Facilitation and Ca²⁺-dependent Inactivation Are Modified by Mutation of the Ca_v1.2 Channel IQ Motif

Abstract

The heart muscle responds to physiological needs with a short-term modulation of cardiac contractility. This process is determined mainly by properties of the cardiac L-type Ca²⁺ channel (Ca_v1.2), including facilitation and Ca²⁺-dependent inactivation (CDI). Both facilitation and CDI involve the interaction of calmodulin with the IQ motif of the Ca_v1.2 channel, especially with Ile-1624. To verify this hypothesis, we created a mouse line in which Ile-1624 was mutated to Glu (Ca_v1.2^I1624E mice). Homozygous Ca_v1.2^I1624E mice were not viable. Therefore, we inactivated the floxed Ca_v1.2 gene of heterozygous Ca_v1.2^I1624E mice by the α-myosin heavy chain-MerCreMer system. The resulting I/E mice were studied at day 10 after treatment with tamoxifen. Electrophysiological recordings in ventricular cardiomyocytes revealed a reduced Ca_v1.2 current (I_Ca) density in I/E mice. Steady-state inactivation and recovery from inactivation were modified in I/E versus control mice. In addition, voltage-dependent facilitation was almost abolished in I/E mice. The time course of I_Ca inactivation in I/E mice was not influenced by the use of Ba²⁺ as a charge carrier. Using 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid as a chelating agent for intracellular Ca²⁺, inactivation of I_Ca was slowed down in control but not I/E mice. The results show that the I/E mutation abolishes Ca²⁺/calmodulin-dependent regulation of Ca_v1.2. The Ca_v1.2^I1624E mutation transforms the channel to a phenotype mimicking CDI.

Introduction

The heart muscle adapts to physiological needs with an autonomic modulation of cardiac contractility. This process is determined mainly by the intrinsic properties of the cardiac L-type Ca²⁺ channel (Ca_v1.2). These properties include Ca²⁺-dependent facilitation (CDF)³ and Ca²⁺-dependent inactivation (CDI). CDF serves to potentiate Ca²⁺ influx through Ca_v1.2 channels during repeated activity, which contributes to increased force-frequency relationship of the heart muscle during exercise (1, 2). CDI terminates Ca²⁺ entry after opening of the channel to avoid Ca²⁺ overload and arrhythmias (3, 4). Both types of regulation involve, as a key step, direct binding of the Ca²⁺ sensor protein calmodulin (CaM) to the Ca_v1.2 channel (4–7), alongside with the action of CaM on Ca²⁺/CaM-dependent protein kinase II (CaMKII) (2, 6, 8–11).

Strong evidence exists that CaM binds to the IQ motif of the Ca_v1.2 channel that is located at amino acids 1624–1635 of the Ca_v1.2 C terminus (5, 6, 12, 13). Especially Ile-1624 determines the amount of CaM binding. For example, the mutation I1624E decreases the affinity of the IQ sequence for CaM by ∼100-fold (6, 11). In addition, the I/E mutation abrogates CDF and CDI of L-type Ca²⁺ currents expressed in Xenopus oocytes (11).

It is unclear if the binding of CaM to the Ca_v1.2 channel is relevant for the function of the cardiac calcium channel in the behaving mouse. Therefore, we mutated Ile-1624 to Glu in the IQ motif of the murine Ca_v1.2 channel gene. The results show that this mutation is lethal. To overcome this problem, we generated mice with a conditional heart-specific I/E mutation in the Ca_v1.2 channel gene. Electrophysiological analysis of Ca_v1.2 channel currents in cardiomyocytes (CMs) from I/E mice revealed that the I/E mutation blocks CaM/CaMKII-mediated regulation of the Ca_v1.2 channel in the heart and induces a channel phenotype with permanent CDI characteristics.

EXPERIMENTAL PROCEDURES

All substances used were of the highest purity available. The Ca_v1.2-specific antibody used in this study has been described previously (14). Amino acid numbering is according to the Oryctolagus cuniculus Ca_v1.2 sequence (GenBank^TM accession number X60782.1).

