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PubMed1993

Virulence of a Bordetella pertussis strain expressing a mutant adenylyl cyclase with decreased calmodulin affinity.

Bordetella pertussis, the pathogen responsible for whooping cough, produces a toxic calmodulin-sensitive adenylyl cyclase which enters animal cells and increases intracellular cAMP. A point mutant of B. pertussis with abolished adenylyl cyclase catalytic activity was over 1000-fold less pathogenic to newborn mice than wild-type bacteria, demonstrating the importance of the adenylyl cyclase for B. pertussis virulence (Gross et al.). The B. pertussis adenylyl cyclase is highly sensitive to calmodulin with an apparent Kd for calmodulin of approximately 1 nM. The importance of this high-affinity calmodulin binding for virulence in vivo was examined by the creation of a B. pertussis point mutant (Trp-242 to Glu-242) with 200-fold lower calmodulin affinity than the native enzyme. This mutant B. pertussis strain retained its virulence in a newborn mouse model of pertussis, but the time course for establishment of a lethal infection in vivo was significantly delayed for the mutant strain. These data illustrate that high-affinity calmodulin binding is not obligatory for the activity of this toxin but is important for the rate for establishment of a lethal infection.

Adenylyl Cyclases↗

PubMed2001

Fumonisin B(1) induces apoptosis in LLC-PK(1) renal epithelial cells via a sphinganine- and calmodulin-dependent pathway.

Fumonisins are a family of mycotoxins produced by Fusarium moniliforme, which is the most common mold found on corn throughout the world. These compounds are both toxic and carcinogenic for animals, and perhaps humans, with the kidney being the most sensitive organ to fumonisin toxicity. The molecular mechanism of fumonisin toxicity appears to involve disruption of de novo biosynthesis of sphingolipids and accumulation of sphinganine. The goals of this study were to determine whether fumonisin B(1) kills LLC-PK(1) renal kidney epithelial cells by inducing apoptosis and to identify genes affected by sphinganine that mediate fumonisin B(1)-induced cell death. Fumonisin B(1) produced morphological changes (i.e., cell shrinkage, membrane blebbing) and time-dependent increases in DNA fragmentation demonstrating that the toxin induces apoptosis. Simultaneously, fumonisin B(1) blocked sphingolipid biosynthesis and caused accumulation of sphinganine. To further investigate the role of sphinganine in fumonisin B(1)-induced apoptosis, beta-fluoroalanine (betaFA) was used to inhibit serine palmitoyltransferase, which catalyzes an earlier step in the sphingolipid biosynthetic pathway. betaFA blocked sphinganine accumulation and prevented fumonisin B(1)-induced DNA fragmentation, confirming that apoptosis induced by fumonisin B(1) is dependent upon accumulation of sphinganine. To examine gene expression, differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) was applied to RNA isolated after 16 h of exposure to fumonisin B(1). Differential expression in response to fumonisin B(1) of a gene identified as calmodulin has been verified by Northern analysis. Sphinganine appears to mediate the effect because betaFA reduces induction of calmodulin mRNA by fumonisin B(1). Fumonisin B(1) also increases calmodulin protein in a concentration-dependent manner and the calmodulin antagonist W7 blocks fumonisin B(1)-induced DNA fragmentation, supporting a role for calmodulin in fumonisin B(1)-induced apoptosis. In contrast, fumonisin B(1) had no effect on expression of bcl-2 family genes (bax, bcl-2, and bcl-x). These findings demonstrate that fumonisin B(1) kills LLC-PK(1) kidney cells by inducing apoptosis. Further, the results establish a sequence of events for fumonisin B(1)-induced apoptosis involving initial disruption of sphingolipid metabolism and accumulation of sphinganine (or a metabolite), which, in turn, induces expression of calmodulin.

Alanine↗

PubMed1993

Effects of various calmodulin antagonists on Na/Ca exchange current of single ventricular cells of guinea-pig.

The effects of various calmodulin inhibitors were examined on the Na/Ca exchange current in single cardiac ventricular cells of the guinea-pig using the whole-cell patch-clamp technique. External application of W-7 and trifluoperazine inhibited Na/Ca exchange current in a dose-dependent manner with IC50 values of 13 and 7 microM, respectively. W-5 inhibited the exchange current but less potently than W-7. More specific calmodulin inhibitors such as CGS 9343B and calmidazolium did not, however, decrease the current as significantly as expected. All these drugs inhibited the Na current more strongly than the Na/Ca exchange current. Ruthenium red (RR), another type of calmodulin inhibitor, did not decrease the exchange current by internal application. Neither mastoparan or melittin (calmodulin-binding peptides) inhibited the exchange current appreciably. RR and the peptides did not affect the Na current either. These results indicate that calmodulin may not involved in the activation of cardiac Na/Ca exchange or the Na current. Internal application of chymotrypsin inhibited the blocking effect of W-7 on the Na/Ca exchange current but not that on the Na current. These results indicate that W-7 blocks the Na/Ca exchange current not by binding to calmodulin but possibly by directly affecting an internal site of the exchanger itself and that the inhibitory action of W-7 is different on the Na/Ca exchange current and the Na current.

