Secondary literature sources for the Ca_chan_IQ domain

For each of the hand selected primary papers associated with the Ca_chan_IQ domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

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

Zhou H, Yu K, McCoy KL, Lee A
J Biol Chem. 2005; 280: 29612-9

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.

A self-sequestered calmodulin-like Ca(2)(+) sensor of mitochondrial SCaMC carrier and its implication to Ca(2)(+)-dependent ATP-Mg/P(i) transport.

Yang Q, Bruschweiler S, Chou JJ
Structure. 2014; 22: 209-17

The mitochondrial carriers play essential roles in energy metabolism. The short Ca(2)(+)-binding mitochondrial carrier (SCaMC) transports ATP-Mg in exchange for Pi and is important for activities that depend on adenine nucleotides. SCaMC adopts, in addition to the transmembrane domain (TMD) that transports solutes, an extramembrane N-terminal domain (NTD) that regulates solute transport in a Ca(2)(+)-dependent manner. Crystal structure of the Ca(2)(+)-bound NTD reveals a compact architecture in which the functional EF hands are sequestered by an endogenous helical segment. Nuclear magnetic resonance (NMR) relaxation rates indicated that removal of Ca(2)(+) from NTD results in a major conformational switch from the rigid and compact Ca(2)(+)-bound state to the dynamic and loose apo state. Finally, we showed using surface plasmon resonance and NMR titration experiments that free apo NTDs could specifically interact with liposome-incorporated TMD, but that Ca(2)(+) binding drastically weakened the interaction. Our results together provide a molecular explanation for Ca(2)(+)-dependent ATP-Mg flux in mitochondria.

Switching of Ca2+-dependent inactivation of Ca(v)1.3 channels by calcium binding proteins of auditory hair cells.

Yang PS, Alseikhan BA, Hiel H, Grant L, Mori MX, Yang W, Fuchs PA, Yue DT
J Neurosci. 2006; 26: 10677-89

Ca(V)1.3 channels comprise a vital subdivision of L-type Ca2+ channels: Ca(V)1.3 channels mediate neurotransmitter release from auditory inner hair cells (IHCs), pancreatic insulin secretion, and cardiac pacemaking. Fitting with these diverse roles, Ca(V)1.3 channels exhibit striking variability in their inactivation by intracellular Ca2+. IHCs show generally weak-to-absent Ca2+-dependent inactivation (CDI), potentially permitting audition of sustained sounds. In contrast, the strong CDI seen elsewhere likely provides critical negative feedback. Here, we explore this mysterious CDI malleability, particularly its comparative weakness in hair cells. At baseline, heterologously expressed Ca(V)1.3 channels exhibit intense CDI, wherein each lobe of calmodulin (CaM) contributes a distinct inactivation component. Because CaM-like molecules (bearing four recognizable but not necessarily functional Ca2+-binding EF hands) can perturb the Ca2+ response of molecules regulated by CaM, we asked whether such CaM-like entities could influence CDI. We find that CaM-like calcium-binding protein (CaBP) molecules are clearly expressed within the organ of Corti. In particular, the rare subtype CaBP4 is specific to IHCs, and CaBP4 proves capable of eliminating even the potent baseline CDI of Ca(V)1.3. CaBP4 thereby represents a plausible candidate for moderating CDI within IHCs.

Engineering proteins for custom inhibition of Ca(V) channels.

Xu X, Colecraft HM
Physiology (Bethesda). 2009; 24: 210-8

The influx of Ca(2+) ions through voltage-dependent calcium (Ca(V)) channels links electrical signals to physiological responses in all excitable cells. Not surprisingly, blocking Ca(V) channel activity is a powerful method to regulate the function of excitable cells, and this is exploited for both physiological and therapeutic benefit. Nevertheless, the full potential for Ca(V) channel inhibition is not being realized by currently available small-molecule blockers or second-messenger modulators due to limitations in targeting them either to defined groups of cells in an organism or to distinct subcellular regions within a single cell. Here, we review early efforts to engineer protein molecule blockers of Ca(V) channels to fill this crucial niche. This technology would greatly expand the toolbox available to physiologists studying the biology of excitable cells at the cellular and systems level.

A Ca2+-binding domain in RyR1 that interacts with the calmodulin binding site and modulates channel activity.

Xiong L, Zhang JZ, He R, Hamilton SL
Biophys J. 2006; 90: 173-82

A fragment of RyR1 (amino acids 4064-4210) is predicted to fold to at least one lobe of calmodulin and to bind Ca(2+). This fragment of RyR1 (R4064-4210) was subcloned, expressed, refolded, and purified. Consistent with the predicted folding pattern, R4064-4210 was found to bind two molecules of Ca(2+) and undergo a structural change upon binding Ca(2+) that exposes hydrophobic amino acids. R4064-4210 also binds to RyR1, the L-type Ca(2+) channel (Cav(1.1)), and several synthetic calmodulin binding peptides. Both R4064-4210 and a peptide representing the calmodulin-binding region of RyR1 (R3614-3643) alter the Ca(2+) dependence of ((3)H)ryanodine binding to RyR1, suggesting that they may both be interfering with an intramolecular interaction between amino acids 4064-4210 and amino acids 3614-3643 in the native RyR1 to alter or regulate the response of the channel to changes in Ca(2+) concentration. The finding that a domain within RyR1 binds Ca(2+) and interacts with calmodulin-binding motifs may provide insights into the mechanism for calcium- and calmodulin-dependent regulation of this channel and perhaps for its regulation by the L-type Ca(2+) channel.

Structural analyses of Ca(2)(+)/CaM interaction with NaV channel C-termini reveal mechanisms of calcium-dependent regulation.

Wang C, Chung BC, Yan H, Wang HG, Lee SY, Pitt GS
Nat Commun. 2014; 5: 4896-4896

Ca(2+) regulates voltage-gated Na(+) (NaV) channels, and perturbed Ca(2+) regulation of NaV function is associated with epilepsy syndromes, autism and cardiac arrhythmias. Understanding the disease mechanisms, however, has been hindered by a lack of structural information and competing models for how Ca(2+) affects NaV channel function. Here we report the crystal structures of two ternary complexes of a human NaV cytosolic C-terminal domain (CTD), a fibroblast growth factor homologous factor and Ca(2+)/calmodulin (Ca(2+)/CaM). These structures rule out direct binding of Ca(2+) to the NaV CTD and uncover new contacts between CaM and the NaV CTD. Probing these new contacts with biochemical and functional experiments allows us to propose a mechanism by which Ca(2+) could regulate NaV channels. Further, our model provides hints towards understanding the molecular basis of the neurologic disorders and cardiac arrhythmias caused by NaV channel mutations.

Three-dimensional structure of CaV3.1: comparison with the cardiac L-type voltage-gated calcium channel monomer architecture.

