HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 550/3/731    most recent 2003.043778v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dzhura, I. Articles by Anderson, M. E. Search for Related Content PubMed PubMed Citation Articles by Dzhura, I. Articles by Anderson, M. E.

J Physiol (2003), 550.3, pp. 731-738
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.043778

C terminus L-type Ca²⁺ channel calmodulin-binding domains are 'auto-agonist' ligands in rabbit ventricular myocytes

Igor Dzhura*, Yuejin Wu*, Rong Zhang*, Roger J. Colbran†, Susan L. Hamilton‡ and Mark E. Anderson*§

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

L-type Ca²⁺ channel C terminus calmodulin (CaM)-binding domains are molecular determinants for Ca²⁺-CaM-dependent increases in L-type Ca²⁺ current (I_Ca), and a CaM-binding IQ domain mimetic peptide (IQmp) increases L-type Ca²⁺ channel current by promoting a gating mode with prolonged openings (mode 2), suggesting the intriguing possibility that CaM-binding domains are 'auto-agonist' signalling molecules. In order to test the breadth of this concept, we studied the effect of a second C terminus CaM-binding domain (CB) mp (CBmp), in conjunction with IQmp, on single L-type Ca²⁺ channel currents in excised cell membrane patches from rabbit ventricular myocytes. Here we show that both CBmp and IQmp are agonist ligands that non-additively increase L-type Ca²⁺ channel opening probability (P_o) by inducing mode 2 gating. CBmp and IQmp agonist effects were lost under conditions favouring calcification of CaM (Ca²⁺-CaM, 150 nM free Ca²⁺ and 10-20 µM CaM), but persisted in the presence of CaM (0-20 µM) under conditions adverse to Ca²⁺-CaM (20 mM BAPTA), indicating that CaM-binding domains increase L-type Ca²⁺ channel P_o by a low Ca²⁺-CaM activity mechanism. Increasing Ca²⁺-CaM in the bath (cytosol) reduced the efficacy of CBmp and IQmp signals with Ba²⁺ as charge carrier, suggesting that CaM binding motifs target a site outside of the pore region. We measured the combined effects of CBmp and Ca²⁺-CaM-dependent protein kinase II (CaMKII) on L-type Ca²⁺ channels by using an engineered Ca²⁺-CaM-independent form of CaMKII that remains active under low Ca²⁺-CaM conditions, permissive for CBmp signalling. CBmp and CaMKII increased L-type Ca²⁺ channel P_o in a non-additive manner, suggesting that low and high Ca²⁺-CaM-dependent L-type Ca²⁺ channel facilitation pathways converge upon a common signalling mechanism.

(Received 26 March 2003; accepted after revision 2 May 2003; first published online 13 June 2003)
Corresponding author M. E. Anderson: Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, TN 37232, USA. Email: mark.anderson{at}vanderbilt.edu

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

L-type Ca²⁺ current (I_Ca) is the primary portal for Ca²⁺ entry into heart, and I_Ca serves as the trigger for release of intracellular Ca²⁺ during cardiac excitation-contraction coupling. Increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) modulate I_Ca by engaging opposing processes of enhanced inactivation (reduces I_Ca) (Plant et al. 1983), and facilitation (increases I_Ca) (Marban & Tsien, 1982). Both Ca²⁺-dependent I_Ca inactivation and facilitation require Ca²⁺ binding to the Ca²⁺-sensing protein calmodulin (CaM) (Zuhlke et al. 1999). Ca²⁺-CaM can facilitate I_Ca by activating the multifunctional Ca²⁺-CaM-dependent protein kinase II (CaMKII) (Yuan & Bers, 1994; Anderson et al. 1994; Xiao et al. 1994) and inducing L-type Ca²⁺ channels (Cav1.2) to enter a gating mode (mode 2) with prolonged openings (Dzhura et al. 2000). However, multiple lines of evidence indicate that L-type Ca²⁺ channel C terminus CaM-binding domains are also critical for Ca²⁺-dependent regulation of L-type Ca²⁺ channel currents (Zuhlke et al. 1999; Qin et al. 1999; Peterson et al. 1999; Pate et al. 2000).

Dialysis of a peptide modelled after the IQ (Qin et al. 1999) or CB (Pate et al. 2000) CaM-binding motifs (Fig. 1A) into cardiac myocytes enhances the normal pattern of I_Ca facilitation (Wu et al. 2001a), suggesting the intriguing possibility that these CaM-binding domains may serve as 'auto-facilitation' ligands. The IQ mimetic peptide (IQmp) increases L-type Ca²⁺ channel opening probability (P_o) by driving channels into a gating mode 2 (Hess et al. 1984; Wu et al. 2001a; Dzhura et al. 2002), a property that is also shared by CaMKII (Dzhura et al. 2000). IQmp and CaMKII increase L-type Ca²⁺ channel P_o in a non-additive fashion (Wu et al. 2001a), possibly indicating that diverse Ca²⁺-CaM-driven signals operate through a common facilitation pathway.