Generation of Mice with the I1624E Mutation

To construct the targeting vector, a 8.0-kb fragment containing exons 39–44 of CACNA1C was isolated from 129/Svj mouse genomic DNA. The targeting vector was composed of a 1.1-kb short 5′-arm, a 4.5-kb fragment containing the neo-tk and loxP sequences, a 743-base fragment including the I/E mutation at position 1624, and the long a 5.5-kb long 3′-arm. All mutation procedures were carried out by site-directed PCR mutagenesis (Stratagene). The targeting construct was electroporated into R1 embryonic stem cells (129/Sv × 129/Sv-CP F1) (15). Positive clones were identified by PCR and confirmed by Southern blotting. The neo-tk cassette was removed from the germ line through cre/loxP-mediated excision in a second targeting step. A positive embryonic stem cell clone was injected into C57BL/6 blastocysts, and chimeras were crossed with C57BL/6 mice. After confirmation of successful targeting by PCR and Southern blot analysis, heterozygous mice were bred with mice expressing Cre under the control of the α-myosin heavy chain promoter (MerCreMer) and with Ca_v1.2 L2 mice (16) to produce inducible adult mice bearing the I1624E mutation. The intercross of the three mouse lines resulted in production of Ca_v1.2^I1624E/L2 × MerCreMer (identified as I/E) and Ca_v1.2^L2/+ × MerCreMer (Ctr) offspring at the expected Mendelian ratio. The experiments were performed with litter-matched mice aged 8–18 weeks on a mixed C57BL6/129/Sv background. The mice were injected with 2 mg of tamoxifen/mouse/day for 4 days. Experiments were performed 10 days after the first tamoxifen injection. All animals were maintained and bred in the animal facility of the Institut für Pharmakologie und Toxikologie, Technische Universität München, and had access to water and standard chow ad libitum. All procedures relating to animal care and treatment conformed to the institutional and governmental guidelines.

Cell Preparation

Ventricular myocytes were isolated as described (Alliance for Cellular Signaling (AfCS) Procedure Protocol PP00000125), maintained at 37 °C, and aerated with 5% CO₂.

Electrophysiological Recordings

The whole cell L-type Ca²⁺ channel current (I_Ca) was measured at 35 °C. Stimulation and data acquisition were performed as described (17). Time constants of I_Ca inactivation were obtained by a fit from the peak current to the current value at the end of the voltage pulse by a two-exponential function using pCLAMP 9 (Molecular Devices). All fits showed a correlation coefficient >0.98.

Statistics

Data plotting and statistical analysis were carried out using Prism 5 (GraphPad Software). The null hypothesis was rejected if p was <0.05. Data are presented as means ± S.E.

RESULTS

Ile-1624 of the CACNA1C gene was mutated to glutamate using transgenic gene knock-in techniques (Fig. 1A). The resulting homozygous mice (genotype Ca_v1.2^I1624E on both alleles) were not viable. Therefore, we crossbred heterozygous Ca_v1.2^I1624E mice with mice expressing the floxed Ca_v1.2 gene and α-myosin heavy chain-MerCreMer (16, 18), allowing tissue- and time-dependent inactivation of the Cav1.2 gene by the tamoxifen-controlled α-myosin heavy chain-MerCreMer recombinase. The mutation in the resulting I/E mice (genotype Ca_v1.2^−/I1624E) was confirmed by genomic sequencing (Fig. 1B). I/E mice had a reduced life span and died within 3 weeks after treatment with tamoxifen (Fig. 1C). Western blot analysis of cardiac muscle using anti-Ca_v1.2 antibody detected reduced protein levels in the ventricles of I/E mice compared with litter-matched Ctr mice (genotype Ca_v1.2^−/+) at day 10 (Fig. 1D). Reduced expression of the Ca_v1.2^I1624E cDNA was confirmed in the HEK293 expression system. For further studies, I/E mice were studied at day 10 after treatment with tamoxifen.

FIGURE 1.

Generation of I/E mice. A, first row, sequence around the IQ motif of CACNA1C. Second row, genomic DNA structure of CACNA1C. boxes represent exons 39–44 encoding part of the C terminus of Ca_v1.2. Third row, targeting vector. neo, neomycin resistance gene; tk, thymidine kinase gene with loxP sequence (triangles) at both sides. The I1624E substitution is shown. Fourth row, knock-in locus after homologous recombination and Cre-mediated deletion of resistance markers. N, NotI; B, BamHI; C, ClaI; X, XhoI; E, EcoRI; H, HindIII. B, sequence analysis of genomic DNA in the region coding for Ile-1624 from one WT Ca_v1.2 and one Ca_v1.2^I1624E (Cav1.2 I/E) mouse. C, survival curve of I/E and litter-matched Ctr mice. t = 0 represents the start of the treatment of the mice with tamoxifen (2 mg/animal on 4 consecutive days). D, immunoblot of cardiac membrane preparations. Expression of Ca_v1.2 protein was reduced in ventricular but not atrial preparations from I/E versus Ctr mice at day 10. GAPDH was used as a loading control. 20 μg of protein was loaded per slot and separated on a 7.5% SDS-polyacrylamide gel.

