The cardiac L-type calcium channel distal carboxy terminus autoinhibition is regulated by calcium

Abstract

The L-type calcium channel (LTCC) provides trigger Ca²⁺ for sarcoplasmic reticulum Ca-release, and LTCC function is influenced by interacting proteins including the LTCC distal COOH terminus (DCT) and calmodulin. DCT is proteolytically cleaved and reassociates with the LTCC complex to regulate calcium channel function. DCT reduces LTCC barium current (I_Ba,L) in reconstituted channel complexes, yet the contribution of DCT to LTCC Ca²⁺ current (I_Ca,L) in cardiomyocyte systems is unexplored. This study tests the hypothesis that DCT attenuates cardiomyocyte I_Ca,L. We measured LTCC current and Ca²⁺ transients with DCT coexpressed in murine cardiomyocytes. We also heterologously coexpressed DCT and Ca_V1.2 constructs with truncations corresponding to the predicted proteolytic cleavage site, Ca_V1.2Δ1801, and a shorter deletion corresponding to well-studied construct, Ca_V1.2Δ1733. DCT inhibited I_Ba,L in cardiomyocytes, and in human embryonic kidney (HEK) 293 cells expressing Ca_V1.2Δ1801 and Ca_V1.2Δ1733. Ca²⁺-CaM relieved DCT block in cardiomyocytes and HEK cells. The selective block of I_Ba,L combined with Ca²⁺-CaM effects suggested that DCT-mediated blockade may be relieved under conditions of elevated Ca²⁺. We therefore tested the hypothesis that DCT block is dynamic, increasing under relatively low Ca²⁺, and show that DCT reduced diastolic Ca²⁺ at low stimulation frequencies but spared high frequency Ca²⁺ entry. DCT reduction of diastolic Ca²⁺ and relief of block at high pacing frequencies and under conditions of supraphysiological bath Ca²⁺ suggests that a physiological function of DCT is to increase the dynamic range of Ca²⁺ transients in response to elevated pacing frequencies. Our data motivate the new hypothesis that DCT is a native reverse use-dependent inhibitor of LTCC current.

the l-type calcium channel (LTCC) is the main pathway for Ca²⁺ entry into cardiomyocyte cytosol (2). LTCC Ca²⁺ current (I_Ca,L) triggers a larger Ca²⁺ release from intracellular stores ultimately leading to cardiac contraction. Thus minor increases of LTCC function can trigger larger changes in cytosolic Ca²⁺ entry; this process is commonly called calcium-induced calcium release (CICR). During diastole LTCC open probability (P_o) is relatively low minimizing CICR under normal conditions; however, P_o does not reach an absolute zero level. Elevation of diastolic Ca²⁺ is strongly associated with pathologies (10). Therefore, mechanisms preventing LTCC initiated CICR during diastole are of potential importance.

The LTCC in cardiomyocytes is composed of a multi-protein complex (3) and has been widely studied in cardiomyocytes and heterologous expression systems. Studies of I_Ca,L in cardiomyocytes are necessary because it is not possible to completely reconstitute a native LTCC complex in a heterologous expression system. In contrast, heterologous expression systems are advantageous because they allow the control of selected components of the LTCC complex. The minimal components to produce I_Ca,L in heterologous express systems include the Ca_V1.2 pore-forming α-subunit and Ca_Vβ (28). Additional components of the complex modify I_Ca,L gating and kinetics in nonmyocyte expression systems (29).

Cardiac Ca_V1.2, the main pore-forming subunit, is an ∼2,171 amino acid protein with a predicted molecular mass of ∼250 kDa (5, 25, 34). The precise length varies with species (34) and splice variants (24). A conserved feature of Ca_V1.2 is that the carboxy terminus is located in the cytosolic space. Spectroscopy studies show that the closely related Ca_V1.1 isoform is proteolytically cleaved at a site in the carboxy terminus. This site corresponds to a predicted calpain cleavage site that is conserved with the Ca_V1.2 channel (21). The Ca_V1.2 distal carboxy terminus (DCT) is a protein with a predicted mass of ∼37 kDa (21, 22), leaving a <200 kDa Ca_V1.2 protein in the cardiomyocyte LTCC complex. It should be noted here that Ca_V1.2 heterologously expressed in nonexcitable cells is not subject to proteolytic processing as in cardiomyocytes, rather functions as an intact ∼250 kDa protein. Western blot studies of Ca_V1.2 in cardiomyocytes consistently show Ca_V1.2 migrating as a 190- and a 240-kDa protein (1, 9). This suggests that not all channels are processed. Regardless, DCT is partially localized to the nucleus where it can modify gene expression in neurons (17) or cardiomyocytes (31). DCT also localizes to the cytosol and surface membrane, presumably in complex with the LTCC.