		View larger version [in this window] [in a new window]
Figure 1. L-type Ca2+ channel C terminus CaM-binding domains increase L-type Ca2+ channel opening probability (Po) A, schematic representation of the L-type Ca2+ channel with the amino acid sequences and relative positions of the IQ and CB CaM-binding motifs. Numerals I-IV indicate homolgous domains in 1C. B, representative L-type Ca2+ channel current recordings under control conditions and after addition of CB or IQ mimetic peptides (mp) to the bath (cytoplasmic) solution. The scale bars indicate 20 ms (horizontal) and 2 pA (vertical). C, mean L-type Ca2+ channel Po under basal conditions (control) or after stepwise increases in CBmp or IQmp (indicated by horizontal bars, with concentrations on the abscissa). The rightmost bar represents L-type Ca2+ channel Po after 10 µM CBmp and 10 µM IQmp added to the bath solution. * P < 0.001 versus control; † P < 0.05 versus CBmp. Differences between 10 and 20 µM CBmp and IQmp were not significant.

We tested the general applicability of the concept that L-type Ca²⁺ channel C terminus CaM-binding motifs are auto-agonist ligands, by introducing IQmp and CBmp to the cytoplasmic face of L-type Ca²⁺ channels under controlled Ca²⁺-CaM and CaMKII activity conditions, in excised cell membrane patches from rabbit ventricular myocytes. Our results show that IQmp and CBmp induce mode 2 gating in cardiac L-type Ca²⁺ channels only under low Ca²⁺-CaM activity conditions, by a novel mechanism that is independent of CaMKII. These findings support the concept that L-type Ca²⁺ channel C terminus CaM-binding domains are also 'auto-agonist' signals for increasing L-type Ca²⁺ channel P_o during periods of low Ca²⁺-CaM activity.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Electrophysiology

Ventricular myocytes were isolated from New Zealand White rabbits killed by pentobarbital (50 mg kg^-1, I.V.) overdose prior to excising the heart, as previously described (Wu et al. 2001a). The Vanderbilt University Animal Care Committee approved all experiments. Single Ca²⁺ channel measurements were performed with excised cell membrane patches using the inside-out configuration (Hamill et al. 1981) in response to depolarizing steps to 0 mV (200 ms) from a holding potential of -70 mV (0.5 Hz), sampled at 20 kHz, and low-pass filtered at 2 kHz (4 pole Bessel). While most (~80 %) Ca²⁺ channels exhibited rundown that prevented inclusion in the experimental results, ~20 % of channels remained sufficiently active for experimental measurements. Blank sweeps were averaged and subtracted from all other sweeps to eliminate uncompensated capacitative transients. Subtracted records were then idealized and analysed using TRANSIT software (VanDongen, 1996). Only cell membrane patches containing a single Ca²⁺ channel were analysed. Analysis of modal gating was performed as described (Yue et al. 1990).

The bath (intracellular) solution was (mM): KCl 150, EGTA 10, Hepes 10, CaCl₂ 7.5, glucose 5.5, EDTA 1, ATP 0.01 and pH was adjusted to 7.4 with 10 M KOH. Experiments were performed at a free [Ca²⁺]_i concentration ~150 nM (Bers et al. 1994). The pipette (extracellular) solution was (mM): BaCl₂ 110, Hepes 5, TTX 0.03 and pH was adjusted to 7.4 with trizma base. Ba²⁺ was used as charge carrier to eliminate high Ca²⁺ concentration microdomains that could activate endogenous CaM anchored in the vicinity of the L-type Ca²⁺ channel cytoplasmic pore (Pitt et al. 2001; Erickson et al. 2001), and to increase the percentage of channels without rundown.

Peptides

IQmp (FLIQEYFRKFKKRKEQ) and CBmp (NEELRAIIKKTWKRTSMKLL) were modelled after CaM-binding domains on the C terminus of the cardiac L-type Ca²⁺ channel subunit (Mikami et al. 1989). A control peptide (FLIQEYFRKSHKRKEG) that does not bind CaM (Wu et al. 2001a) was used in some experiments. The CaMKII inhibitory peptide AC3-I (KKALHRQEAVDCL, IC₅₀ ~3 µM) (Braun & Schulman, 1995a) is a modified CaMKII substrate and the amino acid sequence HRQEAVDCL corresponds to the autophosphorylation site (T286/287) on CaMKII, except T is modified to A to prevent phosphorylation. AC3-I, IQmp, and control peptides (Macromolecular Resources, Fort Collins, CO, USA) and CBmp (Protein Facility at Baylor College of Medicine, TX, USA) were synthesized and isolated to > 95 % purity by reverse phase high performance liquid chromatography.

Constitutively active CaMKII

Constitutively active, Ca²⁺-CaM-independent, monomeric CaMKII (amino acid residues 1-380 of mouse type II, isoform) was expressed in baculovirus and purified with a CaM affinity column as previously described (Wu et al. 1999). The purified CaMKII was made Ca²⁺-CaM-independent 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 CaMKII substrate, autocamtide. Constitutively active CaMKII was used at a final concentration of 0.9 µM, to approximate physiological activity (Gupta & Kranias, 1989). Ca²⁺-CaM-independent CaMKII activity was 35-50 % of total activity and this activity level persisted at > 75 % of initial levels during the course of these experiments.

