Inhibition of human ether à go-go potassium channels by Ca(2+)/calmodulin

Intracellular Ca(2+) inhibits voltage-gated potassium channels of the ether à go-go (EAG) family. To identify the underlying molecular mechanism, we expressed the human version hEAG1 in XENOPUS: oocytes. The channels lost Ca(2+) sensitivity when measured in cell-free membrane patches. However, Ca(2+) sensitivity could be restored by application of recombinant calmodulin (CaM). In the presence of CaM, half inhibition of hEAG1 channels was obtained in 100 nM Ca(2+). Overlay assays using labelled CaM and glutathione S-transferase (GST) fusion fragments of hEAG1 demonstrated direct binding of CaM to a C-terminal domain (hEAG1 amino acids 673-770). Point mutations within this section revealed a novel CaM-binding domain putatively forming an amphipathic helix with both sides being important for binding. The binding of CaM to hEAG1 is, in contrast to Ca(2+)-activated potassium channels, Ca(2+) dependent, with an apparent K(D) of 480 nM. Co-expression experiments of wild-type and mutant channels revealed that the binding of one CaM molecule per channel complex is sufficient for channel inhibition.     

Calmodulin binding to the Fas death domain. Regulation by Fas activation

Fas (APO-1/CD95) is a cell surface receptor that initiates apoptotic pathways, and its cytoplasmic domain interacts with various molecules suggesting that Fas signaling is complex and regulated by multiple proteins. Calmodulin (CaM) is an intracellular Ca(2+)-binding protein, and it mediates many of the effects of Ca2+. Here, we demonstrate that CaM binds to Fas directly and identify the CaM-binding site on the cytoplasmic death domain (DD) of Fas. Fas binds to CaM-Sepharose and is co-immunoprecipitated with CaM. Other death receptors, such as tumor necrosis factor receptor, DR4, and DR5 do not bind to CaM. The interaction between Fas and CaM is Ca(2+)-dependent. Deletion mapping analysis with various GST-fused Fas cytoplasmic domain fragments revealed that the fragment containing helices 1, 2, and 3 of the Fas DD has the CaM-binding ability. Sequence analysis of this fragment predicted a potential CaM-binding site in helix 2 and connected loops. A valine 254 to asparagine mutation in this region, which is analogous to the identified mutant allele of Fas in lpr mice that have a deficiency in Fas-mediated apoptosis, showed reduced CaM binding. Computer modeling of the interaction between CaM and helix 2 of the Fas DD predicted that amino acids, which are important for Fas-CaM binding, and point mutations of these amino acids caused reduced Fas-CaM binding. The interaction between Fas and CaM is increased approximately 2-fold early upon Fas activation (at 30 min) and is decreased to approximately 50% of control at 2 h. These findings suggest a novel function of CaM in Fas-mediated apoptosis.     

Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+/calmodulin

Calmodulin modulation of ion channels has emerged as a prominent theme in biology. The sensitivity of KCNQ1-5 K+ channels to modulation by Ca2+/calmodulin (CaM) was studied using patch-clamp, Ca2+ imaging, and biochemical and pharmacological approaches. Coexpression of CaM in Chinese hamster ovary (CHO) cells strongly reduced currents of KCNQ2, KCNQ4, and KCNQ5, but not KCNQ1 or KCNQ3. In simultaneous current recording/Ca2+ imaging experiments, CaM conferred Ca2+ sensitivity to KCNQ4 and KCNQ5, but not to KCNQ1, KCNQ3, or KCNQ1/KCNE1 channels. A chimera constructed from the carboxy terminus of KCNQ4 and the rest KCNQ1 displayed Ca2+ sensitivity similar to KCNQ4. Chimeras constructed from different lengths of the KCNQ4 carboxy terminal and the rest KCNQ3 localized a region that confers sensitivity to Ca2+/CaM. Lobe-specific mutations of CaM revealed that its amino-terminal lobe mediates the Ca2+ sensitivity of the KCNQ/CaM complex. The site of CaM action within the channel carboxy terminus overlaps with that of the KCNQ opener N-ethylmaleimide (NEM). We found that CaM overexpression reduced NEM augmentation of KCNQ2, KCNQ4, and KCNQ5, and NEM pretreatment reduced Ca2+/CaM-mediated suppression of M current in sympathetic neurons by bradykinin. We propose that two functionally distinct types of carboxy termini underlie the observed differences among this channel family.     

Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases

The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca(2+)-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca(2+) with mutations in the N-lobe (CaM(12)), the C-lobe (CaM(34)), or both lobes of CaM (CaM(1234)) to determine their effect on the binding and activation of the Ca(2+)-dependent neuronal (nNOS) and Ca(2+)-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein-protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca(2+). Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.     

Structural uncoupling between opposing domains of oxidized calmodulin underlies the enhanced binding affinity and inhibition of the plasma membrane Ca-ATPase

Stabilization of the plasma membrane Ca-ATPase (PMCA) in an inactive conformation upon oxidation of multiple methionines in the calcium regulatory protein calmodulin (CaM) is part of an adaptive cellular response to minimize ATP utilization and the generation of reactive oxygen species (ROS) under conditions of oxidative stress. To differentiate oxidant-induced structural changes that selectively modify the amino-terminal domain of CaM from those that modulate the conformational coupling between the opposing domains, we have engineered a tetracysteine binding motif within helix A in the amino-terminal domain of calmodulin (CaM) that permits the selective and rigid attachment of the conformationally sensitive fluorescent probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)(2) (FlAsH-EDT(2)). The position of the FlAsH label in the amino-terminal domain provides a signal for monitoring its binding to the CaM-binding sequence of the PMCA. Following methionine oxidation, there is an enhanced binding affinity between the amino-terminal domain and the CaM-binding sequence of the PMCA. To identify oxidant-induced structural changes, we used frequency domain fluorescence anisotropy measurements to assess the structural coupling between helix A and the amino- and carboxyl-terminal domains of CaM. Helix A undergoes large amplitude motions in apo-CaM; following calcium activation, helix A is immobilized as part of a conformational switch that couples the opposing domains of CaM to stabilize the high-affinity binding cleft associated with target protein binding. Methionine oxidation disrupts the structural coupling between opposing globular domains of CaM, without affecting the calcium-dependent immobilization of helix A associated with activation of the amino-terminal domain to promote high-affinity binding to target proteins. We suggest that this selective disruption of the structural linkage between the opposing globular domains of CaM relieves steric constraints associated with high-affinity target binding, permitting the formation of new contact interactions between the amino-terminal domain and the CaM-binding sequence that stabilizes the PMCA in an inhibited conformation.     

Small-angle X-ray scattering study of calmodulin bound to two peptides corresponding to parts of the calmodulin-binding domain of the plasma membrane Ca2+ pump.

The interaction between calmodulin (CaM) and two synthetic peptides, C20W and C24W, corresponding to parts of the calmodulin-binding domain of the Ca2+ pump of human erythrocytes, has been studied by using small-angle X-ray scattering (SAXS). The total length of the CaM-binding domain of the enzyme is estimated to be 28 amino acids. C20W contains the 20 N-terminal amino acids of this domain, C24W the 24 C-terminal amino acids. The experiments have shown that the binding of either peptide results in a complex with a radius of gyration (Rg) smaller than that of CaM. The complex between CaM and C20W revealed an interatomic length distribution function, P(r), similar to that of calmodulin alone, indicating that the complex retains an extended, dumbbell-shaped structure. By contrast, the binding of C24W resulted in the formation of a globular structure similar to those observed with many other CaM-binding peptides.     

Pivoting between Calmodulin Lobes Triggered by Calcium in the Kv7.2/Calmodulin Complex

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 Ca²⁺. 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 ¹⁵N-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 Ca²⁺ 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, Ca²⁺ makes CaM binding to Q2AB more difficult and, reciprocally, the channel weakens the association of CaM with Ca²⁺.     

Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit from the Endoplasmic Reticulum

The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K⁺ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an “active” conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca²⁺ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca²⁺ sensor, conferring Ca²⁺ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.M-type channels are generated by the KCNQ (Kv7) family of voltage-gated subtypes (1), and they are found throughout the nervous system where they fulfill dominant roles in the control of excitability and neural discharges (2). Like all Kv channels, the KCNQ α subunits share a common core structure of six transmembrane segments with a voltage sensing domain (S1–S4) and a pore domain (S5 and S6; see Fig. 1) (3). Sequence analysis predicts the presence of four helical regions (A–D) in all family members (4), and helices A and B constitute the binding site for calmodulin (CaM).⁸ CaM is a prototypical Ca²⁺ sensor that confers Ca²⁺ sensitivity to a wide array of proteins, including ion channels (5). CaM is thought to mediate Ca²⁺-dependent inhibition of KCNQ channels (6), and in addition, we have postulated that a direct association with CaM is required for KCNQ2 channels to exit the endoplasmic reticulum (7).Open in a separate windowFIGURE 1.Topological representation of a KCNQ subunit. The consensus IQ residues are shown in bold. Circles and squares correspond to the residues mutated here (the squares indicate the mutations causing BFNC). The boxes indicate the regions with a high probability of adopting an α helix configuration, and the thick lines delineate the region fused to GST.To gain a deeper understanding into the involvement of CaM in channel trafficking, we have studied this interaction in vitro with a set of CaM-binding site-specific mutants. Although we had previously explored the impact of some of these mutants on channel trafficking (7), here we have extended this study to the mutant L339R located in helix A. This mutation, as well as the R353G mutation in helix A, has been linked to benign familial neonatal convulsions (BFNC), a human epileptic syndrome of newborn children (2). Accordingly, we demonstrate that there is a strong correlation between the impact of these mutations on the adoption of an “active” conformation by CaM and channel subunit exit from the ER, lending further support to the concept that CaM is a critical regulator for the exit of the channel from the ER.     

Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein

Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.     

Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation

TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.     

Dynamic light scattering study of calmodulin-target peptide complexes

Dynamic light scattering (DLS) has been used to assess the influence of eleven different synthetic peptides, comprising the calmodulin (CaM)-binding domains of various CaM-binding proteins, on the structure of apo-CaM (calcium-free) and Ca(2+)-CaM. Peptides that bind CaM in a 1:1 and 2:1 peptide-to-protein ratio were studied, as were solutions of CaM bound simultaneously to two different peptides. DLS was also used to investigate the effect of Ca(2+) on the N- and C-terminal CaM fragments TR1C and TR2C, and to determine whether the two lobes of CaM interact in solution. The results obtained in this study were comparable to similar solution studies performed for some of these peptides using small-angle x-ray scattering. The addition of Ca(2+) to apo-CaM increased the hydrodynamic radius from 2.5 to 3.0 nm. The peptides studied induced a collapse of the elongated Ca(2+)-CaM structure to a more globular form, decreasing its hydrodynamic radius by an average of 25%. None of the peptides had an effect on the conformation of apo-CaM, indicating that either most of the peptides did not interact with apo-CaM, or if bound, they did not cause a large conformational change. The hydrodynamic radii of TR1C and TR2C CaM fragments were not significantly affected by the addition of Ca(2+). The addition of a target peptide and Ca(2+) to the two fragments of CaM, suggest that a globular complex is forming, as has been seen in nuclear magnetic resonance solution studies. This work demonstrates that dynamic light scattering is an inexpensive and efficient technique for assessing large-scale conformational changes that take place in calmodulin and related proteins upon binding of Ca(2+) ions and peptides, and provides a qualitative picture of how this occurs. This work also illustrates that DLS provides a rapid screening method for identifying new CaM targets.     