Walsh CP, Davies A, Butcher AJ, Dolphin AC, Kitmitto A
J Biol Chem. 2009; 284: 22310-21

Calcium entry through voltage-gated calcium channels has widespread cellular effects upon a host of physiological processes including neuronal excitability, muscle excitation-contraction coupling, and secretion. Using single particle analysis methods, we have determined the first three-dimensional structure, at 23 A resolution, for a member of the low voltage-activated voltage-gated calcium channel family, CaV3.1, a T-type channel. CaV3.1 has dimensions of approximately 115x85x95 A, composed of two distinct segments. The cytoplasmic densities form a vestibule below the transmembrane domain with the C terminus, unambiguously identified by the presence of a His tag being approximately 65 A long and curling around the base of the structure. The cytoplasmic assembly has a large exposed surface area that may serve as a signaling hub with the C terminus acting as a "fishing rod" to bind regulatory proteins. We have also determined a three-dimensional structure, at a resolution of 25 A, for the monomeric form of the cardiac L-type voltage-gated calcium (high voltage-activated) channel with accessory proteins beta and alpha2delta bound to the ion channel polypeptide CaV1.2. Comparison with the skeletal muscle isoform finds a good match particularly with respect to the conformation, size, and shape of the domain identified as that formed by alpha2. Furthermore, modeling of the CaV3.1 structure (analogous to CaV1.2 at these resolutions) into the heteromeric L-type voltage-gated calcium channel complex volume reveals multiple interaction sites for beta-CaV1.2 binding and for the first time identifies the size and organization of the alpha2delta polypeptides.

Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function.

Walker D, De Waard M
Trends Neurosci. 1998; 21: 148-54

Voltage-dependent Ca2+ channels are heteromeric complexes found in the plasma membrane of virtually all cell types and show a high level of electrophysiological and pharmacological diversity. Associated with the pore-forming alpha 1 subunit are the membrane anchored, largely extracellular alpha2-delta, the cytoplasmic beta and sometimes a transmembrane gamma subunit; these subunits dramatically influence the properties and surface expression of these channels. Effects vary depending on subunit isoforms, suggesting that functional diversity of native channels reflects heterogeneity of combinations. Interaction sites between subunits have been identified and advances have been made in our understanding of the molecular basis of functional effects of the auxiliary subunits, their capacity to be regulated by G proteins, and their interaction with related cellular systems.

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

Tang W, Halling DB, Black DJ, Pate P, Zhang JZ, Pedersen S, Altschuld RA, Hamilton SL
Biophys J. 2003; 85: 1538-47

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 (Zuhlke 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.

Functional characterization of alternative splicing in the C terminus of L-type CaV1.3 channels.

Tan BZ, Jiang F, Tan MY, Yu D, Huang H, Shen Y, Soong TW
J Biol Chem. 2011; 286: 42725-35

Ca(V)1.3 channels are unique among the high voltage-activated Ca(2+) channel family because they activate at the most negative potentials and display very rapid calcium-dependent inactivation. Both properties are of crucial importance in neurons of the suprachiasmatic nucleus and substantia nigra, where the influx of Ca(2+) ions at subthreshold membrane voltages supports pacemaking function. Previously, alternative splicing in the Ca(V)1.3 C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), resulting in a pronounced activation at more negative voltages and faster inactivation in the latter. It was further shown that the C-terminal modulator in the Ca(V)1.3(42) isoforms modulates calmodulin binding to the IQ domain. Using splice variant-specific antibodies, we determined that protein localization of both splice variants in different brain regions were similar. Using the transcript-scanning method, we further identified alternative splicing at four loci in the C terminus of Ca(V)1.3 channels. Alternative splicing of exon 41 removes the IQ motif, resulting in a truncated Ca(V)1.3 protein with diminished inactivation. Splicing of exon 43 causes a frameshift and exhibits a robust inactivation of similar intensity to Ca(V)1.3(42A). Alternative splicing of exons 44 and 48 are in-frame, altering interaction of the distal modulator with the IQ domain and tapering inactivation slightly. Thus, alternative splicing in the C terminus of Ca(V)1.3 channels modulates its electrophysiological properties, which could in turn alter neuronal firing properties and functions.

The IQ motif is crucial for Cav1.1 function.

Stroffekova K
J Biomed Biotechnol. 2011; 2011: 504649-504649

Ca(2+)-dependent modulation via calmodulin, with consensus CaM-binding IQ motif playing a key role, has been documented for most high-voltage-activated Ca(2+) channels. The skeletal muscle Ca(v)1.1 also exhibits Ca(2+)-/CaM-dependent modulation. Here, whole-cell Ca(2+) current, Ca(2+) transient, and maximal, immobilization-resistant charge movement (Q(max)) recordings were obtained from cultured mouse myotubes, to test a role of IQ motif in function of Ca(v)1.1. The effect of introducing mutation (IQ to AA) of IQ motif into Ca(v)1.1 was examined. In dysgenic myotubes expressing YFP-Ca(v)1.1(AA), neither Ca(2+) currents nor evoked Ca(2+) transients were detectable. The loss of Ca(2+) current and excitation-contraction coupling did not appear to be a consequence of defective trafficking to the sarcolemma. The Q(max) in dysgenic myotubes expressing YFP-Ca(v)1.1(AA) was similar to that of normal myotubes. These findings suggest that the IQ motif of the Ca(v)1.1 may be an unrecognized site of structural and functional coupling between DHPR and RyR.

The Cav1.2 N terminus contains a CaM kinase site that modulates channel trafficking and function.

Simms BA, Souza IA, Rehak R, Zamponi GW
Pflugers Arch. 2015; 467: 677-86

The L-type voltage-gated calcium channel Cav1.2 and the calcium-activated CaM kinase cascade both regulate excitation transcription coupling in the brain. CaM kinase is known to associate with the C terminus of Cav1.2 in a region called the PreIQ-IQ domain, which also binds multiple calmodulin molecules. Here we identify and characterize a second CaMKII binding site in the N terminus of Cav1.2 that is formed by a stretch of four amino residues (cysteine-isoleucine-serine-isoleucine) and which regulates channel expression and function. By using live cell imaging of tsA-201 cells we show that GFP fusion constructs of the CaMKII binding region, termed N2B-II co-localize with mCherry-CaMKII. Mutating CISI to AAAA ablates binding to and colocalization with CaMKII. Cav1.2-AAAA channels show reduced cell surface expression in tsA-201 cells, but interestingly, display an increase in channel function that offsets the trafficking deficit. Altogether our data reveal that the proximal N terminus of Cav1.2 contains a CaMKII binding region which contributes to channel surface expression and function.

Physical link and functional coupling of presynaptic calcium channels and the synaptic vesicle docking/fusion machinery.

Sheng ZH, Westenbroek RE, Catterall WA
J Bioenerg Biomembr. 1998; 30: 335-45

N- and P/Q-type calcium channels are localized in high density in presynaptic nerve terminals and are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. As outlined in the preceding article, these calcium channels can be purified from brain as a complex with SNARE proteins which are involved in exocytosis. In addition, N-type and P/Q-type calcium channels are co-localized with syntaxin in high-density clusters in nerve terminals. Here we review the role of the synaptic protein interaction (synprint) sites in the intracellular loop II-III (L(II-III)) of both alpha1B and alpha1A subunits of N-type and P/Q-type calcium channels, which bind to syntaxin, SNAP-25, and synaptotagmin. Calcium has a biphasic effect on the interactions of N-type calcium channels with SNARE complexes, stimulating optimal binding in the range of 10-20 microM. PKC or CaM KII phosphorylation of the N-type synprint peptide inhibits interactions with native brain SNARE complexes containing syntaxin and SNAP-25. Introduction of the synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibits EPSPs from synchronous transmitter release by 42%. At physiological Ca2+ concentrations, synprint peptides cause an approximate 25% reduction in transmitter release of injected frog neuromuscular junction in cultures, consistent with detachment of 70% of the docked vesicles from calcium channels based on a theoretical model. Together, these studies suggest that presynaptic calcium channels not only provide the calcium signal required by the exocytotic machinery, but also contain structural elements that are integral to vesicle docking, priming, and fusion processes.

