Calmodulin

EVOLUTIONARY HOMOLOGS (part 3/4)

Calmodulin interaction with miscellaneous proteins

Calmodulin is involved in receptor-mediated endocytosis in yeast. A temperature sensitive calmodulin mutant is completely blocked for alpha-factor internalization almost immediatly upon shift to the restrictive temperature. The uptake step of receptor-mediated endocytosis in yeast is dependent on the calcium binding protein calmodulin (Cmd1p). In order to understand the role that Cmd1p plays, a search was carried out for possible targets among the genes required for the internalization process. Co-immunoprecipitation, two-hybrid and overlay assays demonstrate that Cmd1p interacts with Myo5p, a type I unconventional myosin. Analysis of the endocytic phenotype and the Cmd1p-Myo5p interaction in thermosensitive cmd1 mutants indicates that the Cmd1p-Myo5p interaction is required for endocytosis in vivo. However, the Cmd1p-Myo5p interaction requirement is partially overcome by deleting the calmodulin binding sites (IQ motifs) from Myo5p, suggesting that these motifs inhibit Myo5p function, and implying that endocytosis involves alleviation of calmodulin inhibition of myosin function. Genetic and biochemical evidence obtained with a collection of cmd1 mutant alleles strongly suggests that Cmd1p plays an additional role in the internalization step of receptor-mediated endocytosis in yeast, involving additional calmodulin targets (Geli, 1998).

Functional connections have been identified between Calmodulin and the yeast actin cytoskeleton. cmd1A, one of four intragenic complementing groups of temperature-sensitive yeast calmodulin mutations, results in a characteristic functional defect in actin organization. Among the complementing mutations, a representative cmd1A mutation (cmd1-226: F92A) is synthetically lethal with a mutation in MYO2 that encodes a class V unconventional myosin with calmodulin-binding domains. Gel overlay assay shows that a mutant calmodulin with the F92A alteration has severely reduced binding affinity to a GST-Myo2p fusion protein. Random replacement and site-directed mutagenesis at position 92 of calmodulin indicate that hydrophobic and aromatic residues are allowed at this position, suggests the importance of a hydrophobic interaction between calmodulin and Myo2p. To analyze other components involved in actin organization through calmodulin, mutations were isolated and characterized that show synthetic lethal interaction with cmd1-226; these "cax" mutants fall into five complementation groups. Interestingly, all the mutations themselves affect actin organization. Unlike cax2, cax3, cax4, and cax5 mutations, cax1 shows allele-specific synthetic lethality with the cmd1A allele. CAX1 is identical to ANP1/GEM3/MCD2, which is involved in protein glycosylation. CAX4 is identical to the ORF YGR036c, and CAX5 is identical to MNN10/SLC2/BED1. Several possibilities can be envisioned for the Myo2p function in actin organization. Myo2p itself may be involved in localizing or moving the actin cytoskeleton toward the growing tip. Alternatively, cargoes in secretory vesicles driven by Myo2p may anchor or stabilize the actin cytoskeleton. Myo2p might be capable of cross-linking actin because of the predicted and demonstrated ability of class V myosins to dimerize. There is growing evidence suggesting that myosins regulate the actin network in yeast and other organisms. For example, loss of myosin I function in yeast results in defective actin organization. It has been suggested that 95F myosin (a class VI unconventional myosin) in Drosophila may be involved in the formation of actin furrows through the transport of cytoplasmic components (Sekiya-Kawasaki, 1998).

Activated forms of the GTPases, Rac (See Drosophila Rac) and Cdc42, are known to stimulate formation of microfilament-rich lamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. IQGAP1 is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two approximately 190-kD subunits per molecule plus substoichiometric calmodulin. IQGAP1 contains four potential calmodulin-binding IQ domains and a region homologous to catalytic domains of GTPase-activating proteins, or GAPs. Purified IQGAP1 binds directly to F-actin and cross-links the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Exogenous calmodulin partially inhibits binding of IQGAP1 to F-actin, and is more effective in the absence of calcium than in its presence.. Colocalization of IQGAP1 with cortical microfilaments is cytochalasin-D sensitve. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin (Bashour, 1997).

