Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L-type calcium channels

Elevated intracellular Ca2+ triggers inactivation of L-type calcium channels, providing negative Ca2+ feedback in many cells. Ca2+ binding to the main alpha1c channel subunit has been widely proposed to initiate such Ca2+ -dependent inactivation. Here, we find that overexpression of mutant, Ca2+ -insensitive calmodulin (CaM) ablates Ca2+ -dependent inactivation in a "dominant-negative" manner. This result demonstrates that CaM is the actual Ca2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca2+ -dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of alpha1c. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family.     

Identification of the components controlling inactivation of voltage-gated Ca2+ channels

Ca(2+)-dependent inactivation (CDI) of L-type voltage-gated Ca(2+) channels limits Ca(2+) entry into neurons, thereby regulating numerous cellular events. Here we present the isolation and purification of the Ca(2+)-sensor complex, consisting of calmodulin (CaM) and part of the channel's pore-forming alpha(1C) subunit, and demonstrate the Ca(2+)-dependent conformational shift that underlies inactivation. Dominant-negative CaM mutants that prevent CDI block the sensor's Ca(2+)-dependent conformational change. We show how Ile1654 in the CaM binding IQ motif of alpha(1C) forms the link between the Ca(2+) sensor and the downstream inactivation machinery, using the alpha(1C) EF hand motif as a signal transducer to activate the putative pore-occluder, the alpha(1C) I-II intracellular linker.     

Ca(2+) sensors of L-type Ca(2+) channel

Ca(2+)-induced inactivation of L-type Ca(2+) is differentially mediated by two C-terminal motifs of the alpha(1C) subunit, L (1572-1587) and K (1599-1651) implicated for calmodulin binding. We found that motif L is composed of a highly selective Ca(2+) sensor and an adjacent Ca(2+)-independent tethering site for calmodulin. The Ca(2+) sensor contributes to higher Ca(2+) sensitivity of the motif L complex with calmodulin. Since only combined mutation of both sites removes Ca(2+)-dependent current decay, the two-site modulation by Ca(2+) and calmodulin may underlie Ca(2+)-induced inactivation of the channel.     

Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels

L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca(2+), providing negative Ca(2+) feedback in a diverse array of biological contexts. The dominant Ca(2+) sensor for such Ca(2+)-dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca(2+) sensor induces channel inactivation by Ca(2+)-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca(2+) inactivation appears to be a consensus Ca(2+)-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial since the original report from our lab implicating a critical role for this domain. Here, we demonstrate not only that the consensus EF hand is essential for Ca(2+) inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca(2+) inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca(2+) inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca(2+)-coordinating residues in the consensus EF hand reduce Ca(2+) inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca(2+) sensor, in which Ca(2+) binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca(2+) sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca(2+)-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca(2+)-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca(2+) binding of CaM.     

Regulation of the mouse epithelial Ca2(+) channel TRPV6 by the Ca(2+)-sensor calmodulin

TRPV5 and TRPV6 are members of the superfamily of transient receptor potential (TRP) channels and facilitate Ca(2+) influx in a variety of epithelial cells. The activity of these Ca(2+) channels is tightly controlled by the intracellular Ca(2+) concentration in close vicinity to the channel mouth. The molecular mechanism underlying the Ca(2+)-dependent activity of TRPV5/TRPV6 is, however, still unknown. Here, the putative role of calmodulin (CaM) as the Ca(2+) sensor mediating the regulation of channel activity was investigated. Overexpression of Ca(2+)-insensitive CaM mutants (CaM(1234) and CaM(34)) significantly reduced the Ca(2+) as well as the Na(+) current of TRPV6- but not that of TRPV5-expressing HEK293 cells. By combining pull-down assays and co-immunoprecipitations, we demonstrated that CaM binds to both TRPV5 and TRPV6 in a Ca(2+)-dependent fashion. The binding of CaM to TRPV6 was localized to the transmembrane domain (TRPV6(327-577)) and consensus CaM-binding motifs located in the N (1-5-10 motif, TRPV6(88-97)) and C termini (1-8-14 motif, TRPV6(643-656)), suggesting a mechanism of regulation involving multiple interaction sites. Subsequently, chimeric TRPV6/TRPV5 proteins, in which the N and/or C termini of TRPV6 were substituted by that of TRPV5, were co-expressed with CaM(34) in HEK293 cells. Exchanging, the N and/or the C termini of TRPV6 by that of TRPV5 did not affect the CaM(34)-induced reduction of the Ca(2+) and Na(+) currents. These results suggest that CaM positively affects TRPV6 activity upon Ca(2+) binding to EF-hands 3 and 4, located in the high Ca(2+) affinity CaM C terminus, which involves the N and C termini and the transmembrane domain of TRPV6.     

