Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation.

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase. CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca2+ dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca2+ affinity 8- to 100-fold and slow dissociation kinetics 13- to 132-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca2+ affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca2(2+)-CaM or Ca4(2+)-CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C-terminal domain or the dissociation of Ca2+ loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.     

Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins.

The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3'-5'-cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca2+-ATPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca2+-calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca2+-ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (Kact). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased the Vmax. For MLCK, however, neither Vmax nor Kact were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides.     

Al(3+) interaction sites of calmodulin and the Al(3+) effect on target binding of calmodulin

The interaction between calmodulin (CaM) and Al(3+) was studied by spectroscopic methods. Heteronuclear two-dimensional NMR data indicated that peaks related to the both lobes and middle of the central helix of CaM are largely affected by Al(3+). But chemical shift perturbation suggested that overall conformation of Ca(2+)-loaded CaM is not changed by Al(3+) binding. It is thought that Al(3+) interaction to the middle of the central helix is a key for the property of CaM's target recognition. If the structure and/or flexibility of the central helix are/is changed by Al(3+), target affinity to CaM must be influenced by Al(3+). Thus, we performed surface plasmon resonance experiments to observe the effect of Al(3+) on the target recognition by CaM. The data clearly indicated that target affinity to CaM is reduced by addition of Al(3+). All the results presented here support a hypothesis that Al(3+) may affect on the Ca(2+) signaling pathway in cells.     

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

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.     

Mastoparan/Mastoparan X altered binding behavior of La3+ to calmodulin in ternary complexes

Ca(2+) binds to calmodulin (CaM) and triggers the interaction of CaM with its target proteins; CaM binding proteins (CaMBPs) can also regulate the metal binding to CaM. In the present paper, La(3+) binding to CaM was studied in the presence of the CaM binding peptides, Mastoparan (Mas) and Mas X, using ultrafiltration and titration of fluorescence. Ca(2+) binding was used as an analog to understand La(3+) binding in intact CaM and isolated N/C-terminal CaM domain of metal-CaM binary system and metal-CaM-CaMBPs ternary system. Mas/Mas X increased binding affinity of La(3+) to CaM by 0.5 approximately 3 orders magnitude. The metal ions binding affinity to the C-terminal or the N-terminal CaM domain suggested that in the first phase of binding process both Ca(2+) and La(3+) bind to C-terminal of CaM in the presence of Mas/Mas X. In the presence of CaM binding peptides, La(3+) binding preference was substantially altered from the metal-CaM binary system where La(3+) slightly preferred binding to the N-terminal sites of CaM. Our results will be helpful in understanding La(3+) interactions with CaM in the biological systems.     

Binding of La3+ to calmodulin and its effects on the interaction between calmodulin and calmodulin binding peptide, polistes mastoparan

Binding of La(3+) to calmodulin (CaM) and its effects on the complexes of CaM and CaM-binding peptide, polistes mastoparan (Mas), were investigated by nuclear magnetic resonance (NMR) spectroscopy, fluorescence and circular dichroism spectroscopy, and by the fluorescence stopped-flow method. The four binding sites of La(3+) on CaM were identified as the same as the binding sites of Ca(2+) on CaM through NMR titration of La(3+) to uniformly (15)N-labeled CaM. La(3+) showed a slightly higher affinity to the binding sites on the N-terminal domain of CaM than that to the C-terminal. Large differences between the (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of Ca(4)CaM and La(4)CaM suggest conformational differences between the two complexes. Fluorescence and CD spectra also exhibited structural differences. In the presence of Ca(2+) and La(3+), a hybrid complex, Ca(2)La(2)CaM, was formed, and the binding of La(3+) to the N-terminal domain of CaM seemed preferable over binding to the C-terminal domain. Through fluorescence titration, it was shown that La(4)CaM and Ca(2)La(2)CaM had similar affinities to Mas as Ca(4)CaM. Fluorescence stopped-flow experiments showed that the dissociation rate of La(3+) from the C-terminal domain of CaM was higher than that from the N-terminal. However, in the presence of Mas, the dissociation rate of La(3+) decreased and the dissociation processes from both global domains were indistinguishable. In addition, compared with the case of Ca(4)CaM-Mas, the slower dissociations of Mas from La(4)CaM-Mas and Ca(2)La(2)CaM-Mas complexes indicate that in the presence of La(3+), the CaM-Mas complex became kinetically inert. A possible role of La(3+) in the Ca(2+)-CaM-dependent pathway is discussed.     