Calcium-dependent regulation of the voltage-gated sodium channel hH1: intrinsic and extrinsic sensors use a common molecular switch.

Shah VN, Wingo TL, Weiss KL, Williams CK, Balser JR, Chazin WJ
Proc Natl Acad Sci U S A. 2006; 103: 3592-7

The function of the human cardiac voltage-gated sodium channel Na(V)1.5 (hH1) is regulated in part by binding of calcium to an EF hand in the C-terminal cytoplasmic domain. hH1 is also regulated via an extrinsic calcium-sensing pathway mediated by calmodulin (CaM) via binding to an IQ motif immediately adjacent to the EF-hand domain. The intrinsic EF-hand domain is shown here to interact with the IQ motif, which controls calcium affinity. Remarkably, mutation of the IQ residues has only a minor effect on CaM affinity but drastically reduces calcium affinity of the EF-hand domain, whereas the Brugada mutation A1924T significantly reduces CaM affinity but has no effect on calcium affinity of the EF-hand domain. Moreover, the differences in the biochemical effects of the mutations directly correlate with contrasting effects on channel electrophysiology. A comprehensive model is proposed in which the hH1 IQ motif serves as a molecular switch, coupling the intrinsic and extrinsic calcium sensors.

How "Pharmacoresistant" is Cav2.3, the Major Component of Voltage-Gated R-type Ca2+ Channels?

Schneider T, Dibue M, Hescheler J
Pharmaceuticals (Basel). 2013; 6: 759-76

Membrane-bound voltage-gated Ca2+ channels (VGCCs) are targets for specific signaling complexes, which regulate important processes like gene expression, neurotransmitter release and neuronal excitability. It is becoming increasingly evident that the so called "resistant" (R-type) VGCC Cav2.3 is critical in several physiologic and pathophysiologic processes in the central nervous system, vascular system and in endocrine systems. However its eponymous attribute of pharmacologic inertness initially made in depth investigation of the channel difficult. Although the identification of SNX-482 as a fairly specific inhibitor of Cav2.3 in the nanomolar range has enabled insights into the channels properties, availability of other pharmacologic modulators of Cav2.3 with different chemical, physical and biological properties are of great importance for future investigations. Therefore the literature was screened systematically for molecules that modulate Cav2.3 VGCCs.

Calpastatin binds to a calmodulin-binding site of cardiac Cav1.2 Ca2+ channels.

Saud ZA, Minobe E, Wang WY, Han DY, Horiuchi M, Hao LY, Kameyama M
Biochem Biophys Res Commun. 2007; 364: 372-7

Calpastatin is an endogenous inhibitor of calpain and composed of domain L (CS(L)), which interacts with the Cav1.2 channels, and four repetitive calpain inhibitory domains. We have previously found that CS(L) reprimes activity of the Cav1.2 channels in cell-free patches of cardiac myocytes [L.Y. Hao, A. Kameyama, S. Kuroki, J. Takano, E. Takano, M. Maki, M. Kameyama, Calpastatin domain L is involved in the regulation L-type of Ca2+ channels in guinea pig cardiac myocytes, Biochem. Biophys. Res. Commun. 279 (2000) 756-761; E. Minobe, L.Y. Hao, Z.A. Saud, J.J. Xu, A. Kameyama, M. Maki, K.K. Jewell, T. Parr, R.G. Bardsley, M. Kameyama, A region of calpastatin domain L that reprimes cardiac L-type Ca2+ channels, Biochem. Biophys. Res. Commun. 348 (2006) 288-294]. In this study, we explored the CS(L) interaction site in the Ca2+ channel by the pull-down method, using glutathione-S-transferase-fused fragment peptides of the Cav1.2 channel. CS(L) bound directly to a proximal region of the C-terminal tail of the channel, but not with the N-terminal tail, a distal region of the C-terminal tail or cytoplasmic loops between repeats I-II, II-III or III-IV. Furthermore IQ domain, but not EF-hand-like region or CB domain, in the C-terminal tail was found to bind with CS(L) in a partially Ca2+-dependent manner and in a probably competitive manner with calmodulin. These results suggest that CS(L) modulates Ca2+-channel activity through interacting with the calmodulin-binding site on the C-terminal tail of the Cav1.2 channel.

Crystallographic basis for calcium regulation of sodium channels.

Sarhan MF, Tung CC, Van Petegem F, Ahern CA
Proc Natl Acad Sci U S A. 2012; 109: 3558-63

Voltage-gated sodium channels underlie the rapid regenerative upstroke of action potentials and are modulated by cytoplasmic calcium ions through a poorly understood mechanism. We describe the 1.35 A crystal structure of Ca(2+)-bound calmodulin (Ca(2+)/CaM) in complex with the inactivation gate (DIII-IV linker) of the cardiac sodium channel (Na(V)1.5). The complex harbors the positions of five disease mutations involved with long Q-T type 3 and Brugada syndromes. In conjunction with isothermal titration calorimetry, we identify unique inactivation-gate mutations that enhance or diminish Ca(2+)/CaM binding, which, in turn, sensitize or abolish Ca(2+) regulation of full-length channels in electrophysiological experiments. Additional biochemical experiments support a model whereby a single Ca(2+)/CaM bridges the C-terminal IQ motif to the DIII-IV linker via individual N and C lobes, respectively. The data suggest that Ca(2+)/CaM destabilizes binding of the inactivation gate to its receptor, thus biasing inactivation toward more depolarized potentials.

Interdomain dynamics explored by paramagnetic NMR.

Russo L, Maestre-Martinez M, Wolff S, Becker S, Griesinger C
J Am Chem Soc. 2013; 135: 17111-20

An ensemble-based approach is presented to explore the conformational space sampled by a multidomain protein showing moderate interdomain dynamics in terms of translational and rotational motions. The strategy was applied on a complex of calmodulin (CaM) with the IQ-recognition motif from the voltage-gated calcium channel Ca(v)1.2 (IQ), which adopts three different interdomain orientations in the crystal. The N60D mutant of calmodulin was used to collect pseudocontact shifts and paramagnetically induced residual dipolar couplings for six different lanthanide ions. Then, starting from the crystal structure, pools of conformations were generated by free MD. We found the three crystal conformations in solution, but four additional MD-derived conformations had to be included into the ensemble to fulfill all the paramagnetic data and cross-validate optimally against unused paramagnetic data. Alternative approaches led to similar ensembles. Our "ensemble" approach is a simple and efficient tool to probe and describe the interdomain dynamics and represents a general method that can be used to provide a proper ensemble description of multidomain proteins.

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

Peterson BZ, Lee JS, Mulle JG, Wang Y, de Leon M, Yue DT
Biophys J. 2000; 78: 1906-20

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.

Automated protein model building combined with iterative structure refinement.

Perrakis A, Morris R, Lamzin VS
Nat Struct Biol. 1999; 6: 458-63

In protein crystallography, much time and effort are often required to trace an initial model from an interpretable electron density map and to refine it until it best agrees with the crystallographic data. Here, we present a method to build and refine a protein model automatically and without user intervention, starting from diffraction data extending to resolution higher than 2.3 A and reasonable estimates of crystallographic phases. The method is based on an iterative procedure that describes the electron density map as a set of unconnected atoms and then searches for protein-like patterns. Automatic pattern recognition (model building) combined with refinement, allows a structural model to be obtained reliably within a few CPU hours. We demonstrate the power of the method with examples of a few recently solved structures.

Competitive and non-competitive regulation of calcium-dependent inactivation in CaV1.2 L-type Ca2+ channels by calmodulin and Ca2+-binding protein 1.