The A kinase-anchoring protein AKAP79 coordinates the location of the cAMP-dependent protein kinase (protein kinase A), calcineurin, and protein kinase C (See Drosophila PKC) at the postsynaptic densities in neurons. Individual enzymes in the AKAP79 signaling complex are regulated by distinct second messenger signals; however, both PKC and calcineurin are inhibited when associated with the anchoring protein, suggesting that additional regulatory signals must be required to release active enzyme. This report focuses on the regulation of AKAP79-PKC interaction by calmodulin. AKAP79 binds calmodulin with high affinity in a Ca2+-dependent manner. Both proteins exhibit overlapping staining patterns in cultured hippocampal neurons. Calmodulin reverses the inhibition of PKCbetaII by the AKAP79(31-52) peptide and reduces inhibition by the full-length AKAP79 protein. The effect of calmodulin on inhibition of a constitutively active PKC fragment by the AKAP79(31-52) peptide is shown to be partially dependent on Ca2+. Ca2+/calmodulin reduces PKC coimmunoprecipitated with AKAP79 and results in a 2.6 increase in PKC activity in a preparation of postsynaptic densities. Collectively, these findings suggest that Ca2+/calmodulin competes with PKC for binding to AKAP79, releasing the inhibited kinase from its association with the anchoring protein (Faux, 1997).

The binding of Ca(2+)- and Ba(2+)-calmodulin to caldesmon and its functional consequence has been investigated using three different calmodulin mutants. Two calmodulin mutants have pairs of cysteine residues substituted and oxidized to a disulphide bond in either the N- or C-terminal lobe (C41/75 and C85/112). The third mutant has phenylalanine-92 replaced by alanine (F92A). There is a lower affinity for caldesmon in all the mutants. When Ca2+ is replaced by Ba2+ the affinity of calmodulin for caldesmon is further reduced. The ability of Ca(2+)-calmodulin to release caldesmon's inhibition of the actin-tropomyosin-activated myosin ATPase is virtually abolished by mutation of phenylalanine-92 to alanine or by replacing Ba2+ for Ca2+ in native calmodulin. Both cysteine mutants retain their functional ability, but the increased concentration needed for 50% release of caldesmon inhibition reflectes their decreased affinity. It is concluded that functional binding of Ca(2+)-calmodulin to caldesmon requires multiple interaction sites on both molecules. However, some structural modification in calmodulin does not abolish the caldesmon-related functionality. This suggests that various EF hand proteins can substitute for the calmodulin molecule (Huber, 1996).

Ca2+/calmodulin associates with Src homology 2 domains in the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase, thereby significantly enhancing phosphatidylinositol 3-kinase activity in vitro and in intact cells. In intact cells, CGS9343B, a calmodulin antagonist, inhibits basal and Ca2+-stimulated phosphorylation of phosphatidylinositol. These data demonstrate a novel mechanism for modulating phosphatidylinositol 3-kinase and provide a direct link between components of two fundamental signaling pathways (Joyal, 1997).

G protein-coupled receptor kinases (GRKs) specifically phosphorylate and regulate the activated form of multiple G protein-coupled receptors. Recent studies have revealed that GRKs are also subject to regulation. In this regard, GRK2 and GRK5 can be phosphorylated and either activated or inhibited, respectively, by protein kinase C. Calmodulin, another mediator of calcium signaling, is a potent inhibitor of GRK activity with a selectivity for GRK5 (IC50 approximately 50 nM) > GRK6 >> GRK2 (IC50 approximately 2 &mgr;M) >> GRK1. Calmodulin inhibits GRK5 by caussing a reduced ability of the kinase to bind to both receptor and phospholipid. Interestingly, calmodulin also activates autophosphorylation of GRK5 at sites distinct from the two major autophosphorylation sites on GRK5. Calmodulin-stimulated autophosphorylation directly inhibits GRK5 interaction with receptor, even in the absence of calmodulin. An amino-terminal domain of GRK5 (amino acids 20-39) is sufficient for calmodulin binding. This domain is abundant in basic and hydrophobic residues (characteristics typical of calmodulin binding sites), and is highly conserved in GRK4, GRK5, and GRK6. These studies suggest that calmodulin may serve a general role in mediating calcium-dependent regulation of GRK activity (Pronin, 1997).