The Ca_V1.2 DCT blocks LTCC Ba²⁺ currents (I_Ba,L) in heterologous expression systems (16, 22). Early studies of deletion analysis of Ca_V1.2 expressed in HEK cells presaged the current view that DCT inhibits LTCC current (38). These previous studies showed that the shorter the Ca_V1.2 channel truncation, the larger the resulting current (16). Subsequently, Hulme et al. showed that coexpression of DCT with truncated Ca_V1.2 reduced I_Ba,L (22). Moreover, these studies indicate that DCT reassociates with Ca_V1.2 near the CB/IQ domain on the proximal carboxy terminus (PCT). It is notable that the DCT interaction site overlaps the well-characterized Ca_V1.2 PCT CaM interaction domains (35). Therefore it is plausible to suggest that CaM may modulate DCT-LTCC function. To date, there are no reports of DCT effects on I_Ca,L, nor is there a demonstration of DCT effects on LTCC function in cardiomyocytes (14).

The purpose of this study was to test the hypothesis that DCT inhibits I_Ca,L in cardiomyocytes. Cardiomyocyte LTCC current was tested, as well as a Ca_V1.2Δ1733+β2a+DCT complex in HEK 293 cells. DCT inhibited I_Ba,L but not I_Ca,L. To further explore implications of a selective I_Ba,L blockade we tested the effect of DCT on Ca²⁺ transients and discovered that DCT suppresses diastolic Ca²⁺ in a low frequency-dependent fashion. At high-pacing frequency DCT blockade was not significant. This reveals a new mechanism of action by DCT as a native reverse use-dependent inhibitor (RUDI) of LTCC function.

MATERIALS AND METHODS

Plasmids and mutagenesis.

Full-length Ca_V1.2 plasmid (provided by Dr. T. Kamp, University of Wisconsin) was identical to the cloned full-length rabbit cardiac α_1C-subunit (26, 33) except for alternative splicing in domain IV S3(33). Rat Ca_Vβ2a plasmid (provided by Dr. E. Perez-Reyes, University of Virginia) (28) contains two cysteine residues within the D1 domain associated with palmitoylation. CaM₁₂₃₄ was provided by David T. Yue (Johns Hopkins University), and Ca_V1.2Δ1801 was provided by Henry Colecraft (Columbia University).

Animals and electrophysiology.

Mice were housed in a pathogen-free facility and handled in accordance with standard use protocols, animal welfare regulations, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols were approved by the University of Kentucky Institutional Animal Care and Use Committee (IACUC protocol No. 00963M). Fetal ventricular cardiomyocytes were isolated as described previously (23). Timed pregnant dams of embryonic day 16-19 (E16–19) were anesthetized with ketamine (90 mg/kg) + xylazine (10 mg/kg) injected intraperitoneally. Litters were removed. Fetuses were decapitated and hearts excised. While under anesthesia the dam was euthanatized by heart excision. HEK 293 cells were transiently transfected with plasmids 4–6 h using Lipofectamine 2000 transfection (Invitrogen); then recordings were performed 24–48 h after transfection, as previously described (8). Transfected cells were identified by the expression of enhanced green fluorescent protein (eGFP). The whole cell configuration of the patch-clamp technique was used to measure ionic current. Patch electrodes with resistances of 1–2.5 MΩ contained pipette solution consisting of (in mM) 110 K-gluconate (or 110 TEA-Cl), 40 CsCl, 3 EGTA, 1 MgCl₂, 5 Mg-ATP, and 5 HEPES, pH to 7.36, with CsOH. The bath solution for HEK cells consisted of (in mM) 130 (or 112.5) CsCl, 2.5 (or 30) BaCl₂ or CaCl₂, 1 MgCl₂, 10 tetraethylammonium-Cl, 5 HEPES, and 5 glucose, pH 7.4 with CsOH. For fetal ventricular myocytes, the physiological salt solution contained 140 NaCl, 1.8 CaCl₂, 1 MgCl₂, 5.4 KCl, 10 glucose, and 10 HEPES, pH 7.4. The Na-free bath was 150 N-methyl-d-glucamine, 2.5 BaCl₂ (or CaCl₂), 1 MgCl₂, 10 HEPES, 10 glucose, and 5 4-aminopyridine, pH 7.4. Signals were amplified with an Axopatch 200B amplifier, with series resistance compensation, and captured at 333 kHz A/D system (Axon Instruments, Union City, CA). For myocytes, current-voltage curves were generated by voltage clamp protocols consisting of V_hold = −80 mV, V_prepulse = −40 mV for 100 ms, followed by V_test for 310 ms ranging from −60 mV to +80 mV in 10-mV increments. The 100 ms V_prepulse to −40 mV inactivated voltage-gated Na⁺ currents. For analysis of voltage dependence of steady state inactivation, myocytes were held at −80 mV and subjected to a 50-ms prepulse at 0 mV, followed by a 2-s steady state inactivation pulse from −80 mV to +20 mV in 10-mV increments, then a test-pulse to 0 mV for 300 ms. Eight-second intervals elapsed between each recording sweep. For HEK 293 cells, current-voltage curves were generated by voltage clamp protocols consisting of V_hold = −80 mV followed by 320 ms V_test pulse ranging from −80 mV to +80 mV in 5-mV increments. Whole cell recordings used P/4 leak subtraction. Activation voltage dependence parameters were obtained by fitting current-voltage curves to a modified Boltzmann distribution of the form: I-V = G_max*(V − E_rev)/(1 + exp(V_½ − V)/k), where G_max is maximal conductance, E_rev is reversal potential, V_½ is activation midpoint potential, and k is the slope factor. Data were analyzed with Clampfit 9.2 (Axon Instruments), Origin v7 (OriginLab, Northampton, MA), and unpaired t-test with Welch's correction performed with Prism 5 (GraphPad Software). Sample sizes are listed in the figure and table legends.