CaM binding assays

CaM binding 'gel shift' assays were performed as previously described (Wu et al. 2001a), except CBmp was used in place of IQmp.

Chemical reagents

All other chemicals were reagent grade and purchased from Sigma unless otherwise indicated.

Statistical analysis

The null hypothesis was rejected for P < 0.05 using ANOVA, and data were expressed as means ± S.E.M. Student's t test with the Bonferroni correction was used for repeated measures after ANOVA.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

CaM-binding domains are L-type Ca²⁺ channel agonist ligands

CBmp and IQmp both increased L-type Ca²⁺ channel P_o in a concentration-dependent manner (Fig. 1). CBmp and IQmp both produced an apparent maximum agonist response at ~10 µM, but IQmp increased L-type Ca²⁺ channel P_o to a significantly greater extent than CBmp (Fig. 1). CBmp and IQmp produced a non-additive effect on L-type Ca²⁺ channel P_o at a maximal activity concentration. These findings are consistent with the concept that C terminus CaM-binding domains can operate through a common pathway to increase channel opening.

CBmp increases L-type Ca²⁺ channel opening by inducing mode 2 gating behavior

IQmp increases L-type Ca²⁺ channel P_o by inducing mode 2 gating (Wu et al. 2001a; Dzhura et al. 2002), so we analysed L-type Ca²⁺ channel openings to determine if CBmp operated by a similar biophysical mechanism. CBmp did increase L-type Ca²⁺ channel P_o (Fig. 2A and B) by increasing the percentage of sweeps with prolonged openings, but without changing the time constants for short and long openings (Fig. 2C and D), consistent with an effect on gating mode. CBmp induced L-type Ca²⁺ channels to partition into gating mode 2 (Fig. 2E and F) (Yue et al. 1990), similar to IQmp (Wu et al. 2001a; Dzhura et al. 2002) and CaMKII (Dzhura et al. 2000; Wu et al. 2001a), while a scrambled control peptide was without effect (Wu et al. 2001a; Dzhura et al. 2002). Consistent induction of mode 2 gating indicates that these diverse molecular signals can converge upon a common biophysical mechanism to increase L-type Ca²⁺ channel P_o.

		View larger version [in this window] [in a new window]
Figure 2. The CaM-binding domain mimetic peptide (CBmp) induces mode 2 gating A and B, representative L-type Ca2+ channel recordings (top), ensemble-averaged currents (200-300 tracings, middle), and opening probability (Po) diary plots (bottom). C and D, histograms plot logarithmically binned L-type Ca2+ channel open times (abscissa) against the number of events (ordinate). Open-time distributions were best fitted with the sum of two exponentials, and the long (L) and short (S) time constants for these fits are shown. The percentage of total openings described by L and S are shown in brackets. E and F, quantification of gating modes was performed by plotting Po (top) against the maximum open time (Tomax, bottom) for each non-blank sweep. A vertical line is placed at the minimum of two Gaussian distributions between long and short L-type Ca2+ channel openings, while the horizontal line marks Po = 2 %. Numerals indicate gating modes 0-2 (Yue et al. 1990). Representative gating mode analysis is shown for E, the inactive control peptide, and F, CBmp (10 µM). Data from panels A, C and E are taken from the same cell membrane excised patch treated with inactive control peptide (10 µM) while panels B, D and F are results from a different excised cell membrane patch treated with CBmp (10 µM).

CBmp-induced mode 2 gating is occluded by Ca²⁺-CaM

CBmp (Pate et al. 2000) and IQmp (Qin et al. 1999; Zuhlke et al. 1999; Peterson et al. 1999) are known Ca²⁺-CaM-binding domains, and Ca²⁺-CaM binding prevents IQmp agonist action at L-type Ca²⁺ channels (Wu et al. 2001a). In order to determine the effect of Ca²⁺-CaM on CBmp signalling, we repeated L-type Ca²⁺ channel modal gating analysis in the presence of CaM-enriched (20 µM) bath solution. CBmp-mediated increases in the percentage of sweeps with long channel openings were occluded by high Ca²⁺-CaM bath (cytoplasm) conditions (Fig. 3A-C), while the time constants for short and long L-type Ca²⁺ channel openings were unaffected by low (2 µM) or high (20 µM) CaM in the presence of CBmp (Fig. 3E). The CaM-enriched bath condition, in 150 nM Ca²⁺, prevented the increase in mode 2 gating seen with CBmp alone and with 2 µM CaM, indicating that CBmp-dependent L-type Ca²⁺ channel facilitation was lost under conditions favouring Ca²⁺-CaM binding to CBmp.