Molecular characterization of calmodulin trapping by calcium/calmodulin-dependent protein kinase II

Autophosphorylation of alpha-Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) at Thr(286) results in calmodulin (CaM) trapping, a >10,000-fold decrease in the dissociation rate of CaM from the enzyme. Here we present the first site-directed mutagenesis study on the dissociation of the high affinity complex between CaM and full-length CaM kinase II. We measured dissociation kinetics of CaM and CaM kinase II proteins by using a fluorescently modified CaM that is sensitive to binding to target proteins. In low [Ca(2+)], the phosphorylated mutant kinase F293A and the CaM mutant E120A/M124A exhibited deficient trapping compared with wild-type. In high [Ca(2+)], the CaM mutations E120A, M124A, and E120A/M124A and the CaM kinase II mutations F293A, F293E, N294A, N294P, and R297E increased dissociation rate constants by factors ranging from 2.3 to 116. We have also identified residues in CaM and CaM kinase II that interact in the trapped state by mutant cycle-based analysis, which suggests that interactions between Phe(293) in the kinase and Glu(120) and Met(124) in CaM specifically stabilize the trapped CaM-CaM kinase II complex. Our studies further show that Phe(293) and Asn(294) in CaM kinase II play dual roles, because they likely destabilize the low affinity state of CaM complexed to unphosphorylated kinase but stabilize the trapped state of CaM bound to phosphorylated kinase.     

Calcium-dependent and -independent binding of soybean calmodulin isoforms to the calmodulin binding domain of tobacco MAPK phosphatase-1

The recent finding of an interaction between calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca(2+) signaling and the MAPK cascade, two of the most important signaling pathways in plant cells. Here we have used different biophysical techniques, including fluorescence and NMR spectroscopy as well as microcalorimetry, to characterize the binding of soybean CaM isoforms, SCaM-1 and -4, to synthetic peptides derived from the CaM binding domain of NtMKP1. We find that the actual CaM binding region is shorter than what had previously been suggested. Moreover, the peptide binds to the SCaM C-terminal domain even in the absence of free Ca(2+) with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca(2+), the peptides bind first to the C-terminal lobe of the SCaMs with a nanomolar affinity, and at higher peptide concentrations, a second peptide binds to the N-terminal domain with lower affinity. Thermodynamic analysis demonstrates that the formation of the peptide-bound complex with the Ca(2+)-loaded SCaMs is driven by favorable binding enthalpy due to a combination of hydrophobic and electrostatic interactions. Experiments with CaM proteolytic fragments showed that the two domains bind the peptide in an independent manner. To our knowledge, this is the first report providing direct evidence for sequential binding of two identical peptides of a target protein to CaM. Discussion of the potential biological role of this interaction motif is also provided.     

Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex

The four integral delta subunits of the phosphorylase kinase (PhK) complex are identical to calmodulin (CaM) and confer Ca(2+) sensitivity to the enzyme, but bind independently of Ca(2+). In addition to binding Ca(2+), an obligatory activator of PhK's phosphoryltransferase activity, the delta subunits transmit allosteric signals to PhK's remaining alpha, beta, and gamma subunits in activating the enzyme. Under mild conditions about 10% of the delta subunits can be exchanged for exogenous CaM. In this study, a CaM double-mutant derivatized with a fluorescent donor-acceptor pair (CaM-DA) was exchanged for delta to assess the conformational substates of PhKdelta by single molecule fluorescence resonance energy transfer (FRET) +/-Ca(2+). The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca(2+), as observed by alterations of the compact, mid-length, and extended populations of their FRET distance distributions. Specifically, the combined predominant mid-length and less common compact conformations of PhKdelta became less abundant in the presence of Ca(2+), with the delta subunits assuming more extended conformations. This behavior is in contrast to the compact forms commonly observed for many of CaM's Ca(2+)-dependent interactions with other proteins. In addition, the conformational distributions of the exchanged PhKdelta subunits were distinct from those of CaM-DA free in solution, +/-Ca(2+), as well as from exogenous CaM bound to the PhK complex as delta'. The distinction between delta and delta' is that the latter binds only in the presence of Ca(2+), but stoichiometrically and at a different location in the complex than delta.     

