Calmodulin

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca 2+/CaM) in living cells. If two chemically different inhibitors ...

Active calcium transport by cardiac sarcoplasmic reticulum assumes a central role in the excitation-concentration coupling of the myocardium, in that Ca2+-dependent ATPase (mol.wt. 100 000) of cardiac sarcoplasmic reticulum serves as an energy transducer and a translocator of Ca2+ across the membrane. During the translocation of Ca2+, the ATPase undergoes a complex series of reactions during which the phosphorylated intermediate EP is formed. We documented how the elementary steps of the ATPase reaction are coupled with calcium translocation, and provided evidences to indicate that two key steps of ATPase correspond to the conformational change of the enzyme, and appear to alter the affinity of the enzyme for Ca2+. A line of evidence also indicated that Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum is regulated by a specific protein named phospholamban (mol.wt. 22 000), which serves as a substrate for cyclic AMP-dependent protein kinase. Cyclic AMP-dependent phosphorylation of phospholamban resulted in a marked increase in the rate of turnover of the ATPase, by enhancing the rates of the key elementary steps, i.e. the steps at which the intermediate EP is formed and decomposed. Thus phospholamban is putatively thought to serve as a modulator of Cat2+-dependent ATPase of cardiac sarcoplasmic reticulum. A working model was proposed to interpret the mechanism. Also documented is a possibility that another protein kinase activatable by Ca2+ and calmodulin is functional in regulating the phospholamban-ATPase system, thus suggesting the existence of a dual control system, in which both cyclic AMP- and calmodulin-dependent phosphorylation are in control of the Cat2+-dependent ATPase. Such a control mechanism may provide the interpretation, at the cellular level, that catecholamines exert actions on myocardial contractility. Thus, catecholamine-mediated increases in intracellular cyclic AMP could enhance calcium fluxes across the membrane of sarcoplasmic reticulum, thus resulting in the increased rates of relaxation and, at the same time, the increased rate and extent of contraction. Such a mechanism could also be operational in the tissues, other than the myocardium, in which catecholamines and other hormones serve as the ‘first messenger’, producing intracellular cyclic AMP as the ‘second messenger’.

Hydra feeding response is a very primitive olfactory-like behavior present in a multicellular organism. We investigated the role of nitric oxide (NO) in the induction and control of hydra feeding response. Under basal conditions, hydra specimens produce detectable amounts of nitrite (NO2-), the breakdown product of NO. When hydra were incubated with reduced glutathione (GSH), the typical activator of feeding response, an increase of basal NO production was observed. This effect was inhibited by glutamic or alpha-aminoadipic acids, two GSH antagonists, which block GSH-induced feeding response, and by the NO synthase (NOS) inhibitor L-NAME. Moreover, we found that hydra possess a calcium-dependent (but calmodulin-independent) NOS isoform. By using exogenous NO donors and NOS inhibitors, we demonstrated that NO stimulus can participate both in triggering tentacular movements and in recruiting neighbor tentacles during hydra feeding response. By using dbt2-cGMP, an analog to cGMP, we ob...

Modification of calmodulin by protein carboxyl methyltransferase requires deamidation of one or more labile asparagine residues (Johnson, B.A., Freitag, N. E., and Aswad, D. W. (1985) J. Biol. Chem. 260, 10913-10916). We now show that deamidation results in the generation of two altered forms of calmodulin, designated A and B, which can be separated by electrophoresis under nondenaturing conditions. The A form is characterized by a larger apparent molecular radius, has only 10% the activity of native calmodulin when assayed for its ability to activate a Ca2+/calmodulin-dependent protein kinase from rat brain, and serves as an excellent substrate for the methyltransferase. The B form more closely resembles native calmodulin: it has an apparent molecular radius more like the native, exhibits about 40% the activity of native calmodulin, and is a relatively poor methyl acceptor. Evidence suggests that the A and B forms probably contain isoaspartate (A) and aspartate (B) in place of Asn-...

In the last decade, the physiology of cerebellar neurons and synapses has been extended to a considerable extent. We have found that the mossy fiber-granule cell relay can generate a complex form of long-term potentiation (mf-GrC LTP) following high-frequency mf discharge. Induction. Mf-GrC LTP depends on NMDA and mGlu receptor activation, intracellular Ca(2+) increase, PKC activation, and NO production. The preventative action of intracellular agents (BAPTA, PKC-inhibitors) and of membrane hyperpolarization, and the correlated increase in intracellular Ca(2+) observed using fluorescent dyes, indicate that induction occurs postsynaptically. Expression. Expression includes three components: (a) an increase of synaptic currents, (b) an increase of intrinsic excitability in GrC, and (c) an increase of intrinsic excitability in mf terminals. Based on quantal analysis, the EPSC increase is mostly explained by enhanced neurotransmitter release. NO is a candidate retrograde neurotransmitte...

Neurohormones similar to those of mammals are carried in fish by hypothalamic nerve fibers to regulate directly follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Gonadotropin-releasing hormone (GnRH) stimulates the secretion of FSH and LH and the expression of the glycoprotein hormone alpha (GPalpha), FSHbeta, and LHbeta, as well as their secretion. Its signal transduction leading to LH release is similar to that in mammals although the involvement of cyclic AMP-protein kinase A (cAMP-PKA) cannot be ruled out. Dopamine (DA) acting through DA D2 type receptors may inhibit LH release, but not that of FSH, at sites distal to activation of protein kinase C (PKC) and PKA. GnRH increases the steady-state levels of GPalpha, LHbeta, and FSHbeta mRNAs. Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and neuropeptide Y (NPY) potentiate GnRH effect on gonadotropic cells, and also act directly on the pituitary cells. Whereas PACAP increases all three subunit mRNAs, ...

In this and the associated article 'BioBlender: A Software for Intuitive Representation of Surface Properties of Biomolecules', (Andrei et al) we present BioBlender as a complete instrument for the elaboration of motion (here) and the visualization (Andrei et al) of proteins and other macromolecules, using instruments of computer graphics. A vast number of protein (if not most) exert their function through some extent of motion. Despite recent advances in higly performant methods, it is very difficult to obtain direct information on conformational changes of molecules. However, several systems exist that can shed some light on the variability of conformations of a single peptide chain; among them, NMR methods provide collections of a number of static 'shots' of a moving protein. Starting from this data, and assuming that if a protein exists in more than 1 conformation it must be able to transit between the different states, we have elaborated a system that makes ample use of the computational power of 3D computer graphics technology. Considering information of all (heavy) atoms, we use animation and game engine of Blender to obtain transition states. The model we chose to elaborate our system is Calmodulin, a protein favorite among structural and dynamic studies due to its (relative) simplicity of structure and small dimension. Using Calmodulin we show a procedure that enables the building of a 'navigation map' of NMR models, that can help in the identification of movements. In the process, a number of intermediate conformations is generated, all of which respond to strict bio-physical and bio-chemical criteria. The BioBlender system is available for download from the website www.bioblender.net, together with examples, tutorial and other useful material.