		View larger version [in this window] [in a new window]
Figure 3. Elevated Ca2+-CaM prevents CBmp actions at L-type Ca2+ channels A-C, L-type Ca2+ channel open time histograms with two exponential fits, and long (L) and short (S) time constants describing opening times are shown, as in Fig. 2C and D. While CBmp (10 µM) with CaM (2 µM) increased the percentage of long L-type Ca2+ channel openings (B), this effect was prevented by increased CaM (20 µM, C). Data in panels A and B are the same as panels C and D in Fig. 2), and are shown for comparison with panel C. Summary data show the percentage of long L-type Ca2+ channel openings (D) and L and S (E) with control peptide + CaM (2 µM) (Control, open bar), CBmp + CaM (2 µM) (hatched bar), and CBmp + CaM (20 µM) (black bar), where the number of L-type Ca2+ channels studied is indicated in parentheses. F, summary data show the distribution of gating modes from excised cell membrane patches (n = 3 for each group) in response to control + CaM (2 µM) (open bars), CBmp + CaM (2 µM) (hatched bars) and CBmp + CaM (20 µM) (black bars). The numbers of sweeps (ordinate) with L-type Ca2+ channel openings representative of each gating mode are shown. * P = 0.01 for mode 2 sweeps after CBmp + CaM (2 µM) versus control + CaM (2 µM) and CBmp + CaM (20 µM).

CaM and L-type Ca²⁺ channels can co-localize even under conditions adverse to Ca²⁺-CaM (Erickson et al. 2001; Pitt et al. 2001), raising the possibility that CaM could inhibit CBmp agonist action in the absence of Ca²⁺ binding. However, CaM did not occlude CBmp signalling to L-type Ca²⁺ channels under a condition adverse to Ca²⁺-CaM (20 mM BAPTA, Fig. 4), indicating that Ca²⁺-CaM and not CaM alone is required for inhibition of CBmp signalling and that L-type Ca²⁺ channel facilitation is not due to CaM sequestration. CaMKII inhibition with AC3-I did not affect CBmp increases in L-type Ca²⁺ channel P_o, indicating that CBmp actions were CaMKII-independent (Fig. 4). Considered together with the finding that L-type Ca²⁺ channel facilitation by IQmp is also CaMKII independent and occurs in low CaM conditions (Wu et al. 2001a), these results support the concept that L-type Ca²⁺ channel C terminus CaM-binding domain mimetic peptides are auto-agonist ligands exclusively under low Ca²⁺ activity conditions.

		View larger version [in this window] [in a new window]
Figure 4. CBmp increases L-type Ca2+ channel Po only under low Ca2+-CaM activity conditions A, mean L-type Ca2+ channel Po in the presence of a control peptide (Control) + CaM, CBmp + CaM, or CBmp + AC3-I + CaM. CBmp, Control, and AC3-I were all used at 10 µM, and CaM concentrations are indicated (abscissa) in B. The bath free (cytosolic) Ca2+ = 150 nM before CaM addition in A, while the Ca2+ was buffered with BAPTA (20 mM) in B. The number of excised cell membrane patches studied in each group is indicated by numerals (abscissa). * P < 0.001 versus control (A) and * P = 0.02 versus control (B).

CBmp and CaMKII increase L-type Ca²⁺ channel P_o by low- and high-Ca²⁺-CaM mechanisms

The finding that CBmp was a CaMKII-independent L-type Ca²⁺ channel agonist that operated in low-Ca²⁺-CaM activity conditions (Figs 2-4), while endogenous CaMKII is activated under increased Ca²⁺-CaM conditions (Dzhura et al. 2002), adverse to CBmp activity (Fig. 3 and Fig. 4), led us to test whether L-type Ca²⁺ channels can be differentially activated by low- and high-Ca²⁺-CaM conditions. We measured L-type Ca²⁺ channel P_o in the presence and absence of CBmp while probing the cytoplasmic face of the cell membrane with increasing concentrations of CaM (Fig. 5). CBmp (10 µM) significantly increased L-type Ca²⁺ channel openings at a low (2 µM) CaM concentration, and this agonist action of CBmp was diminished in a stepwise fashion by increasing CaM up to 20 µM. A further increase in CaM to 40 µM revealed a non-significant secondary increase in L-type Ca²⁺ channel P_o in the continued presence of CBmp, suggesting activation of endogenous, cell membrane-associated CaMKII (Dzhura et al. 2002).

		View larger version [in this window] [in a new window]
Figure 5. CBmp and CaMKII increase L-type Ca2+ channel Po by distinct low- and high-Ca2+-CaM activity mechanisms Mean L-type Ca2+ channel Po increases significantly from control (0 µM CaM) after CBmp (10 µM, P = 0.008), but this increase is reversed in step-wise fashion by increasing concentrations of CaM up to 20 µM, in the continued presence of CBmp. This trend reverses at CaM = 40 µM (but does not reach significance versus control) in the presence of CBmp. Mean L-type Ca2+ channel Po significantly increases (P = 0.002) with increasing CaM (from 10-20 µM) in the absence of CBmp, and this increase is occluded by the CaMKII inhibitory peptide AC3-I (10 µM), indicating endogenous CaMKII is localized near the L-type Ca2+ channel in excised cell membrane patches (Dzhura et al. 2002). Labelled horizontal bars indicate the presence of CBmp and AC3-I. CaM concentrations and the number of excised cell membrane patches studied are indicated (abscissa). * P < 0.05 compared to the 0 µM CaM-0 µM CBmp condition, and ** P < 0.05 compared to the 2 µM CaM-0 µM CBmp condition.