Calcium-induced refolding of the calmodulin V136G mutant studied by NMR spectroscopy: evidence for interaction between the two globular domains

The Ca(2+) titration of the (15)N-labeled mutant V136G calmodulin has been monitored using (1)H-(15)N HSQC NMR spectra. Up to a [Ca(2+)]/[CaM] ratio of 2, the Ca(2+) ions bind predominantly to sites I and II on the N-domain in contrast with the behavior of the wild-type calmodulin where the C-terminal domain has the higher affinity for Ca(2+). Surprisingly, the Ca(2+)-binding affinity for the N-domain in the mutant calmodulin is greater than that for the N-domain in the wild-type protein. The mutated C-domain is observed as a mixture of unfolded, partially folded (site III occupied), and native-like folded (sites III and IV occupied) conformations, with relative populations dependent on the [Ca(2+)]/[CaM] ratio. The occupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca(2+) binding in the C-domain is mediated by the integrity of the domain structure. Several NH signals from residues in the Ca(2+)-bound N-domain appear as two signals during the Ca(2+) titration indicating separate species in slow exchange, and it can be deduced that these result from the presence and absence of interdomain interactions in the mutant. It is proposed that an unfolded part of the mutated C-domain interacts with sites on the N-domain that normally bind to target proteins. This would also account for the increase in the Ca(2+) affinity for the N-domain in the mutant compared with the wild-type calmodulin. The results therefore show the wide-ranging effects of a point mutation in a single Ca(2+)-binding site, providing details of the involvement of individual residues in the calcium-induced folding reactions.     

Calmodulin bridging of IQ motifs in myosin-V

Ca(2+)-saturated calmodulin binds to double-length IQ lever-arm sequences from murine myosin-V, forming a 1:1 "bridging" complex with very high affinity, (K9d)<10 pM for double motifs, IQ34, IQ45 and IQ56). Such a 1:1 complex involves interaction of one calmodulin (CaM) molecule with two adjacent IQ-motifs, providing a molecular mechanism for the observed Ca(2+)-dependent CaM dissociation from the IQ-region. Structural considerations suggest that formation of the 1:1 complex requires a severe distortion of the lever-arm, potentially regulating functional motility. This would be consistent with a recent report of diverse, irregular shapes of the lever arm of myosin-V induced by the presence of Ca(2+).     

The carboxy-terminal calcium binding sites of calmodulin control calmodulin's switch from an activator to an inhibitor of RYR1

Calcium and calmodulin both regulate the skeletal muscle calcium release channel, also known as the ryanodine receptor, RYR1. Ca(2+)-free calmodulin (apocalmodulin) activates and Ca(2+)-calmodulin inhibits the ryanodine receptor. The conversion of calmodulin from an activator to an inhibitor is due to Ca(2+) binding to calmodulin. We have previously shown that the binding sites for apocalmodulin and Ca(2+)-calmodulin on RYR1 are overlapping with the Ca(2+)-calmodulin site located slightly N-terminal to the apocalmodulin binding site. We now show that mutations of the calcium binding sites in either the N-terminal or the C-terminal lobes of calmodulin decrease the affinity of calmodulin for the ryanodine receptor, suggesting that both lobes interact with RYR1. Mutation of the two C-terminal Ca(2+) binding sites of calmodulin destroys calmodulin's ability to inhibit ryanodine receptor activity at high calcium concentrations. The mutated calmodulin, however, can still bind to RYR1 at both nanomolar and micromolar Ca(2+) concentrations. Mutating the two N-terminal calcium binding sites of calmodulin does not significantly alter calmodulin's ability to inhibit ryanodine receptor activity. These data suggest that calcium binding to the two C-terminal calcium binding sites within calmodulin is responsible for the switching of calmodulin from an activator to an inhibitor of the ryanodine receptor.     

Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.     

Participation of the Ca(2+)-calmodulin-activated Kinase II in the control of metaphase-anaphase transition in human cells

Treatment of HeLa S3 cells growing in suspension, and of endothelial cells ECV 304 growing as a monolayer, with the inhibitor of the Ca(2+)-calmodulin activated Kinase II KN-93, blocks cells at metaphase for 15 min (HeLa cells) and 30 min (ECV cells). Thereafter cells resume mitosis and enter anaphase. The inactive isomer KN-92 does not show such effects. The results show the involvement of the CaM K II system in the regulation of the metaphase-anaphase transition whereby the activation of the Kinase II, in particular by calmodulin appears to be affected, the residual autophosphorylation of the CaM K II system apparently sufficing after 15 to 30 min to release the cells into anaphase. The results are compared with the metaphase-blocking effects of the noble gas xenon, where the xenon-induced block can be overcome by small intracellular increases of Ca(2+), thus indicating the CaM K II system as a possible target for xenon.     

Phosphorylation of dystrophin and alpha-syntrophin by Ca(2+)-calmodulin dependent protein kinase II.

