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Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels
Igor Dzhura*, Yuejin Wu†, Roger J. Colbran‡, Jeffrey R. Balser*§ and Mark E. Anderson†§¶*Department of Anesthesiology, Vanderbilt University, Nashville, Tennessee 37232, USA
†Department of Internal Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA‡Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA
§Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA¶e-mail: [email protected]
A dynamic positive feedback mechanism, known as ‘facilitation’, augments L-type calcium-ion currents (ICa) in response to increased intracellular Ca2+ concentrations. The Ca2+-binding protein calmodulin (CaM) has been implicated in facilitation, but the single-channel signature and the signalling events underlying Ca2+/CaM-dependent facilitation are unknown. Here we show that the Ca2+/CaM-dependent protein kinase II (CaMK) is necessary and possibly sufficient for ICa facilitation. CaMK induces a channel-gating mode that is characterized by frequent, long openings of L-type Ca2+ channels. We conclude that CaMK-mediated phosphorylation is an essential signalling event in triggering Ca2+/CaM-dependent ICa facilitation.
oltage-gated L-type Ca2+ channels are pore-forming mem-brane-bound proteins that govern diverse physiological func-tions, including gene transcription1, hormone secretion2 and
excitation–contraction coupling in cardiac muscle3. A dynamic ele-ment of the regulation of Ca2+ channels is the unique positive feed-back mechanism, known as ‘facilitation’, that augments inwardcurrents through L-type Ca2+ channels as the intracellular Ca2+ con-centration ([Ca2+]i) rises4–9. Facilitation of ICa is an important mech-anism for grading Ca2+ entry to control intracellular Ca2+ activity.The Ca2+-binding protein CaM has been implicated in Ca2+-dependent facilitation10, but the fundamental signalling events thatunderlie CaM-dependent facilitation have not been elucidated, andthe single-channel signature of Ca2+/CaM-dependent facilitation isunknown. Here we show that CaMK plays a fundamental part in ICa
facilitation in cardiac myocytes. Using a constitutively active engi-neered Ca2+/CaM-independent CaMK, we show that CaMK-medi-ated phosphorylation of unknown, cell-membrane-associatedtarget proteins induces single L-type Ca2+ channels to enter a gatingmode that is characterized by frequent, long openings. These effectsare independent of CaM, but are eliminated by a selective CaMK-inhibitory peptide. We conclude that CaMK-mediated phosphor-ylation is an essential signalling event that triggers Ca2+/CaM-dependent facilitation in L-type Ca2+ channels.
ResultsCa2+-dependent ICa facilitation. Figure 1 illustrates Ca2+-dependentICa facilitation in cardiac myocytes under whole-cell voltage-clampconditions. Ca2+ entry through L-type Ca2+ channels results in Ca2+-dependent ICa facilitation and inactivation (Fig. 1a). Substitution ofBa2+ for Ca2+ in the bath medium supports current through L-typeCa2+ channels, but eliminates ICa facilitation and markedly slowsinactivation (Fig. 1b). Previous studies have shown that CaM-inhibitory peptides competitively inhibit activation of CaMK bypreventing binding of Ca2+-activated CaM to the kinase11. Figure 1cshows that addition of a CaM-inhibitory peptide to the intracellularpipette solution also prevents ICa facilitation. The specific CaMK-inhibitory peptide AC3-I (Fig. 1d), which acts as a pseudosubstrateto inhibit CaMK activity12, similarly ablated ICa facilitation. Becauseother multifunctional serine/threonine kinases, including proteinkinase A (PKA)13 and protein kinase C (PKC)14, also modulate ICa,
in separate experiments we used inhibitory peptides directedagainst these kinases. Peptide inhibitors of PKA (n = 4) and PKC (n= 3) did not affect ICa facilitation (Fig. 1e). These results indicate thatCa2+-dependent ICa facilitation in cardiac myocytes may requireactivation of CaMK by Ca2+-bound CaM.Generation of constitutively active CaMK. These results are notsufficient to prove that CaMK directly activates the L-type Ca2+-channel complex, because CaMK activates several protein targets incardiac myocytes that alter Ca2+ mobilization from intracellularstores15,16, and so could indirectly influence ICa facilitation17. Thus, insubsequent experiments we measured single L-type Ca2+-channelcurrents from isolated cell-membrane patches18, to exclude effectsfrom intracellular Ca2+ stores. The effects of CaMK