Increases in CaM concentration (10-20 µM), in the absence of CBmp, resulted in significant increases in L-type Ca²⁺ channel P_o. The L-type Ca²⁺ channel response to enriched CaM solution was due to activation of endogenous CaMKII present on the excised patches, because it was prevented by the specific CaMKII inhibitory peptide AC3-I, which does not bind to CBmp (data not shown). Addition of CBmp with a Ca²⁺-independent form of CaMKII in 2 µM CaM (L-type Ca²⁺ channel P_o = 27.2 ± 4.8 %, n = 12) did not increase L-type Ca²⁺ channel P_o compared to CaMKII in 2 µM CaM without CBmp (L-type Ca²⁺ channel P_o = 26.6 ± 2.4, n = 10). This result is similar to previous findings with CaMKII and IQmp (Wu et al. 2001a), and indicates that C terminus CaM-binding motifs and CaMKII non-additively facilitate L-type Ca²⁺ channels. These findings show that both Ca²⁺-CaM- and CaMKII-dependent and -independent pathways for increasing L-type Ca²⁺ channel P_o are operative in excised cell membrane patches from cardiac myocytes.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

C terminus Ca²⁺-CaM-binding domains are L-type Ca²⁺ channel agonist ligands

The present findings show that L-type Ca²⁺ channel C terminus CaM-binding domains potentially serve a dual purpose as I_Ca facilitation agonist ligands and molecular determinants for Ca²⁺-CaM-dependent I_Ca inactivation (Zuhlke et al. 1999). CBmp is a novel L-type Ca²⁺ channel agonist ligand that operates by a similar biophysical mechanism to IQmp (Wu et al. 2001a; Dzhura et al. 2002), suggesting the intriguing possibility that L-type Ca²⁺ channel C terminus CaM-binding motifs are signals for I_Ca 'auto-facilitation'. CaM-binding domain mimetic peptides presumably operate as agonist ligands by engaging an allosteric effector site on the L-type Ca²⁺ channel complex to enhance I_Ca facilitation (Fig. 6), but the molecular identity of this site is presently unknown.

		View larger version [in this window] [in a new window]
Figure 6. A model for increasing L-type Ca2+ channel Po in low- and high-Ca2+-CaM conditions Under basal conditions (centre panel) CaMKII is largely inactive (dark folded, membrane-associated bar) and CaM is bound to the L-type Ca2+ channel C terminus by a low-Ca2+ activity mechanism (Pitt et al. 2001) that does not permit Ca2+-CaM-dependent binding with IQ or CB motifs, or significant interaction of these motifs with a facilitation site (fs). Increased Ca2+-CaM (left panel) results in Ca2+-CaM binding to CaMKII, activation and autophosphorylation of endogenous CaMKII (dark, extended bar), which binds the L-type Ca2+ channel C terminus (Hudmon & Schulman, 2002). CaMKII binding to the L-type Ca2+ channel C terminus disrupts CaM binding to the C terminus CaM-binding domains (Hudmon et al. 2002) and improves the interaction of IQ and CB with the facilitation site (fs). CaMKII activation and C terminus binding results in increased L-type Ca2+ channel Po (Dzhura et al. 2000; Wu et al. 2001a; Dzhura et al. 2002). During low-Ca2+-CaM conditions (right panel), C terminus CaM-binding domain mimetic peptides (CBmp and IQmp) are 'enhanced' auto-agonist ligands, by virtue of reduced steric constraint compared to the endogenous counterparts, for interacting with the facilitation site (fs) to induce mode 2 gating. CBmp (Figs 3 and 4) and IQmp (Wu et al. 2001a) cannot interact with the facilitation site while bound to Ca2+-CaM, and so are ineffective during increased Ca2+-CaM activity conditions.

We hypothesize that endogenous CB and IQ can also operate as L-type Ca²⁺ channel auto-facilitation ligands under conditions when they are not bound to Ca²⁺-CaM, and that Ca²⁺-CaM-dependent I_Ca inactivation occurs when these domains are occluded by Ca²⁺-CaM binding. It will be important to further test this hypothesis using other experimental approaches, such as C terminus deletion mutants lacking CB and IQ, because it is possible that CBmp and IQmp are superior agonists to their endogenous counterparts due to reduced steric constraint. We have proposed a model whereby CBmp and IQmp engage a hypothetical facilitation site located within the L-type Ca²⁺ channel complex. In our model, CaMKII and CaM-binding domain mimetic peptides operate through a common pathway; CaMKII activation participates in the facilitation process by improving the interaction of endogenous C terminus CaM-binding domains (CB and IQ) with the facilitation site (Fig. 6).

CaM-binding domains and CaMKII-dependent L-type Ca²⁺ channel regulation

We were able to test the combined effects of CaM-binding domain mimetic peptides and CaMKII on L-type Ca²⁺ channel P_o, in a manner that would be impossible using conventional CaMKII, because the constitutively active CaMKII used in these experiments remains operative under the low Ca²⁺-CaM activity conditions required for IQmp and CBmp actions (Fig. 4). Our studies show that CaMKII, IQmp (Wu et al. 2001a) and CBmp (Fig. 1) increase L-type Ca²⁺ channel activity in a non-additive fashion, suggesting these signals may ultimately converge upon a common facilitation site. The finding that endogenous CaMKII was activated by elevated Ca²⁺-CaM in the presence of Ba²⁺ as a charge carrier (Fig. 4), suggests that CaMKII is anchored and activated outside of the L-type Ca²⁺ channel pore region. Likewise, the occlusion of CBmp increases in L-type Ca²⁺ channel Ba²⁺ current by elevated bath (cytoplasm) Ca²⁺-CaM suggests that the hypothesized facilitation site (Fig. 6) is also not located within the L-type Ca²⁺ channel pore, where calcification of CaM is anticipated to be ineffective due to the high local Ba²⁺ activity.