Ca²⁺ imaging.

E18 fetal mouse cardiomyocytes were used 24–72 h after Lipofectamine 2000 transfection (Invitrogen). Myocytes plated on Poly-L-Lysine 25-mm square glass coverslips were incubated with 2 μM of freshly mixed fura-2 AM (Molecular Probes; F-1221) + 10% FBS media for 8 min at 37°C. Coverslips were transferred to a custom-designed microscope chamber system containing physiological salt solution with 1.8 mM CaCl₂. Transfected cells were identified by the eGFP or eGFP fused Ca_V1.2 (1821–2171) at the NH₂ terminus. Cells were measured initially for 20 s without pacing, and then paced at 1, 2, 3, 0.5, and 0 Hz for 20–60 s at each frequency. Diastolic F₃₄₀/F₃₈₀ ratios were determined using Clampfit 9.2. Ca²⁺ calibrations were performed as in reference (13). All recordings were performed at room temperature (20–22°C) using IonOptix Myocyte Calcium Recording System, Myopacer Field Stimulator, and IonWizard 4.4 revision 13 (IonOptix, Milton, MA). We used unpaired Student's t-test with Welch's correction to test for significance between control and experimental groups.

RESULTS

DCT regulation of Ca_V1.2 current in a heterologous expression system.

Ca_V1.2 truncation at amino acid position 1733 (Ca_V1.2Δ1733) provided early clues to the potential inhibitory contribution of DCT (16, 38). Subsequent studies demonstrated that DCT blocked I_Ba,L when DCT was coexpressed with Ca_V1.2Δ1801(15) (22). We tested both Ca_V1.2Δ1733 and Ca_V1.2Δ1801 coexpressed with Ca_Vβ2a and DCT in HEK 293 cells. Ca_V1.2Δ1733-expressing cells displayed DCT blockade in a similar pattern to that for DCT in cardiomyocytes (see below). DCT reduced peak I_Ba,L but not I_Ca,L (Fig. 1, A, i and ii). DCT interacts with the proximal carboxy terminus of Ca_V1.2 in a region that overlaps with CaM-Ca_V1.2 interaction (22). We therefore tested the effect of coexpression of DCT and CaM. DCT inhibition of I_Ba,L was reversed by coexpression of CaM with DCT (Fig. 1, A, iii and iv, and B). I_Ca,L and I_Ba,L were recorded in all cells, and the relative peak current difference between I_Ca,L and I_Ba,L serves as an internal check that DCT selectively interferes with I_Ba,L but spares I_Ca,L (Fig. 1B). Note that maximal I_Ba,L relative to I_Ca,L is distinct with DCT expression compared with other conditions (Fig. 1A, ii to i, iii, and iv). Ca_V1.2Δ1801-expressing cells also exhibited DCT inhibition of peak I_Ba,L; however, this was accompanied by a commensurate decrease of I_Ca,L (Fig. 2). We will show below that the Ca_V1.2Δ1733 construct tracks with data from cardiomyocytes. In heterologous expression, both Ca_V1.2 truncation constructs mimic DCT attenuation of cardiomyocyte peak I_Ba,L. The absence of I_Ca,L block of peak cardiomyocyte current is also captured by the Ca_V1.2Δ1733 construct used in HEK recordings.

Fig. 1.

L-type calcium channel (LTCC) distal COOH terminus (DCT) decreases LTCC barium current (I_Ba,L) but not LTCC Ca²⁺ current (I_Ca,L) from Δ1733 truncation of Ca_V1.2 expressed in human embryonic kidney (HEK) 293 cells in parallel with that observed in cardiomyocytes. A: current-voltage relationships for I_Ca,L (■) and I_Ba,L (○) for control (i), DCT overexpression (ii), CaM overexpression (iii), and DCT + CaM dual overexpression (iv). Note the difference in the relative I_Ba,L vs. I_Ca,L curves for DCT in contrast with all other conditions. Ai and Aii, insets: expanded views of I_Ca,L at low voltages. Note that the Boltzmann distribution fit deviates from the data particularly for V_test ranging from −35 to −20 mV. B, left: peak I_Ca,L density for potential eliciting maximal current (+20 mV) shows no significant difference by DCT. B, right: peak I_Ba,L density for potential eliciting maximal current (+5 mV) is significantly reduced by DCT, and DCT + CaM restores current amplitude. CaM alone has no significant effect. Sample sizes are listed in Table 1. *P = 0.007 and **P = 0.006 for DCT vs. control and DCT vs. DCT + CaM, respectively. eGFP, enhanced green fluorescent protein.