Oz S, Benmocha A, Sasson Y, Sachyani D, Almagor L, Lee A, Hirsch JA, Dascal N
J Biol Chem. 2013; 288: 12680-91

CaV1.2 interacts with the Ca(2+) sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, alpha1C. Ca(2+)/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca(2+) ions (Ca(2+)-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the alpha1C- subunit using Forster resonance energy transfer (FRET) and recording of Cav1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of alpha1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for alpha1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaM altered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15-20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated alpha1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that the NT is not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in alpha1C.

Crystal structure of the CaV2 IQ domain in complex with Ca2+/calmodulin: high-resolution mechanistic implications for channel regulation by Ca2+.

Mori MX, Vander Kooi CW, Leahy DJ, Yue DT
Structure. 2008; 16: 607-20

Calmodulin (CaM) regulation of Ca(2+) channels is central to Ca(2+) signaling. Ca(V)1 versus Ca(V)2 classes of these channels exhibit divergent forms of regulation, potentially relating to customized CaM/IQ interactions among different channels. Here we report the crystal structures for the Ca(2+)/CaM IQ domains of both Ca(V)2.1 and Ca(V)2.3 channels. These highly similar structures emphasize that major CaM contacts with the IQ domain extend well upstream of traditional consensus residues. Surprisingly, upstream mutations strongly diminished Ca(V)2.1 regulation, whereas downstream perturbations had limited effects. Furthermore, our Ca(V)2 structures closely resemble published Ca(2+)/CaM-Ca(V)1.2 IQ structures, arguing against Ca(V)1/2 regulatory differences based solely on contrasting CaM/IQ conformations. Instead, alanine scanning of the Ca(V)2.1 IQ domain, combined with structure-based molecular simulation of corresponding CaM/IQ binding energy perturbations, suggests that the C lobe of CaM partially dislodges from the IQ element during channel regulation, allowing exposed IQ residues to trigger regulation via isoform-specific interactions with alternative channel regions.

Regulatory interaction of sodium channel IQ-motif with calmodulin C-terminal lobe.

Mori M, Konno T, Morii T, Nagayama K, Imoto K
Biochem Biophys Res Commun. 2003; 307: 290-6

An increasing number of ion channels have been found to be regulated by the direct binding of calmodulin (CaM), but its structural features are mostly unknown. Previously, we identified the Ca(2+)-dependent and -independent interactions of CaM to the voltage-gated sodium channel via an IQ-motif sequence. In this study we used the trypsin-digested CaM fragments (TR(1)C and TR(2)C) to analyze the binding of Ca(2+)-CaM or Ca(2+)-free (apo) CaM with a sodium channel-derived IQ-motif peptide (NaIQ). Circular dichroic spectra showed that NaIQ peptide enhanced alpha-helicity of the CaM C-terminal lobe, but not that of the CaM N-terminal lobe in the absence of Ca(2+), whereas NaIQ enhanced the alpha-helicity of both the N- and C-terminal lobes in the presence of Ca(2+). Furthermore, the competitive binding experiment demonstrated that Ca(2+)-dependent CaM binding of target peptides (MLCKp or melittin) with CaM was markedly suppressed by NaIQ. The results suggest that IQ-motif sequences contribute to prevent target proteins from activation at low Ca(2+) concentrations and may explain a regulatory mechanism why highly Ca(2+)-sensitive target proteins are not activated in the cytoplasm.

Progress in the structural understanding of voltage-gated calcium channel (CaV) function and modulation.

Minor DL Jr, Findeisen F
Channels (Austin). 2010; 4: 459-74

Voltage-gated calcium channels (CaVs) are large, transmembrane multiprotein complexes that couple membrane depolarization to cellular calcium entry. These channels are central to cardiac action potential propagation, neurotransmitter and hormone release, muscle contraction, and calcium-dependent gene transcription. Over the past six years, the advent of high-resolution structural studies of CaV components from different isoforms and CaV modulators has begun to reveal the architecture that underlies the exceptionally rich feedback modulation that controls CaV action. These descriptions of CaV molecular anatomy have provided new, structure-based insights into the mechanisms by which particular channel elements affect voltage-dependent inactivation (VDI), calciumdependent inactivation (CDI), and calciumdependent facilitation (CDF). The initial successes have been achieved through structural studies of soluble channel domains and modulator proteins and have proven most powerful when paired with biochemical and functional studies that validate ideas inspired by the structures. Here, we review the progress in this growing area and highlight some key open challenges for future efforts.

Separate elements within a single IQ-like motif in adenylyl cyclase type 8 impart ca2+/calmodulin binding and autoinhibition.

Macdougall DA, Wachten S, Ciruela A, Sinz A, Cooper DM
J Biol Chem. 2009; 284: 15573-88

The ubiquitous Ca(2+)-sensing protein calmodulin (CaM) fulfills its numerous signaling functions through a wide range of modular binding and activation mechanisms. By activating adenylyl cyclases (ACs) 1 and 8, Ca(2+) acting via calmodulin impacts on the signaling of the other major cellular second messenger cAMP. In possessing two CaM-binding domains, a 1-5-8-14 motif at the N terminus and an IQ-like motif (IQlm) at the C terminus, AC8 offers particularly sophisticated regulatory possibilities. The IQlm has remained unexplored beyond the suggestion that it bound CaM, and the larger C2b region of which it is part was involved in the relief of autoinhibition of AC8. Here we attempt to distinguish the function of individual residues of the IQlm. From a complementary approach of in vitro and cell population AC activity assays, as well as CaM binding, we propose that the IQlm alone, and not the majority of the C2b, imparts CaM binding and autoinhibitory functions. Moreover, this duality of function is spatially separated and depends on amino acid side-chain character. Accordingly, residues critical for CaM binding are positively charged and clustered toward the C terminus, and those essential for the maintenance of autoinhibition are hydrophobic and more N-terminal. Secondary structure prediction of the IQlm supports this separation, with an ideally placed break in the alpha-helical nature of the sequence. We additionally find that the N and C termini of AC8 interact, which is an association specifically abrogated by fully Ca(2+)-bound, but not Ca(2+)-free, CaM. These data support a sophisticated activation mechanism of AC8 by CaM, in which the duality of the IQlm function is critical.

The motif of human cardiac myosin-binding protein C is required for its Ca2+-dependent interaction with calmodulin.

Lu Y, Kwan AH, Jeffries CM, Guss JM, Trewhella J
J Biol Chem. 2012; 287: 31596-607

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 DeltaS2. 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) approximately 10 muM), which is similar to the affinity previously determined for myosin DeltaS2. However, unlike the interaction with myosin DeltaS2, 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.

Roles of molecular regions in determining differences between voltage dependence of activation of CaV3.1 and CaV1.2 calcium channels.