Both CaM (Erickson et al. 2001; Pitt et al. 2001) and CaMKII (Dzhura et al. 2002; Hudmon et al. 2002) are targeted to L-type Ca²⁺ channels, and the association between CaMKII and L-type Ca²⁺ channels is maintained following excision of cell membrane patches in ventricular myocytes (Fig. 5; Dzhura et al. 2002). In contrast to CBmp and IQmp, endogenous CaMKII increases L-type Ca²⁺ channel P_o under conditions of increased Ca²⁺-CaM (Fig. 5). Based on recent findings that activated CaMKII can bind to the L-type Ca²⁺ channel C terminus in a region that involves CB and IQ (Hudmon et al. 2002), we hypothesize that CaMKII prevents Ca²⁺-CaM binding to CB and IQ to reduce I_Ca inactivation and enhance the interaction of these domains with a facilitation site (Fig. 6).

I_Ca regulation by the distal C terminus

The L-type Ca²⁺ channel C terminus also contains inhibitory elements that reside distal to the IQ and CB domains. Truncation of the distal C terminus (beyond the IQ and CB domains) results in increased peak I_Ca, and segments of the distal C terminus can associate with L-type Ca²⁺ channel truncation mutants to reduce current (Gao et al. 2001). At present it is unclear if CB/IQ signalling interacts with these more distal C terminus segments, but, taken together, these findings indicate that the C terminus is a rich store of regulatory elements for controlling cell Ca²⁺ entry.

Is all facilitation the same?

Macroscopic and unitary Ca²⁺ current can increase in response to multiple and diverse interventions, by a process often termed facilitation. Despite a substantial body of work in this area, the mechanistic interrelationship of these different types of facilitation and the role of facilitation in cardiac physiology is uncertain. Recently, facilitation of Ca²⁺ channels, with Ca²⁺ as the charge carrier, by CaMKII was linked to arrhythmias in a model of cardiac hypertrophy (Wu et al. 2002), while increased protein kinase A (PKA) activity can also facilitate Ca²⁺ current and favour cellular arrhythmia triggers called afterdepolarizations (Priori & Corr, 1990). Taken together, these findings suggest that disordered facilitation can participate in important pathophysiological responses. Positive cell membrane conditioning pre-pulses (Pietrobon & Hess, 1990; Dzhura et al. 2002), CaMKII (Yuan & Bers, 1994; Anderson et al. 1994; Xiao et al. 1994; Dzhura et al. 2000), and PKA (Yue et al. 1990; Dzhura et al. 2002) have all been implicated in the process of facilitation. It is interesting to note that the subunit is required for pre-pulse facilitation (Kamp et al. 2000) and is also an important element for PKA responses (Bünemann et al. 1999). These findings suggest that the subunit could contribute to the hypothesized facilitation site in our model (Fig. 6).

L-type Ca²⁺channel recordings in excised cell membrane patches

Both CaM and CaMKII can modulate multiple targets critical for cytoplasmic Ca²⁺ homeostasis (Braun & Schulman, 1995b; Saimi & Kung, 2002), complicating interpretation of I_Ca measurements in cellular studies. Specifically, CaM and CaMKII can modulate I_Ca through direct actions at the L-type Ca²⁺ channel complex (Peterson et al. 1999; Dzhura et al. 2000; Zuhlke et al. 2000; Wu et al. 2001a), and through indirect actions on cytoplasmic Ca²⁺ homeostasis (Braun & Schulman, 1995b; Li et al. 1997; Wu et al. 2001b; Balshaw et al. 2001). The excised cell membrane patch preparation has the advantage of measuring direct actions of signalling molecules at the L-type Ca²⁺ channel complex, and this approach was used to initially demonstrate that CaMKII directly facilitates L-type Ca²⁺ channels by inducing a shift to mode 2 gating (Dzhura et al. 2000) and to show that endogenous CaMKII is functionally targeted to L-type Ca²⁺ channels (Dzhura et al. 2002). However, excised patches lack critical cellular components, such as sarcoplasmic reticulum, which are important for integrated signalling responses, including I_Ca inactivation (Adachi-Akahane et al. 1996; Wu et al. 2001a) and facilitation (Wu et al. 2001a), and unitary currents are very difficult to resolve using Ca²⁺ as the charge carrier. The IQ domain is located within a cell membrane-targeting sequence (Gao et al. 2000), but single channel measurements do not provide information about a potential role of the IQ or CB domains in cell membrane targeting. New experimental approaches with L-type Ca²⁺ channel-targeted CaM and CaMKII will be helpful to better understand the effects of these signal elements for determining I_Ca in intact cardiomyocytes.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

