Caldendrin, a neuron-specific modulator of Cav/1.2 (L-type) Ca2+ channels

EF-hand Ca2+-binding proteins such as calmodulin and CaBP1 have emerged as important regulatory subunits of voltage-gated Ca2+ channels. Here, we show that caldendrin, a variant of CaBP1 enriched in the brain, interacts with and distinctly modulates Cav1.2 (L-type) voltage-gated Ca2+ channels relative to other Ca2+-binding proteins. Caldendrin binds to the C-terminal IQ-domain of the pore-forming alpha1-subunit of Cav1.2 (alpha(1)1.2) and competitively displaces calmodulin and CaBP1 from this site. Compared with CaBP1, caldendrin causes a more modest suppression of Ca2+-dependent inactivation of Cav1.2 through a different subset of molecular determinants. Caldendrin does not bind to the N-terminal domain of alpha11.2, a site that is critical for functional interactions of the channel with CaBP1. Deletion of the N-terminal domain inhibits CaBP1, but spares caldendrin modulation of Cav1.2 inactivation. In contrast, mutations of the IQ-domain abolish physical and functional interactions of caldendrin and Cav1.2, but do not prevent channel modulation by CaBP1. Using antibodies specific for caldendrin and Cav1.2, we show that caldendrin coimmunoprecipitates with Cav1.2 from the brain and colocalizes with Cav1.2 in somatodendritic puncta of cortical neurons in culture. Our findings reveal functional diversity within related Ca2+-binding proteins, which may enhance the specificity of Ca2+ signaling by Cav1.2 channels in different cellular contexts.     

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.     

CaBP1 regulates voltage-dependent inactivation and activation of Ca(V)1.2 (L-type) calcium channels

CaBP1 is a Ca(2+)-binding protein that regulates the gating of voltage-gated (Ca(V)) Ca(2+) channels. In the Ca(V)1.2 channel α(1)-subunit (α(1C)), CaBP1 interacts with cytosolic N- and C-terminal domains and blunts Ca(2+)-dependent inactivation. To clarify the role of the α(1C) N-terminal domain in CaBP1 regulation, we compared the effects of CaBP1 on two alternatively spliced variants of α(1C) containing a long or short N-terminal domain. In both isoforms, CaBP1 inhibited Ca(2+)-dependent inactivation but also caused a depolarizing shift in voltage-dependent activation and enhanced voltage-dependent inactivation (VDI). In binding assays, CaBP1 interacted with the distal third of the N-terminal domain in a Ca(2+)-independent manner. This segment is distinct from the previously identified calmodulin-binding site in the N terminus. However, deletion of a segment in the proximal N-terminal domain of both α(1C) isoforms, which spared the CaBP1-binding site, inhibited the effect of CaBP1 on VDI. This result suggests a modular organization of the α(1C) N-terminal domain, with separate determinants for CaBP1 binding and transduction of the effect on VDI. Our findings expand the diversity and mechanisms of Ca(V) channel regulation by CaBP1 and define a novel modulatory function for the initial segment of the N terminus of α(1C).     

Ca2+-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca(2+) channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca(2+)-binding proteins are of particular importance as sensors of presynaptic Ca(2+), and a multiple of them are indeed utilized in the signaling of Ca(2+) channels. However, despite its conserved structure, CaM is the only known EF-hand Ca(2+)-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca(2+) channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca(2+)-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca(2+)-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca(2+), PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca(2+) channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

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.     

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.     

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.     

