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Calcineurin Controls Voltage-Dependent-Inactivation (VDI) of the Normal and Timothy Cardiac Channels

Scientific Reports volume 2, Article number: 366 (2012) | Download Citation


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.


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.


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.

Figure 1: Single and double amino acid mutations within the CaN binding motif at the C-tail of α11.2 subunit restore VDI of Cav1.2.
Figure 1

(Ba) Schematic presentation of: the minimal CaN-binding site (upper), Ala 1929 (middle) and His1926, His1927 residues within this minimal CaN- binding domain (lower). (b), Schematic illustration of channel mutants: α11.2/A1929P (right, upper), and α11.2/H1926A/H1927A (right, lower). (c) Normalized IBa records from representative oocytes expressing Cav1.2 (wt) and Cav1.2A1929P (left) and Normalized IBa records from representative oocytes expressing Cav1.2 (wt) and Cav1.2H1926A/H1927A (right) (d) Steady-state inactivation was recorded in stimulated oocytes according to a multistep protocol of VDI (inset). Smooth curves were generated from a single Boltzmann function of wt and the mutated channels as indicated. Values are displayed as mean ± S.E.M (n = 10−12). Peak currents were: For HH/AA experiment: α11.2, −1140±98 nA (n = 12); α11.2/HH/AA, −1560±162 nA (n = 14); The A1929P experiment: α11.2, −398 ± 58 nA (n = 8); α11.2/A1929P, −316±33 (n = 21).

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.

Table 1: Steady-state inactivation parameters of wt CaV1.2 and TS channel CaV1.2G406R

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.

Figure 2: Cyclosporine A (CsA) increases VDI of Cav1.2.
Figure 2

(a) Normalized IBa and ICa records from representative oocytes expressing Cav1.2 (wt) in the presence of 2 µM CsA (ICa) (left), or 0.6 and 2 µM of CsA (IBa)(right). (b) The voltage-dependence of Cav1.2 inactivation in the presence or absence of CsA (protocol in Fig 1d). Normalized ICa (left) and IBa (right) were fitted to the Boltzmann equation. Values are displayed as mean ±S.E.M (n = 10−12). Peak currents for: ICa, α11.2, −700 ± 57 nA (n = 13); α11.2 +2 µMCsA, −625 ± 76 nA (n = 12); IBa, α11.2, −774 ± 34 nA (n = 10) ;α11.2 +2 µMCsA, −670 ± 69 nA (n = 8).

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).

Figure 3: Single point mutation at CaN binding motif of α11.2/G406R subunit increases VDI of the Timothy channel.
Figure 3

(a) Schematic illustration and location (b) of the G406R Timothy mutation, and the A1929P mutation within the CaN binding motif of the human α11.2 subunit (c) Normalized ICa (left) and IBa (right) recordings from representative oocytes expressing Cav1.2/G406R and Cav1.2/G406R/A1929P (d) The voltage-dependence of inactivation of Cav1.2G406R and Cav1.2G406R/A1929P in Ca2+ (left) and Ba2+ (right). Values are displayed as mean ± S.E.M (n = 10−12). Peak currents for ICa, α11.2/G406R, −493 ± 74 nA (n = 13); α11.2/A1929P −661 ± 115nA (n = 8); IBa, α11.2, −643 ± 57 nA (n = 16); α11.2G406R/A1929P, −1132±18 nA (n = 21).

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).

Figure 4: A mutation within CaN binding motif of TS α11.2/G406R subunit increases VDI of the Timothy channel.
Figure 4

(a) Schematic illustration and location (b) of the Timothy α11.2G406R subunit and the His1926A, and His1927A mutations within the CaN binding motif of the human α11.2 subunit (c) Normalized ICa (left) and IBa (right) records from representative oocytes expressing Cav1.2/G406R and Cav1.2/G406R/HH/AA (d) The voltage-dependence of inactivation of Cav1.2G406R and Cav1.2G406R/HH/AA in Ca2+ (left) and Ba2+ (right). Values are displayed as mean ± S.E.M (n = 10−12). Peak currents for ICa α11.2/G406R, −493 ± 74 nA (n = 13); α11.2/G406R/HH/AA, −338 ± 52 nA (n = 14) For IBa, α11.2/G406R, −377 ± 61 nA (n = 13); α11.2G406R/HH/AA −518 ± 23 nA (n = 10).

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.

Figure 5: Cyclosporine A effectively restores VDI of Timothy channel G406R.
Figure 5

(a) Normalized ICa (n = 10) (left) and IBa (n = 11) (right) records from representative oocytes expressing Cav1.2/G406R, in the presence or in the absence of 1 µM CsA. (b) Steady-state inactivation of Cav1.2G406R in the presence of increasing CsA concentrations, as indicated for ICa (left) and IBa (right). (c) Efficacy of CsA on VDI. Normalized maximal steady-state inactivating ICa at +10 mV (left) and IBa at 0 mV (right) plotted at increasing CsA concentrations. Data displayed as mean ± S.E.M (n = 8−12). Peak currents for ICa α11.2/G406R, −377 ± 40 nA (n = 13); α11.2/G406R + 50 nM CsA, −229 ± 43 nA (n = 11); IBa, α11.2/G406R, −377 ± 61 nA (n = 12); α11.2/G406R + 50 nM CsA, −194 ± 38 nA (n = 10).

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.

Figure 6: FK-506 restores Timothy channel inactivation.
Figure 6

ICa was elicited in oocytes expressing α11.2G406R/α2δ12b (Cav1.2G406R) from a holding potential of -80 mV to various test potentials in 5 mV increments, in response to 1200 ms pulse (a) representative ICa traces elicited in the absence and in the presence of 0.1, 1.0 and 10 µM FK-506 (b) Current–voltage relationship of G406R in the absence and in the presence of FK-506, at the indicated concentrations (c) Peak current amplitudes normalized to maximum current (taken from (b)) shown as I/Imax ratios of inward currents of G406R channel in the presence and in the absence of FK-506 are plotted against test potentials and displayed with a Boltzmann fit (mean ± S.E.M; n = 10–12; more details in supporting online text) (d) The time constant of activation (τact, mean±S.E.M., n = 10), fitted by a single exponent (e) Steady-state inactivation recorded in stimulated wt (left) and G406R channel (right) with or without 1 µM CsA, according to a multistep protocol of VDI measurements (Fig. 1C inset). Normalized currents were fitted to the Boltzmann equation (Methods).


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.



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(tact)], 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).


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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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    • Moshe Cohen-Kutner
    •  & Yfat Yahalom

    Equal contribution.


  1. The Hebrew University of Jerusalem Institute of Life Sciences Dept. of Biological Chemistry Givat-Ram, Jerusalem, 91904, Israel

    • Moshe Cohen-Kutner
    • , Yfat Yahalom
    • , Michael Trus
    •  & Daphne Atlas


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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Daphne Atlas.

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