Abstract
Ca2+-entry in the heart is tightly controlled by Cav1.2 inactivation, which involves Ca2+-dependent inactivation (CDI) and voltage-dependent inactivation (VDI) components. Timothy syndrome, a subtype-form of congenital long-QT syndrome, results from a nearly complete elimination of VDI by the G406R mutation in the α11.2 subunit of Cav1.2. Here, we show that a single (A1929P) or a double mutation (H1926A-H1927A) within the CaN-binding site at the human C-terminal tail of α11.2, accelerate the inactivation rate and enhances VDI of both wt and Timothy channels. These results identify the CaN-binding site as the long-sought VDI-regulatory motif of the cardiac channel. The substantial increase in VDI and the accelerated inactivation caused by the selective inhibitors of CaN, cyclosporine A and FK-506, which act at the same CaN-binding site, further support this conclusion. A reversal of enhanced-sympathetic tone by VDI-enhancing CaN inhibitors could be beneficial for improving Timothy syndrome complications such as long-QT and autism.
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Introduction
The inactivation of the L-type voltage-gated calcium channel Cav1.2 plays a central role in controlling excitation-contraction (E–C) coupling in cardiac myocytes and consists of two independent mechanisms, CDI and VDI (for reviews, see1,2,3,4,5,6).
Early studies revealed CDI signaling to be predominantly mediated by Ca2+/calmodulin binding to an IQ motif at the proximal C-tail of the α11.2 subunit of Cav1.27,8,9,10,11. A proteolytically cleaved distal domain has been suggested to form a non-covalent complex with the proximal C-tail acting like an autoinhibitory motif12. While CDI has been extensively studied and is well documented, the exact mechanism by which VDI regulates Cav1.2 remains largely unknown. The importance of VDI became apparent by the discovery of a mis-sense mutation G406R at the α11.2 pore forming subunit of Cav1.2 that manifests itself in the Timothy syndrome (TS). In TS, VDI impairment resulted in prolonged QT interval and severe arrhythmias in the heart, accompanied by an autism spectrum disorder13,14.
The well documented participation of calcineurin (CaN), a Ca2+/calmodulin-dependent protein IIB phosphatase, in controlling Ca2+ signaling in mouse ventricular myocytes15,16,17,18,19,20, has prompted a search for CaN binding site(s) within the α11.2 subunit.
Previous in vitro binding experiments have demonstrated that CaN binds to the C-tail of rabbit α11.2 at a minimal site spanning amino acids D1943-L197121 and to undefined sites downstream of L197122. Deletion of large segments comprising 285, 405 or 438 amino acids at the C-terminus, which comprise of the CaN-binding site, increased channel activity23, are consistent with the elimination of a VDI regulatory interface at the rabbit α11.2 C-terminus24,25,12.
Here we have combined molecular, electrophysiological and pharmacological approaches to establish CaN as a VDI-regulatory protein of the human Cav1.2 channel. Although CaN binding site at the C-tail of α11.2 was established21,22, the functional effect of CaN on Cav1.2 properties is not known. We show that a single point mutation A1929P at CaN binding motif accelerates Cav1.2 inactivation and increases the extent of VDI. The A1929P mutation, previously shown to abrogate binding of CaN to the channel21,22, accelerated channel inactivation most likely by preventing CaN binding to the CaN-binding motif within the C-tail of the channel. The mutation also restored the impaired voltage inactivation of the Timothy G406R-channel. These results identify the CaN binding motif as a negative modulator of VDI. VDI restoration by CaN selective inhibitors cyclosporine A and FK506 confirm CaN as a regulator of VDI and implicate the CaN-binding site in the α11.2 subunit as a potential therapeutic target for TS and other arrhythmias.
Results
Mutations within the CaN binding motif, A1929P, or H1926A/H1927A, accelerate the rate of Cav1.2 voltage-dependent inactivation
The most distal C-terminal domain of rabbit α11.2 comprises a putative CaN minimal binding motif D1943-L197121, corresponding to D1913-L1941 of the human α11.2 subunit ( Fig. 1a , upper), a phosphatase 2A (PP2A)-binding site at L1935−R1941 and an additional CaN binding-site downstream of R194122. Both CaN and PP2A sites are located downstream to the PKA interaction domain26,27,28.
The functional impact of the CaN binding motif on CaV1.2 properties was tested in BAPTA-injected Xenopus oocytes, using the two-electrode-voltage-clamped assay. Both CaN29 and cyclophilin30 are natively expressed in Xenopus oocytes.