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

L-type Ca(2+) channels are unusual in displaying two opposing forms of autoregulatory feedback, Ca(2+)-dependent inactivation and facilitation. Previous studies suggest that both involve direct interactions between calmodulin (CaM) and a consensus CaM-binding sequence (IQ motif) in the C terminus of the channel's alpha(1C) subunit. Here we report the functional effects of an extensive series of modifications of the IQ motif aimed at dissecting the structural determinants of the different forms of modulation. Although the combined substitution by alanine at five key positions (Ile(1624), Gln(1625), Phe(1628), Arg(1629), and Lys(1630)) abolished all Ca(2+) dependence, corresponding single alanine replacements behaved similarly to the wild-type channel (77wt) in four of five cases. The mutant I1624A stood out in displaying little or no Ca(2+)-dependent inactivation, but clear Ca(2+)- and frequency-dependent facilitation. An even more pronounced tilt in favor of facilitation was seen with the double mutant I1624A/Q1625A: overt facilitation was observed even during a single depolarizing pulse, as confirmed by two-pulse experiments. Replacement of Ile(1624) by 13 other amino acids produced graded and distinct patterns of change in the two forms of modulation. The extent of Ca(2+)-dependent facilitation was monotonically correlated with the affinity of CaM for the mutant IQ motif, determined in peptide binding experiments in vitro. Ca(2+)-dependent inactivation also depended on strong CaM binding to the IQ motif, but showed an additional requirement for a bulky, hydrophobic side chain at position 1624. Abolition of Ca(2+)-dependent modulation by IQ motif modifications mimicked and occluded the effects of overexpressing a dominant-negative CaM mutant.     

Calmodulin mediates Ca2+ sensitivity of sodium channels

Ca2+ has been proposed to regulate Na+ channels through the action of calmodulin (CaM) bound to an IQ motif or through direct binding to a paired EF hand motif in the Nav1 C terminus. Mutations within these sites cause cardiac arrhythmias or autism, but details about how Ca2+ confers sensitivity are poorly understood. Studies on the homologous Cav1.2 channel revealed non-canonical CaM interactions, providing a framework for exploring Na+ channels. In contrast to previous reports, we found that Ca2+ does not bind directly to Na+ channel C termini. Rather, Ca2+ sensitivity appears to be mediated by CaM bound to the C termini in a manner that differs significantly from CaM regulation of Cav1.2. In Nav1.2 or Nav1.5, CaM bound to a localized region containing the IQ motif and did not support the large Ca(2+)-dependent conformational change seen in the Cav1.2.CaM complex. Furthermore, CaM binding to Nav1 C termini lowered Ca2+ binding affinity and cooperativity among the CaM-binding sites compared with CaM alone. Nonetheless, we found suggestive evidence for Ca2+/CaM-dependent effects upon Nav1 channels. The R1902C autism mutation conferred a Ca(2+)-dependent conformational change in Nav1.2 C terminus.CaM complex that was absent in the wild-type complex. In Nav1.5, CaM modulates the Cterminal interaction with the III-IV linker, which has been suggested as necessary to stabilize the inactivation gate, to minimize sustained channel activity during depolarization, and to prevent cardiac arrhythmias that lead to sudden death. Together, these data offer new biochemical evidence for Ca2+/CaM modulation of Na+ channel function.     

FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation

L-type Ca(2+) channels possess a Ca(2+)-dependent inactivation (CDI) mechanism, affording feedback in diverse neurobiological settings and serving as prototype for unconventional calmodulin (CaM) regulation emerging in many Ca(2+) channels. Crucial to such regulation is the preassociation of Ca(2+)-free CaM (apoCaM) to channels, facilitating rapid triggering of CDI as Ca(2+)/CaM shifts to a channel IQ site (IQ). Progress has been hindered by controversy over the preassociation site, as identified by in vitro assays. Most critical has been the failure to resolve a functional signature of preassociation. Here, we deploy novel FRET assays in live cells to identify a 73 aa channel segment, containing IQ, as the critical preassociation pocket. IQ mutations disrupting preassociation revealed accelerated voltage-dependent inactivation (VDI) as the functional hallmark of channels lacking preassociated CaM. Hence, the alpha(1C) IQ segment is multifunctional-serving as ligand for preassociation and as Ca(2+)/CaM effector site for CDI.     

