrease in the rates of Ca2+ dissociation and the observed increases in Ca2+ affinities, the decreases observed in the rates of Ca2+ dissociation are not alone large enough to account for the observed changes in Ca2+ affinities. Alterations in the Ca2+ association rates (k1) are also likely to be occurring. We estimated the Ca2+ association rates for the N and C-lobes of F19W and F92W, respectively, in the presence and absence of the IQ peptides (Table 5) using the relationship kon = koff/Kd. In all cases the calculated Ca2+ association rate to the N-lobe was decreased when F19W was complexed with an IQ peptide. The binding of CaM to the peptides, therefore, apparently inhibits the access of Ca2+ to its binding site. The smallest decrease in association rate was obtained with F19W bound to the Lc-IQ peptide. These data suggest that Ca2+ will associate with the N-lobe of CaM bound to Lc-IQ faster than to the N-lobes of the other IQ peptides. However, for both Lc-IQ and P/Q-IQ, the rate of Ca2+ association with the C-lobe of F92W suggest that Ca2+ will also bind faster to the C-lobe of CaM bound to Lc-IQ compared with CaM bound to the other peptides. If this same difference in Ca2+ association to the C-lobe exists in the native channels, it could contribute to the lack of an effect of added Ca2+ buffers on CDI of L-type calcium channels and on CDF of P/Q type channels.
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DISCUSSION |
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The L-type channel is less sensitive than the other voltage-dependent Ca2+ channels to intracellular Ca2+ buffering (8). We think the reason for this lies in the relative rates at which Ca2+ binds to the lobes of CaM bound to the different IQ motifs. CaM bound Lc-IQ binds Ca2+ faster and with higher affinity at both lobes than the other complexes. If this is also true for CaM bound to the channel, this is likely to make the L-type channel less sensitive to intracellular Ca2+ buffers.
All of the peptides, with the exception of N-IQ, increase the Ca2+ affinity of both lobes of CaM and, in doing so, make the apparent Ca2+ affinities of the two lobes approximately equivalent. Kinetic analyses, however, show that Ca2+ association and dissociation with the N-lobe is faster than with the C-lobe when CaM is bound to the IQ peptides. If the interaction of CaM with the peptides mimics the in vivo situation, the differential binding by the lobes of CaM could have profound consequences for functional outcomes of changes in intracellular Ca2+. An increase in intracellular Ca2+ concentrations would lead to Ca2+ binding first to the N-lobe of CaM and then the C-lobe. In this scenario, the outcome of the Ca2+-dependent events would depend on the nature of the Ca2+ signal (amplitude, duration, and location) and the rate at which the Ca2+-driven signal is transduced into the functional change in the channel. Rapid events (occurring on a time scale that is less than or equal to the time that Ca2+ remains elevated) would be likely to be driven either by Ca2+ binding to N-lobe or, if Ca2+ is elevated long enough to saturate the C-lobe, by Ca2+ binding to both lobes. Events that occur during or after the decrease in intracellular Ca2+ are more likely to be driven by the Ca2+ bound C-lobe because the N-lobe will release its Ca2+ before the C-lobe. For the N-lobe to remain Ca2+ bound, the intracellular Ca2+ levels would need to remain elevated, and, therefore, events that require sustained Ca2+ levels would be likely to be driven by Ca2+ binding to the N-lobe. In summary, we propose that the ability of calmodulin to regulate both Ca2+-dependent facilitation and inactivation is related to its ability when in complex with the IQ motifs to assume multiple conformational states controlled by differential binding of Ca2+ at its N- and C-lobes in response to different types of Ca2+ signals.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
* D. J. Black and D. B. Halling contributed equally to this study.
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