A Ca(2+)-calmodulin dependent protein kinase activity (DGC-PK) was previously shown to associate with skeletal muscle dystrophin glycoprotein complex (DGC) preparations, and phosphorylate dystrophin and a protein with the same electrophoretic mobility as alpha-syntrophin (R. Madhavan, H.W. Jarrett, Biochemistry 33 (1994) 5797-5804). Here, we show that DGC-PK and Ca(2+)-calmodulin dependent protein kinase II (CaM kinase II) phosphorylate a common site (RSDS(3616)) within the dystrophin C terminal domain that fits the consensus CaM kinase II phosphorylation motif (R/KXXS/T). Furthermore, both kinase activities phosphorylate exactly the same three fusion proteins (dystrophin fusions DysS7 and DysS9, and the syntrophin fusion) out of a panel of eight fusion proteins (representing nearly 100% of syntrophin and 80% of dystrophin protein sequences), demonstrating that DGC-PK and CaM kinase II have the same substrate specificity. Complementing these results, anti-CaM kinase II antibodies specifically stained purified DGC immobilized on nitrocellulose membranes. Renaturation of electrophoretically resolved DGC proteins revealed a single protein kinase band (M(r) approximately 60,000) that, like CaM kinase II, underwent Ca(2+)-calmodulin dependent autophosphorylation. Based on these observations, we conclude DGC-PK represents a dystrophin-/syntrophin-phosphorylating skeletal muscle isoform of CaM kinase II. We also show that phosphorylation of the dystrophin C terminal domain sequences inhibits their syntrophin binding in vitro, suggesting a regulatory role for phosphorylation.     

Novel interaction of the voltage-dependent sodium channel (VDSC) with calmodulin: does VDSC acquire calmodulin-mediated Ca2+-sensitivity?

The voltage-dependent sodium channel (VDSC) interacts with intracellular molecules to modulate channel properties and localizations in neuronal cells. To study protein interactions, we applied yeast two-hybrid screening to the cytoplasmic C-terminal domain of the main pore-forming alpha-subunit. We found a novel interaction between the C-terminal domain and calmodulin (CaM). By two-hybrid interaction assays, we specified the interaction site of VDSC in a C-terminal region, which is composed of 38 amino acid residues and contains both IQ-like and Baa motifs. Using a fusion protein of the C-terminal domain, we showed that interaction with CaM occurred in the presence and absence of Ca(2+). Two synthetic peptides, each covering the IQ-like (NaIQ) or the Baa motifs (NaBaa), were used to examine the binding property by a gel mobility shift assay. Although the NaIQ and NaBaa sequences are overlapped, NaBaa binds only to Ca(2+)-bound Ca(2+)CaM, whereas NaIQ binds to both Ca(2+)CaM and Ca(2+)-free apoCaM. Fluorescence spectroscopy of dansylated CaM showed Ca(2+)-dependent spectral changes not only for NaBaa.CaM but also for NaIQ.CaM. The results, taken together with other results, indicate that whereas the NaBaa.CaM complex is formed in a Ca(2+)-dependent manner, the NaIQ.CaM complex has two conformational states, distinct with respect to the peptide binding site and the CaM conformation, depending on the Ca(2+) concentration. These observations suggest the possibility that VDSC is functionally modulated through the direct CaM interaction and the Ca(2+)-dependent conformational transition of the complex.     

Converting troponin C into calmodulin: effects of mutations in the central helix and of changes in temperature

Calmodulin (CaM) and troponin C (TnC) are the most similar members of EF-hand family and show few differences in the primary structure. Here, we use mutants of troponin that mimic calmodulin and changes in temperature to investigate the factors that determine their specificity as regulatory proteins. Using a double mutant of troponin that resembles calmodulin in lacking both the N-terminal helix and KGK(91-93) we observe a small difference from troponin in binding to the erythrocyte Ca(2+)-ATPase, and an improvement in enzyme activation. A triple mutant, where in addition, the residues 88-90 are replaced with the corresponding sequence from calmodulin is equivalent to calmodulin in maximal activation, and it restores protein ability to increase Ca(2+) affinity for the enzyme. However, this mutant also binds less tightly (1/100) than calmodulin. Remarkably, a decrease in temperature has a more marked effect in protein binding than either mutation, reducing the difference in affinities to 18-fold, but without any improvement in their ability to increase Ca(2+) affinity for the enzyme. Spectroscopic analysis of hydrophobic domain exposure in EF-hand proteins was carried out using 8-anilino-1-naphthalenesulfonic acid (ANS). The probe shows a much higher fluorescence when bound to the complex Ca(4)-calmodulin than to Ca(4)-troponin. Decreasing the temperature exposes additional hydrophobic regions of troponin. Changing the Mg(2+) concentration does not affect their bindings to the enzyme. It is suggested that the requirements for troponin to mimic calmodulin in binding to the target enzyme, and those for activating it, are met by different regions of the protein.     