on single L-typeCa2+ channels have not been reported previously, in part becauseBa2+, used as a charge carrier to improve the resolution of single-channel openings, cannot form complexes with CaM19 to activateCaMK. We overcame this experimental obstacle to CaMK activa-tion by generating a constitutively active CaMK for use in thesestudies in order to measure CaMK effects on L-type Ca2+ channelsin the presence of Ba2+. CaM is present with the constitutively activeCaMK used in these studies, but neither CaM nor Ca2+ is requiredfor kinase activity17, thus allowing separate control of [Ca2+]i andCaMK activity in the presence of Ba2+.CaMK reconstitutes ICa facilitation. Under control conditions, sin-gle L-type Ca2+ channels in excised, inside-out patches exhibitedintermittent brief openings (Fig. 2a). The probability of opening ofL-type Ca2+ channels increased markedly when constitutively activeCaMK was present in the bath (Fig. 2c), but not when CaM alonewas present (Fig. 2b). Addition of constitutively active CaMKresulted in a rapid transition to sweeps with a high probability ofchannel opening (Fig. 2g). Ensemble-averaged single-channel cur-rent tracings showed that this CaMK effect reconstituted the ICa
facilitation seen in whole cells (Fig. 1a). As shown previously20, theensemble-averaged currents recorded from excised patches, withBa2+ as the charge carrier, lacked the decay phase seen in whole-cellICa recordings (Fig. 1a, c, d), presumably as a result of a decrease inCa2+-dependent inactivation. Facilitation of whole-cell peak ICa,with Ca2+ as the charge carrier, was modest compared with facilita-tion in the ensemble currents in the presence of constitutively activeCaMK with Ba2+ as the charge carrier. It is likely that the competingprocess of Ca2+-dependent inactivation diminishes the effect of
V
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articles
facilitation. Reduction in the activity of endogenous phosphatasesin the ripped-off membrane patches may also contribute to thegreater magnitude of the CaMK effect in the experiments using sin-gle Ca2+ channels. The observed magnitude of Ca2+-dependent ICa
facilitation in whole cells is, nevertheless, probably sufficient toincrease the strength of cardiac contractions21,22 and contribute tothe development of life-threatening arrhythmias17,23.
Addition of a CaM-inhibitory peptide (Fig. 2e, h) to the cyto-plasmic side of the voltage-clamped cell membrane did not inhibitthe effect of the constitutively active CaMK, but the specific CaMK-inhibitory peptide AC3-I (Fig. 2d, h) did prevent facilitation.Although the engineered CaMK that we used is CaM independent,the inability of a CaM inhibitor to oppose CaMK action (Fig. 2e, h)confirms that the effects of membrane-bound endogenous CaM donot complicate the results. Addition of exogenous CaM (2 µM) inthe absence of CaMK also failed to alter the activity of L-type Ca2+
channels (Fig. 2b, h). Likewise, PKA- or PKC-inhibitory peptidesdid not oppose the effects of CaMK (Fig. 2h). The effects of CaMKwere prevented by addition of a tenfold excess (in comparison withthe amount of ATP) of the non-hydrolysable ATP analogue AMP-PNP (Fig. 2f, h), indicating that ATP is required for CaMK facilita-tion. This result indicates that a phosphorylation mechanism,rather than a nonspecific action, may underlie CaMK-mediated ICa
facilitation. Taken together, these findings show that CaM alone isunable to support ICa facilitation and that constitutively activeCaMK is sufficient to induce the activity of L-type Ca2+ channelsthat underlies ICa facilitation in cardiac myocytes.CaMK induces mode-2 gating. We next considered the functionaleffects of CaMK on gating of L-type Ca2+ channels. Figure 3 illus-trates quantification of the open times of single L-type Ca2+ chan-nels. Open-time histograms were best described by long (τL) andshort (τS) time constants. Addition of CaM (2 µM) to the cyto-plasmic face of the channels (Fig. 3b) did not change the distri-bution of open or closed times compared with controls (Fig. 3a).In contrast, CaMK increased the probability of long openings (τL)(Fig. 3c, d), but did not significantly change the duration of eitheropen-time component (Fig. 3e). The finding that CaMK changesthe relative probability of long versus short openings withoutaltering the open times indicates that CaMK may operatethrough a modal gating mechanism20,24. Enhanced β-adrenergicsignalling causes the gating of L-type Ca2+ channels to shift