To test the physiological significance of the mutation of Ile to Glu in the cardiac Ca_v1.2 channel, I_Ca was recorded in isolated ventricular CMs from I/E and Ctr mice using the patch-clamp technique (Fig. 2A). Cell capacitance was similar in CMs from I/E and Ctr mice (221 ± 14 (n = 52) and 213 ± 10 (n = 60) picofarads, respectively). The current-voltage relation of I_Ca was significantly reduced in CMs from I/E mice versus CMs from Ctr mice (Fig. 2B). These findings indicate expression of Ca_v1.2^I1624E channels in the membrane of CMs from I/E mice and an even functional availability of these channels.

FIGURE 2.

Analysis of L-type I_Ca in ventricular CMs. A, original recordings of I_Ca in a CM from a Ctr mouse and from an I/E mouse. CMs were stimulated by the voltage protocol depicted at 0.2 Hz. Current traces and voltage protocols are superimposed. A prepulse from −80 to −40 mV was used to inactivate fast Na⁺ currents. Traces are corrected for cell capacitance. pF, picofarad. B, current-voltage relation of I_Ca. Peak current density is plotted against the voltage pulse. Data points represent means ± S.E. with n = 60 for Ctr and n = 52 for I/E mice. Data sets from Ctr and I/E mice were statistically different as revealed by two-way analysis of variance (repeated measurement design, p < 0.05). C, recovery from inactivation of I_Ca. CMs were stimulated by the twin-pulse protocol depicted at 0.03 Hz. Fractions of current (I₂/I₁) are plotted against the interval duration. Data points represent means ± S.E. with n = 13 for Ctr, n = 14 for I/E mice, and n = 3 for I/E mice with KN-93 (1 μm). Data sets from Ctr and I/E mice were statistically different as revealed by two-way analysis of variance (repeated measurement design, p < 0.01). D, steady-state inactivation of I_Ca. CMs were stimulated by the twin-pulse protocol depicted at 0.03 Hz. Fractions of current (I₂/I₁) are plotted against the prepulse voltage. Data points represent means ± S.E. E_0.5 was calculated to be −16 ± 1 mV (n = 11) for Ctr mice and −27 ± 4 mV (n = 17) for I/E mice after fitting the data sets with a Boltzmann equation. Some data points from I/E mice (n = 3) were obtained in the presence of KN-93 (1 μm). Data sets from Ctr and I/E mice were statistically different as revealed by two-way analysis of variance (repeated measurement design, p < 0.01).

CaM regulates CDF and CDI of Ca_v1.2 (4–7). Facilitation is mainly due to activation of multifunctional CaMKII (8, 10, 17, 19). Inhibition of CaMKII has been shown to influence recovery from inactivation and steady-state inactivation of I_Ca (10). In accordance with these studies, recovery from inactivation of I_Ca was slowed down in CMs from I/E mice compared with CMs from Ctr mice (Fig. 2C). Fits of the recovery from inactivation by one-exponential functions revealed time constants of 54 ms (Ctr) and 72 ms (I/E). Likewise, the steady-state inactivation curve of I_Ca was shifted to the left in CMs from I/E mice compared with CMs from Ctr mice (Fig. 2D). Using the Boltzmann equation, the voltage for half-maximal inactivation was calculated to be −16 mV (Ctr) and −27 mV (I/E). KN-93 (1 μm) did not influence recovery from inactivation and steady-state inactivation in CMs from I/E mice. These results indicate that the I/E mutation in the Ca_v1.2 channel mimics the effects of CaMKII inhibitors on Ca_v1.2 channel properties (10).

Several studies have shown that facilitation of Ca_v1.2 currents depends on CaM/CaMKII (6, 9, 17, 19–21). Facilitation of I_Ca was almost abolished in CMs from I/E mice (Fig. 3, A–C). As suggested previously (9, 17, 20, 21), facilitation depended on CaMKII. Inhibition of CaMKII activity by KN-93 (1 μm) abolished facilitation in CMs from Ctr mice but had no effect in CMs from I/E mice, supporting a specific effect of KN-93 in Ctr CMs (Fig. 3D). Taken together, these results support the notion that the I/E mutation of the Ca_v1.2 channel abolishes CaM/CaMKII-mediated effects on facilitation.

FIGURE 3.

