Presence Of Calcium-calmodulin Research Articles

Allosteric Changes in the NMDA Receptor Associated with Calcium-Dependent Inactivation

N-methyl-D-aspartate (NMDA) receptors mediate synaptic excitatory signaling in the mammalian central nervous system by forming calcium-permeable transmembrane channels upon binding glutamate and coagonist glycine. Ca2+ influx through NMDA receptors leads to channel inactivation through a process mediated by resident calmodulin bound to the intracellular C-terminal segment of the GluN1 subunit of the receptor. Using single-molecule FRET investigations, we show that in the presence of calcium-calmodulin, the distance across the two GluN1 subunits at the entrance of the first transmembrane segment is shorter and the bilobed cleft of the glycine-binding domain in GluN1 is more closed when bound to glycine and glutamate relative to what is observed in the presence of barium-calmodulin. Consistent with these observations, the glycine deactivation rate is slower in the presence of calcium-calmodulin. Taken together, these results show that the binding of calcium-calmodulin to the C-terminus has long-range allosteric effects on the extracellular segments of the receptor that may contribute to the calcium-dependent inactivation.

The Potassium Channel Interacting Protein 3 (DREAM/KChIP3) Heterodimerizes with and Regulates Calmodulin Function

Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca(2+)) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca(2+), DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca(2+). In the absence of Ca(2+), DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca(2+)-bound DREAM/KChIP3 binds CaM with a dissociation constant of ∼3 μM as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional (1)H,(15)N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to (15)N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca(2+)-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca(2+) also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH(2)-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.

Modulation of rod, but not cone, cGMP-gated photoreceptor channels by calcium-calmodulin.

Inside-out patches containing cGMP-gated channels were excised from catfish rod or cone outer segments and held under voltage clamp. The net cGMP-dependent currents elicited by saturating and subsaturating concentrations of cGMP at +/-30 mV were measured and the dependence of current upon cGMP concentration was determined. The apparent affinity of the channel for its ligand was estimated by fitting these data with the Hill equation. The concentration of cGMP required to give half the maximum current (K1/2) in rod and cone channels at +30 mV was approximately 28 microM and approximately 37 microM, respectively, and was weakly voltage dependent. Thus, cone channels have an intrinsically higher K1/2 than rod channels. For both types of channel, the Hill coefficient was approximately 2.3. In the presence of calcium-calmodulin, the apparent affinity of the rod channel for cGMP decreased by about twofold, but the apparent affinity of the cone channels was unaffected. These results indicate that the open probability of the cone channel for its ligand cannot be modulated by calmodulin. This represents the first significant departure between rod and cone photoreceptors in mechanisms used by phototransduction and suggests that the beta subunit of the cone channel must be different from that of the rod channel.

The mammalian pineal expresses the cone but not the rod cyclic GMP phosphodiesterase.

To determine the presence of cone or rod cyclic GMP phosphodiesterase (EC 3.1.4.17) in the mammalian pineal, extracts from adult rat and bovine pineals were injected onto a Mono Q anion-exchange HPLC column and eluted with an NaCl linear gradient. Fractions were immunoadsorbed with monoclonal antibodies specific to rod and cone phosphodiesterases (ROS-1) and to calmodulin-phosphodiesterase complexes (ACC). Profiles were assayed with 10 mumol/L [3H]cyclic GMP in the presence of calcium-calmodulin, histone, or trypsin. Rat and bovine pineals displayed a single peak of activity recognized by ROS-1, which corresponded to the activity of the cone but not to the rod in bovine retina. ROS-1 immunoadsorbed approximately 80% of the activity in the 60-day-old rat pineal but only 26% of the activity in bovine pineal. ACC immunoadsorbed the remaining activity in both species. Western blot analysis of rat pineal extracts revealed three polypeptides of approximately 87, 15, and 10 kDa when probed with a rod/cone phosphodiesterase-specific antiserum. The specific activity of the cone-like phosphodiesterase in 10-day-old rat pineals was twice that of this isozyme in the bovine retina and 150 times that in the bovine pineal. The specific activity of phosphodiesterase in rat pineals decreased with age. We conclude that an enzyme with biochemical and antigenic characteristics similar to cone, but distinct from rod phosphodiesterase, is present in bovine and rat pineals.

Relationship between cyclic nucleotide levels and 5-methyl-6-(4-pyridyl)-2H-1,4-thiazin-3(4H)-one (ZSY-27), a new positive inotropic agent with a vasodilatory action, -induced relaxation of rabbit thoracic aorta.

The aim of this investigation was to substantiate the hypothesis that the vasorelaxant effects of 5-methyl-6-(4-pyridyl)-2H-1,4-thiazin-3(4H)-one (ZSY-27) are mediated by accumulation of intracellular cyclic nucleotides as a consequence of inhibition of cyclic nucleotide phosphodiesterase activity. Both activities of adenosine 3',5'-monophosphate-phosphodiesterase (cAMP-PDE) in the presence of ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) and guanosine 3',5'-monophosphate-phosphodiesterase (cGMP-PDE) in the presence of calcium-calmodulin from rabbit thoracic aorta were inhibited in a concentration-dependent manner by ZSY-27 (10(-5) - 10(-3) M). The IC50 values for ZSY-27 on cAMP- and cGMP-PDE activity were 2.1 x 10(-4) and 8.8 x 10(-4) M, respectively. Furthermore, ZSY-27 antagonized competitively cAMP-PDE (Ki = 1.9 x 10(-4) M). On the other hand, ZSY-27 exhibited a mixed-type inhibitory pattern, with reduction of both maximum velocity and affinity for the substrate of the cGMP-PDE, with a Ki value of 1.0 x 10(-3) M. Spontaneous myogenic tone of rabbit thoracic aorta was significantly attenuated from 1 min after addition of ZSY-27 (3 x 10(-4) M). Contents of cAMP and cGMP were significantly increased from 1 and 3 min after addition of ZSY-27, respectively. Temporally, relaxant effects of ZSY-27 were associated with increases of cAMP content, but not with that of cGMP content.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased phosphorylation of a membrane protein consequent to amygdaloid kindling.

The phosphorylation of both particulate and soluble proteins in the amygdala was examined in electrically kindled rats. In animals receiving electrical stimulation in the left amygdala for 5-6 days that displayed electrical after-discharges but no motor seizures, no changes were observed in the phosphorylation of either particulate or soluble proteins. In animals stimulated for 20-21 days where major motor seizures were produced, the phosphorylation of a protein having a molecular weight of 45,000 ( 45K ) was markedly increased. The phosphorylation of this protein was increased in both the right (unstimulated) and left (stimulated) amygdala. Major motor seizures induced by electroconvulsive shocks, however, did not alter phosphorylation of this protein. Phosphorylation of the 45K protein was stimulated by calcium and calmodulin. The 45K protein is a major phosphoprotein of amygdala, representing 3.2% of the total particulate phosphoproteins in control animals and 7.4% in the kindled animals. In the presence of calcium-calmodulin, 16.2% of net protein phosphorylation was accounted for by the 45K protein.