Li J, Stevens L, Klugbauer N, Wray D
J Biol Chem. 2004; 279: 26858-67

Voltage-dependent calcium channels are classified into low voltage-activated and high voltage-activated channels. We have investigated the molecular basis for this difference in voltage dependence of activation by constructing chimeras between a low voltage-activated channel (Ca(V)3.1) and a high voltage-activated channel (Ca(V)1.2), focusing on steady-state activation properties. Wild type and chimeras were expressed in oocytes, and two-electrode voltage clamp recordings were made of calcium channel currents. Replacement of domains I, III, or IV of the Ca 3.1 channel with the corresponding domain of Ca(V)1.2 led (V)to high voltage-activated channels; for these constructs the current/voltage (I/V) curves were similar to those for Ca(V)1.2 wild type. However, replacement of domain II gave only a small shift to the right of the I/V curve and modulation of the activation kinetics but did not lead to a high voltage-activating channel with an I/V curve like Ca 1.2. We also investigated the role of the voltage sensor (V)S4 by replacing the S4 segment of Ca(V)3.1 with that of Ca 1.2. For domain I, there was no shift in the I/V curve (V)as compared with Ca(V)3.1, and there were relatively small shifts to the right for domains III and IV. Taken together, these results suggest that domains I, III, and IV (rather than domain II) are apparently critical for channel opening and, therefore, contribute strongly to the difference in voltage dependence of activation between Ca 3.1 and Ca(V)1.2. However, the S4 segments in domains I, (V)III, and IV did not account for this difference in voltage dependence.

Apo calmodulin binding to the L-type voltage-gated calcium channel Cav1.2 IQ peptide.

Lian LY, Myatt D, Kitmitto A
Biochem Biophys Res Commun. 2007; 353: 565-70

The influx of calcium through the L-type voltage-gated calcium channels (LTCCs) is the trigger for the process of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum, an essential step for cardiac contraction. There are two feedback mechanisms that regulate LTCC activity: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF), both of which are mediated by calmodulin (CaM) binding. The IQ domain (aa 1645-1668) housed within the cytoplasmic domain of the LTCC Cav1.2 subunit has been shown to bind both calcium-loaded (Ca2+CaM ) and calcium-free CaM (apoCaM). Here, we provide new data for the structural basis for the interaction of apoCaM with the IQ peptide using NMR, revealing that the apoCaM C-lobe residues are most significantly perturbed upon complex formation. In addition, we have employed transmission electron microscopy of purified LTCC complexes which shows that both apoCaM and Ca2+CaM can bind to the intact channel.

Molecular determinants of Ca(2+)/calmodulin-dependent regulation of Ca(v)2.1 channels.

Lee A, Zhou H, Scheuer T, Catterall WA
Proc Natl Acad Sci U S A. 2003; 100: 16059-64

Ca2+-dependent facilitation and inactivation (CDF and CDI) of Cav2.1 channels modulate presynaptic P/Q-type Ca2+ currents and contribute to activity-dependent synaptic plasticity. This dual feedback regulation by Ca2+ involves calmodulin (CaM) binding to the alpha1 subunit (alpha12.1). The molecular determinants for Ca2+-dependent modulation of Cav2.1 channels reside in CaM and in two CaM-binding sites in the C-terminal domain of alpha12.1, the CaM-binding domain (CBD) and the IQ-like domain. In transfected tsA-201 cells, CDF and CDI were both reduced by deletion of CBD. In contrast, alanine substitution of the first two residues of the IQ-like domain (IM-AA) completely prevented CDF but had little effect on CDI, and glutamate substitutions (IM-EE) greatly accelerated voltage-dependent inactivation but did not prevent CDI. Mutational analyses of the Ca2+ binding sites of CaM showed that both the N- and C-terminal lobes of CaM were required for full development of facilitation, but only the N-terminal lobe was essential for CDI. In biochemical assays, CaM12 and CaM34 were unable to bind CBD, whereas CaM34 but not CaM12 retained Ca2+-dependent binding to the IQ-like domain. These findings support a model in which Ca2+ binding to the C-terminal EF-hands of preassociated CaM initiates CDF via interaction with the IQ-like domain. Further Ca2+ binding to the N-terminal EF-hands promotes secondary CaM interactions with CBD, which enhance facilitation and cause a conformational change that initiates CDI. This multifaceted mechanism allows positive regulation of Cav2.1 in response to local Ca2+ increases (CDF) and negative regulation during more global Ca2+ increases (CDI).

Ca2+/calmodulin-dependent facilitation and inactivation of P/Q-type Ca2+ channels.

Lee A, Scheuer T, Catterall WA
J Neurosci. 2000; 20: 6830-8

Trains of action potentials cause Ca(2+)-dependent facilitation and inactivation of presynaptic P/Q-type Ca(2+) channels that can alter synaptic efficacy. A potential mechanism for these effects involves calmodulin, which associates in a Ca(2+)-dependent manner with the pore-forming alpha(1A) subunit. Here, we report that Ca(2+) and calmodulin dramatically enhance inactivation and facilitation of P/Q-type Ca(2+) channels containing the auxiliary beta(2a) subunit compared with their relatively small effects on channels with beta(1b). Tetanic stimulation causes an initial enhancement followed by a gradual decline in P/Q-type Ca(2+) currents over time. Recovery of Ca(2+) currents from facilitation and inactivation is relatively slow (30 sec to 1 min). These effects are strongly inhibited by high intracellular BAPTA, replacement of extracellular Ca(2+) with Ba(2+), and a calmodulin inhibitor peptide. The Ca(2+)/calmodulin-dependent facilitation and inactivation of P/Q-type Ca(2+) channels observed here are consistent with the behavior of presynaptic Ca(2+) channels in neurons, revealing how dual feedback regulation of P/Q-type channels by Ca(2+) and calmodulin could contribute to activity-dependent synaptic plasticity.

Lobe specific Ca2+-calmodulin nano-domain in neuronal spines: a single molecule level analysis.

Kubota Y, Waxham MN
PLoS Comput Biol. 2010; 6: 1000987-1000987

Calmodulin (CaM) is a ubiquitous Ca(2+) buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca(2+)-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca(2+)-CaM-dependent enzymes: Ca(2+)/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca(2+) and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca(2+) ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca(2+) and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca(2+) signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca(2+)-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca(2+) channels, and to the microscopic injection rate of Ca(2+) ions. We also demonstrate that Ca(2+) saturation takes place via two different pathways depending on the Ca(2+) injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca(2+) sensors that can differentially transduce Ca(2+) influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca(2+)-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity.

PEP-19, an intrinsically disordered regulator of calmodulin signaling.

Kleerekoper QK, Putkey JA
J Biol Chem. 2009; 284: 7455-64

PEP-19 is a small calmodulin (CaM)-binding protein that greatly increases the rates of association and dissociation of Ca(2+) from the C-domain of CaM, an effect that is mediated by an acidic/IQ sequence in PEP-19. We show here using NMR that PEP-19 is an intrinsically disordered protein, but with residual structure localized to its acidic/IQ motif. We also show that the k(on) and k(off) rates for binding PEP-19 to apo-CaM are at least 50-fold slower than for binding to Ca(2+)-CaM. These data indicate that intrinsic disorder confers plasticity that allows PEP-19 to bind to either apo- or Ca(2+)-CaM via different structural modes, and that complex formation may be facilitated by conformational selection of residual structure in the acidic/IQ sequence.

Multiple C-terminal tail Ca(2+)/CaMs regulate Ca(V)1.2 function but do not mediate channel dimerization.