Fig. 2.

DCT decreases I_Ba,L, and I_Ca,L from Ca_V1.2Δ1801 truncation of Ca_V1.2 expressed in HEK 293 cells. A: current-voltage relationships for I_Ca,L (■) and I_Ba,L (○) for control (i), DCT overexpression (ii), CaM overexpression (iii), and DCT + CaM dual overexpression (iv). B, left: DCT reduces the peak I_Ca,L density at V_test +20 mV (P < 0.05). B, right: DCT reduces the peak I_Ba,L density, and DCT + CaM restores current amplitude. CaM alone has no significant effect. *P = 0.01 and **P = 0.007 for DCT vs. control and DCT vs. DCT + CaM, respectively.

DCT did not significantly alter the voltage dependence of activation for I_Ca,L or I_Ba,L (Table 1), with one notable exception. Ca_V1.2Δ1733 I_Ca,L required a second Boltzmann distribution to fit data negative to −20 mV (Fig. 1Aii, inset). Seven of eight DCT-expressing cells appeared with data points that fell below the fit line for V_test ranging from −20 to −35 mV, whereas a single Boltzmann distribution approximated the data as shown in Fig. 1Ai for all cells in the eGFP control group (P < 0.001; Fishers exact test). There is no voltage threshold for LTCC opening. Rather, at relatively negative voltages, P_o is at very low levels. These results are consistent with the notion that DCT preferentially inhibits LTCC when P_o is relatively low.

Table 1.

Voltage dependence of current activation Boltzmann fit of V_1/2 and k for Ca_V1.2Δ1733 and Ca_V1.2Δ1801

	eGFP	DCT	CaM	DCT + CaM
CaV1.2Δ1733+β2a+
30 Calcium
n	8	8‡	5	10
V1/2, mV	7.0 ± 0.7	9.8 ± 1.7	8.2 ± 3.0	8.7 ± 0.9
k, mV	10 ± 1.1	9.7 ± 0.5	10.1 ± 0.4	9.0 ± 0.2
30 Barium
n	10	8	5	13
V1/2	−3.1 ± 1.1	−1.3 ± 1.6	−6.5 ± 1.5	−2.4 ± 1.8
k	6.7 ± 0.2	7.7 ± 0.3	7.4 ± 0.6	6.8 ± 0.4
CaV1.2Δ1801+β2a+
30 Calcium
n	4	10	7	11
V1/2	8.6 ± 0.95	3.4 ± 0.69	11.7 ± 0.65	11.0 ± 0.6
k	8.8 ± 0.43	9.8 ± 0.28	9.2 ± 0.31	8.9 ± 0..22
30 Barium
n	4	10	7	11
V1/2	2.2 ± 0.43	−1.4 ± 0.44	4.6 ± 0.35	−1.5 ± 0.50
k	6.7 ± 0.25	6.9 ± 0.25	7.2 ± 0.17	6.8 ± 0.3

Values are means ± SE. A double Boltzmann equation was required to satisfy all points for distal COOH terminus (DCT) coexpression in 7 of 8 cells with 30 mM Ca.

^‡Similar behavior with barium currents was noted by Hulme et al. (22). eGFP, enhanced green fluorescent protein; V_1/2, activation midpoint potential; k, slope factor.

Role of Ca²⁺-CaM in CaM-DCT current restoration.

To distinguish between Ca²⁺ and Ca²⁺-CaM requirement for relief of DCT blockade we coexpressed CaM₁₂₃₄ (Ca²⁺ binding-deficient mutant) (29) with Ca_VΔ1733 and measured I_Ba,L and I_Ca,L. We focused on Ca_VΔ1733 for this experiment because this construct captured DCT effects on cardiomyocyte I_Ba,L and I_Ca,L (see below). Figure 3A shows the response of I_Ba,L and I_Ca,L to CaM₁₂₃₄ with or without DCT. CaM₁₂₃₄ did not restore current in cells coexpressing DCT. Rather, both the Ba²⁺ and Ca²⁺ current amplitudes in control and DCT group were indistinguishable. CaM₁₂₃₄ reduced the control I_Ba,L, and DCT + CaM₁₂₃₄ did not result in an additive block (Fig. 3B). Comparison of I_Ba,L and I_Ca,L I-V curves for CaM₁₂₃₄ expression (Fig. 3) to I_Ba,L and I_Ca,L I-V curves without CaM₁₂₃₄ (Figs. 1A, i to ii, and 3A, i to ii) illustrates that CaM₁₂₃₄ preferentially blocked I_Ba,L compared with I_Ca,L. Taken together, these results show that CaM₁₂₃₄ and DCT individually exert similar action on LTCC current. These results are consistent with a model that limiting channel P_o and limiting Ca²⁺ favors increased DCT blockade.