Animals↗

PubMed1986

Endogenous dopamine release from tuberoinfundibular neurons: does calmodulin play any role?

The possible involvement of calmodulin in the process of endogenous dopamine (DA) release from arcuate-periventricular nuclei-median eminence fragments, containing tuberoinfundibular dopaminergic (TIDA) neurons, has been investigated in an in vitro incubation system. For this purpose the basal and K+-stimulated DA release was examined in the presence and in the absence of the different putative calmodulin antagonists, pimozide, trifluoperazine, penfluridol and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Trifluoperazine and pimozide in concentrations up to 100 microM were both uneffective in blocking K+-evoked DA release. Penfluridol in doses of 5 and 10 microM, did not prevent 35 mM K+-induced endogenous DA release. It was able to reduce K+-stimulated DA release only at the very large concentration of 100 microM. W-7 added in vitro to the hypothalamic fragments, prevented endogenous DA release evoked by 35 mM K+ in a dose-dependent manner. W-5, a chlorine deficient analogue of W-7, that interacts only weakly with calmodulin, failed to modify K+-stimulated endogenous DA release in doses up to 200 microM. All the putative calmodulin antagonists used in the present study did not induce any change of basal DA release. In conclusion the fact that most of the agents, except W-7, known to antagonize calmodulin-dependent processes in many biological systems failed to interfere with the release of endogenous DA from TIDA neurons seems to suggest that calmodulin does not play a crucial role in the process of DA release and that the inhibitory effect of W-7 on endogenous DA release may be better attributed to other mechanisms different from its anticalmodulin action.

Animals↗

PubMed1986

Calmodulin-dependency of human neutrophil phosphodiesterase.

A recent study has reported that the phosphodiesterases of human neutrophils are calmodulin-insensitive (Smolen and Geosits, Inflammation 8:193-199, 1984). In the present study, two forms of human neutrophil phosphodiesterase were separated by chromatography on DEAE-52. Peak I phosphodiesterase is activated 2.3-fold by calcium and calmodulin but is not stimulated by either calcium or calmodulin alone. Calmodulin-dependent activation of the phosphodiesterase is blocked by both 20 microM trifluoperazine and 20 microM W-7. Peak II is not stimulated by calmodulin. These findings suggest that calmodulin may play an important role in regulating alterations in cyclic nucleotide metabolism that accompany neutrophil activation.

Calcium↗

PubMed1991

The regulatory role of calmodulin in the proteolysis of individual neurofilament proteins by calpain.

The in vitro degradation of individual neurofilament proteins by calpain and the effects of calmodulin on this proteolysis were studied. Two major results are reported. First, in the presence of calcium, calmodulin binds to the 200-kD neurofilament protein, but only weakly associates with the 150-kD neurofilament protein. The 70-kD neurofilament protein shows no specific calmodulin-binding. Second, calmodulin inhibits the calpain-mediated degradation of the 200-kD neurofilament protein, but does not alter the hydrolysis of the 150-kD and 70-kD neurofilament proteins. In addition, calmodulin is able to bind to the 200-kD neurofilament protein in the presence of other neurofilament subunits, indicating that calmodulin may play a role in the regulation of the metabolism of the 200-kD neurofilament protein in vivo.

Animals↗

PubMed1993

[125I]calmodulin binding to synaptic plasma membrane from rat brain: kinetic and Arrhenius analysis.

Binding of [125I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca(2+)-dependent membrane binding of [125I]calmodulin was found to have a Bmax of 284 pmol/mg protein and an apparent affinity with a Kd of 131 nM. Kinetic analysis indicates that at 37 degrees C, the dissociation of [125I]calmodulin-membrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7 x 10(-1) min-1 and a half-time (t1/2) of 1.8 min for the fast component, and a k of 4.8 x 10(-2) min-1 and a t1/2 of 14.5 min for the slow component. At 0 degrees C, substantial dissociation still occurred, with a k of 4.5 x 10(-2) min-1 and a t1/2 of 15.3 min for the fast component, and a k of 5.5 x 10(-3) min-1 and a t1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associate and dissociate calmodulin. In Arrhenius analysis of temperature effects, [125I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (Td) estimated to be 23.8 degrees C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [125I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10(-7)-10(-6) M), a steroid hormone, and decreased by ethanol (50-200 mM), a centrally acting drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Goat testis calmodulin: purification and physicochemical characterization.