Adachi-Akahane S, Cleemann L & Morad, M (1996). Cross-signaling between L-type Ca2+ channels and ryanodine receptors in rat ventricular myocytes. J Gen Physiol 108, 435-454	[Abstract]
Anderson ME, Braun,AP, Schulman H & Premack BA (1994). Multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca(2+)-induced enhancement of the L-type Ca2+ current in rabbit ventricular myocytes. Circ Res 75, 854-861	[Abstract]
Balshaw DM, Xu L, Yamaguchi N, Pasek DA & Meissner G (2001). Calmodulin binding and inhibition of cardiac muscle calcium release channel (ryanodine receptor). J Biol Chem 276, 20144-20153	[Abstract/Full Text]
Bers DM, Patton CW & Nuccitelli R (1994). A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol 40, 3-29	[Medline]
Braun AP , & Schulman H (1995a). A non-selective cation current activated via the multifunctional Ca(2+)-calmodulin-dependent protein kinase in human epithelial cells. J Physiol 488, 37-55	[Abstract]
Braun AP , & Schulman H (1995b). The multifunctional calcium/calmodulin-dependent protein kinase: from form to function. Annu Rev Physiol 57, 417-445	[Medline]
B, ünemann M, Gerhardstein BL, Gao T & Hosey MM (1999). Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the beta(2) subunit. J Biol Chem 274, 33851-33854	[Abstract/Full Text]
Dzhura I, Wu Y, Colbran RJ, Balser Jr & Anderson ME (2000). Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels. Nat Cell Biol 2, 173-177	[Medline]
Dzhura I, Wu YJ, Colbran RJ, Corbin JD, Balser JR & Anderson ME (2002). Cytoskeletal disrupting agents prevent calmodulin kinase, IQ domain and voltage-dependent facilitation of L-type Ca2+ channels. J Physiol 545, 399-406	[Abstract/Full Text]
Erickson MG, Alseikhan BA, Peterson BZ & Yue DT (2001). Preassociation of calmodulin with voltage-gated Ca2+ channels revealed by FRET in single living cells. Neuron 31, 973-985	[Medline]
Gao T, Bünemann M, Gerhardstein BL, Ma H & Hosey MM (2000). Role of the C terminus of the 1c(Cav1. 2) subunit in membrane targeting of cardiac L-type calcium channels. J Biol Chem 275, 25436-25444	[Abstract/Full Text]
Gao T, Cuadra AE, Ma H, Bünemann M, Gerhardstein BL, Cheng T, Ten Eick R & Hosey MM (2001). C-terminal fragments of the 1C (Cav1. 2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated 1C subunits. J Biol Chem 276, 21089-21097	[Abstract/Full Text]
Gupta RC , & Kranias EG (1989). Purification and characterization of a calcium-calmodulin- dependent phospholamban kinase from canine myocardium. Biochemistry 28, 5909-5916	[Medline]
Hamill OP, Marty A, Neher E, Sakmann B & Sigworth FJ (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391, 85-100	[Medline]
Hess P, Lansman JP & Tsien RW (1984). Different modes of Ca channel gating behavior favoured by dihydropyridine Ca agonists and antagonists. Nature 311, 538-544	[Medline]
Hudmon A, Pitt GS, Tsien RW & Schulman H (2002). Molecular mechanism and regulation of the interaction between calcium-calmodulin dependent protein kinase II and the L-type calcium channel. Biophys J 82, 32 (abstract)
Hudmon A , & Schulman, H. (2002). Structure/function of the multifunctional Ca2+/calmodulin -dependent protein kinase II. Biochem J 364, 593-611	[Medline]
Kamp TJ, Hu H & Marban E (2000). Voltage-dependent facilitation of cardiac L-type Ca channels expressed in HEK-293 cells requires beta-subunit. Am J Physiol Heart Circ Physiol 278, H126-136	[Abstract/Full Text]
Li L, Satoh H, Ginsburg KS & Bers DM (1997). The effect of Ca(2+)-calmodulin-dependent protein kinase II on cardiac excitation-contraction coupling in ferret ventricular myocytes. J Physiol 501, 17-31	[Abstract]
Marban, E & Tsien, RW (1982). Enhancement of calcium current during digitalis inotropy in mammalian heart: positive feed-back regulation by intracellular calcium? J Physiol 329, 589-614	[Medline]
Pate P, Mochca-Morales J, Wu Y, Zhang JZ, Rodney GG, Serysheva II, Williams BY, Anderson ME & Hamilton SL (2000). Determinants for calmodulin binding on voltage-dependent Ca2+ channels. J Biol Chem 275, 39786-39792	[Abstract/Full Text]
Peterson BZ, Demaria CD, Adelman JP & Yue DT (1999). Calmodulin is the Ca2+ sensor for Ca2+-dependent inactivation of L-type calcium channels. Neuron 22, 549-558	[Medline]
Pietrobon D , & Hess P (1990). Novel mechanism of voltage-dependent gating in L-type calcium channels. Nature 346, 651-655	[Medline]
Pitt GS, Zuhlke RD, Hudmon A, Schulman H, Reuter H & Tsien RW (2001). Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels. J Biol Chem 276, 30794-30802	[Abstract/Full Text]
Plant TD, Standen NB & Ward TA (1983). The effects of injection of calcium ions and calcium chelators on calcium channel inactivation in Helix neurones. J Physiol 334, 189-212	[Abstract]
Priori SG , & Corr PB (1990). Mechanisms underlying early and delayed afterdepolarizations induced by catecholamines. Am J Physiol 258, H1796-1805	[Medline]
Qin N, Olcese R, Bransby M, Lin T & Birnbaumer L (1999). Ca2+-induced inhibition of the cardiac Ca2+ channel depends on calmodulin. Proc Natl Acad Sci U S A 96, 2435-2438	[Abstract/Full Text]
Saimi Y , & Kung C (2002). Calmodulin as an ion channel subunit. Annu Rev Physiol 64, 289-311	[Medline]
Van Dongen AM, (1996). A new algorithm for idealizing single ion channel data containing multiple unknown conductance levels. Biophys J 70, 1303-1315	[Abstract]
Wu Y, Colbran RJ & Anderson ME (2001b). Calmodulin kinase is a molecular switch for cardiac excitation-contraction coupling. Proc Natl Acad Sci U S A 98, 2877-2881	[Abstract/Full Text]
Wu Y, Dzhura I, Colbran RJ & Anderson ME (2001a). Calmodulin kinase and a calmodulin-binding 'IQ' domain facilitate L-type Ca(2+) current in rabbit ventricular myocytes by a common mechanism. J Physiol 535, 679-687	[Abstract/Full Text]
Wu Y, Macmillan LB, McNeill RB, Colbran RJ & Anderson ME. (1999). CaM kinase augments cardiac L-type Ca2+ current: a cellular mechanism for long Q-T arrhythmias. Am J Physiol 276, H2168-2178	[Medline]
Wu Y, Temple J, Zhang R, Dzhura I, Zhang W, Trimble RW, Roden DM, Passier R, Olson EN, Colbran RJ & Anderson ME (2002). Calmodulin kinase II and arrhythmias in a mouse model of cardiac hypertrophy. Circulation 106, 1288-1293	[Abstract/Full Text]
Xiao RP, Cheng H, Lederer WJ, Suzuki T & Lakatta, EG (1994). Dual regulation of Ca2+/calmodulin-dependent kinase II activity by membrane voltage and by calcium influx. Proc Natl Acad Sci U S A 91, 9659-9663	[Abstract]
Yuan W , & Bers DM (1994). Ca-dependent facilitation of cardiac Ca current is due to Ca-calmodulin-dependent protein kinase. Am J Physiol 267, H982-993	[Medline]
Yue DT, Herzig S & Marban E (1990). Beta-adrenergic stimulation of calcium channels occurs by potentiation of high-activity gating modes. Proc Natl Acad Sci U S A 87, 753-757	[Abstract]
Zuhlke RD, Pitt GS, Deisseroth K, Tsien RW & Reuter H (1999). Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 399, 159-162	[Medline]
Zuhlke RD, Pitt GS, Tsien RW & Reuter H (2000). Ca2+-sensitive inactivation and facilitation of L-type Ca2+ channels both depend on specific amino acid residues in a consensus calmodulin-binding motif in the (alpha)1C subunit. J Biol Chem 275, 21121-21129	[Abstract/Full Text]

