Reduced Calmodulin Binding Research Articles

Reduced axonal surface expression and phosphoinositide sensitivity in Kv7 channels disrupts their function to inhibit neuronal excitability in Kcnq2 epileptic encephalopathy

Neuronal Kv7/KCNQ channels are voltage-gated potassium channels composed of Kv7.2/KCNQ2 and Kv7.3/KCNQ3 subunits. Enriched at the axonal membrane, they potently suppress neuronal excitability. De novo and inherited dominant mutations in Kv7.2 cause early onset epileptic encephalopathy characterized by drug resistant seizures and profound psychomotor delay. However, their precise pathogenic mechanisms remain elusive. Here, we investigated selected epileptic encephalopathy causing mutations in calmodulin (CaM)-binding helices A and B of Kv7.2. We discovered that R333W, K526N, and R532W mutations located peripheral to CaM contact sites decreased axonal surface expression of heteromeric channels although only R333W mutation reduced CaM binding to Kv7.2. These mutations also altered gating modulation by phosphatidylinositol 4,5-bisphosphate (PIP2), revealing novel PIP2 binding residues. While these mutations disrupted Kv7 function to suppress excitability, hyperexcitability was observed in neurons expressing Kv7.2-R532W that displayed severe impairment in voltage-dependent activation. The M518 V mutation at the CaM contact site in helix B caused most defects in Kv7 channels by severely reducing their CaM binding, K+ currents, and axonal surface expression. Interestingly, the M518 V mutation induced ubiquitination and accelerated proteasome-dependent degradation of Kv7.2, whereas the presence of Kv7.3 blocked this degradation. Furthermore, expression of Kv7.2-M518V increased neuronal death. Together, our results demonstrate that epileptic encephalopathy mutations in helices A and B of Kv7.2 cause abnormal Kv7 expression and function by disrupting Kv7.2 binding to CaM and/or modulation by PIP2. We propose that such multiple Kv7 channel defects could exert more severe impacts on neuronal excitability and health, and thus serve as pathogenic mechanisms underlying Kcnq2 epileptic encephalopathy.

Ca2+-dependent regulation and binding of calmodulin to multiple sites of Transient Receptor Potential Melastatin 3 (TRPM3) ion channels

TRPM3 proteins assemble to Ca2+-permeable cation channels in the plasma membrane, which act as nociceptors of noxious heat and mediators of insulin and cytokine release. Here we show that TRPM3 channel activity is strongly dependent on intracellular Ca2+. Conceivably, this effect is attributed to the Ca2+ binding protein calmodulin, which binds to TRPM3 in a Ca2+-dependent manner. We identified five calmodulin binding sites within the amino terminus of TRPM3, which displayed different binding affinities in dependence of Ca2+. Mutations of lysine residues in calmodulin binding site 2 strongly reduced calmodulin binding and TRPM3 activity indicating the importance of this domain for TRPM3-mediated Ca2+ signaling. Our data show that TRPM3 channels are regulated by intracellular Ca2+ and provide the basis for a mechanistic understanding of the regulation of TRPM3 by calmodulin.

Novel regulations of the angiotensin II receptor type 1 by calmodulin

The angiotensin II receptor type 1 (AT1R) mediates many Ca2+-dependent actions of angiotensin II (AngII). Calmodulin (CaM) is a key transducer of Ca2+ signals in cells. Two locations on the receptor’s submembrane domains (SMD) 3 and 4 are known to interact with CaM. However, the binding sites for CaM, biochemical properties of the interactions, and their functional impact are not fully understood. Using a FRET-based screening method, we identified a new binding site for CaM on SMD2 (a.a. 125–141), in addition to SMD3 and the juxtamembranous region of SMD4 (SMD4JM, a.a., 309–327). Simultaneous measurements of CaM binding and free Ca2+ show that the interactions are Ca2+-dependent, with disparate Kd and EC50(Ca2+) values within the physiological range of cytoplasmic Ca2+. Full interaction between CaM and SMD3 requires the entire domain (a.a. 215–242) and has an EC50(Ca2+) value in the range of resting cytoplasmic Ca2+, suggesting AT1R-CaM interaction can occur in resting conditions in cells. AngII induces robust ERK1/2 phosphorylation in primary vascular smooth muscle cells. This effect is suppressed by AT1R inhibitor losartan and virtually abolished by CaM antagonist W-7. AngII-induced ERK1/2 phosphorylation is suppressed in cells expressing mutant AT1R with reduced CaM binding at each identified binding domain. AngII triggers transient Ca2+ signals in cells expressing wild-type AT1R. These signals are reduced in cells expressing mutant AT1R with reduced CaM binding at SMD3 or SMD4JM, but are very slow-rising, low amplitude signal in cells expressing AT1R with reduced CaM binding at SMD2. The data indicate that CaM interactions with AT1R can occur at various domains, with different affinities, at different physiological Ca2+ levels, and are important for AT1R-mediated signaling.