Apocalmodulin and Ca2+ calmodulin-binding sites on the CaV1.2 channel

The cardiac L-type voltage-dependent calcium channel is responsible for initiating excitation-contraction coupling. Three sequences (amino acids 1609-1628, 1627-1652, and 1665-1685, designated A, C, and IQ, respectively) of its alpha(1) subunit contribute to calmodulin (CaM) binding and Ca(2+)-dependent inactivation. Peptides matching the A, C, and IQ sequences all bind Ca(2+)CaM. Longer peptides representing A plus C (A-C) or C plus IQ (C-IQ) bind only a single molecule of Ca(2+)CaM. Apocalmodulin (ApoCaM) binds with low affinity to the IQ peptide and with higher affinity to the C-IQ peptide. Binding to the IQ and C peptides increases the Ca(2+) affinity of the C-lobe of CaM, but only the IQ peptide alters the Ca(2+) affinity of the N-lobe. Conversion of the isoleucine and glutamine residues of the IQ motif to alanines in the channel destroys inactivation (Zühlke et al., 2000). The double mutation in the peptide reduces the interaction with apoCaM. A mutant CaM unable to bind Ca(2+) at sites 3 and 4 (which abolishes the ability of CaM to inactivate the channel) binds to the IQ, but not to the C or A peptide. Our data are consistent with a model in which apoCaM binding to the region around the IQ motif is necessary for the rapid binding of Ca(2+) to the C-lobe of CaM. Upon Ca(2+) binding, this lobe is likely to engage the A-C region.     

Molecular mechanism for divergent regulation of Cav1.2 Ca2+ channels by calmodulin and Ca2+-binding protein-1

Ca(2+)-binding protein-1 (CaBP1) and calmodulin (CaM) are highly related Ca(2+)-binding proteins that directly interact with, and yet differentially regulate, voltage-gated Ca(2+) channels. Whereas CaM enhances inactivation of Ca(2+) currents through Ca(v)1.2 (L-type) Ca(2+) channels, CaBP1 completely prevents this process. How CaBP1 and CaM mediate such opposing effects on Ca(v)1.2 inactivation is unknown. Here, we identified molecular determinants in the alpha(1)-subunit of Ca(v)1.2 (alpha(1)1.2) that distinguish the effects of CaBP1 and CaM on inactivation. Although both proteins bind to a well characterized IQ-domain in the cytoplasmic C-terminal domain of alpha(1)1.2, mutations of the IQ-domain that significantly weakened CaM and CaBP1 binding abolished the functional effects of CaM, but not CaBP1. Pulldown binding assays revealed Ca(2+)-independent binding of CaBP1 to the N-terminal domain (NT) of alpha(1)1.2, which was in contrast to Ca(2+)-dependent binding of CaM to this region. Deletion of the NT abolished the effects of CaBP1 in prolonging Ca(v)1.2 Ca(2+) currents, but spared Ca(2+)-dependent inactivation due to CaM. We conclude that the NT and IQ-domains of alpha(1)1.2 mediate functionally distinct interactions with CaBP1 and CaM that promote conformational alterations that either stabilize or inhibit inactivation of Ca(v)1.2.     

Matrin 3 is a Ca2+/calmodulin-binding protein cleaved by caspases

Matrin 3 is a nuclear matrix protein that has been implicated in interacting with other nuclear proteins to anchor hyperedited RNAs to the nuclear matrix, in modulating the activity of proximal promoters, and as the main PKA substrate following NMDA receptor activation. In our proteome-wide selections for calmodulin (CaM) binding proteins and for caspase substrates using mRNA-displayed human proteome libraries, matrin 3 was identified as both a Ca(2+)-dependent CaM-binding protein and a downstream substrate of caspases. We report here, the in vitro characterization of the CaM-binding motif and the caspase cleavage site on matrin 3. Significantly, the Ca(2+)/CaM-binding motif is partially overlapped by the RRM of matrin 3 and is also very close to the bipartite NLS that is essential for its nuclear localization. The caspase cleavage site is downstream of the NLS but upstream of the second U1-like zinc finger. Our results suggest that the functions of matrin 3 could be regulated by both Ca(2+)-dependent interaction with CaM and caspase-mediated cleavage.     