Previously we showed that a truncated human α11.2, in which the CaN binding motif was deleted by a premature stop codon inserted at G1911, led to an increase in VDI (data not shown). To define a specific correlation between CaN-binding site at the human α11.2 and VDI, we used a single point mutant of α11.2 ( Fig. 1a , middle). In vitro studies, carried out in rabbit α11.2, showed that mutating Ala to Pro (A1959P) at the CaN-site abrogated CaN binding21,22. According to these studies, the corresponding human Ala1929 is located within a minimal CaN binding site spanning L1920 and L1935 of the human α11.2 tail ( Fig. 1b , upper).
Whole-cell currents were recorded from oocytes injected with cRNA corresponding either to the human α11.2/A1929P or the full-length human α11.2 subunit together with the auxiliary subunits α2δ1 and β2b.
Depolarization produced decaying inward barium current (IBa) in both Cav1.2 and Cav1.2/A1929P ( Fig. 1c , left). Superimposed exemplary IBa traces showed that A1929P mutant was inactivated faster than Cav1.2 ( Fig. 1c , left). Quantitatively, the smaller ratio of inward current at the end of a 1,000 ms test pulse relative to the peak current (r1000) was 0.43 ± 0.06 for Cav1.2/A1929P compared to 0.62±0.01 for the wt channel, which indicates faster inactivation of the mutant channel ( Supplementary Table S1 online).
The steady-state voltage inactivation of A1929P mutant was tested and compared to wt channel ( Fig. 1d , inset). The CaV1.2/A1929P mutant exhibited virtually complete voltage-dependence of IBa inactivation, compared to VDI of Cav1.2 ( Fig. 1d , left, Table 1 ). The Boltzmann curve of the A1929P mutant was shifted to hyperpolarizing potentials, compared to the full-length native Cav1.2 ( Table 1 ). A plausible explanation for a stronger VDI is that the A1929P mutation interferes with CaN-binding to the α11.2 C-tail21,22 and alleviates an inhibitory effect of CaN on VDI.
To further confirm the CaN binding site a VDI-negative regulatory site, we examined two neighboring vicinal His residues, H1926, H1927, positioned within the minimal CaN binding domain21,22 for their effect on VDI ( Fig. 1a lower; Fig. 1b lower).
Whole-cell IBa were recorded from channels co-expressing either wt α11.2 or α11.2/HH/AA together with the auxiliary α2δ1 and β2b. Superimposed exemplary IBa traces showed that Cav1.2/HH/AA was inactivated faster than Cav1.2 ( Fig. 1c right). Similar to the enhanced inactivation displayed by the A1929P mutant, the smaller ratio of inward current at the end of a 1,000 ms test pulse to the peak current (r1000) of Cav1.2/HH/AA indicated 0.37±0.02 compared to 0.62±0.01 of wt channel, showing faster inactivation than the wt Cav1.2 ( Supplementary Table S1 online). The CaV1.2/HH/AA mutant exhibited virtually complete voltage-dependence of inactivation, compared to the partial VDI of Cav1.2 ( Fig. 1d , right; Table 1 ). Although the effect of the H1926A/H1927A mutation on CaN binding was not shown through a direct binding, it seemed to up regulate VDI, similar to A1929.
Direct inhibition of CaN increases the extent of voltage inactivation
To confirm that CaN operates as a VDI-regulatory protein, we employed a pharmacological approach, using cyclosporin A (CsA), a potent and selective CaN inhibitor31. CsA was used in a concentrations range of 0.01–2 µM, since at a level of 20 μM it increases the probability of channel openings32.
The ICa of wt Cav1.2 decayed fast, as shown by the current traces ( Fig. 2a left) and by the nearly complete voltage-dependent steady-state inactivation at +10 mV ( Fig. 2b , left). The presence of 2 µM CsA did not change the inactivating profile ( Fig. 2a , left; Supplementary Table S1 online) or the extent of ICa inactivation ( Fig. 2b , left). Hence, 2 µM CsA does not affect the wt channel, when it is fully inactivated by the combined action of CDI + VDI.
In the absence of CDI, when Ba2+ is the charge carrier, the Cav1.2 currents decayed slowly, driven by the limited VDI mechanism ( Fig. 2b , right). The presence of 0.6 µM or 2 µM CsA dramatically increased this partial inactivation of wt Cav1.2 ( Fig. 2b , right; Supplementary Table S1 online) and shifted the Boltzmann curves towards hyperpolarizing voltages ( Fig. 2b , right; Table 1 ). This shift was not seen in the channel mutants and could be mediated by CsA inhibiting an additional CaN binding motif21. Hence, the presence of CsA imposed a full VDI upon the native Cav1.2. In contrast with the considerable effects on inactivation, the activation kinetics of wt channel was only marginally affected by 0.2 or 1 µM CsA (ICa) (see Supplementary Fig. S1 online). Current amplitude was decreased in the presence of 2 µM CsA (IBa), but there were no significant changes in the time constant of activation, or I/Imax (see Supplementary Fig. S2 online).