The HMG box domain of the testis determining factor, SRY, includes a basic amphiphilic sequence common to calmodulin (CaM) binding proteins. SRY exhibits calcium-dependent binding to CaM. Binding occurs via the HMG box; an SRY peptide of residues 57-80 binds CaM like the intact domain. SRY/CaM complex formation is specifically inhibited by the SRY DNA binding site sequence, AACAAT, but not by a mutated sequence. Fluorescence spectra of the SRY/CaM complex indicate 1:1 stoichiometry and that binding is accompanied by a conformational change in SRY. The A domain of HMG1 also binds CaM. It is proposed that CaM binding is a property of the wider HMG box family, including SOX and TCF/LEF proteins. These results suggest that CaM may regulate the DNA binding activity of HMG box transcription factors (Harley, 1996).

In addition to the well-characterized GTP-dependent nuclear transport observed in permeabilized cells, a mode of nuclear transport occurs that is GTP-independent at elevated cytoplasmic calcium concentrations. Nuclear transport under these conditions is blocked by calmodulin inhibitors. Recombinant calmodulin restores ATP-dependent nuclear transport in the absence of cytosol. Calmodulin-dependent transport is inhibited by wheat germ agglutinin consistent with transport proceeding through nuclear pores. It is proposed that release of intracellular calcium stores upon cell activation inhibits GTP-dependent nuclear transport; the elevated cytosolic calcium then acts through calmodulin to stimulate the novel GTP-independent mode of import (Sweitzer, 1996).

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases (See Drosophila Nos). The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains, termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin. Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." Co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS, expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin (Michel, 1997).

The protection against apoptosis provided by growth factors in several cell lines is due to stimulation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, which results in activation of protein kinase B (PKB; also known as c-Akt) and phosphorylation and sequestration to protein 14-3-3 of the proapoptotic Bcl-2-family member BAD. A modest increase in intracellular Ca2+ concentration also promotes survival of some cultured neurons through a pathway that requires calmodulin but is independent of PI(3)K and the MAP kinases. Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) activates PKB directly, resulting in phosphorylation of BAD on serine residue 136 and the interaction of BAD with protein 14-3-3. Serum withdrawal induces a three- to fourfold increase in cell death of NG108 neuroblastoma cells, and this apoptosis is largely blocked by increasing the intracellular Ca2+ concentration with NMDA (N-methyl-D-aspartate) or KCl or by transfection with constitutively active CaM-KK. The effect of NMDA on cell survival is blocked by transfection with dominant-negative forms of CaM-KK or PKB. These results identify a Ca2+-triggered signaling cascade in which CaM-KK activates PKB, which in turn phosphorylates BAD and protects cells from apoptosis (Yano, 1998).

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends of N-methyl-D-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic alpha-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin, with a Kd value of 44 nM, was detected. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 does not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. The region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. A pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses (Masuko, 1999).

Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP-25. Calcium influx is thought to control this process via Ca2+-binding proteins that associate with components of the SNARE complex. Ca2+/calmodulin or phospholipids bind in a mutually exclusive fashion to a C-terminal domain of VAMP (VAMP_77-90), and residues involved were identified by plasmon resonance spectroscopy. Microinjection of wild-type VAMP_77-90, but not mutant peptides, inhibits catecholamine release from chromaffin cells monitored by carbon fiber amperometry. Pre-incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibits Ca2+-dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppresses secretion. In the presence of TeNT, exocytosis is restored by transfection of TeNT-resistant (Q76V, F77W) VAMP, but additional targeted mutations in VAMP_77-90 abolishes its ability to rescue release. The calmodulin- and phospholipid-binding domain of VAMP 2 is thus required for Ca2+-dependent exocytosis, possibly to regulate SNARE complex assembly (Quetglas, 2002).