Kinetics of calmodulin binding to calcineurin

Calcineurin (CaN) binds Ca(2+)-saturated calmodulin (CaM) with relatively high affinity; however, an accurate steady-state K(d) value has not been determined. In this report, we describe, using steady-state and stopped-flow fluorescence techniques, the rates of association and dissociation of Ca(2+)-saturated CaM from CaN heterodimer (CaNA/CaNB) and CaNA only. The rate of Ca(2+)/CaM association was determined to be 4.6 x 10(7) M(-1)s(-1). The rate of Ca(2+)/CaM dissociation from CaN was slower than previously reported and was approximately 0.0012 s(-1). In preparations of CaNA alone (no regulatory CaNB subunit), the dissociation rate was slowed further to 0.00026 s(-1). From these data we calculate a K(d) for binding of Ca(2+)-saturated CaM to CaN of 28 pM. This K(d) is significantly lower than previously reported estimates of approximately 1 nM and indicates that CaN is one of the highest affinity CaM-binding proteins identified to date.     

A new role for IQ motif proteins in regulating calmodulin function

IQ motifs are found in diverse families of calmodulin (CaM)-binding proteins. Some of these, like PEP-19 and RC3, are highly abundant in neuronal tissues, but being devoid of catalytic activity, their biological roles are not understood. We hypothesized that these IQ motif proteins might have unique effects on the Ca2+ binding properties of CaM, since they bind to CaM in the presence or absence of Ca2+. Here we show that PEP-19 accelerates by 40 to 50-fold both the slow association and dissociation of Ca2+ from the C-domain of free CaM, and we identify the sites of interaction between CaM and PEP-19 using NMR. Importantly, we demonstrate that PEP-19 can also increase the rate of dissociation of Ca2+ from CaM when bound to intact CaM-dependent protein kinase II. Thus, PEP-19, and presumably similar members of the IQ family of proteins, has the potential to alter the Ca2+-binding dynamics of free CaM and CaM that is bound to other target proteins. Since Ca2+ binding to the C-domain of CaM is the rate-limiting step for activation of CaM-dependent enzymes, the data reveal a new concept of importance in understanding the temporal dynamics of Ca2+-dependent cell signaling.     

Regulation of TRPM2 by extra- and intracellular calcium

TRPM2 is a calcium-permeable nonselective cation channel that is opened by the binding of ADP-ribose (ADPR) to a C-terminal nudix domain. Channel activity is further regulated by several cytosolic factors, including cyclic ADPR (cADPR), nicotinamide adenine dinucleotide phosphate (NAADP), Ca(2+) and calmodulin (CaM), and adenosine monophosphate (AMP). In addition, intracellular ions typically used in patch-clamp experiments such as Cs(+) or Na(+) can alter ADPR sensitivity and voltage dependence, complicating the evaluation of the roles of the various modulators in a physiological context. We investigated the roles of extra- and intracellular Ca(2+) as well as CaM as modulators of ADPR-induced TRPM2 currents under more physiological conditions, using K(+)-based internal saline in patch-clamp experiments performed on human TRPM2 expressed in HEK293 cells. Our results show that in the absence of Ca(2+), both internally and externally, ADPR alone cannot induce cation currents. In the absence of extracellular Ca(2+), a minimum of 30 nM internal Ca(2+) is required to cause partial TRPM2 activation with ADPR. However, 200 microM external Ca(2+) is as efficient as 1 mM Ca(2+) in TRPM2 activation, indicating an external Ca(2+) binding site important for proper channel function. Ca(2+) facilitates ADPR gating with a half-maximal effective concentration of 50 nM and this is independent of extracellular Ca(2+). Furthermore, TRPM2 currents inactivate if intracellular Ca(2+) levels fall below 100 nM irrespective of extracellular Ca(2+). The facilitatory effect of intracellular Ca(2+) is not mimicked by Mg(2+), Ba(2+), or Zn(2+). Only Sr(2+) facilitates TRPM2 as effectively as Ca(2+), but this is due to Sr(2+)-induced Ca(2+) release from internal stores rather than a direct effect of Sr(2+) itself. Together, these data demonstrate that cytosolic Ca(2+) regulates TRPM2 channel activation. Its facilitatory action likely occurs via CaM, since the addition of 100 microM CaM to the patch pipette significantly enhances ADPR-induced TRPM2 currents at fixed [Ca(2+)](i) and this can be counteracted by calmidazolium. We conclude that ADPR is responsible for TRPM2 gating and Ca(2+) facilitates activation via calmodulin.