froma mode characterized by brief, infrequent openings (mode 0) toone of two more active modes that feature either frequent, briefopenings (mode 1) or very long openings (mode 2)20,24. With thisin mind, we analysed our data by plotting maximum open times(To,max) against opening probability (Po) (Fig. 4) in each sweep tostudy CaMK-induced partitioning between these three modes.We defined sweeps with a Po of more than 2% (indicated by thehorizontal lines in Fig. 4) as mode-0 sweeps, similar to thosedescribed in ref. 24. Under all conditions there was a nadirbetween short and long To,max values at 8 ms (Fig. 4a–c). Thisinflection (indicated by the vertical lines) was used to dividemode-1 gatings (high Po, brief openings) from mode-2 gatings(high Po, long openings). This analysis showed that CaMK signif-
Figure 1 Paired depolarizing pulses (–80 mV to +10 mV at 0.5 Hz) lead to Ca2+-dependent facilitation of whole-cell ICa. a, ICa facilitation is seen under control conditions in cardiac myocytes when Ca2+ is the charge carrier. The current trace labelled 3 in the inset shows that all active current is eliminated by nifedipine, an antagonist of L-type Ca2+-channels, under these conditions. b, ICa facilitation is eliminated and inactivation is slowed when extracellular Ba2+ is substituted for Ca2+, using the same cell as that used for a. c, d, ICa facilitation is prevented by intracellular dialysis of a CaM-inhibitory peptide (290–309, 100 µM, n = 3) or a specific CaMK-inhibitory peptide (AC3-I, 20 µM, n = 8), but not by an inactive control peptide (AC3-C, 20 µM, n = 8; this peptide was used as the control in e). All currents were scaled to the second pulse and the holding current is equal to 0 pA; the vertical scale bar indicates 450 pA (a), 500 pA (b), 1,000 pA (c), or 400 pA (d), and the horizontal bar indicates 150 ms in all panels. e, Summary graph of ICa facilitation under the conditions described in a–d and following dialysis with inhibitory peptides against PKA (PKI) and PKC. The ordinate shows peak ICa from the first (ICa1) and second (ICa2) stimulation pulses, expressed as a ratio. Asterisk indicates P = 0.0002 compared to AC3-I; error bars indicate s.e.m.
Control Baa b
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Figure 2 Constitutively active CaMK causes prolonged openings of single L-type Ca2+ channels in excised inside-out patches. a–c, Addition of engineered Ca2+/CaM-independent CaMK (0.9 µM) to the recording chamber markedly increased the duration of openings of L-type Ca2+ channels compared with the duration in controls or in the presence of CaM alone. In all cases (a–g) the concentration of free Ca2+ in the bath solution was 10 µM. CaM was required to generate the constitutively active CaMK used in these experiments and so was present (1.8–2.0 µM) alone (b) and in experiments with CaMK (c–g). d–f, Prolongation of single channel openings by CaMK is prevented by adding a CaMK-inhibitory peptide (AC3-I, 10 µM; d) or a tenfold excess of the non-hydrolysable ATP analogue AMP-PNP (100 µM; f) to the recording chamber. Addition of a CaM-inhibitory peptide (290–309, 10 µM; e) did not prevent CaMK-mediated prolonged openings of L-type Ca2+ channels. The ensemble-averaged currents shown below each set of five current traces were calculated from 365–370 non-blank sweeps for each membrane patch. An all-sweep probability diary is shown at the bottom of each panel; each bar represents the percentage of time that the channel was open during the 200-ms depolarization. g, Addition of constitutively active CaMK to the recording chamber evokes a rapid transition to sweeps with a high Po. h, Mean Po values obtained when using bath solutions of low Ca2+ concentration (100 nM, white bars) or high Ca2+ concentration (10 µM, black bars), in the presence or absence of CaM and/or constitutively active CaMK as indicated. Inhibitory peptides (10 µM each) added to the bath solution did not affect the mean Po in the presence or absence of CaM in low or high bath Ca2+ concentrations compared with controls. Addition of constitutively active CaMK to the bath solution significantly increased the mean Po as compared to controls (asterisk indicates P < 0.001) and this effect was not prevented by inhibitory peptides against PKA (PKI), PKC, or CaM (290–309). The effects of CaMK on mean Po were, however, occluded by a tenfold excess (100 mM) of AMP-PNP and by a CaMK inhibitory peptide (AC3-I) added to the bath solution (see Methods).