Calmodulin has been purified in large quantities from goat (Capra hiscus) testis. The procedure includes heat treatment, hydrophobic interaction chromatography, and gel filtration. Goat testis calmodulin closely resembles other mammalian testis calmodulin studied so far. The protein has an extinction coefficient value (E1%1cm) of 2.09 at 280 nm, a Stokes radius of 23.2 A at 0.15 M KCl, and a frictional ratio of 1.38. Ca2+, and Tb3+ binding studies demonstrate that the protein has four Ca2(+)-binding sites with a Kd of 52.5 microM. Goat testis calmodulin shows close similarity to other calmodulins in the amino acid composition and in demonstrating an altered migration on SDS/PAGE upon Ca2+ binding. The protein also exhibits anomalously high values for molecular weight and Stokes radius as determined from the analytical gel chromatography and a change in its elution volume with the change of salt concentration in the eluant. These results have been discussed in view of the recently available knowledge from the crystallographic studies of rat testis calmodulin.

Amino Acids↗

PubMed1985

Lens cell-to-cell channel protein: II. Conformational change in the presence of calmodulin.

Lens fibers are coupled by communicating junctions, clusters of cell-to-cell channels composed of a 28-kD intrinsic membrane protein (MIP26). Evidence suggests that these and other cell-to-cell channels may close as a result of protein conformational change induced by activated calmodulin. To test the validity of this hypothesis, we have measured the intrinsic fluorescence emission and far-ultraviolet circular dichroism of the isolated components MIP26, calmodulin, and the MIP26-calmodulin complex, both in the absence and presence of Ca++, an uncoupling agent. MIP26 shows no change in either fluorescence emission (primarily tryptophan and a measure of aromatic constitutivity) or in its circular dichroism spectrum. Calmodulin exhibits a 32% increase in fluorescence emission intensity with constant emission wavelength, entirely tyrosine, and a 44% increase in alpha-helicity, changes previously described. The MIP26-calmodulin complex, on the other hand, displays fluorescence emission and circular dichroism spectra which are slightly different from the sum of the two single components, but shows marked differences in both spectra upon Ca++ addition. This indicates a change in conformation in one or both of the two components. Spectral changes include a 5-nm blue-shift, a 50% increase in tyrosine fluorescence emission, a 25% decrease in tryptophan fluorescence emission, and a 5% increase in the alpha-helicity of the complex. These changes also occur about an isosbestic point and are fully reversible. These data provide additional evidence that activated calmodulin may modulate gating of cell-to-cell channels by affecting channel protein.

Animals↗

PubMed1985

Interaction of smooth muscle relaxant drugs with calmodulin and cyclic nucleotide phosphodiesterase.

Some smooth muscle relaxant drugs with an unknown mechanism of action have been tested for their interaction with calmodulin and with calmodulin-induced cyclic nucleotide phosphodiesterase (PDE) activity. The affinity of these drugs for calmodulin does not parallel their inhibitory effect on the calmodulin activation of PDE. The lack of parallelism could be due to a binding of the drugs to different sites on calmodulin; furthermore a binding of papaverine, octylonium bromide and felodipine to PDE molecule might also be considered to explain their inhibitory effect on PDE basal activity. The myolytic effect of octylonium bromide and pinaverium bromide may be due to their interaction with calmodulin-dependent systems.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1996

Calmodulin increases the initial rate of exocytosis in adrenal chromaffin cells.

The effect of calmodulin on exocytosis in bovine adrenal medullary chromaffin cells was examined by the use of patch-clamp capacitance recording. Calmodulin was dialysed into cells via the patch-pipette and cells stimulated by depolarisation. Following a test stimulation, cells were dialysed with a control or with a calmodulin containing buffer for 10 mins and then were stimulated at 2 min intervals thereafter. The inclusion of calmodulin in the pipette did not increase Ca2+ currents which instead decreased during dialysis. The presence of calmodulin, however, resulted in a 2-fold increase in the initial rate of exocytosis during the 10 min depolarisation step. These results demonstrate the utility of the patch-clamp capacitance technique for the examination of the effect of soluble proteins on exocytosis and in conjunction with previous work on permeabilised chromaffin cells suggest that calmodulin regulates late steps in Ca2+-dependent exocytosis.