Myosin VI (Drosophila homolog: Jaguar) contains an inserted sequence that is unique among myosin superfamily members and has been suggested to be a determinant of the reverse directionality and unusual motility of the motor. It is thought that each head of a two-headed myosin VI molecule binds one calmodulin (CaM) by means of a single 'IQ motif'. Using truncations of the myosin VI protein and electrospray ionization(ESI)-MS, it has been demonstrated that in fact each myosin VI head binds two CaMs. One CaM binds to a conventional IQ motif either with or without calcium and likely plays a regulatory role when calcium binds to its N-terminal lobe. The second CaM binds to a unique insertion between the converter region and IQ motif. This unusual CaM-binding site normally binds CaM with four Ca2+ and can bind only if the C-terminal lobe of CaM is occupied by calcium. Regions of the MD outside of the insert peptide contribute to the Ca(2+)-CaM binding; truncations that eliminate elements of the MD alter CaM binding and allow calcium dissociation. It is suggested that the Ca(2+)-CaM bound to the unique insert represents a structural CaM, and not a calcium sensor or regulatory component of the motor. This structure is likely an integral part of the myosin VI 'converter' region and repositions the myosin VI 'lever arm' to allow reverse direction (minus-end) motility on actin (Bahloul, 2004).

Calmodulin (CaM) is a major effector for the intracellular actions of Ca2+ in nearly all cell types. CaM-binding protein, designated regulator of calmodulin signaling (RCS), has been identified. G protein-coupled receptor (GPCR)-dependent activation of protein kinase A (PKA) led to phosphorylation of RCS at Ser55 and increased its binding to CaM. Phospho-RCS acts as a competitive inhibitor of CaM-dependent enzymes, including protein phosphatase 2B (PP2B, also called calcineurin). Increasing RCS phosphorylation blocks GPCR- and PP2B-mediated suppression of L-type Ca2+ currents in striatal neurons. Conversely, genetic deletion of RCS significantly increases this modulation. Through a molecular mechanism that amplifies GPCR- and PKA-mediated signaling and attenuates GPCR- and PP2B-mediated signaling, RCS synergistically increases the phosphorylation of key proteins whose phosphorylation is regulated by PKA and PP2B (Rakhilin, 2004).

AlphaII-spectrin is a major cortical cytoskeletal protein contributing to membrane organization and integrity. The Ca2+-activated binding of calmodulin to an unstructured insert in the 11th repeat unit of alphaII-spectrin enhances the susceptibility of spectrin to calpain cleavage but abolishes its sensitivity to several caspases and to at least one bacterially derived pathologic protease. Other regulatory inputs including phosphorylation by c-Src also modulate the proteolytic susceptibility of alphaII-spectrin. These pathways, acting through spectrin, appear to control membrane plasticity and integrity in several cell types. To provide a structural basis for understanding these crucial biological events, the crystal structure of a complex between bovine calmodulin and the calmodulin-binding domain of human alphaII-spectrin has been solved. The structure revealed that the entire calmodulin-spectrin-binding interface is hydrophobic in nature. The spectrin domain is also unique in folding into an amphiphilic helix once positioned within the calmodulin-binding groove. The structure of this complex provides insight into the mechanisms by which calmodulin, calpain, caspase, and tyrosine phosphorylation act on spectrin to regulate essential cellular processes (Simonovic, 2006).

Interaction of Calmodulin with CaMKII

Changes in synaptic strength that underlie memory formation in the CNS are initiated by pulses of Ca2+ flowing through NMDA-type glutamate receptors into postsynaptic spines. Differences in the duration and size of the pulses determine whether a synapse is potentiated or depressed after repetitive synaptic activity. Calmodulin (CaM) is a major Ca2+ effector protein that binds up to four Ca2+ ions. CaM with bound Ca2+ can activate at least six signaling enzymes in the spine. In fluctuating cytosolic Ca2+, a large fraction of free CaM is bound to fewer than four Ca2+ ions. Binding to targets increases the affinity of CaM's remaining Ca2+-binding sites. Thus, initial binding of CaM to a target may depend on the target's affinity for CaM with only one or two bound Ca2+ ions. To study CaM-dependent signaling in the spine, mutant CaMs were designed that bind Ca2+ only at the two N-terminal or two C-terminal sites by using computationally designed mutations to stabilize the inactivated Ca2+-binding domains in the 'closed' Ca2+-free conformation. Their interactions with CaMKII, a major Ca2+/CaM target that mediates initiation of long-term potentiation, were measured. CaM with two Ca2+ ions bound in its C-terminal lobe not only binds to CaMKII with low micromolar affinity but also partially activates kinase activity. These results support the idea that competition for binding of CaM with two bound Ca2+ ions may influence significantly the outcome of local Ca2+ signaling in spines and, perhaps, in other signaling pathways (Shifman, 2006).