Fig. 3.

CaM₁₂₃₄ blocks I_Ba,L, and DCT blockade is not additive in Cav1.2Δ1733 coexpressed in HEK 293 cells. A: DCT coexpression with CaM₁₂₃₄ decreases I_Ba,L, but the effect is not additive with I_Ca,L (■) and I_Ba,L (○). BAPTA (10 mM) in the pipette does not affect the I_Ba,L inhibition by DCT (data not shown). There is no apparent synergistic effect of DCT + CaM₁₂₃₄. B, left: peak I_Ca,L density for potential eliciting maximal current (+20 mV) shows no significant difference by DCT. B, right: peak I_Ba,L density for potential eliciting maximal current (+5 mV). Light gray data points show eGFP and DCT data from Fig. 1B, right, for reference. *P < 0.01.

DCT and LTCC current kinetics.

Voltage-dependent inactivation (VDI) was measured as the fractional remaining I_Ba,L 300 ms after V_test. Ca_V1.2Δ1733 or Ca_V1.2 Δ1801 channels coexpressed with DCT showed indistinguishable VDI (Fig. 4, A and B; Ca_V1.2Δ1801 not shown). CaM coexpression also did not alter VDI. CaM-Ca_V1.2 PCT interaction leads to calcium-dependent inactivation (CDI) (29). Given that CaM antagonized DCT inhibition of current, we asked whether DCT reciprocally interferes with CDI. I_Ca,L decay kinetics, a measure of CDI, was also not significantly altered by DCT (Fig. 4, A and B; Ca_V1.2Δ1801 not shown). CaM₁₂₃₄ overexpression has a dominant negative effect on CDI imposed by endogenous HEK CaM. Consistent with earlier findings CaM₁₂₃₄ reduced CDI (29). Coexpression of CaM₁₂₃₄ + DCT further reduced CDI. In summary, for heterologously expressed LTCC, DCT blocks I_Ba,L but not I_Ca,L; this effect can be masked by CaM overexpression, and CaM₁₂₃₄ and DCT show similar, nonsynergistic effects on LTCC function.

Fig. 4.

Current kinetics in HEK 293 cells. A: Ca_V1.2Δ1733 current traces for V_hold −80 mV stepped to 0 mV for control (left), DCT (center), and DCT + CaM (right). B: remaining fractional current 300 ms after the peak for I_Ca,L (left) and I_Ba,L (right), respectively. There are no significant differences for I_Ba,L voltage-dependent inactivation (VDI). C: Ca_V1.2Δ1733 + CaM₁₂₃₄ current traces for V_hold −80 mV stepped to 0 mV for control and DCT (right). D: remaining fractional current 300 ms after the peak for I_Ca,L (left) and I_Ba,L (right), respectively. *P < 0.01 for calcium-dependent inactivation (CDI).

DCT does not alter CDI.

VDI and CDI require a functional multimeric LTCC complex—a situation that can never fully be reproduced in a heterologous expression system. Therefore, we explored the effect of DCT in ventricular cardiomyocytes. Figure 5A shows representative current traces for I_Ba,L and I_Ca,L in control, DCT, and DCT + CaM-expressing cardiomyocytes. The fraction of remaining Ca²⁺ current 25 ms after I_Ca,L peak was not significantly different among control, DCT, and DCT + CaM overexpressing cardiomyocytes (Fig. 5B). I_Ba,L decay kinetics at 50 ms reflects VDI. DCT accelerated VDI (Fig. 5B), and this effect was reversed by coexpression of DCT and CaM. To determine the effect on steady-state inactivation, we voltage-clamped cells to a range of potentials for 2 s and assayed the available current. Steady-state inactivation curves are described by single Boltzmann distributions. For I_Ca,L there are no significant differences between control and DCT-expressing myocytes (Table 2). For I_Ba,L the midpoint of inactivation is not different, but the slope factor is significantly steeper in the presence of DCT (Table 2).

Fig. 5.

DCT enhances VDI in cardiomyocytes. A: current traces for V_hold −50 mV stepped to 0 mV for control (left), DCT (center), and DCT + CaM (right) in cardiomyocytes. B: remaining fractional current 25 and 50 ms after the peak for I_Ca,L (left) and I_Ba,L (right), respectively. There are no significant differences for I_Ca,L CDI. DCT overexpressing cells showed a significantly greater VDI than control or DCT + CaM. *P = 0.04 for control vs. DCT and **P = 0.01 for CaM + DCT vs. DCT. C: VDI of Ca²⁺ channel current in cardiomyocytes. Midpoint of activation was shifted positive for cardiomyocytes + DCT in Ba²⁺. Steepness of the slope increases with overexpression of DCT compared with control eGFP. Boltzmann fit parameters and sample sizes are in Table 2.

Table 2.

Steady state inactivation of cardiomyocyte barium current

Cardiomyocytes	eGFP	DCT
2.5 mM barium
n	8	7
V1/2	−31 ± 1.1	−28 ± 1.1
k	7.3 ± 0.52	6.1 ± 0.25∗
Offset	0.035 ± 0.01	0.1 ± 0.021∗∗

Values are means ± SE.