Thermodynamics of apocalmodulin and nitric oxide synthase II peptide interaction

The Ca2+-free form of calmodulin (CaM), apocalmodulin (ApoCaM), regulates a variety of target proteins including nitric oxide synthase II (NOS-II). The CaM-binding site of NOS-II can bind ApoCaM with high affinity. Substitution of hydrophobic amino acids by charged amino acids at crucial positions 3, 9 and 13 within the CaM-binding motif did not abolish the ApoCaM interaction that occurred with significant affinity, though the affinity of the interaction was decreased remarkably. Isothermal titration calorimetry revealed that interaction of ApoCaM and synthetic NOS-II peptides was driven entropically.     

Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels

To quantify the modulation of KCNQ2/3 current by [Ca2+]i and to test if calmodulin (CaM) mediates this action, simultaneous whole-cell recording and Ca2+ imaging was performed on CHO cells expressing KCNQ2/3 channels, either alone, or together with wild-type (wt) CaM, or dominant-negative (DN) CaM. We varied [Ca2+]i from <10 to >400 nM with ionomycin (5 microM) added to either a 2 mM Ca2+, or EGTA-buffered Ca2+-free, solution. Coexpression of wt CaM made KCNQ2/3 currents highly sensitive to [Ca2+]i (IC50 70 +/- 20 nM, max inhibition 73%, n = 10). However, coexpression of DN CaM rendered KCNQ2/3 currents largely [Ca2+]i insensitive (max inhibition 8 +/- 3%, n = 10). In cells without cotransfected CaM, the Ca2+ sensitivity was variable but generally weak. [Ca2+]i modulation of M current in superior cervical ganglion (SCG) neurons followed the same pattern as in CHO cells expressed with KCNQ2/3 and wt CaM, suggesting that endogenous M current is also highly sensitive to [Ca2+]i. Coimmunoprecipitations showed binding of CaM to KCNQ2-5 that was similar in the presence of 5 mM Ca2+ or 5 mM EGTA. Gel-shift analyses suggested Ca2+-dependent CaM binding to an "IQ-like" motif present in the carboxy terminus of KCNQ2-5. We tested whether bradykinin modulation of M current in SCG neurons uses CaM. Wt or DN CaM was exogenously expressed in SCG cells using pseudovirions or the biolistic "gene gun." Using both methods, expression of both wt CaM and DN CaM strongly reduced bradykinin inhibition of M current, but for all groups muscarinic inhibition was unaffected. Cells expressed with wt CaM had strongly reduced tonic current amplitudes as well. We observed similar [Ca2+]i rises by bradykinin in all the groups of cells, indicating that CaM did not affect Ca2+ release from stores. We conclude that M-type currents are highly sensitive to [Ca2+]i and that calmodulin acts as their Ca2+ sensor.     

The plant plasma membrane Ca2+ pump ACA8 contains overlapping as well as physically separated autoinhibitory and calmodulin-binding domains

In plant Ca(2+) pumps belonging to the P(2B) subfamily of P-type ATPases, the N-terminal cytoplasmic domain is responsible for pump autoinhibition. Binding of calmodulin (CaM) to this region results in pump activation but the structural basis for CaM activation is still not clear. All residues in a putative CaM-binding domain (Arg(43) to Lys(68)) were mutagenized and the resulting recombinant proteins were studied with respect to CaM binding and the activation state. The results demonstrate that (i) the binding site for CaM is overlapping with the autoinhibitory region and (ii) the autoinhibitory region comprises significantly fewer residues than the CaM-binding region. In a helical wheel projection of the CaM-binding domain, residues involved in autoinhibition cluster on one side of the helix, which is proposed to interact with an intramolecular receptor site in the pump. Residues influencing CaM negatively are situated on the other face of the helix, likely to face the cytosol, whereas residues controlling CaM binding positively are scattered throughout. We propose that early CaM recognition is mediated by the cytosolic face and that CaM subsequently competes with the intramolecular autoinhibitor in binding to the other face of the helix.     