The A1929P mutation at the CaN-binding site restores inactivation of the Timothy channel
A single missense mutation, G406R in exon 8a of the cardiac L-type calcium channel (CACNA1C, Cav1.2, α11.2) was shown to cause the Timothy syndrome (TS), which is as characterized by a prolongation of QT intervals (designated LQT8) due to a significant loss of voltage inactivation of Cav1.213,14,33,34,35.
Since the major effect of the G406R mutation on the TS channel was a loss of VDI, we tested VDI regulation by this well-defined CaN-regulatory site in the TS channel
Initially, the α11.2/G406R subunit of the Timothy channel was mutated to α11.2/G406R/A1929P and the kinetics of inactivation were tested, using voltage-clamped oocytes (Experimental Procedures; Fig. 3a ).
Representative normalized ICa or IBa traces showed faster inactivation of Cav1.2/G406R/A1929P compared to unmodified TS Cav1.2/G406R ( Fig. 3b , ( Supplementary Table S1 online). Similarly, the steady-state inactivation of both of ICa and IBa, displayed elevated Cmax values and a shift in V1/2 towards positive potentials ( Fig. 3c , Table 1 ). Hence, a single point mutation that abrogates CaN binding to the C-tail of α11.2 subunit, significantly restored VDI of TS channel.
The HH/AA double mutation at the CaN-binding site of the Timothy channel restores voltage-dependent inactivation
The double mutation HH/AA, inserted in the native human α11.2 subunit of Cav1.2 channel resulted in a distinctive increase in VDI (see above). To consider the effects of the HH/AA mutation on TS, we mutated the α11.2/G406R subunit to α11.2/G406R/HH/AA ( Fig. 4a ). Representative normalized ICa or IBa traces showed faster inactivation of Cav1.2/G406R/HH/AA compared to the unmodified Cav1.2/G406R channel ( Fig. 4b ; Supplementary Table S1 online). Similarly, the steady-state inactivation of both of ICa and IBa, displayed elevated Cmax values and a shift in V1/2 towards positive potentials ( Fig. 4c , Table 1 ).
Consistent with a previous study36, the ratio of inactivation rates of inward currents (ICa/IBa) based on exemplar normalized traces was significantly different between wt and G406R channels (see Supplementary Fig. S3 online). In contrast, there was no significant difference in ICa /IBa of wt and the channel mutants, G406R/H1926A/H1927A or G406R/A1929P. This data suggests that in the Timothy channel the motif(s) that regulate CDI proceed independently of VDI regulation by the CaN-binding motif. Since the A1929P and HH/AA mutations in the G406R background, change the Ica/IBa ratio back to the WT levels, it further suggests that the A1929P and HH/AA mutations also affect the net CDI. A small effect on net CDI was also visible in wt channel mutants (see Supplementary Fig. S3 online).
CaN inhibitors increase VDI of Timothy channel
Finally, to further confirm VDI regulation by CaN-binding motif and to establish a prospective strategy for TS treatment, we evaluated the effects of CsA and FK-506 (Tacrolimus) two structurally unrelated CaN inhibitors, on Cav1.2G406R inactivation. The normalized ICa and IBa of the Timothy channel showed marked acceleration of inactivation in the presence of 1 µM CsA ( Fig. 5a ). Moreover, the steady-state voltage-dependence of ICa and IBa inactivation of the TS channel ( Fig. 5b ) was greater in the presence of CsA. The effects of CsA, at the concentrations as indicated, were accompanied by a strong shift of the Boltzmann curves towards negative voltages ( Fig. 5b , Table 1 ). When plotted against CsA concentration, the Cmax of Cav1.2/G406R at +10 mV displayed an apparent Kd of 27 nM for ICa, ( Fig. 5c , upper) and 37 nM, for IBa ( Fig. 5c , lower). These values correspond to the physiological affinity constant of CsA for CaN31.
The effects of CsA on maximal inactivation of wt channel (Fig. 2) and TS channel were compared (Fig. 5d). From these results it appears that the CaN binding site is a distinct and specific negative regulator of VDI.
A good control for the convergent effects of CaN using the mutations (A1929P or HH1926-1927AA) or CsA is the lack of additive effect on VDI. In the mutated channels only one of the CaN binding sites is eliminated, while an additional CaN binding site21, could still bind CaN. Therefore, combining CsA with A1929P or HH/AA mutants might show an effect mediated either by a second CaN binding site, or by CsA binding at another site.