Mutation of the calmodulin binding motif IQ of the L-type Ca(v)1.2 Ca2+ channel to EQ induces dilated cardiomyopathy and death

Cardiac excitation-contraction coupling (EC coupling) links the electrical excitation of the cell membrane to the mechanical contractile machinery of the heart. Calcium channels are major players of EC coupling and are regulated by voltage and Ca(2+)/calmodulin (CaM). CaM binds to the IQ motif located in the C terminus of the Ca(v)1.2 channel and induces Ca(2+)-dependent inactivation (CDI) and facilitation (CDF). Mutation of Ile to Glu (Ile1624Glu) in the IQ motif abolished regulation of the channel by CDI and CDF. Here, we addressed the physiological consequences of such a mutation in the heart. Murine hearts expressing the Ca(v)1.2(I1624E) mutation were generated in adult heterozygous mice through inactivation of the floxed WT Ca(v)1.2(L2) allele by tamoxifen-induced cardiac-specific activation of the MerCreMer Cre recombinase. Within 10 days after the first tamoxifen injection these mice developed dilated cardiomyopathy (DCM) accompanied by apoptosis of cardiac myocytes (CM) and fibrosis. In Ca(v)1.2(I1624E) hearts, the activity of phospho-CaM kinase II and phospho-MAPK was increased. CMs expressed reduced levels of Ca(v)1.2(I1624E) channel protein and I(Ca). The Ca(v)1.2(I1624E) channel showed "CDI" kinetics. Despite a lower sarcoplasmic reticulum Ca(2+) content, cellular contractility and global Ca(2+) transients remained unchanged because the EC coupling gain was up-regulated by an increased neuroendocrine activity. Treatment of mice with metoprolol and captopril reduced DCM in Ca(v)1.2(I1624E) hearts at day 10. We conclude that mutation of the IQ motif to IE leads to dilated cardiomyopathy and death.     

Voltage- and calcium-dependent inactivation in high voltage-gated Ca(2+) channels

Calcium influx into cardiac myocytes via voltage-gated Ca channels is a key step in initiating the contractile response. During prolonged depolarizations, toxic Ca(2+) overload is prevented by channel inactivation occurring through two different processes identified by their primary trigger: voltage or intracellular Ca(2+). In physiological situations, cardiac L-type (Ca(V)1.2) Ca(2+) channels inactivate primarily via Ca(2+)-dependent inactivation (CDI), while neuronal P/Q (Ca(V)2.1) Ca(2+) channels use preferentially voltage-dependent inactivation (VDI). In certain situations however, these two types of channels have been shown to be able to inactivate by both processes. From a structural view point, the rearrangement occurring during CDI and VDI is not precisely known, but functional studies have underlined the role played by at least 2 channel sequences: a C-terminal binding site for the Ca(2+) sensor calmodulin, essential for CDI, and the loop connecting domains I and II, essential for VDI. The conserved regulation of VDI and CDI by the auxiliary channel beta subunit strongly suggests that these two mechanisms may use a set of common protein-protein interactions that are influenced by the auxiliary subunit. We will review our current knowledge of these interactions. New data are presented on L-P/Q (Ca(V)1.2/Ca(V)2.1) channel chimera that confirm the role of the I-II loop in VDI and CDI, and reveal some of the essential steps in Ca(2+) channel inactivation.     

RyRCa2+ leak limits cardiac Ca2+ window current overcoming the tonic effect of calmodulinin mice

Ca(2+) mediates the functional coupling between L-type Ca(2+) channel (LTCC) and sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca(2+)-induced Ca(2+)-release (CICR) mechanism triggered by Ca(2+) influx, but also as the retrograde Ca(2+)-dependent inactivation (CDI) of LTCC, which depends on both Ca(2+) permeating through the LTCC itself and on SR Ca(2+) release through the RyR. This latter effect has been suggested to rely on local rather than global Ca(2+) signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca(2+) leak, we evidence here that increased occurrence of the discrete local SR Ca(2+) releases through the RyRs (Ca(2+) sparks) cause a depolarizing shift in activation and a hyperpolarizing shift in isochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca(2+)](i) buffer capacity or depleting SR Ca(2+) store blunted these changes, which could be reproduced in WT cells by RyRCa(2+) leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca(2+) control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca(2+) signals and CaM function.     