Facilitation of I_Ca in ventricular CMs. A and B, original recordings of I_Ca in a CM from a Ctr mouse and from an I/E mouse, respectively. CMs were stimulated by the twin-pulse protocol depicted at 0.03 Hz. Current traces, obtained at time intervals (Δt) of 50–350 ms in 50-ms increments, are superimposed. C, facilitation of I_Ca. Fractions of current (I₂/I₁) are plotted against the interval duration (Δt). Data points represent means ± S.E. with n = 24 for Ctr and n = 26 for I/E mice. Data sets from Ctr and I/E mice were statistically different as revealed by two-way analysis of variance (repeated measurement design, p < 0.001). D, effect of KN-93 (1 μm) on peak facilitation of I_Ca in CMs from Ctr and I/E mice. Error bars represent means ± S.E. Numbers indicate the number of experiments. Each experiment was performed with and without KN-93. **, p < 0.01.

In general, CDI of I_Ca is attenuated by the use of Ba²⁺ as a charge carrier or by high concentrations of intracellular Ca²⁺ buffers (22). Consequently, we recorded current through L-type Ca²⁺ channels in the same CMs using Ca²⁺ and Ba²⁺ as the charge carrier. As expected, the fast component of inactivation observed with Ca²⁺ was slowed down with Ba²⁺ as the charge carrier, resulting in poorly inactivating currents in CMs from Ctr mice (Fig. 4, A and C). In contrast, a fast component of inactivation was still present in CMs from I/E mice with both Ca²⁺ and Ba²⁺ as the charge carrier (Fig. 4, B and C). Next, we compared the effects of buffering intracellular Ca²⁺ by the Ca²⁺ chelators EGTA and BAPTA. BAPTA has been shown to bind Ca²⁺ more efficiently than EGTA, thus attenuating CDI of I_Ca (23). Indeed, inactivation of I_Ca was slowed down in BAPTA- versus EGTA-dialyzed CMs from Ctr mice (Fig. 4, D and F). However, inactivation of I_Ca was not slowed down in BAPTA- versus EGTA-dialyzed CMs from I/E mice (Fig. 4, E and F), in which the fast component of inactivation was even faster in BAPTA- versus EGTA-dialyzed CMs. Slow components of inactivation were not different in CMs from Ctr and I/E mice. These results suggest that the mutation of Ile to Glu at position 1624 of the Ca_v1.2 channel abolishes the effects of Ca²⁺ on inactivation of I_Ca, most likely because the channel has already been transformed to a phenotype mimicking CDI.

FIGURE 4.

Inactivation of I_Ca in ventricular CMs. A and B, original recordings of I_Ca in a CM from a Ctr mouse and from an I/E mouse, respectively, activated by a voltage pulse to 0 mV. Currents were normalized to the maximal inward current. Both displayed recordings were obtained from the same CM. C, time constants of current inactivation. Error bars represent means ± S.E. Numbers indicate the number of experiments. *, p < 0.05; ***, p < 0.001; n.s, non-significant. D and E, original recordings of I_Ca in CMs from Ctr mice and from I/E mice, respectively, activated by a voltage pulse to 0 mV. Currents were normalized to the maximal inward current. Cells were dialyzed with 10 mm BAPTA or 10 mm EGTA. F, time constants of current inactivation. Error bars represent means ± S.E. Numbers indicate the number of experiments. *, p < 0.05; ***, p < 0.001; n.s, non-significant (C and F).

DISCUSSION

In this study, we have shown that exchange of Ile with Glu in the CaM-binding motif (IQ) of the Ca_v1.2 channel gene is lethal to mice. Electrophysiological analysis of CMs from mice with a conditional heart-specific I/E mutation in the Ca_v1.2 gene revealed that the mutation abolishes CaM/CaMKII-mediated regulation of the Ca_v1.2 channel. In addition, the mutation transforms the Ca_v1.2 channel to a phenotype that recapitulates the properties of a Ca²⁺-inactivated channel.

In heart muscle, Ca_v1.2 is strongly associated with a number of regulator proteins, building up a macromolecular signaling complex (24, 25). Among the association partners, CaM is permanently bound to the channel and acts as a resident Ca²⁺ sensor (26, 27). Ca²⁺-bound CaM regulates both CDI and CDF of Ca_v1.2 (5), the latter by regulating the activity of CaMKII that is tethered to the Ca_v1.2 channel (21, 28). The major binding site for CaM is the IQ motif (amino acids 1624–1635) located in the C-terminal tail of the Ca_v1.2 channel (5, 7). Mutations in the IQ motif have been shown to inhibit CaM binding to the Ca_v1.2 channel, thus reducing facilitation and CDI (11). Especially exchange of Ile-1624 with Glu in the IQ motif of the Ca_v1.2 channel reduces CaM affinity by ∼100-fold and prohibits effectively facilitation and CDI of I_Ca in the Xenopus oocyte expression system (11). This work clearly demonstrated that exchange of Ile-1624 with Glu in the cardiac murine Ca_v1.2 channel gene likewise altered the electrophysiological properties of I_Ca in CMs and reduced the life span of the mutant mice.