Kim EY, Rumpf CH, Van Petegem F, Arant RJ, Findeisen F, Cooley ES, Isacoff EY, Minor DL Jr
EMBO J. 2010; 29: 3924-38

Interactions between voltage-gated calcium channels (Ca(V)s) and calmodulin (CaM) modulate Ca(V) function. In this study, we report the structure of a Ca(2+)/CaM Ca(V)1.2 C-terminal tail complex that contains two PreIQ helices bridged by two Ca(2+)/CaMs and two Ca(2+)/CaM-IQ domain complexes. Sedimentation equilibrium experiments establish that the complex has a 2:1 Ca(2+)/CaM:C-terminal tail stoichiometry and does not form higher order assemblies. Moreover, subunit-counting experiments demonstrate that in live cell membranes Ca(V)1.2s are monomers. Thus, contrary to previous proposals, the crystallographic dimer lacks physiological relevance. Isothermal titration calorimetry and biochemical experiments show that the two Ca(2+)/CaMs in the complex have different properties. Ca(2+)/CaM bound to the PreIQ C-region is labile, whereas Ca(2+)/CaM bound to the IQ domain is not. Furthermore, neither of lobes of apo-CaM interacts strongly with the PreIQ domain. Electrophysiological studies indicate that the PreIQ C-region has a role in calcium-dependent facilitation. Together, the data show that two Ca(2+)/CaMs can bind the Ca(V)1.2 tail simultaneously and indicate a functional role for Ca(2+)/CaM at the C-region site.

Structures of CaV2 Ca2+/CaM-IQ domain complexes reveal binding modes that underlie calcium-dependent inactivation and facilitation.

Kim EY, Rumpf CH, Fujiwara Y, Cooley ES, Van Petegem F, Minor DL Jr
Structure. 2008; 16: 1455-67

Calcium influx drives two opposing voltage-activated calcium channel (Ca(V)) self-modulatory processes: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Specific Ca(2+)/calmodulin (Ca(2+)/CaM) lobes produce CDI and CDF through interactions with the Ca(V)alpha(1) subunit IQ domain. Curiously, Ca(2+)/CaM lobe modulation polarity appears inverted between Ca(V)1s and Ca(V)2s. Here, we present crystal structures of Ca(V)2.1, Ca(V)2.2, and Ca(V)2.3 Ca(2+)/CaM-IQ domain complexes. All display binding orientations opposite to Ca(V)1.2 with a physical reversal of the CaM lobe positions relative to the IQ alpha-helix. Titration calorimetry reveals lobe competition for a high-affinity site common to Ca(V)1 and Ca(V)2 IQ domains that is occupied by the CDI lobe in the structures. Electrophysiological experiments demonstrate that the N-terminal Ca(V)2 Ca(2+)/C-lobe anchors affect CDF. Together, the data unveil the remarkable structural plasticity at the heart of Ca(V) feedback modulation and indicate that Ca(V)1 and Ca(V)2 IQ domains bear a dedicated CDF site that exchanges Ca(2+)/CaM lobe occupants.

Calmodulin wraps around its binding domain in the plasma membrane Ca2+ pump anchored by a novel 18-1 motif.

Juranic N, Atanasova E, Filoteo AG, Macura S, Prendergast FG, Penniston JT, Strehler EE
J Biol Chem. 2010; 285: 4015-24

Using solution NMR spectroscopy, we obtained the structure of Ca(2+)-calmodulin (holoCaM) in complex with peptide C28 from the binding domain of the plasma membrane Ca(2+)-ATPase (PMCA) pump isoform 4b. This provides the first atomic resolution insight into the binding mode of holoCaM to the full-length binding domain of PMCA. Structural comparison of the previously determined holoCaM.C20 complex with this holoCaM.C28 complex supports the idea that the initial binding step is represented by (holoCaM.C20) and the final bound complex by (holoCaM.C28). This affirms the existing multi-step kinetic model of PMCA4b activation by CaM. The complex exhibits a new binding motif in which holoCaM is wrapped around helical C28 peptide using two anchoring residues from the peptide at relative positions 18 and 1. The anchors correspond to Phe-1110 and Trp-1093, respectively, in full-length PMCA4b, and the peptide and CaM are oriented in an anti-parallel manner. This is a greater sequence distance between anchors than in any of the known holoCaM complexes with a helical peptide. Analysis of the geometry of holoCaM-peptide binding for the cases where the target peptide adopts an alpha(D)-helix with its anchors buried in the main hydrophobic pockets of the two CaM lobes establishes that only relative sequential positions of 10, 14, 17, and 18 are allowed for the second anchor.

Lobe-related concentration- and Ca(2+)-dependent interactions of calmodulin with C- and N-terminal tails of the CaV1.2 channel.

He G, Guo F, Zhu T, Shao D, Feng R, Yin D, Sun X, Hu H, Hwang A, Minobe E, Kameyama M, Hao L
J Physiol Sci. 2013; 63: 345-53

This study examined the bindings of calmodulin (CaM) and its mutants with the C- and N-terminal tails of the voltage-gated Ca(2+) channel CaV1.2 at different CaM and Ca(2+) concentrations ([Ca(2+)]) by using the pull-down assay method to obtain basic information on the binding mode, including its concentration- and Ca(2+)-dependencies. Our data show that more than one CaM molecule could bind to the CaV1.2 C-terminal tail at high [Ca(2+)]. Additionally, the C-lobe of CaM is highly critical in sensing the change of [Ca(2+)] in its binding to the C-terminal tail of CaV1.2, and the binding between CaM and the N-terminal tail of CaV1.2 requires high [Ca(2+)]. Our data provide new details on the interactions between CaM and the CaV1.2 channel.

Calmodulin- and Ca2+-dependent facilitation and inactivation of the Cav1.2 Ca2+ channels in guinea-pig ventricular myocytes.

Han DY, Minobe E, Wang WY, Guo F, Xu JJ, Hao LY, Kameyama M
J Pharmacol Sci. 2010; 112: 310-9

The L-type Ca(2+) channel (Ca(V)1.2) shows clear Ca(2+)-dependent facilitation and inactivation. Here we have examined the effects of calmodulin (CaM) and Ca(2+) on Ca(2+) channel in guinea-pig ventricular myocytes in the inside-out patch mode, where rundown of the channels was controlled. At a free [Ca(2+)] of 0.1 microM, CaM (0.15, 0.7, 1.4, 2.1, 3.5, and 7.0 microM) + ATP (2.4 mM) induced channel activities of 27%, 98%, 142%, 222%, 65%, and 20% relative to the control activity, respectively, showing a bell-shaped relationship. Similar results were observed at a free [Ca(2+)] <0.01 microM or with a Ca(2+)-insensitive mutant, CaM(1234), suggesting that apoCaM may induce facilitation and inactivation of the channel activity. The bell-shaped curve of CaM was shifted to the lower concentration side with increasing [Ca(2+)]. A simple model for CaM- and Ca(2+)-dependent modulations of the channel activity, which involves two CaM-binding sites, was proposed. We suggest that both apoCaM and Ca(2+)/CaM can induce facilitation and inactivation of Ca(V)1.2 Ca(2+) channels and that the basic role of Ca(2+) is to accelerate CaM-dependent facilitation and inactivation.

Both N- and C-lobes of calmodulin are required for Ca2+-dependent regulations of CaV1.2 Ca2+ channels.

Guo F, Minobe E, Yazawa K, Asmara H, Bai XY, Han DY, Hao LY, Kameyama M
Biochem Biophys Res Commun. 2010; 391: 1170-6

We investigated the concentration- and Ca(2+)-dependent effects of CaM mutants, CaM(12) and CaM(34), in which Ca(2+)-binding to its N- and C-lobes was eliminated, respectively, on the Ca(V)1.2 Ca(2+) channel by inside-out patch clamp in guinea-pig cardiomyocytes. Both CaM(12) and CaM(34) (0.7-10muM) applied with 3mM ATP produced channel activity after "rundown". Concentration-response curves were bell-shaped, similar to that for wild-type CaM. However, there was no obvious leftward shift of the curves by increasing [Ca(2+)], suggesting that both functional lobes of CaM were necessary for the Ca(2+)-dependent shift. However, channel activity induced by the CaM mutants showed Ca(2+)-dependent decrease, implying a Ca(2+) sensor existing besides CaM. These results suggest that both N- and C-lobes of CaM are required for the Ca(2+)-dependent regulations of Ca(V)1.2 Ca(2+) channels.