Calmodulin and vacuole fusion and endocytosis

The basic reaction mechanisms for membrane fusion in the trafficking of intracellular membranes and in exocytosis are probably identical. But in contrast to regulated exocytosis, intracellular fusion reactions are referred to as 'constitutive', since no final Ca2+-dependent triggering step has been observed. Although transport from the endoplasmic reticulum to the Golgi apparatus in the cell depends on Ca2+, as does endosome fusion and assembly of the nuclear envelope, it is unclear whether Ca2+ triggers these events. Membrane fusion involves several subreactions: priming, tethering and docking. Proteins that are needed for fusion include p115, SNAPs, NSF, SNAREs and small GTPases, all of which operate in these early reactions, but the machinery that catalyses the final mixing of biological membranes is still unknown. Ca2+ is released from the vacuolar lumen following completion of the docking step. Calmodulin is identified as the putative Ca2+ sensor and as the first component required in the post-docking phase of vacuole fusion. Calmodulin binds tightly to vacuoles upon Ca2+ release. Unlike synaptotagmin or syncollin in exocytosis, calmodulin does not act as a fusion clamp but actively promotes bilayer mixing. Hence, activation of SNAREs is not sufficient to drive bilayer mixing between physiological membranes. It is proposed that Ca2+ control of the latest phase of membrane fusion may be a conserved feature, relevant not only for exocytosis, but also for intracellular, 'constitutive' fusion reactions. However, the origin of the Ca2+ signal, its receptor and its mode of processing differ (Peters, 1998).

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin [5-hydroxytryptamine (5-HT)] 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation is attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation. This is because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src, but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase have no effect on 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis is required for 5-HT1A receptor-mediated Erk1/2 activation, a role for CAM in receptor endocytosis is postulated. Inhibition of receptor endocytosis by use of sequestration-defective mutants of beta-arrestin1 and dynamin attenuates 5-HT1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevents agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. It is concluded that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects CAM's role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2 (Della Rocca, 1999).

Calmodulin and ion channels

NMDA (N-methyl-D-aspartate) receptors are excitatory neurotransmitter receptors in the brain critical for synaptic plasticity and neuronal development. These receptors are Ca2+-permeable glutamate-gated ion channels whose physiological properties are regulated by intracellular Ca2+. Calmodulin interacts with the NR1 subunit of the NMDA receptor. Calmodulin binding to the NR1 subunit is Ca2+ dependent and occurs with homomeric NR1 complexes, heteromeric NR1/NR2 subunit complexes, and NMDA receptors from brain. Calmodulin binding to NR1 causes a 4-fold reduction in NMDA channel open probability. These results demonstrate that NMDA receptor function can be regulated by direct binding of calmodulin to the NR1 subunit, and suggest a possible mechanism for activity-dependent feedback inhibition and Ca2+-dependent inactivation of NMDA receptors (Ehlers, 1996).

Ca2+ influx through N-methyl-d-aspartate (NMDA) receptors activates signal transduction pathways critical for many forms of synaptic plasticity in the brain. NMDA receptor-mediated Ca2+ influx also downregulates the gating of NMDA channels through a process called Ca2+-dependent inactivation (CDI). Recent studies have demonstrated that the calcium binding protein calmodulin directly interacts with NMDA receptors, suggesting that calmodulin may play a role in CDI. The mutation of a specific calmodulin binding site in the C0 region of the NR1 subunit of the NMDA receptor blocks CDI. Intracellular infusion of a calmodulin inhibitory peptide markedly reduces CDI of both recombinant and neuronal NMDA receptors. This inactivating effect of calmodulin can be prevented by coexpressing a region of the cytoskeletal protein alpha-actinin2, known to interact with the C0 region of NR1. Taken together, these results demonstrate that the binding of Ca2+/calmodulin to NR1 mediates CDI of the NMDA receptor and suggest that inactivation occurs via Ca2+/calmodulin-dependent release of the receptor complex from the neuronal cytoskeleton (Zhang, 1998).