^∗P = 0.03 for DCT vs. eGFP k;

^∗∗P < 0.01 for DCT vs. eGFP offset.

DCT inhibits I_Ba,L but not I_Ca,L in ventricular cardiomyocytes.

Heterologous expression studies predict that cardiomyocyte L-type current will be inhibited by DCT (Fig. 1) (22). Current-voltage curves for Ca²⁺ currents (I_Ca,L) and Ba²⁺ currents (I_Ba,L) were measured using 10-mV depolarizing steps from a −50-mV prepulse to inactivate sodiume current. I_Ca,L and I_Ba,L were measured in the same cell (Fig. 6). As expected, DCT inhibited I_Ba,L; however, DCT did not significantly reduce I_Ca,L (Fig. 6Aii). Vertical scatter plots of maximal current illustrate the lack of significant difference for I_Ca,L between eGFP (control) and eGFP-DCT transfected cells (Fig. 6B). By contrast, I_Ba,L is significantly reduced by DCT expression (Fig. 6C). By extension from heterologous expression studies, we postulated that CaM coexpression with DCT can then mitigate DCT block of I_Ba,L. CaM overexpression alone has no significant effect on peak cardiomyocyte I_Ca,L or I_Ba,L (Fig. 6Aiii); however, exogenous-CaM-expression interfered with the DCT inhibition of I_Ba,L (Fig. 6, A and C), and CaM shifted reversal potential, suggesting possible effects on contaminating outward current activated at large positive potentials. Consistent with heterologous expression studies, I_Ca,L was not significantly different following DCT, or DCT + CaM overexpression (Fig. 6, A and B). To test whether the channel number is changed by DCT overexpression, we measured gating charge movement. There was no significant difference among control, DCT, CaM, or DCT + CaM groups (data not shown). Similarly, gating charge normalized to ionic current was unchanged (data not shown). These results are consistent with the notion that DCT has no significant effect on surface expression of Ca_V1.2. Taken together, common findings from heterologous expression and cardiomyocytes reveal that DCT block is strongest under conditions when Ca²⁺-CaM is lowest.

Fig. 6.

DCT decreases I_Ba,L but not I_Ca,L in ventricular cardiomyocytes. A: current voltage relationships for I_Ca,L (■) and I_Ba,L (○) for control (i), DCT overexpression (ii), CaM overexpression (iii), and DCT + CaM dual overexpression (iv). Note the difference in the relative peak I_Ba,L vs. I_Ca,L curves for DCT in contrast with all other conditions. B: peak I_Ca,L density for potential eliciting maximal current (+10 mV) shows no significant difference by DCT. C: peak I_Ba,L density for potential eliciting maximal current (0 mV) is significantly reduced by DCT, and DCT + CaM restores current amplitude. *P = 0.01 and **P = 0.01; n = 5, 6, and 9 for control, DCT, and DCT + CaM, respectively.

DCT increases the dynamic range of Ca²⁺ transients by lowering diastolic Ca²⁺.

Our results are consistent with DCT, causing a preferential inhibition of LTCC under conditions of relatively low Ca²⁺. Another way to view this is that DCT blocks best when the channel is relatively inactive. We define this as a RUDI. It is very difficult to directly measure LTCC blockade at low open probabilities. However, LTCC preamplifies CICR in cardiomyocytes. We surmised that during quiescence, or at low pacing frequencies, diastolic Ca²⁺ might be sufficiently low to create a detectable DCT block. To test this we measured cytosolic Ca²⁺ from fura-2 loaded cardiomyocytes. Cardiomyocytes transfected with DCT have significantly lower quiescent F₃₄₀/F₃₈₀ ratios during quiescence (Fig. 7A). Cells were then field paced at frequencies ranging from 0.5 to 3 Hz (Fig. 7B). In all cases a positive staircase for Ca²⁺ transients was observed. Normalizing the diastolic Ca²⁺ level in paced cells to that during quiescence reveals that DCT significantly increased the dynamic range of responses (Fig. 7C). The diastolic Ca²⁺ difference between 0.5 and 3 Hz is significantly greater in DCT transfected cardiomyocytes (Fig. 7D). To more rigorously test the supposition that DCT progressively unblocked as cytosolic Ca²⁺ increased, we measured diastolic Ca²⁺ varying bath Ca²⁺ (13). In 1.8 mM bath Ca²⁺ DCT significantly reduces diastolic intracellular Ca²⁺ concentration (Fig. 8). Raising bath Ca²⁺ to 6 mM resulted in no significant difference of diastolic or systolic intracellular Ca²⁺ concentration in DCT versus control (Fig. 8). We also superimposed data from Frampton et al. (13) for reference. Taken together, DCT reduction of quiescent Ca²⁺ levels and increased dynamic range of Ca²⁺ transients are consistent with our new hypothesis that DCT block is regulated by cytosolic Ca²⁺.