Animals↗

PubMed1986

The high affinity calcium inhibition of parathyroid adenylate cyclase is not calmodulin dependent.

To investigate the possible role of calmodulin in the calcium sensitivity of parathyroid adenylate cyclase (AC), the effect of the calmodulin inhibitor trifluoperazine hydrochloride (TFP) on the calcium sensitivity of forskolin-stimulated AC activity was investigated in membranes prepared from normal porcine parathyroid glands. TFP inhibited AC in a concentration-dependent manner, the IC50 being approximately 100 microM. The inhibition of the enzyme occurred at roughly the same concentration of TFP in the presence and absence of calcium. Another calmodulin inhibitor, N-(6-aminohexyl)-chloro-1-naphthalenesulfonamide (W-7), also inhibited AC in a calcium-independent manner with a IC50 of approximately 200 microM. The pattern of calcium inhibition of AC was compared in membranes prewashed with either EGTA or 2 microM ionic calcium plus 100 microM TFP in an attempt to remove endogenous calmodulin. Neither treatment significantly altered the apparent affinities of the two previously reported calcium inhibition sites, nor did they alter the relative contribution of the individual calcium inhibition sites to the overall calcium inhibition. Inclusion of 100 microM TFP in the incubation mixture resulted in no change in the apparent affinities of the calcium inhibition site although it did result in a significant decrease in the relative contribution of the high affinity site (P less than 0.05). Addition of exogenous calmodulin (5-50 micrograms/ml) had no significant effect on AC. We conclude from these studies that the inhibition of parathyroid AC by calcium is independent of calmodulin and that this enzyme has intrinsic high sensitivity to calcium.

Adenylyl Cyclases↗

PubMed1997

The actions of calmodulin antagonists W-7 and TFP and of calcium on the gating kinetics of the calcium-activated large conductance potassium channel of the chara protoplasmic drop: a substate-sensitive analysis.

The effects of the calmodulin antagonists W-7 and trifluoperazine have been measured on the Ca2+-activated potassium channel in the membrane surrounding protoplasmic drops expressed from internodal cells of charophyte plants. The large-conductance (170 pS), voltage- and Ca2+-dependent gating, and prominent conductance substrate of this channel shows a strong kinetic resemblance to those of the Maxi-K channel from animal cells. This is the first study of the action of calmodulin antagonists which measures their effects on the most populated substates as well as the closed and main open states of Maxi-K channels. The substate analysis provides new evidence for different modes of action of- and different bindings sites for these calmodulin antagonists. Neither antagonist produces the simple closure of the channel reported previously as its effect on the Maxi-K channel, though both do induce flicker-block, reducing the mean current to near zero at high concentrations following an inverted Michaelis-Menten curve. W-7 reduces residence time in the fully open state, thus raising, in the same proportions, the probabilities of finding the channel in the closed state or a pre-existing substate. Its binding to the channel is voltage- and calcium-dependent. In contrast, trifluoperazine reduces residence in the open state and promotes an apparently new substate which overlaps the closed state at -50 mV but is distinguishable from it at voltages more negative than -100 mV. This substate may represent times that trifluoperazine is bound to the channel. Both antagonists have effects clearly distinguishable from that of withdrawing calcium from the channel, which does not affect open state residence time but increases closed state residence time. Thus neither antagonist reverses the activating effect of Ca2-. This is good kinetic evidence against the view that the channel is activated by Ca2+-calmodulin and that the effect of a calmodulin antagonist is to reverse this process by making Ca2--calmodulin less available.

Calcium↗

PubMed2003

Time-resolved fluorescence anisotropy studies show domain-specific interactions of calmodulin with IQ target sequences of myosin V.

Single cysteine mutants of calmodulin, Cam(S38C) and Cam(N111C), have been specifically labelled with Alexa488 maleimide to study the effects of calcium on the structural dynamics of calmodulin complexed with IQ3, IQ4 and IQ34 target peptide motifs of mouse unconventional myosin-V. Using phase fluorometry, the time-resolved anisotropy shows well-separated global and segmental correlation times. The calcium-sensitive global motion of either calmodulin domain can be independently monitored in domain-specific interactions of either apo- or Ca(4).calmodulin with IQ3 or IQ4 peptides. C-domain interactions predominate, and apo-N-domain interactions are unexpectedly weak. The 1:1 complex of Ca(4).calmodulin with IQ34 behaves as a compact globular species. The results demonstrate novel dynamic aspects of calmodulin-IQ interactions relating to the calcium regulation of motility of unconventional myosin.