We showed that 0.2 µM CsA did not affect the rate of inactivation of G406R/A1929P mutant, measured using Ba2+ as the charge carrier ( Supplementary Table S1 online). Also Cmax values, 61±5% compared to 60 ± 7% or V1/2, -14.1±5.5 mV compared to 16.6 ± 6.8 mV, were not affected by the addition of CsA. No additive effect of VDI was observed when 80 µM CsA was applied to the HH/AA/G406R mutant, using Ca2+ as the charge carrier ( Supplementary Table S1 online). Similar to G406R/A1929P and G406R/HH/AA mutants, CsA changed the Ica/IBa ratio of G406R back to WT level and appeared to have an effect also on net CDI (see Supplementary Fig. S3 online).
Together, these results support the view that CsA acts through inhibiting CaN-binding at the C-tail of α11.2 to restore the VDI of the channel.
Unlike the strong effects on inactivation, CsA only marginally affected the ICa current amplitude and activation properties of Cav1.2/G406R (see Supplementary Fig. S1 online). CsA displayed a significant increase in the rate of activation of IBa, slightly affecting I/Imax (see Supplementary Fig. S2 online).
Like CsA, FK-506 accelerated ICa inactivation of the Timothy channel ( Fig. 6a ). It increased current amplitude ( Fig. 6b ) without shifting the I/Imax ratio ( Fig. 6c ) and accelerated activation ( Fig. 6d ). Similar to CsA, with Ca2+ as the charge carrier, FK-506 displayed no effect on the steady-state inactivation of the wt channel ( Fig. 6e , left) but increased the steady state of IBa voltage-inactivation of the TS channel ( Fig. 6e , right). The restoration of VDI of the Timothy channel by yet another CaN inhibitor, further confirms that this site is a CaN-specific binding motif that acts as a negative regulatory-motif of Cav1.2.
Discussion
Tandan et al21 identified a CaN-binding sites on the CaV1.2 N- and C- termini. The inhibition of CaN by cyclosporine (CsA) induced an immediate potentiation of CaV1.2 currents in neonatal myocytes. This led the authors to suggest that the channel is a CaN substrate where CaV1.2 is affected directly by CaN binding rather than through a transcriptional regulation.
The present study identifies the CaN binding-site within the Cav1.2 C-terminus as a regulatory domain of voltage-dependent inactivation (VDI). First, the CaN binding site is demonstrated as a negative regulatory site of normal cardiac channel inactivation and secondly, the slow VDI, which is a hallmark of Cav1.2 channels bearing the Timothy syndrome (TS) mutation, can be converted into faster wt like VDI by selective inhibition of CaN.
The impact of the distal C terminus on VDI of CaV1.2 was tested initially by deleting 227 amino acids from the C terminus of the human α11.2 subunit (data not shown). The slight increase in VDI observed could result from multiple effects due to the deletion of several regulatory sites, known to reside at the C-terminal of the α11.2 subunit1. Therefore, a more specific target was chosen, which is located within the CaN binding site, previously identified by direct biochemical experiments21. In these studies mutating Ala to Pro (A1959P) within the minimal CaN binding site L1920-L1935, at the C-tail of α11.2, abrogated CaN binding. In the present work, the CaV1.2/A1929P mutant exhibited virtually complete voltage-dependence of inactivation, compared to the partial VDI of Cav1.2. The importance of this CaN site in regulating VDI was further confirmed by mutating two vicinal histidine residues, which are located within the CaN minimal binding domain at the α11.2 C-terminus. Although no direct effect of the HH/AA mutation on CaN binding was reported, the CaV1.2/HH/AA mutant displayed almost a complete voltage-dependence of inactivation similar to that of the A1929P mutant. Hence, the accelerated rate of inactivation and the increase in the extent of VDI demonstrated by the single and the double mutants, results from interference of CaN binding to the mutated CaN-binding site at the α11.2 C-tail. Furthermore, it demonstrates that mutating two His residues can prevent CaN function, consistent with it’s being part of the minimal binding site for CaN21. The stronger VDI establishes the CaN-interaction site as a VDI-negative regulatory motif of Cav1.2. Moreover, like the truncated channel, these results suggest a relief of an inhibitory component, reminiscent of the auto-inhibitory, PKA-dependent regulation of the cardiac channel37.
The regulation VDI through the CaN binding site was further supported pharmacologically, using CsA. This potent and selective CaN inhibitor significantly increased VDI, suggesting that CsA alleviates an inhibitory constitutive constraint of CaN on VDI. Presumably, an altered interaction of the catalytically-inactive CaN, which remains bound to the CaN-motif at the C-tail21, interfered with the normal constitutive binding of CaN to the C-tail domain, thereby eliciting complete VDI. The pharmacological approach thus confirmed the results of mutagenesis (A1929P, HH/AA), demonstrating the role of CaN as a negative-regulator of VDI of the cardiac channel. The convergent effects of CsA at the CaN binding-site at α11.2 C-terminus, further confirms this motif as being a VDI-regulatory site of Cav1.2. This conclusion is congruent with previous findings in mouse ventricular myocytes that CaN inhibition by CaN inhibition by CsA increases the rate of decay of evoked [Ca2+] transients18.