The emerging role of calmodulin regulation of RyR2 in controlling heart rhythm, the progression of heart failure and the antiarrhythmic action of dantrolene.

Cardiac output and rhythm depend on the release and the take-up of calcium from the sarcoplasmic reticulum (SR). Excessive diastolic calcium leak from the SR due to dysfunctional calcium release channels (RyR2) contributes to the formation of delayed after-depolarizations, which underlie the fatal arrhythmias that occur in heart failure and inherited syndromes. Calmodulin (CaM) is a calcium-binding protein that regulates target proteins and acts as a calcium sensor. CaM is comprised of two calcium-binding EF-hand domains and a flexible linker. CaM is an accessory protein that partially inhibits RyR2 channel activity. CaM is critical for normal cardiac function, and altered CaM binding and efficacy may contribute to defects in SR calcium release. The present paper reviews CaM binding to RyR2 and how it regulates RyR2 channel activity. It then goes on to review how mutations in the CaM amino acid sequence give rise to inherited syndromes such as Catecholaminergic Polymorphic Ventricular Tachychardia (CPVT) and long QT syndrome (LQTS). In addition, the role of reduced CaM binding to RyR2 that results from RyR2 phosphorylation or from oxidation of either RyR2 or CaM contributes to the progression of heart failure is reviewed. Finally, this manuscript reviews recent evidence that CaM binding to RyR2 is required for the inhibitory action of a pharmaceutical agent (dantrolene) on RyR2. Dantrolene is a clinically used muscle relaxant that has recently been found to exert antiarrhythmic effects against SR Ca2+ overload arrhythmias.

Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-coupled Estrogen Receptor 1

Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca(2+) signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17β-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca(2+) and Ca(2+)-CaM indicated that E2 also increases free Ca(2+)-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca(2+)-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gβγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca(2+) efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca(2+) signals and promote Ca(2+)-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca(2+)/CaM signals and functional linkage in the endothelial CaM target network.

CaMKII and Heart Failure Promote a Pathological Ryanodine Receptor Conformation that Reduces Calmodulin Binding and Enhances SR Ca2+ Leak

Increased diastolic SR Ca2+ (Ca) leak via cardiac ryanodine receptors (RyR2) can cause cardiac arrhythmias and dysfunction in heart failure (HF). RyR2 phosphorylation (at Ser2814) by Ca/calmodulin-dependent protein kinase II (CaMKII) is thought to be a critical promoter of leaky RyR2 in HF and arrhythmias. A conformational change in RyR2 (“domain unzipping”) observed in HF can increase SR Ca leak. Calmodulin (CaM) quiets RyR2 gating, and CaM binding affinity for RyR2 is reduced in HF. Here, we tested the hypothesis that CaMKII-dependent phosphorylation of RyR2 increases Ca leak by favoring the same unzipped-conformation that exhibits reduced CaM-RyR2 binding affinity. Using non-phosphorylatable RyR2-S2814A and phosphomimetic RyR2-S2814D knock-in mice, fluorescence resonance energy transfer (FRET) was measured to directly detect binding of fluorescently labeled DPc10 (RyR2 unzipping peptide), FK506-binding protein12.6, and CaM to RyR2 in permeabilized cardiomyocytes. In S2814D vs. S2814A: (1) Ca sparks and waves were increased; (2) CaM-RyR2 binding affinity was reduced 3-fold, but FRETmax was unaltered; and (3) F-DPc10 accessibility was 2-fold faster, reflecting unzipped-conformation. Dantrolene (1 μM), which stabilizes the zipped conformation: (1) restored normal CaM-RyR2 binding affinity; (2) reduced DPc10 access (zipped-conformation); and (3) reduced frequency of Ca sparks and waves in S2814D. Furthermore, in rabbit non-ischemic HF myocytes, in which RyR2 is more highly phosphorylated by CaMKII, vs. age-matched control: (1) The CaM-RyR2 binding affinity was reduced by 2.5-fold; and (2) F-DPc10 accessibility was 1.5-fold faster. Dantrolene also restored CaM-RyR2 binding affinity and kept zipped conformation in rabbit HF. We conclude that CaMKII-dependent phosphorylation of RyR2 promotes a pathological RyR2 conformation with reduced CaM binding affinity and elevated Ca leakiness. Dantrolene reverses this conformational change and restores normal CaM binding affinity in S2814D mouse and rabbit HF.

Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter, RyR structure and calmodulin affinity

Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain–domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM–RyR2 and FK506-binding protein (FKBP12.6)–RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1–3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca2+ sparks and spontaneous Ca2+ waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM–RyR2 binding, but had no effect on FKBP12.6–RyR2 binding. Dantrolene restored CaM–RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10–RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM–RyR2 affinity, has no effect on FKBP12.6–RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM–RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.

CaMKII-Dependent Phosphorylation of RyR2 Causes Domain Unzipping and Reduced Calmodulin Binding, But Dantrolene Reverses These Effects

Increased diastolic SR Ca2+ (Ca) leak via cardiac ryanodine receptors (RyR2) can cause cardiac arrhythmias and dysfunction in heart failure (HF). CaMKII-dependent RyR2 phosphorylation (at S2814) is thought to be a critical promoter of leaky RyR2 in HF. A conformational change in RyR2 (“domain unzipping”) observed in HF can also increase SR Ca leak. Calmodulin (CaM) quiets RyR2 gating, and the CaM affinity for RyR2 is reduced in HF. Further, we have shown reciprocal negative coupling between CaM binding to RyR2 and accessibility of the unzipping RyR peptide DPc10 (Oda et al. 2013). That is, unzipping with DPc10 reduced CaM binding and increased Ca leak, whereas CaM quieted leak and inhibited DPc10 access. Here, we tested the hypothesis that RyR2 phosphorylation by CaMKII increases Ca leak by favoring the same unzipped-conformation that exhibits reduced CaM affinity. We either activated endogenous myocyte CaMKII or used knock-in mice expressing non-phosphorylatable RyR2-S2814A or phosphomimetic RyR2-S2814D. We used FRET to directly detect binding of fluorescently labeled DPc10, FKBP12.6, and CaM to RyR2 in permeabilized cardiomyocytes. In phosphomimetic S2814D vs. S2814A myocytes, CaM-RyR2 affinity was reduced 3-fold (Kd= 72 ± 9 nM vs. 20 ± 2 nM), but FRETmax was unaltered. Access of DPc10 was also 2-fold faster in S2814D versus S2814A myocytes. Dantrolene (1 μM), which stabilizes the zipped conformation, restored CaM-RyR2 binding affinity in S2814D, slowed DPc10 wash-in, and lowered Bmax. Forced RyR2-S2814D saturation with 500 nM CaM also slowed DPc10 wash-in and reduced Bmax. We conclude that RyR2 phosphorylation by CaMKII causes pathological conformation changes (unzipping) that reduce RyR2-CaM affinity and ability to quiet SR Ca leak. Dantrolene effectively reverses these effects.

Kinase activity and calmodulin binding are essential for growth signaling by the phytosulfokine receptor PSKR1

The cell growth-promoting peptide phytosulfokine (PSK) is perceived by leucine-rich repeat (LRR) receptor kinases. To elucidate PSK receptor function we analyzed PSKR1 kinase activity and binding to Ca(2+) sensors and evaluated the contribution of these activities to growth control in planta. Ectopically expressed PSKR1 was capable of auto- and transphosphorylation. Replacement of a conserved lysine within the ATP-binding region by a glutamate resulted in the inhibition of auto- and transphosphorylation kinase activities. Expression of the kinase-inactive PSKR1(K762E) receptor in the pskr null background did not restore root or shoot growth. Instead, the mutant phenotype was enhanced suggesting that the inactive receptor protein exerts growth-inhibitory activity. Bioinformatic analysis predicted a putative calmodulin (CaM)-binding site within PSKR1 kinase subdomain VIa. Bimolecular fluorescence complementation analysis demonstrated that PSKR1 binds to all isoforms of CaM, more weakly to the CaM-like protein CML8 but apparently not to CML9. Mutation of a conserved tryptophan (W831S) within the predicted CaM-binding site strongly reduced CaM binding. Expression of PSKR1(W831S) in the pskr null background resulted in growth inhibition that was similar to that of the kinase-inactive receptor. We conclude that PSK signaling requires Ca(2+) /CaM binding and kinase activity of PSKR1 in planta. We further propose that the inactivated kinase interferes with other growth-promoting signaling pathway(s).