Amino Acid Sequence↗

PubMed1997

Cisplatin-induced inhibition of the calcium-calmodulin complex, neuronal nitric oxide synthase activation and their role in stomach distention.

Cisplatin (8 mg/kg; i.p.) treatment of Wistar rats produced no change in nitric oxide synthase (NOS) localization or its intensity for up to 5 days. However, immunohistochemically the levels of L-citrulline and the Ca(2+)-calmodulin complex were decreased after only 3 days. An in vitro experiment using an analog of calmodulin. Mero-Calmodulin-1, showed that cis-diammine-diaquacisplatinum(II), a hydrolyzed form of cisplatin, inhibited the calmodulin conformational shift from occurring through a direct interaction with the calmodulin molecule. The results indicate that distention of the stomach was due to inhibition of neuronal NOS activation by a direct interaction between cisplatin and the calcium binding sites of the calmodulin molecule.

Animals↗

PubMed2002

Calmodulin immunolocalization in outer segments of Xenopus laevis photoreceptors.

Because adaptation of vertebrate photoreceptors to light is mediated by changes in the level of calcium in their outer segments (OS), proteins that bind calcium are important in phototransduction. This study has used immunofluorescence to investigate the distribution of the calcium-binding protein calmodulin within photoreceptor OS dissociated from amphibian ( Xenopus laevis) retinas. The OS of rods and cones had a streak of fluorescence to calmodulin at the ciliary axoneme. The OS of rods (but not cones) also displayed regularly spaced puncta of anti-calmodulin fluorescence along longitudinal lines coinciding with their multiple incisures. This location of calmodulin immunofluorescence closely matches the known location of microtubules within the OS of amphibian rods and cones. These findings provide evidence that calmodulin is closely associated with the microtubules of both the axonemal and incisural cytoskeletal systems in OS, and suggest that this association is important for calmodulin function in photoreceptors.

Animals↗

PubMed1983

Radiometric assay of S-adenosylmethionine:calmodulin(lysine)N-methyltransferase by calcium-dependent hydrophobic interaction chromatography.

A radiometric assay for S-adenosylmethionine:calmodulin(lysine)N-methyltransferase in tissue extracts has been developed. This assay utilizes the non-N-methylated calmodulin from Dictyostelium discoideum as a substrate and calcium-dependent hydrophobic interaction chromatography to isolate the reaction product, N-methylated calmodulin. The assay measures the transfer of 3H-labeled methyl groups from [methyl-3H]S-adenosylmethionine to calmodulin. Methylated calmodulin is eluted from phenyl-Sepharose columns with EDTA and protein carboxylmethyl esters are removed by heat treatment prior to liquid scintillation counting of [methyl-3H]calmodulin. This assay is more specific, more sensitive, and more precise than the acid-precipitation assay previously employed and lends itself more readily to the assay of large numbers of samples.

Animals↗

PubMed1984

Regulatory aspects of rat liver mitochondrial phospholipase A2: effects of calcium ions and calmodulin.

A comparative study was made of the metal ion requirement of rat liver mitochondrial phospholipase A2 in purified and membrane-associated forms. Membrane-bound enzyme was assayed using either exogenous or endogenous phosphatidylethanolamine. Although several divalent metal ions caused increased activity of the membrane-associated enzyme, only Ca2+ and Sr2+ activated the purified phospholipase A2. The activity in the presence of Sr2+ amounted to about 25% of that found with Ca2+. When the Ca2+ concentration was varied two activity plateaus were observed. The corresponding dissociation constants varied from 6 to 20 microM Ca2+ and from 1.4 to 12 mM Ca2+ for the high- and low-affinity binding sites, respectively, depending on the assay conditions and whether purified or membrane-bound enzyme was used. A kSr2+ of 60 microM was found for the high-affinity binding site. The effect of calmodulin and its antagonist trifluoperazine was also investigated using purified and membrane-associated enzyme. When membrane-bound enzyme was measured with exogenous phosphatidylethanolamine, small stimulations by calmodulin were found. However, these were not believed to indicate a specific role for calmodulin in the Ca2+ dependency of the phospholipase A2, since trifluoperazine did not lower the activity of the membrane-bound enzyme to levels below those found in the presence of Ca2+ alone. Membrane-bound enzyme in its action toward endogenous phosphatidylethanolamine was neither stimulated by calmodulin nor inhibited by trifluoperazine. Purified enzyme was also not stimulated by calmodulin, while trifluoperazine caused small stimulations, presumably due to interactions at the substrate level. These results indicate that calmodulin involvement in phospholipase A2 activation should not be generalized.

Animals↗