Plasma membrane calcium pump (PMCA) isoforms 4CII (generated by splicing at the C-terminus) and 4BICI (a pump version lacking the 10th transmembrane domain) were expressed in Sf9 cells using the baculovirus system. The purified PMCA4CII has a 20-fold lower affinity for calmodulin than the PMCA4CI, the PMCA4 isoform of the erythrocytes' membranes, but manifests a higher activity in the absence of calmodulin. The amount of phosphoenzyme intermediate formed by PMCA4CII in the presence of Ca2+ alone is almost 3 times higher than in PMCA4CI. The isoform lacking the 10th transmembrane domain (PMCA4BICI) has no Ca2+-dependent ATPase activity, but is still able to form the phosphoenzyme intermediate starting from phosphate. When expressed in COS cells, this isoform is retained in the endoplasmic reticulum; changes in membrane architecture apparently occur during its expression; the C-terminal portion of the isoform is located in the cytosol, indicating that the deletion of the 10th transmembrane domain results in the loss of at least another transmembrane domain (Preiano, 1996).

In primary cultures of cerebellar neurons, glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca2+ and Na+ into the neuron. To maintain Na+ homeostasis, the excess Na+ entering through the ion channel should be removed by Na+,K(+)-ATPase. Incubation of primary cultured cerebellar neurons with glutamate results in activation of the Na+,K(+)-ATPase. The effect is rapid, peaking between 5 and 15 min (85% activation), and is maintained for at least 2 h. Glutamate-induced activation of Na+,K(+)-ATPase is dose dependent: it is appreciable (37%) at 0.1 microM and peaks (85%) at 100 microM. The increase in Na+,K(+)-ATPase activity by glutamate is prevented by MK-801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase is reversed by phorbol ester, an activator of protein kinase C, indicating that activation of Na+,K(+)-ATPase is due to decreased phosphorylation by protein kinase C. Either W-7 or cyclosporin (both inhibitors of calcineurin) prevents the activation of Na+,K(+)-ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na+,K(+)-ATPase that has been previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na+,K(+)-ATPase (Marcaida, 1996).

Neonatal rat sympathetic neurons developing in tissue culture contain 5 nicotinic acetylcholine receptor transcripts: alpha 3, alpha 5, alpha 7, beta 2, and beta 4. When examined in culture, neurons express four of these transcripts (alpha 3, alpha 5, beta 2, and beta 4) at levels similar to those in neurons developing in vivo: alpha 3 mRNA levels increase two- to threefold over the first week, whereas the levels for alpha 5, beta 2, and beta 4 remain essentially constant. In contrast, alpha 7 mRNA levels drop by 60-75% within the first 48 hr and remain low. During the first week, the ACh-evoked current densities on these cultured neurons increase twofold and correlate well with the increase in alpha 3 mRNA levels. Depolarizing the neurons with 40 mM KCl for 1-2 d upregulates the alpha 7 gene; this specific change in alpha 7 mRNA level correlates with an increase in alpha-bungarotoxin (alpha-BTX) binding on the surface of the neurons. Depolarization has little effect on the expression of the other four transcripts, or on either the magnitude or kinetics of the ACh-evoked currents. Furthermore, activators or inhibitors of protein kinase A (PKA), protein kinase C (PKC), or tyrosine kinase do not affect nAChR transcript levels in these cultured neurons. The effect of membrane depolarization on alpha 7 expression is a result of Ca2+ influx through L-type Ca2+ channels, and it has been shown that alpha 7 is upregulated through a Ca2+/calmodulin-dependent protein kinase (CaM kinase) pathway. The identification of CaM kinase as a link between activity and neurotransmitter receptor expression may indicate a novel mechanism that underlies some forms of synaptic plasticity (De Koninck, 1995).

Intracellular Ca2+ inhibits voltage-gated potassium channels of the ether a go-go (EAG) family. To identify the underlying molecular mechanism, the human version hEAG1 was expressed in Xenopus oocytes. The channels lose Ca2+ sensitivity when measured in cell-free membrane patches. However, Ca2+ sensitivity can be restored by application of recombinant calmodulin (CaM). In the presence of CaM, half inhibition of hEAG1 channels was obtained in 100 nM Ca2+. Overlay assays using labelled CaM and glutathione S-transferase (GST) fusion fragments of hEAG1 demonstrate direct binding of CaM to a C-terminal domain (hEAG1 amino acids 673-770). Point mutations within this section reveal 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 Ca2+-activated potassium channels, Ca2+ dependent, with an apparent KD 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 (Schonherr, 2000).

Calmodulin and calcium channels

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