Fig. 7.

DCT reduces quiescent cytosolic Ca²⁺ and increases the dynamic frequency response range. A: quiescent cytosolic fura-2 fluorescent ratio (R_q) is reduced by DCT expression (*P = 0.03; n = 9 control, n = 14 DCT). B: representative Ca²⁺ transients for 3 and 0.5 Hz stimulation. The horizontal dashed line is the R_q from A. C: pooled mean diastolic fura-2 ratio (R_diastolic; R_d) normalized to R_q for 0.5, 1, 2, and 3 Hz. Note the increase in dynamic response range for DCT-transfected cardiomyocytes. D: normalized R_diastole/R_q response range manifested as difference between 3 and 0.5 Hz. DCT significantly increased response range *(P = 0.01).

Fig. 8.

DCT decreases diastolic calcium in cardiomyocytes. A: calcium transients induced by 0.5-Hz electrical field stimulation in 1.8 and 6 mM bath Ca²⁺. Horizontal dashed lines shown for referencing diastolic Ca²⁺ level. B: relationship between diastolic and systolic calcium transients in cardiomyocytes overexpressing DCT (●, dashed line) vs. control (□, solid line) at 1.8 (symbol at left) and 6 (symbol at right) mM calcium, respectively. The light gray solid squares show data from Frampton et al. (13) for 2 and 6 mM Ca²⁺ for reference. *P = 0.04 for diastolic DCT vs. control (n = 12 and n = 6, respectively).

DISCUSSION

Cardiomyocytes contain DCT separate from the LTCC (31). In the present study we report novel mechanisms of action of DCT on I_Ca,L in cardiomyocytes. Our first major finding was that DCT inhibited cardiomyocyte I_Ba,L but not I_Ca,L. HEK 293 cells responded similarly to overexpressed DCT. DCT blockade of I_Ba,L was consistent with earlier studies in HEK 293 cells (22, 38); however, to our knowledge earlier studies did not consider I_Ca,L. Second, our data showed that DCT blockade was antagonized by Ca²⁺-CaM. A third major finding is that our results are consistent with the novel model that DCT blocks channels best at conditions of low Ca²⁺, and at potentials corresponding to low open probabilities. In essence, the data suggest that DCT is a RUDI of LTCC in cardiomyocytes. The potential physiological significance of DCT RUDI is revealed by the increase of dynamic range of Ca²⁺ transients as a function of stimulation frequency.

The first major finding of the present study, DCT blocks I_Ba,L but not I_Ca,L leads to the conundrum: how can I_Ba,L selective block be physiologically relevant? Closer examination of activation of I_Ca,L shows that at relatively low voltages DCT prevents detection of measurable macroscopic I_Ca,L (Fig. 1Aii, inset). Use-dependent inhibition can be generalized as blockade of a voltage-gated ion channel that is in a depolarization-induced open or inactivated state. Conversely, RUDI posits blockade of a channel in a relatively low P_o state. RUDI is commonly associated with the rate-dependent action of sotatol (18, 20), although increases of late sodium current can underlie RUDI (39). Blockade of I_Ca,L, preferentially at low potentials, and low frequency-dependent attenuation of Ca²⁺ transients is consistent with RUDI. Thus we now speculate that DCT has a contribution as an intrinsic modulator of rate-dependent Ca²⁺ handling. This is an important point for follow-up studies.

DCT and RGK proteins share the common feature of interfering with I_Ca,L and interacting with the Ca_V1.2 PCT. Excess CaM interferes with RGK blockade of I_Ca,L manifested as alterations of peak current and slowing CDI (27). The CaM-RGK findings motivated assessment of CaM-DCT interaction with respect to LTCC function. In common with RGK modulation, CaM also interfered with DCT blockade; however, there was no effect on CDI nor on I_Ca,L. Our results highlight the importance of multiple proteins in the native heteromultimeric protein complex that comprises cardiomyocyte LTCC. In this light HEK 293 cells serve as reduced systems, whereby specific protein expression can be controlled simultaneously; however, consideration of missing components from native complexes must be weighed on interpretations of results. The correspondence of DCT blockade of I_Ba,L and the differential correspondence between HEK 293 I_Ca,L for Ca_V1.2Δ1733 but not Ca_VΔ1801 is not completely surprising nor easily interpreted. It is important that in all cases DCT effects are competed by excess Ca-CaM. An important limitation of data interpretation is the absence of control of stoichiometry in any given cell. In this vein the apparent increased variability of current amplitude with DCT + CaM expression (Fig. 1B) might be taken as a manifestation of lack of control of individual protein levels. Nonetheless, as first reported by Fuller et al. (15), we also report DCT blockade of I_Ba,L for Ca_V1.2Δ1801 expressed in HEK 293 cells (Fig. 2). Despite quantitative differences, the present results for DCT effects are in common with previous reports confirming an autoinhibitory function of DCT that may be propagated from DCT through the proximal carboxy terminus EF-hand to control channel gating (6).