Regulation of the NaV1.5 cytoplasmic domain by calmodulin.

Gabelli SB, Boto A, Kuhns VH, Bianchet MA, Farinelli F, Aripirala S, Yoder J, Jakoncic J, Tomaselli GF, Amzel LM
Nat Commun. 2014; 5: 5126-5126

Voltage-gated sodium channels (Na(v)) underlie the rapid upstroke of action potentials in excitable tissues. Binding of channel-interactive proteins is essential for controlling fast and long-term inactivation. In the structure of the complex of the carboxy-terminal portion of Na(v)1.5 (CTNa(v)1.5) with calmodulin (CaM)-Mg(2+) reported here, both CaM lobes interact with the CTNa(v)1.5. On the basis of the differences between this structure and that of an inactivated complex, we propose that the structure reported here represents a non-inactivated state of the CTNa(v), that is, the state that is poised for activation. Electrophysiological characterization of mutants further supports the importance of the interactions identified in the structure. Isothermal titration calorimetry experiments show that CaM binds to CTNa(v)1.5 with high affinity. The results of this study provide unique insights into the physiological activation and the pathophysiology of Na(v) channels.

Structural and energetic determinants of apo calmodulin binding to the IQ motif of the Na(V)1.2 voltage-dependent sodium channel.

Feldkamp MD, Yu L, Shea MA
Structure. 2011; 19: 733-47

The neuronal voltage-dependent sodium channel (Na(v)1.2), essential for generation and propagation of action potentials, is regulated by calmodulin (CaM) binding to the IQ motif in its alpha subunit. A peptide (Na(v)1.2(IQp), KRKQEEVSAIVIQRAYRRYLLKQKVKK) representing the IQ motif had higher affinity for apo CaM than (Ca(2+))(4)-CaM. Association was mediated solely by the C-domain of CaM. A solution structure (2KXW.pdb) of apo (13)C,(15)N-CaM C-domain bound to Na(v)1.2(IQp) was determined with NMR. The region of Na(v)1.2(IQp) bound to CaM was helical; R1902, an Na(v)1.2 residue implicated in familial autism, did not contact CaM. The apo C-domain of CaM in this complex shares features of the same domain bound to myosin V IQ motifs (2IX7) and bound to an SK channel peptide (1G4Y) that does not contain an IQ motif. Thermodynamic and structural studies of CaM-Na(v)1.2(IQp) interactions show that apo and (Ca(2+))(4)-CaM adopt distinct conformations that both permit tight association with Na(v)1.2(IQp) during gating.

Mechanism of auxiliary beta-subunit-mediated membrane targeting of L-type (Ca(V)1.2) channels.

Fang K, Colecraft HM
J Physiol. 2011; 589: 4437-55

Ca(2+) influx via Ca(V)1/Ca(V)2 channels drives processes ranging from neurotransmission to muscle contraction. Association of a pore-forming alpha(1) and cytosolic beta is necessary for trafficking Ca(V)1/Ca(V)2 channels to the cell surface through poorly understood mechanisms. A prevalent idea suggests beta binds the alpha(1) intracellular I-II loop, masking an endoplasmic reticulum (ER) retention signal as the dominant mechanism for Ca(V)1/Ca(V)2 channel membrane trafficking. There are hints that other alpha(1) subunit cytoplasmic domains may play a significant role, but the nature of their potential contribution is unclear. We assessed the roles of all intracellular domains of Ca(V)1.2-alpha(1C) by generating chimeras featuring substitutions of all possible permutations of intracellular loops/termini of alpha(1C) into the beta-independent Ca(V)3.1-alpha(1G) channel. Surprisingly, functional analyses demonstrated alpha(1C) I-II loop strongly increases channel surface density while other cytoplasmic domains had a competing opposing effect. Alanine-scanning mutagenesis identified an acidic-residue putative ER export motif responsible for the I-II loop-mediated increase in channel surface density. beta-dependent increase in current arose as an emergent property requiring four alpha(1C) intracellular domains, with the I-II loop and C-terminus being essential. The results suggest beta binding to the alpha(1C) I-II loop causes a C-terminus-dependent rearrangement of intracellular domains, shifting a balance of power between export signals on the I-II loop and retention signals elsewhere.

Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway.

Dolmetsch RE, Pajvani U, Fife K, Spotts JM, Greenberg ME
Science. 2001; 294: 333-9

Increases in the intracellular concentration of calcium ([Ca2+]i) activate various signaling pathways that lead to the expression of genes that are essential for dendritic development, neuronal survival, and synaptic plasticity. The mode of Ca2+ entry into a neuron plays a key role in determining which signaling pathways are activated and thus specifies the cellular response to Ca2+. Ca2+ influx through L-type voltage-activated channels (LTCs) is particularly effective at activating transcription factors such as CREB and MEF-2. We developed a functional knock-in technique to investigate the features of LTCs that specifically couple them to the signaling pathways that regulate gene expression. We found that an isoleucine-glutamine ("IQ") motif in the carboxyl terminus of the LTC that binds Ca2+-calmodulin (CaM) is critical for conveying the Ca2+ signal to the nucleus. Ca2+-CaM binding to the LTC was necessary for activation of the Ras/mitogen-activated protein kinase (MAPK) pathway, which conveys local Ca2+ signals from the mouth of the LTC to the nucleus. CaM functions as a local Ca2+ sensor at the mouth of the LTC that activates the MAPK pathway and leads to the stimulation of genes that are essential for neuronal survival and plasticity.

The cardiac L-type calcium channel distal carboxy terminus autoinhibition is regulated by calcium.

Crump SM, Andres DA, Sievert G, Satin J
Am J Physiol Heart Circ Physiol. 2013; 304: 45564-45564

The L-type calcium channel (LTCC) provides trigger Ca(2+) for sarcoplasmic reticulum Ca-release, and LTCC function is influenced by interacting proteins including the LTCC distal COOH terminus (DCT) and calmodulin. DCT is proteolytically cleaved and reassociates with the LTCC complex to regulate calcium channel function. DCT reduces LTCC barium current (I(Ba,L)) in reconstituted channel complexes, yet the contribution of DCT to LTCC Ca(2+) current (I(Ca,L)) in cardiomyocyte systems is unexplored. This study tests the hypothesis that DCT attenuates cardiomyocyte I(Ca,L). We measured LTCC current and Ca(2+) transients with DCT coexpressed in murine cardiomyocytes. We also heterologously coexpressed DCT and Ca(V)1.2 constructs with truncations corresponding to the predicted proteolytic cleavage site, Ca(V)1.2Delta1801, and a shorter deletion corresponding to well-studied construct, Ca(V)1.2Delta1733. DCT inhibited I(Ba,L) in cardiomyocytes, and in human embryonic kidney (HEK) 293 cells expressing Ca(V)1.2Delta1801 and Ca(V)1.2Delta1733. Ca(2+)-CaM relieved DCT block in cardiomyocytes and HEK cells. The selective block of I(Ba,L) combined with Ca(2+)-CaM effects suggested that DCT-mediated blockade may be relieved under conditions of elevated Ca(2+). We therefore tested the hypothesis that DCT block is dynamic, increasing under relatively low Ca(2+), and show that DCT reduced diastolic Ca(2+) at low stimulation frequencies but spared high frequency Ca(2+) entry. DCT reduction of diastolic Ca(2+) and relief of block at high pacing frequencies and under conditions of supraphysiological bath Ca(2+) suggests that a physiological function of DCT is to increase the dynamic range of Ca(2+) transients in response to elevated pacing frequencies. Our data motivate the new hypothesis that DCT is a native reverse use-dependent inhibitor of LTCC current.