Structural and Biophysical Characterization of the Interactions between the Death Domain of Fas Receptor and Calmodulin

The extrinsic apoptotic pathway is initiated by cell surface death receptors such as Fas. Engagement of Fas by Fas ligand triggers a conformational change that allows Fas to interact with adaptor protein Fas-associated death domain (FADD) via the death domain, which recruits downstream signaling proteins to form the death-inducing signaling complex (DISC). Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells, suggesting a novel role of CaM in Fas-mediated signaling. CaM antagonists induce apoptosis through a Fas-related mechanism in cholangiocarcinoma and other cancer cell lines possibly by inhibiting Fas-CaM interactions. The structural determinants of Fas-CaM interaction and the underlying molecular mechanisms of inhibition, however, are unknown. Here we employed NMR and biophysical techniques to elucidate these mechanisms. Our data show that CaM binds to the death domain of Fas (FasDD) with an apparent dissociation constant (Kd) of ~2 μM and 2:1 CaM:FasDD stoichiometry. The interactions between FasDD and CaM are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. We also show that both the N- and C-terminal lobes of CaM are important for binding. NMR and surface plasmon resonance data show that three CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, tamoxifen, and trifluoperazine) greatly inhibit Fas-CaM interactions by blocking the Fas-binding site on CaM. Our findings provide the first structural evidence for Fas-CaM interactions and mechanism of inhibition and provide new insight into the molecular basis for a novel role of CaM in regulating Fas-mediated apoptosis.

Enhanced binding of calmodulin to the ryanodine receptor corrects contractile dysfunction in failing hearts

The channel function of the cardiac ryanodine receptor (RyR2) is modulated by calmodulin (CaM). However, the involvement of CaM in aberrant Ca(2+) release in diseased hearts remains unclear. Here, we investigated the pathogenic role of defective CaM binding to the RyR2 in the channel dysfunction associated with heart failure. The involvement of CaM in aberrant Ca(2+) release was assessed in normal and pacing-induced failing canine hearts. The apparent affinity of CaM for RyR2 was considerably lower in failing sarcoplasmic reticulum (SR) compared with normal SR. Thus, the amount of CaM bound to RyR2 was markedly decreased in failing myocytes. Expression of the CaM isoform Gly-Ser-His-CaM (GSH-CaM), which has much higher binding affinity than wild-type CaM for RyR1, restored normal CaM binding to RyR2 in both SR and myocytes of failing hearts. The Ca(2+) spark frequency (SpF) was markedly higher and the SR Ca(2+) content was lower in failing myocytes compared with normal myocytes. The incorporation of GSH-CaM into the failing myocytes corrected the aberrant SpF and SR Ca(2+) content to normal levels. Reduced CaM binding to RyR2 seems to play a critical role in the pathogenesis of aberrant Ca(2+) release in failing hearts. Correction of the reduced CaM binding to RyR2 stabilizes the RyR2 channel function and thereby restores normal Ca(2+) handling and contractile function to failing hearts.