We then explored whether the CaN regulatory effect could be applied to the slow inactivating Timothy channel13,14,33,34,35. The mechanism by which the G406R mutation decelerates VDI is not yet fully understood14,38,39,40,41,36. Several reports suggested that G406R generates a phosphorylation site for the Ca2+/calmodulin–dependent kinase II and increases the open probability (Po) of CaV1.2-LQT8 (TS) channels see also32,39,42. More recent studies have shown that CaV1.2-LQT8 clusters display a higher probability of coordinated openings and closings41 and a disruption between activation and inactivation of the TS channel43.
Similar to A1929P and the HH/AA mutated wt channels, both the A1929P and the HH/AA mutated Timothy channels showed a significant increase in the rate and the extent of VDI. The impact of either the single or the double mutations on the VDI of the wt and the Timothy channels is consistent with altered CaN binding to the regulatory motif at the human C-tail of α11.2. These data support our model, whereby the CaN binding site at α11.2 serves as a negative-VDI regulatory motif. These results are also consistent with the findings that knockout of the A-kinase anchoring protein AKAP150, an associated scaffolding protein that targets CaN to subcellular sites19 could restore arrythmogenic mechanisms in LQT836.
Furthermore, because the non-TS mutated channel Cav1.2/HH/AA showed a similar increase in VDI ( Fig. 1 ), the results favor the interpretation that the distinct CaN-regulatory inactivation determinant at the α11.2 C-tail acts independently of G406R impairment of VDI.
Finally, using CaN selective inhibitors also showed an increase in VDI of the TS. Similar to the wt channel, CsA was very effective in increasing the rate of inactivation and also FK-506 (Tacrolimus), a structurally unrelated CaN inhibitor also showed acceleration of channel inactivation.
Based on these results, we suggest that CsA or FK-506 have the potential to prevent cardiac associated pathology and be clinically beneficial for correcting the cardiac malfunction of Timothy patients. These highly efficient CaN inhibitors, which are mainly used as immunosuppressant drugs, increase VDI at low concentrations and hardly alter wt ICa kinetics.
Thus, CsA can be added to the list of proposed treatments to ameliorate TS related disorders42,44,45. Since TS patients often suffer from infections that can be fatal, the use of immunosuppressant’s would seem to be contraindicated. Nevertheless, in light of the rather small doses needed to enhance voltage inactivation, the shift in V1/2, the severity of the disease and the absence of other alternative drugs to treat this syndrome, CaN inhibitors could be of potential clinical use.
In summary, detailed characterization of the kinetics of Cav1.2 and Cav1.2G406R inactivation has identified the CaN-binding site at the C-tail of α11.2 as a VDI-negative-regulatory motif. The regulation of VDI by CaN, a cytoplasmic Ca2+/calmodulin-dependent protein phosphatase, is consistent with the model of Findlay4, in which an on/off phosphorylation-dephosphorylation switch is proposed to be responsible for converting a non-inactivating into an inactivating channel. These results also provide a novel rational for “repairing” a non-inactivating Timothy channel by targeting the VDI regulatory motif. The highly selective CaN inhibitors, CsA or FK-506, which effectively increase VDI, offer pharmacologic opportunities as potential candidates for treating Timothy patients.
Methods
Reagents
Cyclosporin A (CsA, M.W 1202.61) was purchased from Novartis. CsA was dissolved in DMSO to a final concentration of 42 mM. FK-506 (Tacrolimus) was purchased from LC Laboratories. FK-506 was dissolved in DMSO to a final concentration of 10 mM. CsA and FK506 were diluted in the appropriate Ba2+ or Ca2+ solutions to the desired concentration prior to the experiment.
cDNA Constructs
The full-length human α1C wt channel (accession # Z34815), the full-length human α1C G406R, rabbit α2δ1 (accession # NM_001082276) and rabbit β2b (accession # X64298) were all cloned in the expression vector pSPORT expression vector. These constructs all were a kind gift from Dr. M. Sanguinetti (University of Utah).
Single mutation
The A1929P mutation was introduced via site-directed mutagenesis of the human a11.2 or a11.2/G406R using the following primers: Forward Primer TTG CAT CTG GTT CAT CAT CAG CCA TTG GCA GTG GCA GGC CTG AGC, reverse primer: GCTCAGGCC TGC CAC TGC CAA TGG CTG ATG ATG AAC CAG ATG CAA.