Acknowledgements

This work was supported by NHLBI, HL03727, HL62494 (to M.E.A.) and AR44864 (to S.L.H.). The authors thank Martha Bass and Jinying Yang for technical assistance, and Linda Selfridge for secretarial assistance. Dr Anderson is an Established Investigator of the American Heart Association.

This article has been cited by other articles:

				Y.-J. Qu, V. E. Bondarenko, C. Xie, S. Wang, M. S. Awayda, H. C. Strauss, and M. J. Morales W-7 modulates Kv4.3: pore block and Ca2+-calmodulin inhibition Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2364 - H2377. [Abstract] [Full Text] [PDF]

				M. E. Anderson Multiple downstream proarrhythmic targets for calmodulin kinase II: Moving beyond an ion channel-centric focus Cardiovasc Res, March 1, 2007; 73(4): 657 - 666. [Abstract] [Full Text] [PDF]

				K. A. Young and J. H. Caldwell Modulation of skeletal and cardiac voltage-gated sodium channels by calmodulin J. Physiol., June 1, 2005; 565(2): 349 - 370. [Abstract] [Full Text] [PDF]

				E. Kobrinsky, S. Tiwari, V. A. Maltsev, J. B. Harry, E. Lakatta, D. R. Abernethy, and N. M. Soldatov Differential Role of the {alpha}1C Subunit Tails in Regulation of the Cav1.2 Channel by Membrane Potential, {beta} Subunits, and Ca2+ Ions J. Biol. Chem., April 1, 2005; 280(13): 12474 - 12485. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 550/3/731    most recent 2003.043778v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dzhura, I. Articles by Anderson, M. E. Search for Related Content PubMed PubMed Citation Articles by Dzhura, I. Articles by Anderson, M. E.