Reduced CaM/FLIP binding by a single point mutation in c-FLIPL modulates Fas-mediated apoptosis and decreases tumorigenesis

We have previously demonstrated that calmodulin (CaM) binds directly to c-FLIPL in a Ca2+-dependent manner. Deletion of the CaM-binding region (amino acid 197–213) results in reduced CaM binding, and increased Fas-mediated apoptosis and decreased tumorigenesis of cholangiocarcinoma cells. The present studies were designed to identify the precise amino acids between 197 and 213 that are responsible for CaM/FLIP binding, and their roles in mediating the anti-apoptotic function of c-FLIPL. Sequence analysis of the CaM-binding region at 197–213 predicted three unique positively charged residues at 204, 207 and 209, which might be responsible for the CaM/FLIP binding. A point mutation at H204 of c-FLIPL was found to markedly reduce CaM binding, whereas point mutation at R207 or K209 did not affect c-FLIPL binding to CaM. Decreased CaM/FLIP binding was confirmed in cholangiocarcinoma cells overexpressing the H204 c-FLIPL mutant. Reduced CaM binding by the H204 mutant resulted in increased sensitivity to Fas-mediated apoptosis and inhibited tumor growth in mice compared with wild-type c-FLIPL. Death-inducing signaling complex (DISC) analysis showed that the reduced CaM binding to H204 mutant resulted in less c-FLIPL recruited into the DISC. Concurrently, increased caspase 8 was recruited to the DISC, which resulted in increased cleavage and activation of caspase 8, activation of downstream caspase 3 and increased apoptosis. Therefore, these results demonstrate that the H204 residue is responsible for c-FLIPL binding to CaM, which mediates the anti-apoptotic function of c-FLIPL, most likely through affecting recruitment of caspase 8 into the DISC and thus caspase 8 activation. These studies further characterized CaM/FLIP interaction and its function in regulating Fas-mediated apoptosis and tumorigenesis, which may provide new therapeutic targets for cancer therapy.

CaMKII-Mediated Phosphorylation of the Myosin Motor Myo1c Is Required for Insulin-Stimulated GLUT4 Translocation in Adipocytes

The unconventional myosin Myo1c has been implicated in insulin-regulated GLUT4 translocation to the plasma membrane in adipocytes. We show that Myo1c undergoes insulin-dependent phosphorylation at S701. Phosphorylation was accompanied by enhanced 14-3-3 binding and reduced calmodulin binding. Recombinant CaMKII phosphorylated Myo1c in vitro and siRNA knockdown of CaMKIIdelta abolished insulin-dependent Myo1c phosphorylation in vivo. CaMKII activity was increased upon insulin treatment and the CaMKII inhibitors CN21 and KN-62 or the Ca(2+) chelator BAPTA-AM blocked insulin-dependent Myo1c phosphorylation and insulin-stimulated glucose transport in adipocytes. Myo1c ATPase activity was increased after CaMKII phosphorylation in vitro and after insulin stimulation of CHO/IR/IRS-1 cells. Expression of wild-type Myo1c, but not S701A or ATPase dead mutant K111A, rescued the inhibition of GLUT4 translocation by siRNA-mediated Myo1c knockdown. These data suggest that insulin regulates Myo1c function via CaMKII-dependent phosphorylation, and these events play a role in insulin-regulated GLUT4 trafficking in adipocytes likely involving Myo1c motor activity.

Fluorescence polarization assay for calmodulin binding to plasma membrane Ca 2+–ATPase: Dependence on enzyme and Ca 2+ concentrations

Calmodulin (CaM) is a Ca 2+ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the K d for the interaction of CaM with the plasma membrane Ca 2+–ATPase (PMCA), a Ca 2+ pump regulated by binding of CaM. Previous assays of PMCA–CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca 2+ dependence of CaM binding to PMCA. FP assays directly detect CaM–target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K d in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K d value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA ( K d = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca 2+ dependence follows a simple Hill plot demonstrating cooperative binding of Ca 2+ to the binding sites in CaM.

Calmodulin binding is dispensable for Rem-mediated Ca2+ channel inhibition

GTPases of the Ras-related RGK family are negative regulators of high voltage-activated (HVA) Ca2+ channel activity. In this study, we examined the role of calmodulin (CaM) association in Rem-mediated Ca2+ channel inhibition. We found that the Rem/CaM interaction is Ca2+-dependent, and that truncation of the Rem C-terminus before position 277 prevents CaM binding. Serial mutagenesis of the Rem C-terminus between residues 265 and 276 to alanine generated two mutants (Rem(L271A) and Rem(L274A)) that displayed reduced CaM binding, and a subset of these mutants displayed significantly lower cell periphery localization than Rem(WT). However, reductions in CaM association or membrane trafficking did not affect function, as all Rem mutants could completely inhibit Ca2+ channels. The Rem(1-275) truncation mutant partially inhibited Ca2+ channel activity despite its inability to bind CaM. Taken together, these studies indicate that CaM association is not essential for either Rem-mediated Ca2+ channel inhibition or plasma membrane localization.