Double mutation
The H1926A, H1927A mutations were introduced via site-directed mutagenesis of the human α11.2 or α11.2/G406R using the following primers: Forward Primer-AGA CAG TCC TGC CCT TGC ATC TGG TTG CTG CTC AGG CAT TGG CAG TGG CAG GCC TGA G Reverse Primer-CTC AGG CCT GCC ACT GCC AAT GCC TGA GCA GCA ACC AGA TGC AAG GGC AGG ACT GTC T.
Mutagenesis was performed by PCR of the human template channel using Phusion enzyme (Finnzymes) followed by cleavage with Dpn I and transformation into XL-10 Gold cells (Startagene).
Two-electrode voltage-clamp recordings in Xenopus oocytes
Stage V and VI oocytes were surgically removed from female Xenopus laevis performed as described46. Capped polyA cRNA from linearized cDNA templates encoding wt or mutant human Cav1.2 subunit α11.2 (5–7 ng), rabbit β2b (2.7 ng) and rabbit α2δ1 (5–7 ng) subunits were co-injected into oocytes and whole-cell currents were recorded using the standard two-electrode voltage-clamp configuration as described previously46. Briefly Voltage and current 1% agar cushioned electrodes (0.3–0.6-megohm tip resistance) were filled with 3 M KCl. Prior to recording for 15 min, oocytes were injected with 40 nl of solution containing 5 mM HEPES, pH 7.0 and 5 mM of the Ca2+ chelator 1,2-bis (2-aminophenoxy) ethane-N, N, N′, N′-tetraacetic acid (potassium salt) final concentration (in oocyte).
Activation kinetics
The activation time constants were determined by fitting the raw current data with the equation: I(t) = I max[1-exp(t/τact)], where I(t) indicates the amplitude of current at time t, I max is the maximum amplitude and τact is the time constant for activation. Each trace was fitted separately according to Boltzmann and the averaged values were plotted. Cav1.2 activation was fitted to single exponential function. All quantitative results are given as the mean ± S.E. (n = 6–10)46.
Steady-state inactivation
A multistep protocol was used to determine the steady state of inactivation. A 250 ms control step was applied to +20 mV, followed by a 150 ms repolarization step to -100 mV. Inactivation was tested by applying 4.5 s conditioning pulses from −100 mV to +20 mV in 10 mV increments, followed by a second test pulse of 250 ms to +20 mV.
Voltage-dependence of Ca2+ and Ba2+ current inactivation was determined with a two-pulse protocol47. The fraction of the non-inactivating current was recorded at the end of the pulse at +20 mV and normalized data were fitted by a single Boltzmann function.
Data acquisition and analyses were performed using pCLAMP9 (Axon Instruments). Currents were filtered at 2 kHz and digitized at 10 kHz. Data were fitted to a Boltzmann function to obtain half point (V1/2) and slope factor (k) for the voltage dependence of CaV1.2 inactivation. Data are presented as mean ± SEM. The protocol of voltage inactivation is shown in Fig. 1C inset. The fraction of the non-inactivating current was recorded at the end of the pulse at +20 mV and normalized data were fitted by a single Boltzmann function.
Voltage clamp and data analysis
Inward calcium current (ICa) and barium current (IBa) were elicited from a holding potential of −80 mV to various test potentials at 5 mV intervals, by a 1500 ms test pulse.
Channel kinetics was recorded using the standard two-electrode voltage clamp method48. Recordings were made in Ba2+ solution (in mM): 5Ba(OH)2, 50 N-methyl d-glucamine, 1 KOH and 5 HEPES (pH 7.5), titrated to pH 7.5 [(CH3)2SO4] or in Ca2+ solution (in mM): 5Ca(OH)2, 50 N-methyl d-glucamine, 1 KOH and 5 HEPES (pH 7.5), titrated to pH 7.5 [(CH3)2SO4]48.
Data analysis
Current inactivation
The fraction of peak current remaining at the end of a 1,000-ms depolarization (r1000) or at 200 ms (r200), divided by the maximal current was used to quantify the level of inactivation.
Steady state inactivation
curves were fitted by a single Boltzmann distribution with I/Imax = C/{1+exp [V1/2−Vm/k]}+(1−C), where C = maximal steady-state inactivating current (Cmax), V1/2 = midpoint of inactivation, Vm is the conditioning voltage and k = slope parameter.
The software packages used was pClampfit 9.0 (Axon Instruments, Foster City, CA, USA) and Origin 7.5 (MicroCal).
References
Catterall, W. A. Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 16, 521-555 (2000).
Stotz, S. C. & Zamponi, G. W. Identification of inactivation determinants in the domain IIS6 region of high voltage-activated calcium channels. J Biol Chem 276, 33001-33010 (2001).
Budde, T., Meuth, S. & Pape, H. C. Calcium-dependent inactivation of neuronal calcium channels. Nat Rev Neurosci 3, 873-883 (2002).