Neurotransmitter modulation of small-conductance Ca2+-activated K+ channels by regulation of Ca2+ gating

Small-conductance Ca2+-activated K+ (SK) channels are widely expressed in neuronal tissues where they underlie post-spike hyperpolarizations, regulate spike-frequency adaptation, and shape synaptic responses. SK channels constitutively interact with calmodulin (CaM), which serves as Ca2+ sensor, and with protein kinase CK2 and protein phosphatase 2A, which modulate their Ca2+ gating. By recording coupled activities of Ca2+ and SK2 channels, we showed that SK2 channels can be inhibited by neurotransmitters independently of changes in the activity of the priming Ca2+ channels. This inhibition involvesSK2-associated CK2 and results from a 3-fold reduction in the Ca2+ sensitivity of channel gating. CK2phosphorylated SK2-bound CaM but not KCNQ2-bound CaM, thereby selectively regulating SK2 channels. We extended these observations to sensory neurons by showing that noradrenaline inhibits SK current and increases neuronal excitability in aCK2-dependent fashion. Hence, neurotransmitter-initiated signaling cascades can dynamically regulate Ca2+ sensitivity of SK channels and directly influence somatic excitability.     

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.     

Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase.

We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions.     

Conformation of the calmodulin-binding domain of metabotropic glutamate receptor subtype 7 and its interaction with calmodulin

Calmodulin (CaM), a Ca(2+)-binding protein, is a well-known regulator of various cellular functions. One of the targets of CaM is metabotropic glutamate receptor 7 (mGluR7), which serves as a low-pass filter for glutamate in the pre-synaptic terminal to regulate neurotransmission. Surface plasmon resonance (SPR), circular dichroism (CD) spectroscopy and nuclear magnetic spectroscopy (NMR) were performed to study the structure of the peptides corresponding to the CaM-binding domain of mGluR7 and their interaction with CaM. Unlike well-known CaM-binding peptides, mGluR7 has a random coil structure even in the presence of trifluoroethanol. Moreover, NMR data suggested that the complex between Ca(2+)/CaM and the mGluR7 peptide has multiple conformations. The mGluR7 peptide has been found to interact with CaM even in the absence of Ca(2+), and the binding is directed toward the C-domain of apo-CaM rather than the N-domain. We propose a possible mechanism for the activation of mGluR7 by CaM. A pre-binding occurs between apo-CaM and mGluR7 in the resting state of cells. Then, the Ca(2+)/CaM-mGluR7 complex is formed once Ca(2+) influx occurs. The weak interaction at lower Ca(2+) concentrations is likely to bind CaM to mGluR7 for the fast complex formation in response to the elevation of Ca(2+) concentration.     

Functionally important calmodulin-binding sites in both NH2- and COOH-terminal regions of the cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit

Whereas an important aspect of sensory adaptation in rod photoreceptors and olfactory receptor neurons is thought to be the regulation of cyclic nucleotide-gated (CNG) channel activity by calcium-calmodulin (Ca2+-CaM), it is not clear that cone photoreceptor CNG channels are similarly modulated. Cone CNG channels are composed of at least two different subunit types, CNGA3 and CNGB3. We have investigated whether calmodulin modulates the activity of these channels by direct binding to the CNGB3 subunit. Heteromeric channels were formed by co-expression of human CNGB3 with human CNGA3 subunits in Xenopus oocytes; CNGB3 subunits conferred sensitivity to regulation by Ca2+-CaM, whereas CaM regulation of homomeric CNGA3 channels was not detected. To explore the mechanism underlying this regulation, we localized potential CaM-binding sites in both NH2- and COOH-terminal cytoplasmic domains of CNGB3 using gel-overlay and glutathione S-transferase pull-down assays. For both sites, binding of CaM depended on the presence of Ca2+. Individual deletions of either CaM-binding site in CNGB3 generated channels that remained sensitive to regulation by Ca2+-CaM, but deletion of both together resulted in heteromeric channels that were not modulated. Thus, both NH2- and COOH-terminal CaM-binding sites in CNGB3 are functionally important for regulation of recombinant cone CNG channels. These studies suggest a potential role for direct binding and unbinding of Ca2+-CaM to human CNGB3 during cone photoreceptor adaptation and recovery.     

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

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.