Regulation of InsP3 receptor activity by neuronal Ca2+-binding proteins

Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) were recently demonstrated to be activated independently of InsP(3) by a family of calmodulin (CaM)-like neuronal Ca(2+)-binding proteins (CaBPs). We investigated the interaction of both naturally occurring long and short CaBP1 isoforms with InsP(3)Rs, and their functional effects on InsP(3)R-evoked Ca(2+) signals. Using several experimental paradigms, including transient expression in COS cells, acute injection of recombinant protein into Xenopus oocytes and (45)Ca(2+) flux from permeabilised COS cells, we demonstrated that CaBPs decrease the sensitivity of InsP(3)-induced Ca(2+) release (IICR). In addition, we found a Ca(2+)-independent interaction between CaBP1 and the NH(2)-terminal 159 amino acids of the type 1 InsP(3)R. This interaction resulted in decreased InsP(3) binding to the receptor reminiscent of that observed for CaM. Unlike CaM, however, CaBPs do not inhibit ryanodine receptors, have a higher affinity for InsP(3)Rs and more potently inhibited IICR. We also show that phosphorylation of CaBP1 at a casein kinase 2 consensus site regulates its inhibition of IICR. Our data suggest that CaBPs are endogenous regulators of InsP(3)Rs tuning the sensitivity of cells to InsP(3).     

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.     

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.     

Endogenous and exogenous Ca2+ buffers differentially modulate Ca2+-dependent inactivation of Ca(v)2.1 Ca2+ channels

Voltage-gated Ca2+ channels undergo a negative feedback regulation by Ca2+ ions, Ca2+-dependent inactivation, which is important for restricting Ca2+ signals in nerve and muscle. Although the molecular details underlying Ca2+-dependent inactivation have been characterized, little is known about how this process might be modulated in excitable cells. Based on previous findings that Ca2+-dependent inactivation of Ca(v)2.1 (P/Q-type) Ca2+ channels is suppressed by strong cytoplasmic Ca2+ buffering, we investigated how factors that regulate cellular Ca2+ levels affect inactivation of Ca(v)2.1 Ca2+ currents in transfected 293T cells. We found that inactivation of Ca(v)2.1 Ca2+ currents increased exponentially with current amplitude with low intracellular concentrations of the slow buffer EGTA (0.5 mm), but not with high concentrations of the fast Ca2+ buffer BAPTA (10 mm). However, when the concentration of BAPTA was reduced to 0.5 mm, inactivation of Ca2+ currents was significantly greater than with an equivalent concentration of EGTA, indicating the importance of buffer kinetics in modulating Ca2+-dependent inactivation of Ca(v)2.1. Cotransfection of Ca(v)2.1 with the EF-hand Ca2+-binding proteins, parvalbumin and calbindin, significantly altered the relationship between Ca2+ current amplitude and inactivation in ways that were unexpected from behavior as passive Ca2+ buffers. We conclude that Ca2+-dependent inactivation of Ca(v)2.1 depends on a subplasmalemmal Ca2+ microdomain that is affected by the amplitude of the Ca2+ current and differentially modulated by distinct Ca2+ buffers.     

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 basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels.

Ca(2+)-dependent inactivation (CDI) of L-type Ca(2+) channels plays a critical role in controlling Ca(2+) entry and downstream signal transduction in excitable cells. Ca(2+)-insensitive forms of calmodulin (CaM) act as dominant negatives to prevent CDI, suggesting that CaM acts as a resident Ca(2+) sensor. However, it is not known how the Ca(2+) sensor is constitutively tethered. We have found that the tethering of Ca(2+)-insensitive CaM was localized to the C-terminal tail of alpha(1C), close to the CDI effector motif, and that it depended on nanomolar Ca(2+) concentrations, likely attained in quiescent cells. Two stretches of amino acids were found to support the tethering and to contain putative CaM-binding sequences close to or overlapping residues previously shown to affect CDI and Ca(2+)-independent inactivation. Synthetic peptides containing these sequences displayed differences in CaM-binding properties, both in affinity and Ca(2+) dependence, leading us to propose a novel mechanism for CDI. In contrast to a traditional disinhibitory scenario, we suggest that apoCaM is tethered at two sites and signals actively to slow inactivation. When the C-terminal lobe of CaM binds to the nearby CaM effector sequence (IQ motif), the braking effect is relieved, and CDI is accelerated.     

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.     

Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin

Five members of a novel Ca(2+)-binding protein subfamily (CaBP), with 46-58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these Ca(2+)-binding proteins and CaM include alterations within their second EF-hand loop that render these motifs inactive in Ca(2+) coordination and the fact that their central alpha-helixes are extended by one alpha-helical turn. CaBP1 and CaBP2 contain a consensus sequence for N-terminal myristoylation, similar to members of the recoverin subfamily and are fatty acid acylated in vitro. The patterns of expression differ for each of the various members. Expression of CaBP5, for example, is restricted to retinal rod and cone bipolar cells. In contrast, CaBP1 has a more widespread pattern of expression. In the brain, CaBP1 is found in the cerebral cortex and hippocampus, and in the retina this protein is found in cone bipolar and amacrine cells. CaBP1 and CaBP2 are expressed as multiple, alternatively spliced variants, and in heterologous expression systems these forms show different patterns of subcellular localization. In reconstitution assays, CaBPs are able to substitute functionally for CaM. These data suggest that these novel CaBPs are an important component of Ca(2+)-mediated cellular signal transduction in the central nervous system where they may augment or substitute for CaM.     

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.     

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.     

Serine residue in the IIIS5-S6 linker of the L-type Ca2+ channel alpha 1C subunit is the critical determinant of the action of dihydropyridine Ca2+ channel agonists

The dihydropyridine (DHP)-binding site has been identified within L-type Ca(2+) channel alpha(1C) subunit. However, the molecular mechanism underlying modulation of Ca(2+) channel gating by DHPs has not been clarified. To search for novel determinants of high affinity DHP binding, we introduced point mutations in the rat brain Ca(2+) channel alpha(1C) subunit (rbCII or Ca(v)1.2c) based on the comparison of amino acid sequences between rbCII and the ascidian L-type Ca(2+) channel alpha(1) subunit, which is insensitive to DHPs. The alpha(1C) mutants (S1115A, S1146A, and A1420S) and rbCII were transiently expressed in BHK6 cells with beta(1a) and alpha(2)/delta subunits. The mutation did not affect the electrophysiological properties of the Ca(2+) channel, or the voltage- and concentration-dependent block of Ca(2+) channel currents produced by diltiazem and verapamil. However, the S1115A channel was significantly less sensitive to DHP antagonists. Interestingly, in the S1115A channel, DHP agonists failed to enhance whole-cell Ca(2+) channel currents and the prolongation of mean open time, as well as the increment of NP(o). Responsiveness to the non-DHP agonist FPL-64176 was also markedly reduced in the S1115A channel. When S1115 was replaced by other amino acids (S1115D, S1115T, or S1115V), only S1115T was slightly sensitive to S-(-)-Bay K 8644. These results indicate that the hydroxyl group of Ser(1115) in IIIS5-S6 linker of the L-type Ca(2+) channel alpha(1C) subunit plays a critical role in DHP binding and in the action of DHP Ca(2+) channel agonists.     

Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).     

Structural basis for the differential effects of CaBP1 and calmodulin on Ca(V)1.2 calcium-dependent inactivation

Calcium-binding protein 1 (CaBP1), a calmodulin (CaM) homolog, endows certain voltage-gated calcium channels (Ca(V)s) with unusual properties. CaBP1 inhibits Ca(V)1.2 calcium-dependent inactivation (CDI) and introduces calcium-dependent facilitation (CDF). Here, we show that the ability of CaBP1 to inhibit Ca(V)1.2 CDI and induce CDF arises from interaction between the CaBP1 N-lobe and interlobe linker residue Glu94. Unlike CaM, where functional EF hands are essential for channel modulation, CDI inhibition does not require functional CaBP1 EF hands. Furthermore, CaBP1-mediated CDF has different molecular requirements than CaM-mediated CDF. Overall, the data show that CaBP1 comprises two structural modules having separate functions: similar to CaM, the CaBP1 C-lobe serves as a high-affinity anchor that binds the Ca(V)1.2 IQ domain at a site that overlaps with the Ca2+/CaM C-lobe site, whereas the N-lobe/linker module houses the elements required for channel modulation. Discovery of this division provides the framework for understanding how CaBP1 regulates Ca(V)s.