Findlay, I. Physiological modulation of inactivation in L-type Ca2+ channels: one switch. J Physiol 554, 275-283 (2004).
Morad, M. & Soldatov, N. Calcium channel inactivation: possible role in signal transduction and Ca2+ signaling. Cell Calcium 38, 223-231 (2005).
Cens, T., Rousset, M., Leyris, J. P., Fesquet, P. & Charnet, P. Voltage- and calcium-dependent inactivation in high voltage-gated Ca(2+) channels. Prog Biophys Mol Biol 90, 104-117 (2006).
de Leon, M. et al. Essential Ca(2+)-binding motif for Ca(2+)-sensitive inactivation of L-type Ca2+ channels. Science 270, 1502-1506 (1995).
De Jongh, K. S. et al. Specific phosphorylation of a site in the full-length form of the alpha 1 subunit of the cardiac L-type calcium channel by adenosine 3',5'-cyclic monophosphate-dependent protein kinase. Biochemistry 35, 10392-10402 (1996).
Soldatov, N. M., Zuhlke, R. D., Bouron, A. & Reuter, H. Molecular structures involved in L-type calcium channel inactivation. Role of the carboxyl-terminal region encoded by exons 40-42 in alpha1C subunit in the kinetics and Ca2+ dependence of inactivation. J Biol Chem 272, 3560-3566 (1997).
Peterson, B. Z., DeMaria, C. D., Adelman, J. P. & Yue, D. T. Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L-type calcium channels. Neuron 22, 549-558 (1999).
Zuhlke, R. D. & Reuter, H. Ca2+-sensitive inactivation of L-type Ca2+ channels depends on multiple cytoplasmic amino acid sequences of the alpha1C subunit. Proc Natl Acad Sci U S A 95, 3287-3294 (1998).
Hulme, J. T., Yarov-Yarovoy, V., Lin, T. W., Scheuer, T. & Catterall, W. A. Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain. J Physiol 576, 87-102 (2006).
Splawski, I. et al. Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell 119, 19-31 (2004).
Splawski, I. et al. Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations. Proc Natl Acad Sci U S A 102, 8089-8096; discussion 8086-8088 (2005).
Molkentin, J. D. et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93, 215-228 (1998).
Rusnak, F. & Mertz, P. Calcineurin: form and function. Physiol Rev 80, 1483-1521 (2000).
Bandyopadhyay, A., Shin, D. W., Ahn, J. O. & Kim, D. H. Calcineurin regulates ryanodine receptor/Ca(2+)-release channels in rat heart. Biochem J 352 Pt 1, 61-70 (2000).
Santana, L. F., Chase, E. G., Votaw, V. S., Nelson, M. T. & Greven, R. Functional coupling of calcineurin and protein kinase A in mouse ventricular myocytes. J Physiol 544, 57-69 (2002).
Oliveria, S. F., Dell'Acqua, M. L. & Sather, W. A. AKAP79/150 anchoring of calcineurin controls neuronal L-type Ca2+ channel activity and nuclear signaling. Neuron 55, 261-275 (2007).
Heineke, J. et al. Cardiomyocyte GATA4 functions as a stress-responsive regulator of angiogenesis in the murine heart. J Clin Invest 117, 3198-3210 (2007).
Tandan, S. et al. Physical and functional interaction between calcineurin and the cardiac L-type Ca2+ channel. Circ Res 105, 51-60 (2009).
Xu, H. et al. Targeting of protein phosphatases PP2A and PP2B to the C-terminus of the L-type calcium channel Ca v1.2. Biochemistry 49, 10298-10307 (2010).
Wei, X. et al. Modification of Ca2+ channel activity by deletions at the carboxyl terminus of the cardiac alpha 1 subunit. J Biol Chem 269, 1635-1640 (1994).
Gao, T. et al. cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits. Neuron 19, 185-196 (1997).
Zuhlke, R. D., Pitt, G. S., Deisseroth, K., Tsien, R. W. & Reuter, H. Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 399, 159-162 (1999).
Ono, K. & Fozzard, H. A. Two phosphatase sites on the Ca2+ channel affecting different kinetic functions. J Physiol 470, 73-84 (1993).
Sculptoreanu, A., Rotman, E., Takahashi, M., Scheuer, T. & Catterall, W. A. Voltage-dependent potentiation of the activity of cardiac L-type calcium channel alpha 1 subunits due to phosphorylation by cAMP-dependent protein kinase. Proc Natl Acad Sci U S A 90, 10135-10139 (1993).
Davare, M. A., Horne, M. C. & Hell, J. W. Protein phosphatase 2A is associated with class C L-type calcium channels (Cav1.2) and antagonizes channel phosphorylation by cAMP-dependent protein kinase. J Biol Chem 275, 39710-39717 (2000).