Unified mechanisms of Ca2+ regulation across the Ca2+ channel family

L-type (CaV1.2) and P/Q-type (CaV2.1) calcium channels possess lobe-specific CaM regulation, where Ca2+ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the CaV1-2 channel family, R-type (CaV2.3) and N-type (CaV2.2), have appeared to lack such CaM regulation. We report here that R- and N-type channels undergo Ca(2+)-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca2+ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in CaV1-2 channels-independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca2+ activity, respectively.     

Calmodulin regulates Ca(2+)-dependent feedback inhibition of store-operated Ca(2+) influx by interaction with a site in the C terminus of TrpC1

The mechanism involved in [Ca(2+)](i)-dependent feedback inhibition of store-operated Ca(2+) entry (SOCE) is not yet known. Expression of Ca(2+)-insensitive calmodulin (Mut-CaM) but not wild-type CaM increased SOCE and decreased its Ca(2+)-dependent inactivation. Expression of TrpC1 lacking C terminus aa 664-793 (TrpC1DeltaC) also attenuated Ca(2+)-dependent inactivation of SOCE. CaM interacted with endogenous and expressed TrpC1 and with GST-TrpC1 C terminus but not with TrpC1DeltaC. Two CaM binding domains, aa 715-749 and aa 758-793, were identified. Expression of TrpC1Delta758-793 but not TrpC1Delta715-749 mimicked the effects of TrpC1DeltaC and Mut-CaM on SOCE. These data demonstrate that CaM mediates Ca(2+)-dependent feedback inhibition of SOCE via binding to a domain in the C terminus of TrpC1. These findings reveal an integral role for TrpC1 in the regulation of SOCE.     

Mechanism of calmodulin inhibition of cardiac sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor)

The functional effects of calmodulin (CaM) on single cardiac sarcoplasmic reticulum Ca(2+) release channels (ryanodine receptors) (RyR2s) were determined in the presence of two endogenous channel effectors, MgATP and reduced glutathione, using the planar lipid bilayer method. Single-channel activities, number of events, and open and close times were determined at varying cytosolic Ca(2+) concentrations. CaM reduced channel open probability at <10 micro M Ca(2+) by decreasing channel events and mean open times and increasing mean close times. At >10 micro M Ca(2+), CaM was less effective in inhibiting RyR2. CaM decreased mean open times but increased channel events, without significantly affecting mean close times. A series of voltage pulses was applied to the bilayer from +50 to -50 mV and from -50 mV to +50 mV to rapidly increase and decrease open channel-mediated sarcoplasmic reticulum lumenal to cytosolic Ca(2+) fluxes. CaM decreased the duration of the open events after the voltage switch from -50 mV to +50 mV. In parallel experiments, a Ca(2+)-insensitive calmodulin mutant was without effect on RyR2 activity. The results are discussed in terms of a possible role of CaM in the termination of cardiac sarcoplasmic reticulum Ca(2+) release.     

The Motif of Human Cardiac Myosin-binding Protein C Is Required for Its Ca2+-dependent Interaction with Calmodulin

The N-terminal modules of cardiac myosin-binding protein C (cMyBP-C) play a regulatory role in mediating interactions between myosin and actin during heart muscle contraction. The so-called "motif," located between the second and third immunoglobulin modules of the cardiac isoform, is believed to modulate contractility via an "on-off" phosphorylation-dependent tether to myosin ΔS2. Here we report a novel Ca(2+)-dependent interaction between the motif and calmodulin (CaM) based on the results of a combined fluorescence, NMR, and light and x-ray scattering study. We show that constructs of cMyBP-C containing the motif bind to Ca(2+)/CaM with a moderate affinity (K(D) ～10 μm), which is similar to the affinity previously determined for myosin ΔS2. However, unlike the interaction with myosin ΔS2, the Ca(2+)/CaM interaction is unaffected by substitution with a triphosphorylated motif mimic. Further, Ca(2+)/CaM interacts with the highly conserved residues (Glu(319)-Lys(341)) toward the C-terminal end of the motif. Consistent with the Ca(2+) dependence, the binding of CaM to the motif is mediated via the hydrophobic clefts within the N- and C-lobes that are known to become more exposed upon Ca(2+) binding. Overall, Ca(2+)/CaM engages with the motif in an extended clamp configuration as opposed to the collapsed binding mode often observed in other CaM-protein interactions. Our results suggest that CaM may act as a structural conduit that links cMyBP-C with Ca(2+) signaling pathways to help coordinate phosphorylation events and synchronize the multiple interactions between cMyBP-C, myosin, and actin during the heart muscle contraction.     