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NATURE CELL BIOLOGY | VOL 2 | MARCH 2000 | cellbio.nature.com 175
0 mV
–70 mVControl CaM CaMK
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icantly increased mode-2 activity at the expense of modes 0 and1 (Fig. 4d), in contrast to β-adrenergic signalling which enhancesboth modes 1 and 2 (ref. 24).
DiscussionCa2+-mediated facilitation of ICa is a key feedback mechanism in car-diac and neuronal signal transduction. Recent studies have impli-cated Ca2+-dependent binding of CaM to the carboxy terminus ofthe L-type Ca2+-channel α1c subunit and to P/Q-type Ca2+ channels25
as an essential element in the modulation of Ca2+-dependent ICa
facilitation10 and inactivation10,26. However, our studies of excisedpatches have shown that CaM is not sufficient to stimulate ICa facil-itation (Fig. 2b, e, h). Instead, a CaMK-mediated phosphorylationevent appears to be an essential molecular mechanism underlyingCa2+-dependent ICa facilitation. Nonetheless, binding of CaM to theC terminus of the L-type Ca2+ channel probably has a facilitatory
role, perhaps by anchoring CaM at a site that supports CaMK-mediated phosphorylation or by producing a conformationalchange in the L-type Ca2+ channel that allows CaMK-mediatedphosphorylation. The biophysical mechanism of CaMK signallinginvolves a repartitioning of L-type Ca2+-channel gating modes in amanner that exclusively potentiates long openings, in contrast to β-adrenergic signalling24. This unique gating effect of CaMK is likelyto strategically amplify the role of L-type Ca2+ channels in the cas-cade of molecular signals that regulate intracellular Ca2+. h
Methods Electrophysiology.Electrophysiology using whole cells was done according to published methods9. Murine ventricular
myocytes were freshly isolated as described for rabbit myocytes9. Pure ICa was obtained by adding Cs+ and
tetraethylammonium chloride (TEA) and reducing Na+ and K+ in the pipette and bath solutions.
Elimination of the residual current with nifedipine (10 µM) confirmed the identity of active current as
ICa (Fig. 1a, inset). For whole-cell measurements, the pipette (intracellular) solution contained (in mM)
CsCl 120.0, EGTA 1.0, HEPES 10.0, TEA 10.0, phosphocreatine 5.0, MgATP 1.0, and NaGTP 1.0; the pH
was adjusted to 7.2 with 1.0 N CsOH. The bath (extracellular) solution contained (in mM) choline
chloride or NMDG 137.0, CsCl 25.0, HEPES 10.0, glucose 10.0, CaCl2 1.8, and MgCl2 0.5; the pH was
adjusted to 7.4 with 1.0 N CsOH or HCl. Ba2+ was substituted for Ca2+ for the experiments described in
Fig. 1b. For single-Ca2+-channel measurements, the bath (intracellular) solution contained (in mM) KCl
150, EGTA 10 , HEPES 10, CaCl2 7.5 or 11, glucose 5.5, EDTA 1, and ATP 0.01; the pH was adjusted to
7.4 with 10 N KOH. The calculated resting free [Ca2+] was ~100 nM for the solution with a low Ca2+
concentration, and ~10 µM for the solution with a high Ca2+ concentration27. The pipette (extracellular)
solution contained (in mM) BaCl2 110, HEPES 5, TTX 0.03; the pH was adjusted to 7.4 with trizma base.