Experiments using peptides containing the entire IQ motif of Ca_v1.2 or the I/E mutation showed an ∼100-fold decreased affinity of the I/E mutation for CaM (11). However, no in vivo quantitative measurements of affinity changes are available for the full-length channel with this mutation. Therefore, this number must be viewed with caution. The I/A mutant, which has as strong an effect on CDI as the I/E mutant but leaves CDF intact in heterologous expression studies, showed no measurable changes in its association with CaM, as shown by both biochemical studies (11) and crystal structure (29). Therefore, one cannot rule out a possibility that the effects of the I/E mutation also result from some distortion in the structure and correspondingly in the function of the IQ domain and not only from a reduction in CaM binding.

CaMKII is a major modulator of I_Ca activity (2). Inhibition of CaMKII by inhibitory peptides or blockers such as KN-93 prolongs recovery from inactivation (10, 30), shifts the steady-state inactivation curve to more negative voltages (10, 31), and reduces facilitation of I_Ca (17, 28, 30). In addition, knock-out of CaMKIIδ slows down recovery from inactivation and reduces facilitation of I_Ca (9). The I/E mutation of the Ca_v1.2 channel likewise prolonged recovery from inactivation, shifted the steady-state inactivation curve to more negative voltages, and reduced facilitation of I_Ca. Thus, we conclude that the I/E mutation abolishes the effects of CaMKII on the Ca_v1.2 channel because the I/E mutation shows a reduced affinity for CaM (11), preventing activation of CaMKII. At present, we cannot rule out the possibility that the I/E mutation distorts the C terminus of Ca_v1.2 in vivo and thereby reduces the affinity for CaMKII.

The fundamental role of CaM in mediating CDI has been discussed in several excellent reviews (4, 5, 20, 32). Unfortunately, pharmacological inhibitors of CaM are not useful to characterize the role of CaM in regulating cardiac I_Ca (33). Instead, the role of CaM in cardiac I_Ca is assessed mainly by reducing intracellular [Ca²⁺], namely by the use of Ba²⁺ as the charge carrier for currents through Ca_v1.2 channels, by the use of high concentrations of intracellular Ca²⁺ buffers, or the replacement of intracellular CaM with a CaM that does not bind Ca²⁺ (7). Each experimental condition attenuates CDI, as has been observed in part in this study using CMs from Ctr mice. In contrast, CDI was no longer observed in CMs from I/E mice. Instead, inactivation of I_Ca in I/E CMs was not different from that in Ctr CMs under all conditions tested. Thus, although the I/E mutation decreases significantly the affinity of the IQ motif for CaM, the mutant channel inactivates in a way that recapitulates the binding of a fully activated CaM.

In this study, we have shown that adult mice carrying the I/E mutation in the cardiac Ca_v1.2 channel gene are not viable. Preliminary experiments suggest that these mice develop dilated cardiomyopathy, in concert with a reduced contractility at an unchanged heart rate.⁴ At present, we can only speculate about the reasons for this phenotype. One reason may be that the I/E channel inactivates fast and thereby decreases the amount of Ca²⁺ entry. This lack of Ca²⁺ is not compensated and decreases cardiac contraction, which increases sympathetic tone and initiates cardiac dilation.

Another reason may be that a hindered association of CaM with the Ca_v1.2 channel reduces trafficking of the channel to the membrane during biosynthesis, as shown for cultured neurons (34), which could account for the observed reduction in channel expression and in contractility. A further reason may be the missing facilitation due to the absence of CaMKII-mediated regulation of I_Ca, which may reduce the ability of heart muscle to adapt to exercise. Indeed, mice deficient in CaMKIIδ show a reduced heart rate in response to work load or β-adrenergic stimulation (9). This phenotype, together with a fully inactivated I_Ca, may limit Ca²⁺ entry and thus Ca²⁺-induced Ca²⁺ release, leading to an insufficient contraction and finally to death of the mice. In conclusion, the mutation of Ile to Glu at position 1624 of the Ca_v1.2 channel abolishes CaM/CaMKII-dependent regulation of I_Ca but simultaneously transforms the channel to a phenotype mimicking CDI.