Regulation of the transient receptor potential channel TRPM2 by the Ca2+ sensor calmodulin

TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca(2+)-permeable channel activated by oxidative stress or tumor necrosis factoralpha involved in susceptibility to cell death. TRPM2 activation is dependent on the level of intracellular Ca(2+). We explored whether calmodulin (CaM) is the Ca(2+) sensor for TRPM2. HEK 293T cells were transfected with TRPM2 and wild type CaM or mutant CaM (CaM(MUT)) with substitutions of all four EF hands. Treatment of cells expressing TRPM2 with H(2)O(2) or tumor necrosis factor alpha resulted in a significant increase in intracellular calcium ([Ca(2+)](i)). This was not affected by coexpression of CaM, suggesting that endogenous CaM levels are sufficient for maximal response. Cotransfection of CaM(MUT) with TRPM2 dramatically inhibited the increase in [Ca(2+)](i), demonstrating the requirement for CaM in TRPM2 activation. Immunoprecipitation confirmed direct interaction of CaM and CaM(MUT) with TRPM2, and the Ca(2+) dependence of this association. CaM bound strongly to the TRPM2 N terminus (amino acids 1-730), but weakly to the C terminus (amino acids 1060-1503). CaM binding to an IQ-like motif (amino acids 406-416) in the TRPM2 N terminus was demonstrated utilizing gel shift, immunoprecipitation, biotinylated CaM overlay, and pull-down assays. A substitution mutant of the IQ-like motif of TRPM2 (TRPM2-IQ(MUT1)) reduced but did not eliminate CaM binding to TRPM2, suggesting the presence of at least one other CaM binding site. The functional importance of the TRPM2 IQ-like motif was demonstrated by treatment of TRPM2-IQ(MUT1)-expressing cells with H(2)O(2). The increase in [Ca(2+)](i) observed with wild type TRPM2 was absent and cell viability was preserved. These data demonstrate the requirement for CaM in TRPM2 activation. They suggest that Ca(2+) entering through TRPM2 enhances interaction of CaM with TRPM2 at the IQ-like motif in the N terminus, providing crucial positive feedback for channel activation.     

A Ca2+/calmodulin-binding peroxidase from Euphorbia latex: novel aspects of calcium-hydrogen peroxide cross-talk in the regulation of plant defenses

Calmodulin (CaM) is a ubiquitous Ca(2+) sensor found in all eukaryotes, where it participates in the regulation of diverse calcium-dependent physiological processes. In response to fluctuations of the intracellular concentration of Ca(2+), CaM binds to a set of unrelated target proteins and modulates their activity. In plants, a growing number of CaM-binding proteins have been identified that apparently do not have a counterpart in animals. Some of these plant-specific Ca(2+)/CaM-activated proteins are known to tune the interaction between calcium and H(2)O(2) in orchestrating plant defenses against biotic and abiotic stresses. We previously characterized a calcium-dependent peroxidase isolated from the latex of the Mediterranean shrub Euphorbia characias (ELP) [Medda et al. (2003) Biochemistry 42, 8909-8918]. Here we report the cDNA nucleotide sequence of Euphorbia latex peroxidase, showing that the derived protein has two distinct amino acid sequences recognized as CaM-binding sites. The cDNA encoding for an E. characias CaM was also found and sequenced, and its protein product was detected in the latex. Results obtained from different CaM-binding assays and the determination of steady-state parameters showed unequivocally that ELP is a CaM-binding protein activated by the Ca(2+)/CaM system. To the best of our knowledge, this is the first example of a peroxidase regulated by this classic signal transduction mechanism. These findings suggest that peroxidase might be another node in the Ca(2+)/H(2)O(2)-mediated plant defense system, having both positive and negative effects in regulating H(2)O(2) homeostasis.