The finding that CaM-LTCC PCT tethering is Ca²⁺ dependent (30) raises the idea that apparent DCT block is better conceptualized as a dynamic CaM displacement by DCT. Peptides to the CaM-interacting motif of PCT have autoagonist (11) function. Conversely, in the absence of CaM or in the presence of excess CaM₁₂₃₄ macroscopic LTCC conductance is decreased (29). In the present study CaM₁₂₃₄ and DCT had similar and nonsynergistic effects on LTCC current. We envision that for I_Ca,L, compared with I_Ba,L, sufficient Ca²⁺ entry is achieved to strengthen CaM-PCT interaction and thus prevents DCT from interfering with CaM-PCT function. The exception is low voltage whereby sufficiently low open channel probability limits Ca²⁺ entry (despite an increased driving force). This effect is subtle in patch-clamp recordings, but CICR amplifies Ca²⁺ signaling. Reverse-use dependence manifested as low frequency-dependent DCT attenuation of Ca²⁺ transients illustrates potential physiological relevance DCT modulation of LTCC activity.

The well-described Ca²⁺-dependent CaM-PCT interaction provides a logical framework to explain DCT effects. In this scheme, DCT is expected to be a more effective competitor for PCT than CaM at low Ca²⁺, and CaM-less LTCC have diminished ability to open. DCT blockade is not complete, and the remaining current has unaltered kinetics (present study), consistent with the idea that the open channels have active CaM-PCT complexes.

A developing mouse model system was adopted for studying the contribution of DCT to cardiomyocyte physiology. Specifically, our study addresses the effect of DCT on I_Ca,L in a native cell environment. Developing cardiomyocytes have an advantage over adult myocytes in addressing this specific aim. First, developing myocytes have relatively large I_Ca,L (7) (32). Second, developing cardiomyocytes can be efficiently transfected for exogenous protein expression (8) such as DCT and CaM demonstrated in this article. Finally, developing cardiomyocytes can be cultured in vitro for several days in contrast with adult cardiomyocytes, which undergo a dedifferentiation process. Although developing myocytes are not a substitute for human adult myocytes, they fit the criteria for asking questions about I_Ca,L current regulation by DCT. The physiological attributes of developing cardiomyocytes as a model system are also worthy of discussion. Developing cardiomyocytes have a relatively slow heart rate, compared with mature cells. Consequently the diastolic interval is relatively prolonged (12, 19). Developing cardiomyocytes have a comparatively long action potential duration (7, 36). Secondary to the reduced heart rate and prolonged action potential duration, excitation-contraction coupling is more reliant on external Ca²⁺ in immature compared with adult cardiomyocytes (2, 4, 37). Thus developing cardiomyocytes use a mixture of external and SR Ca²⁺ for excitation-contraction coupling approximating that of mature large mammalian systems.

The notion that DCT is a RUDI has potential physiological and therapeutic significance. The purpose of this study was to test the function of DCT in cardiomyocytes. As a result of our findings, we propose a new mechanism of regulation of LTCC function. Determining the degree of intrinsic RUDI that can be attributed to DCT in a cardiomyocyte will require further careful study. Ca²⁺-CaM antagonism of DCT RUDI requires simultaneous control of stoichiometries of CaM, LTCC, and DCT. Also, consideration of other PCT interacting proteins, such as RGKs (27), akap15, or Ca_Vβ₂, could potentially contribute to LTCC functional regulation. The significance of DCT as a RUDI extends beyond cardiomyocyte physiology. From a therapeutic perspective, overexpression of DCT is posited to have the beneficial impact of decreasing Ca²⁺ entry during diastole, yet sparing Ca²⁺ entry during systole. A key future stumbling block for application of DCT as a therapeutic is, of course, the requirement for cytosolic delivery.

In conclusion, our data identify DCT blockade of LTCC function, but only under conditions when either Ca²⁺ levels are low and at relatively low potentials. We show that DCT increases the stimulation frequency-dependent dynamic range of Ca²⁺ transients in cardiomyocytes, leading us to the new hypothesis that DCT is an intrinsic RUDI of LTCC function. A logical extension of our findings is that DCT may provide a novel therapeutic benefit by controlling Ca²⁺ entry at diastolic potentials while sparing Ca²⁺ entry for systole.

GRANTS

This work was supported by National Institutes of Health Grants HL-074091 (to J. Satin), HL-072936 (to D. A. Andres and J. Satin), and 2P20 RR-020171 from the National Center for Research Resources (to D. A. Andres) and National Heart, Lung, and Blood Institute Training Grant HL-072793 (to S. M. Crump).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: S.M.C. and J.S. conception and design of research; S.M.C., D.A.A., and G.S. performed experiments; S.M.C. analyzed data; S.M.C. and J.S. interpreted results of experiments; S.M.C. prepared figures; S.M.C. and J.S. drafted manuscript; S.M.C., D.A.A., and J.S. edited and revised manuscript; S.M.C., D.A.A., and J.S. approved final version of manuscript.