The C2A domain of synaptotagmin alters the kinetics of voltage-gated Ca2+ channels Ca(v)1.2 (Lc-type) and Ca(v)2.3 (R-type).

Cohen R, Elferink LA, Atlas D
J Biol Chem. 2003; 278: 9258-66

Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.

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

Blaich A, Pahlavan S, Tian Q, Oberhofer M, Poomvanicha M, Lenhardt P, Domes K, Wegener JW, Moosmang S, Ruppenthal S, Scholz A, Lipp P, Hofmann F
J Biol Chem. 2012; 287: 22616-25

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.

Variations at the semiconserved glycine in the IQ domain consensus sequence have a major impact on Ca2+-dependent switching in calmodulin-IQ domain complexes.

Black DJ, Persechini A
Biochemistry. 2010; 49: 78-83

We have replaced the semiconserved Gly in the IQ domain consensus sequence with Ala, Arg, or Met in a reference sequence and determined how this affects its complexes with calmodulin. The K(d) for the Ca(2+)-free reference complex is 2.4 +/- 0.3 microM. The Ala and Arg replacements increase this to 5.4 +/- 0.4 and 6.2 +/- 0.5 microM, while the Met increases it to 26.4 +/- 2.5 microM. When Ca(2+) is bound to both calmodulin lobes, the K(d) for the reference complex is not significantly affected, but the K(d) for the Ala variant decreases to 0.9 +/- 0.04 microM, and the values for the Arg and Met variants decrease to 0.4 +/- 0.03 microM. Using mutant calmodulins, we defined the effect of Ca(2+) binding to each lobe, with the C-terminal preceding the N-terminal (C-->N) or vice versa (N-->C). In the C-->N order the first step increases the reference K(d) approximately 5-fold, while it decreases the values for the variants approximately 2- to approximately 10-fold. The second step decreases the K(d) values for the all of the complexes approximately 5-fold, suggesting that the N-terminal lobe does not interact with the semiconserved position after the first step. In the N-->C order the first step increases the K(d) values for the reference complex and Met and Ala variants approximately 15- to approximately 200-fold but does not affect the value for the Arg variant. The second step decreases the K(d) values for the reference and Arg variant approximately 10- and approximately 15-fold and the Ala and Met variants approximately 2000-fold. Thus, both steps in the N-->C order are sensitive to variations at the semiconserved position, while only the first is in the C-->N order. Due to energy coupling, this order is followed under equilibrium conditions.

The IQ domains in neuromodulin and PEP19 represent two major functional classes.

Black DJ, LaMartina D, Persechini A
Biochemistry. 2009; 48: 11766-72

The affinities of Ca(2+)-saturated and Ca(2+)-free calmodulin for a fluorescent reporter construct containing the PEP19 IQ domain differ by a factor of approximately 100, with K(d) values of 11.0 +/- 1.2 and 1128.4 +/- 176.5 muM, respectively, while the affinities of a reporter containing the neuromodulin IQ domain are essentially identical, with K(d) values of 2.9 +/- 0.3 and 2.4 +/- 0.3 muM, respectively. When Ca(2+) is bound only to the C-terminal pair of Ca(2+)-binding sites in calmodulin, the K(d) value for the PEP19 reporter complex is decreased approximately 5-fold, while the value for the neuromodulin reporter complex is increased by the same factor. When Ca(2+) is bound only to the N-terminal pair of Ca(2+)-binding sites, the K(d) value for the PEP19 reporter complex is unaffected, but the value for the complex with the neuromodulin reporter is increased approximately 12-fold. These functional differences are largely ascribed to three differences in the CaM-binding sequences of the two reporters. Replacement of a central Gly in the neuromodulin IQ domain with a Lys at this position in PEP19 almost entirely accounts for the distinctive patterns of Ca(2+)-dependent stability changes exhibited by the two complexes. Replacement of a Lys immediately before the "IQ" amino acid pair in the neuromodulin sequence with the Ala in PEP19 accounts for the remaining Ca(2+)-dependent differences. Replacement of an Ala in the N-terminal half of the neuromodulin sequence with the Gln in PEP19 accounts for approximately half of the Ca(2+)-independent difference in the stabilities of the two reporter complexes, with the Ca(2+)-independent effect of the Lys replacement accounting for most of the remainder. Since the central Gly in the neuromodulin sequence is conserved in half of all known IQ domains, these results suggest that the presence or absence of this residue defines two major functional classes.

Engineered calmodulins reveal the unexpected eminence of Ca2+ channel inactivation in controlling heart excitation.

Alseikhan BA, DeMaria CD, Colecraft HM, Yue DT
Proc Natl Acad Sci U S A. 2002; 99: 17185-90

Engineered calmodulins (CaMs), rendered Ca2+-insensitive by mutations, function as dominant negatives in heterologous systems, and have revealed mechanisms of ion channel modulation by Ca2+/CaM. The use of these CaMs in native mammalian cells now emerges as a strategy to unmask the biology of such Ca2+ feedback. Here, we developed recombinant adenoviruses bearing engineered CaMs to facilitate their expression in adult heart cells, where Ca2+ regulation may be essential for moment-to-moment control of the heartbeat. Engineered CaMs not only eliminated the Ca2+-dependent inactivation of native calcium channels, but exposed an unexpectedly large impact of removing such feedback: the unprecedented (4- to 5-fold) prolongation of action potentials. This striking result recasts the basic paradigm for action-potential control and illustrates the promise of virally delivered engineered CaM to investigate the biology of numerous other CaM-signaling pathways.

Pivoting between calmodulin lobes triggered by calcium in the Kv7.2/calmodulin complex.

Alaimo A, Alberdi A, Gomis-Perez C, Fernandez-Orth J, Bernardo-Seisdedos G, Malo C, Millet O, Areso P, Villarroel A
PLoS One. 2014; 9: 86711-86711

Kv7.2 (KCNQ2) is the principal molecular component of the slow voltage gated M-channel, which strongly influences neuronal excitability. Calmodulin (CaM) binds to two intracellular C-terminal segments of Kv7.2 channels, helices A and B, and it is required for exit from the endoplasmic reticulum. However, the molecular mechanisms by which CaM controls channel trafficking are currently unknown. Here we used two complementary approaches to explore the molecular events underlying the association between CaM and Kv7.2 and their regulation by Ca(2+). First, we performed a fluorometric assay using dansylated calmodulin (D-CaM) to characterize the interaction of its individual lobes to the Kv7.2 CaM binding site (Q2AB). Second, we explored the association of Q2AB with CaM by NMR spectroscopy, using (15)N-labeled CaM as a reporter. The combined data highlight the interdependency of the N- and C-lobes of CaM in the interaction with Q2AB, suggesting that when CaM binds Ca(2+) the binding interface pivots between the N-lobe whose interactions are dominated by helix B and the C-lobe where the predominant interaction is with helix A. In addition, Ca(2+) makes CaM binding to Q2AB more difficult and, reciprocally, the channel weakens the association of CaM with Ca(2+).