Mochida, S. & Hunt, T. Calcineurin is required to release Xenopus egg extracts from meiotic M phase. Nature 449, 336-340 (2007).
Helekar, S. A., Char, D., Neff, S. & Patrick, J. Prolyl isomerase requirement for the expression of functional homo-oligomeric ligand-gated ion channels. Neuron 12, 179-189 (1994).
Swanson, S. K. et al. Cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins A and B. Proc Natl Acad Sci U S A. 89, 3741-3745 (1992).
Erxleben, C. et al. Cyclosporin and Timothy syndrome increase mode 2 gating of CaV1.2 calcium channels through aberrant phosphorylation of S6 helices. Proc Natl Acad Sci U S A 103, 3932-3937 (2006).
Striessnig, J., Bolz, H. J. & Koschak, A. Channelopathies in Cav1.1, Cav1.3 and Cav1.4 voltage-gated L-type Ca2+ channels. Pflugers Arch 460 (2), 361-74 (2010).
Liao, P. & Soong, T. W. CaV1.2 channelopathies: from arrhythmias to autism, bipolar disorder and immunodeficiency. Pflugers Arch 460, 353-359 (2010).
Bidaud, I. & Lory, P. Hallmarks of the channelopathies associated with L-type calcium channels: a focus on the Timothy mutations in Ca(v)1.2 channels. Biochimie 93, 2080-2086 (2011).
Cheng, E. P. et al. Restoration of normal L-type Ca2+ channel function during Timothy syndrome by ablation of an anchoring protein. Circ Res 109, 255-261 (2011).
Fuller, M. D., Emrick, M. A., Sadilek, M., Scheuer, T. & Catterall, W. A. Molecular mechanism of calcium channel regulation in the fight-or-flight response. Sci Signal 3, ra70 (2010).
Barrett, C. F. & Tsien, R. W. The Timothy syndrome mutation differentially affects voltage- and calcium-dependent inactivation of CaV1.2 L-type calcium channels. Proc Natl Acad Sci U S A 105, 2157-2162 (2008).
Thiel, W. H. et al. Proarrhythmic defects in Timothy syndrome require calmodulin kinase II. Circulation 118, 2225-2234 (2008).
Yazawa, M. et al. Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471, 230-234 (2010).
Navedo, M. F. et al. Increased coupled gating of L-type Ca2+ channels during hypertension and Timothy syndrome. Circ Res 106, 748-756 (2010).
Yarotskyy, V., Gao, G., Peterson, B. Z. & Elmslie, K. S. The Timothy syndrome mutation of cardiac CaV1.2 (L-type) channels: multiple altered gating mechanisms and pharmacological restoration of inactivation. J Physiol 587, 551-565 (2009).
Depil, K. et al. Timothy mutation disrupts link between activation and inactivation in CaV1.2. J Biol Chem 286, 31557 (2011).
Yarotskyy, V. et al. Roscovitine binds to novel L-channel (CaV1.2) sites that separately affect activation and inactivation. J Biol Chem 285, 43-53 (2010).
Shah, D. P., Baez-Escudero, J. L., Weisberg, I. L., Beshai, J. F. & Burke, M. C. Ranolazine Safely Decreases Ventricular and Atrial Fibrillation in Timothy Syndrome (LQT8). Pacing Clin Electrophysiol 35, e62-64 (2012).
Wiser, O. et al. The voltage sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery. Proc Natl Acad Sci U S A 96, 248-253 (1999).
Livneh, A., Cohen, R. & Atlas, D. A novel molecular inactivation determinant of voltage-gated CaV1.2 L-type Ca2+ channel. Neuroscience 139, 1275-1287 (2006).
Wiser, O., Bennett, M. K. & Atlas, D. Functional interaction of syntaxin and SNAP-25 with voltage-sensitive L- and N-type Ca2+ channels. EMBO J 15, 4100-4110 (1996).
Acknowledgements
The authors thank H. L. Lauterbach fund and The Betty Feffer Fund for (D.A.) and The Haya and Shlomo Margalit Fund for M.C-K. The authors thank Dr. S. Klein for critically reading the manuscript
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YY and MCK performed the experiments, analyzed the data and prepared the figures. MT prepared the DNA constructs. DA wrote the main manuscript text and prepared figures. All authors reviewed the manuscript.
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Cohen-Kutner, M., Yahalom, Y., Trus, M. et al. Calcineurin Controls Voltage-Dependent-Inactivation (VDI) of the Normal and Timothy Cardiac Channels. Sci Rep 2, 366 (2012). https://doi.org/10.1038/srep00366
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DOI: https://doi.org/10.1038/srep00366
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