Dueling Regulators

To investigate the mechanisms whereby phosphoinositides (PIs) regulate members of the TRP (transient receptor potential) family of ion channels, Kwon et al . focused on TRPC6, a channel modulated both by phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) and by calmodulin. Various PIs bound directly to a fusion protein containing the C-terminal region of TRPC6, and mutational analysis revealed that the PI-binding site overlapped that for calmodulin (PCaM domain). PIs disrupted calmodulin binding to the C-terminal region of TRPC6 in an in vitro assay, with PIP 3 being most potent; mutational analysis indicated that this interaction involved the PCaM domain. When expressed in human embryonic kidney (HEK) 293T cells, a TRPC6 mutant associated with decreased potency of PIs in disrupting calmodulin binding displayed reduced whole-cell currents relative to wild-type after TRPC6 activation, whereas a TRPC6 mutant associated with enhanced potency of PI disruption of calmodulin binding showed larger peak currents. Experiments in which intracellular calcium was buffered or in which calmodulin was overexpressed supported the idea that calcium-calmodulin inhibits TRPC6 activity and that PIs enhance TRPC6 function by disrupting the TRPC6-calmodulin interaction, as did experiments in which an AKT-PH domain was overexpressed to act as a PIP 3 "sponge." Other TRP family channels thought to bind calmodulin also bound PIs; with some of these, PIs reduced calmodulin binding. PIs also reduced calmodulin binding to the KCNQ1 potassium channel and the Ca v 1.2 calcium channel. Thus, the authors propose that PI regulation of calmodulin binding may represent a common mechanism for modulating ion channel function. Y. Kwon, T. Hofmann, C. Montell, Integration of phosphoinositide- and calmodulin-mediated regulation of TRPC6. Mol. Cell 25 , 491-503 (2007). [Online Journal]

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.

Fas Binding to Calmodulin Regulates Apoptosis in Osteoclasts

Promotion of osteoclast apoptosis is one therapeutic approach to osteoporosis. Calmodulin, the major intracellular Ca(2+) receptor, modulates both osteoclastogenesis and bone resorption. The calmodulin antagonist, trifluoperazine, rescues bone loss in ovariectomized mice (Zhang, L., Feng, X., and McDonald, J. M. (2003) Endocrinology 144, 4536-4543). We show here that a 3-h treatment of mouse osteoclasts with either of the calmodulin antagonists, tamoxifen or trifluoperazine, induces osteoclast apoptosis dose-dependently. Tamoxifen, 10 microm, and trifluoperazine, 10 microm, induce 7.3 +/- 1.8-fold and 5.3 +/- 0.9-fold increases in osteoclast apoptosis, respectively. In Jurkat cells, calmodulin binds to Fas, the death receptor, and this binding is regulated during Fas-mediated apoptosis (Ahn, E. Y., Lim, S. T., Cook, W. J., and McDonald, J. M. (2004) J. Biol. Chem. 279, 5661-5666). In osteoclasts, calmodulin also binds Fas. When osteoclasts are treated with 10 microm trifluoperazine, the binding between Fas and calmodulin is dramatically decreased at 15 min and gradually recovers by 60 min. A point mutation of the Fas death domain in the Lpr(-cg) mouse renders Fas inactive. Using glutathione S-transferase fusion proteins, the human Fas cytoplasmic domain is shown to bind calmodulin, whereas a point mutation (V254N) comparable with the Lpr(-cg) mutation in mice has markedly reduced calmodulin binding. Osteoclasts derived from Lpr(-cg) mice have diminished calmodulin/Fas binding and are more sensitive to calmodulin antagonist-induced apoptosis than those from wild-type mice. Both tamoxifen- and trifluoperazine-induced apoptosis are increased 1.6 +/- 0.2-fold in Lpr(-cg)-derived osteoclasts compared with osteoclasts derived from wild-type mice. In summary, calmodulin antagonists induce apoptosis in osteoclasts by a mechanism involving interference with calmodulin binding to Fas. The effects of calmodulin/Fas binding on calmodulin antagonist-induced apoptosis may open a new avenue for therapy for osteoporosis.

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.