Currents were recorded from isolated cell-membrane patches in response to step depolarizations (0.5-
Hz steps) using the inside-out configuration18, sampled at 20 kHz, and low-pass-filtered at 2 kHz (4 pole
Figure 3 Quantification of the open times of single L-type Ca2+ channels. a–c, Open-time histograms plot logarithmically binned durations of open and closed times (abscissa) against the number of events (ordinate)33. Open-time distributions were best-fit by the sum of two exponentials and the long (τL) and short (τS) time constants from these fits are shown. The percentage of total openings of L-type Ca2+ channels described by τS and τL are indicated in brackets. Data are from the same membrane patches as those shown in Fig. 2a–c. d, The percentage of long channel openings is significantly (P < 0.001) increased by application of CaMK, compared with control or CaM-treated membrane patches. e, The open-time durations (τS and τL) are not changed by addition of CaMK compared with CaM-treated or control cells.
*
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Figure 4 CaMK favours a single, high-open-probability mode of gating of L-type Ca2+ channels, characterized by prolonged channel openings. a–c, Top, Po plotted against the maximum channel open time in each non-blank sweep (To,max), a, in controls; b, after addition of CaM (2 µM); and c, after addition of CaMK (0.9 µM). Bottom, histogram plots of To,max, each fitted with two Gaussian distributions. A vertical time line marks the minimum (obtained from the fitted Gaussian distributions) between long and short openings of L-type Ca2+ channels. The horizontal dashed lines at Po = 2% divide high and low Po sweeps. Bold numbers indicate gating modes (0, 1 or 2). The data in a–c correspond to the cells shown in Fig. 2a–c and Fig. 3a–c. d, Summary data from cell-membrane patches (n = 3) in the control, CaM-treated and CaMK-treated groups, showing the number of sweeps that fall into each of the three gating modes. Asterisk indicates P<0.001 compared with controls.
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Bessel). Blank sweeps were averaged and subtracted from all other sweeps to eliminate uncompensated
capacitative transients. Subtracted records were then idealized and analysed using commercial
(TRANSIT) software28. Only cell-membrane patches containing a single Ca2+ channel were analysed.
Inhibitory peptides.The CaMK inhibitory peptide AC3-I (KKALHRQEAVDCL; half-maximal inhibitory concentration
(IC50) ~3 µM)12 and the inactive control peptide AC3-C (KKALHAQERVDCL; IC50 > 500 µM) were gifts
from H. Schulman. AC3-I is a modified CaMK substrate and the amino-acid sequence HRQEAVDCL
corresponds to the autophosphorylation site (T286/287) on CaMK, except that T is modified to A to
prevent phosphorylation29. The CaM-inhibitory peptide 290–309 (LKKFNARRKLKGAILTTMLA; IC50 =
52 nM; Biomol)11 and the PKA (TYADFIASGRTGRRNAI; Ki = 1.7 nM)30 and PKC
(RFARKGALRQKNVHEVKN; Ki = 150 nM)31 inhibitory peptides were purchased from Gibco.
Generation of constitutively active CaMK.CaMK (amino-acid residues 1–380 of mouse type II, α-isoform) was expressed in baculovirus, purified
with a CaM-affinity column as described17, and used at a final concentration of 0.9 µM to approximate
physiological activity32. The purified CaMK was made independent of Ca2+/CaM by thiophosphorylation
of Thr 286 in the presence of Ca2+, CaM, Mg2+ and adenosine 5′-O-(3-thiotriphosphate); Ca2+/CaM-
independent activity was verified with a phosphorylation assay using a synthetic CaMK substrate,
autocamtide, as described17. Ca2+/CaM-independent CaMK activity was 35–50% of total activity and this
activity level persisted at >75% of initial levels during the course of the experiments.
RECEIVED 6 DECEMBER 1999; REVISED 12 JANUARY 2000; ACCEPTED 26 JANUARY 2000; PUBLISHED 10 FEBRUARY 2000.
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ACKNOWLEDGEMENTS
This work was supported by grants from the NIH (grants NHLBI, HL03727 and HL62494 to M.E.A.) and
the American Heart Association (to M.E.A. and R.J.C.). J.R.B. is an Established Investigator of the
American Heart Association. We thank M. Bass for technical assistance, and D. Roden and A. George for
helpful criticisms and suggestions.
Correspondence and requests for materials should be addressed to M.E.A.
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